HURJ Volume 25 - Spring 2021

HURJ hopkins undergraduate research journal

Hopkins Undergraduate Research Journal

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“THERE ARE NO SMALL JOBS AT SSP” Vice Admiral Johnny R. Wolfe, Jr. Director, Strategic Systems Programs 2 2

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Hopkins Undergraduate Research Journal Hopkins Undergraduate Research Journal

HURJ

hopkins undergraduate research journal

Spring 2021

Table of Contents 06 08 16

Letter from the Editors

STEM & TECHNOLOGY Hospital Operating Rooms Scheduling Problem; Analyzing and Optimizing Utilization Shengwei Zhang

Correlation between Fragile X Mental Retardation Protein (FMRP) and Metabotropic Glutamate Receptor Subtype 5 (mGluR5) in Fragile X Syndrome (FXS) Samuel D. Martin

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The Two-Way Highway Between the Gut and The Brain: How Microbiota Shape Neural Function Sid Kavuturu

Assessment of Biodegradation of Trimethoprim and Sulfamethoxazole in Anaerobic Digestion of Animal Manure Melody Multra

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Subcellular Fractionation of Immune Cell Organelles for Synthetic Cellular Biology Jody Mou, Joshua Devier, Prof. Joshua C. Doloff

HUMANITIES & SOCIAL SCIENCES

43

African Americans in Pharmacogenetics: Opportunities for Care Improvement

48

The Village Panchayat as an Inspiration for the India-Pakistan Conflict

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The Authenticity of the Letter of Thomas Aquinas to Brother John: A Natural Language Processing Analysis

Elise Moore

Riya Jain

John Schindler

62 67 75 83

Understanding the Drop Out Decision: A Dynamic Optimization Model Serena Goldberg

U.S.-India Maritime Strategic Cooperation Avi Kirpekar

The Hybridization of Modernity and Tradition in the Age of Indian Matrimonial Websites Jemina Opman-Auge

HU RJ E di tori a l B oa r d

LETTER from the EDITORS

Hopkins Undergraduate Research Journal (HURJ) is the first undergraduate research journal at Johns Hopkins University and is one of the oldest undergraduate research journals in North America. Through its publications, HURJ aims to celebrate and share undergraduates’ diverse research pursuits in the sciences, humanities, social sciences, and engineering.

Despite these unprecedented times, our editorial staff and student authors have worked collaboratively and industriously throughout this year to showcase authors’ research projects.

From an array of outstanding student submissions, our editorial board selects, edits, and formats articles that emphasize the hard work and passion surrounding undergraduate research at Hopkins. Although the pandemic limited research opportunities, through this issue, we hope to demonstrate the adaptability and resilience of these student researchers and to honor their various intellectual pursuits.

This year, we have made several changes to how our issue was compiled. We implemented copy editing workshops for our editors to ensure consistency and to emphasize the importance of clarity and readability in scientific communication. For the first time, we also have enlisted faculty and graduate students as external reviewers to provide subject matter expertise on the research articles. We hope that these changes will foster discourse within the research community at Hopkins and contribute to the scientific growth of our undergraduate researchers.

We would like to extend a special thanks to our editorial staff and contributing authors, whose dedication and diligence were instrumental in assembling this issue. We would also like to express our sincere gratitude to our faculty and graduate reviewers who volunteered their time to provide valuable feedback during the editing process.

We welcome all comments, feedback, and questions, which can be addressed to thehurj@gmail.com. We hope that you join us to celebrate the achievements of our undergraduate researchers and find inspiration in pursuing your own research endeavors.

Sincerely, Sandeep and Grace Co-Editors-in Chief

Sandeep Kambhampati Co-Editor-in Chief

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Grace Kim Co-Editor-in Chief

STEM & TECHNOLOGY

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Hospital Operating Rooms Scheduling Problem; Analyzing and Optimizing Utilization A B S T R A C T:

Shengwei Zhang

Operating rooms (ORs) are one of the main resources in a hospital, and any disruption in their workflow can have

a cascade effect on the rest of the hospital operations. In this study, the problem of OR scheduling was approached from two perspectives.

First, utilization metrics to comprehensively evaluate OR utilization are defined, and applied to a retrospective OR data set from a large academic medical center. Second, a mathematical optimization model to schedule surgical cases to improve OR utilization is defined. Later the pre-defined utilization metrics are employed to validate the model effectiveness.

1. I N T R O D U C T I O N

Operating rooms (ORs) are among the most critical and in demand resources in hospitals, and any disruption in their workflow can have huge downstream effects on the capacity of differerent units ranging from Post-Anesthesia Care Units (PACUs) to Intensive Care Units (ICUs). This is more important during a pandemic when there is an expected surge in the demand of ORs and different units, especially ICUs. As a result, maximizing efficiency in utilization of such resources is paramount.

Since many factors contributing to the utilization effciency of ORs, investigating methods that effectively improve the effciency in the use of ORs is a complex task.

In this paper, the historical OR utilization data and patterns in various sites of a large academic medical center in the US is explored and a mathematical model that aims to optimize the OR scheduling process and maximize utilization and efficiency is proposed.

Existing data on the utilization of ORs shows how different services adhere to the master block schedule. Analysis of such past 8

utilization data sheds light on the current state of utilization per site, OR, and service. In addition, it highlights services that may need different amount of time on the master block schedule, rooms that may require alternate operating hours, and the extent that OR utilization was planned.

Consequently, it shows whether or not the actual utilization aligns with the planned one. Such analysis provides utilization metrics that can be used as parameters for a mathematical model that can optimize OR utilization and smooth out downstream effects and demand on capacity. A large deviation from the planned utilization signals cultural, operational, and local obstacles that hinder better OR utilization even if the OR scheduling was the best possible.

Utilization analysis of ORs is even more important during a high demand period like the COVID-19 pandemic. Many hospitals experience higher demands as a direct result of the increasing number of COVID-19 cases, which restrict the downstream capacity for ORs and can potentially create more OR

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holds (Murray, 2020). The Institute for Health Metrics and Evaluation’s (IHME) projection indicates that the US experienced a peak usage of beds, ICU and ventilator resources in the mid April 2020 and the trend predicts another peak in the fall (COVID-19, 2020). Even though the demand for elective surgical services has decreased during the pandemic, this decrease is temporary and with the reopening of elective ORs, there will likely be a higher-than-usual demand due to elective cases that were delayed since March 2020.

Additionally, the increased usage of Personal Protective Equipment (PPEs) and heightened precautions has slowed down the operations for surgeons, supporting sta, and the cleaning crew (Al-Jabir et al., 2020). Therefore, increasing OR usage efficiency is essential in the future.

This work provides a retrospective analysis of existing OR utilization data for a large academic medical center and numerous analytical insights to the existing data. Then, metrics that can be used to assess the performance of the OR with respect to utilization efficiency are indicated. Later a

STEM & TECHNOLOGY

case scheduling model that maximizes utilization and efficiency of ORs is developed. Those utilization metrics will be applied to test the case scheduling model to evaluate the model effectiveness.

2. P R O B L E M S E T T I N G

In this section, the general layout of the operating room scheduling problem is outlined.

Specifically, detailed information is provided on the data gathered on a case study hospital for this work. Firstly, the set-up of the hospital considered in this work, including the infrastructure and the OR scheduling strategies and policies, is described. Then, details of the current scheduling scheme and case distribution data of the hospital are provided.

The case study hospital considered in this work is a large academic medical center in the US with 1,154 acute care beds, 2,197 full-time physicians, and 47,903 inpatient admissions per year. The focus is on five operating sites located in this hospital, which are denoted here as Site 1 to Site 5 where Site 1 is the outpatient unit of the hospital. Table 1 represents the number of ORs per site.

The hospital adheres to a master block scheduling strategy. The default operating hours for each room is from 7:30 am to 5:00 pm during weekdays. If any room has different operating hours, this information is included in the master block schedule of that room. Additionally, in the master schedule, some blocks are further divided into sub-blocks.

For example, Room 03 in Site 5 is scheduled for Service F on the 1st and 4th Wednesdays of each month and Service G on the 2nd, 3rd and 5th Wednesdays.

In addition to the above master block schedule,

the hospital adopts a modified block scheduling strategy to improve OR utilization.

The hospital follows the original master block schedule up to seven days with slight variations from service to service before the actual surgery date.

Then, any remaining unscheduled blocks are “freed up”. For instance, if R01 is scheduled for a specific surgeon from 12–5 pm and the actual schedule is filled up from 12–2 pm one week prior to that date, then the block from 2–5 pm is freed for any other operation. This approach is expected to increase the utilization of the operating rooms and add extra degrees of flexibility for surgeons.

In order to contextualize our methods, historical case data spanning over three months from the hospital including each case’s urgency level, the date time of the case, and OR being used, and the service of the case was collected.

Row 2 in Table 2 illustrates that the five sites have a total number of 8, 992 unique cases during the three-month time-frame. It is worth mentioning that it is possible for one patient to attribute to more than one case in this data set. Additionally, cases that occurred during weekends and holidays are excluded, as shown in Table 2 since weekends often follow a different scheduling scheme and mainly consider urgent cases. This exclusion included a total of 348 cases spanning over the weekends and holidays.

Additional general information on the cases is provided in Table 3.

3. U T I L I Z A T I O N ASSESSMENT M E T R I C S

In order to develop an OR performance optimization model, metrics that characterize optimal performance need to be defined.

In this section, two widely used metrics to comprehensively evaluate OR performance are provided. The first metric answers Room Utilization, how well the ORs are utilized during the operating hours. The second metric considers Block Utilization, how well each service adheres to the planned time it has been allocated to. Each of these metrics are further discussed in this section. Then, an optimization problem designed to optimize the performance of the ORs based on these metrics is formulated.

In the setting of the OR scheduling problem, utilization is measuring the efficiency and performance of each OR. Typically, utilization represents what percentage of the operating hours of OR is occupied for service. By analyzing utilization of each room based on specified metrics, it is possible to have a clearer picture of whether the ORs performed efficiently and whether there are any rooms for improvement. This section defines different utilization measures. The utilization is measured in two settings of Room Utilization in Section 3.1, and Block Utilization.

3.1 Room Utilization

Room Utilization focuses on the amount of time that the OR is actually used during operating hours and it is defined in EQUATION 1:

In the equation above, the term Total operating hours refers to the total hours that the OR is usable per day: 9.5 hours if not specified otherwise.

Occupied time denotes the time span during which the room was occupied during the operating hours.

It is the summation of three components: Surgery time, OR hold time, and Turnover time. Surgery time is the sum of the duration of the procedures conducted in the room.

OR hold time refers to the duration between the end of a procedure

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and the time when the patient is moved out of the OR. This time is usually used to specify a PACU/ ICU/bed for the patient based on ongoing downstream resource usage.. Turnover time is the time used for cleaning up after the latest procedure and preparing for the upcoming case. Based on expert advice, 30 minutes is considered for the turnover time between every two cases, except for the last case of the day that starts during the operational hours.

Figure 1 illustrates a sample schematic of the operating hours in an OR. The gray bar represents the total operating hours during which three cases are happening (Case 1, 2, and 3), and this bar will be the denominator term in the room utilization formulation. The three braces in the figure represent the duration each case occupies the room, respectively. However, since only the operating hours from 7:30 am to 5 pm are considered, Case 1 starts early, and Case 3 runs overtime.

Thus, the run-over and start-early time is excluded, and denoted as Over time. Over time represents how much each room used goes past its specified operating time. The average room utilization for each of the 62 ORs is reported in Section 5.1.

3.2 Block Utilization

On a more granular level than the entire operating hours of each room, an additional point of interest is how accurately each room follows the block schedule and how well each service uses its allocated time. To this end,two different types of utilization measures are calculated. The first is for each OR to show how well it adheres to the block schedule, and second for each service in each site to show how accurately this service follows its specified schedule.

3.2.1 Room-Block Schedule

Two types of utilization for block utilization per room are 10

defined. The first one is In-block Utilization which is defined in Eq. (2) and indicates how well each service utilizes the allocated room and time. The second is Out-block Utilization which is defined in Eq. (3) and represents the amount of time that each service utilizes from other blocks specified for other operations.

EQUATIONS 2-3:

3.2.2 Service-Block Utilization

In addition to utilization

at room level, another point of interest is how each service utilizes the OR and the time assigned to that specific service. As a result, two more utilization metrics are defined. The first one is Good Utilization which is defined in Eq. (4) and measures how well each service utilizes the time and room allocated for it. The second one is Total utilization which is defined in Eq. (5) and represents each service’s overall utilization of the room and time, regardless of whether it follows the block schedule or not.

Where (i) Urgent hours refers to the total working hours for all urgent cases; (ii) Spill-over refers to the time that an in-block case runs-over/starts-early. Good Utilization includes urgent hours since the nature of urgent cases makes them hard to be in-block and maintain some tolerance in Block Utilization analysis. Good Utilization includes Spill-over hours because, even though the hours are beyond the block, these hours are part of the in-block cases. Results of In-block/Out block/Good/Total Utilization are reported in Section 5.2.2.

4. P H A S E D OPERATING ROOM SCHEDULING METHODOLOGY

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This study designs a scheduling model focused on midrange time-frames (e.g., two weeks).

Due to the dynamic change of available resources in the course of the analysis period, a phased model that schedules cases on a daily basis was considered. For each day, a List Scheduling Algorithm for case selection is applied based on the limited resource capacities. Then, with the aim of increasing overall in-block Utilization, a Mixed-integer Programming (MIP) formulation that is able to optimally schedule the given cases. The objective function of this MIP is to minimize the maximum OR idle time among all the ORs while following the master block schedule.

It is assumed that the master block schedule and resources capacities are given. In addition, resources including PPEs, ICUs, and beds are taken into account.

The aim is to schedule all cases without conflict by considering the required resources and available times, increase overall in-block utilization and decrease out-block utilization, and ensure comparable room utilization in each OR and day. It is also assumed that case duration times and master block schedules are also given.

The input of the proposed model consists of a list of candidate cases proposed by the surgeons.

After the list of cases to be scheduled in two weeks is determined, each Case Window is specified. The Case Window is defined as the range of operating days in the two-week time frame that the specified case can be scheduled in. Some Case Windows are tight and correspond to cases that must be operated on certain days. On the other hand, some may be flexible and the window can be any day in the two-week timeframe. Case Windows will appear as constraints in a three-phased model. Phases will be considered in sequential order and the output of

STEM & TECHNOLOGY

the last phase will serve as the input to the next phase. At each round, a one-day schedule is produced and sequentially, all subsequent one-day schedules are generated from the first day to the last day of the time frame.

4.1 Phase 1: Selection of Daily To-Be Scheduled Cases

For each daily cycle of analysis, the cases that should be scheduled throughout the day are listed. List Scheduling Algorithm completes this process. This algorithm provides an ordered list of operations by assigning them priorities. Then, cases are chosen for scheduling using the following iterative steps.

(1) Select the operation with the highest priority. (2) Select a resource to accommodate this process. (3) If sucient resources are not found, move to the next operation in the list. The List Scheduling Algorithm is applied to the master block schedule in order to prioritize operations and provide a list of operations to be scheduled for a given day. After all the cases on the list are traversed once, a list of to-be-scheduled cases will serve as input to the next phase.

4.2 Phase 2: Mixed-integer Programming Optimization

Phase 1 results in a list of cases to be scheduled. This list serves as input for an optimization model that aims to maximize utilization. As mentioned earlier, a mixed integer programming formulation schedules all cases to all the operating ORs. The first provides a list of the parameters of the model as follows: • i : case number, i = 1, 2,...,I • r : OR number, r = 1, 2,...,R • Ir : the set of all cases OR r will operate on the day, r = 1, 2,...,R • ti : the duration of case i plus turnover time. The turnover time is considered 30 minutes after each case by standard, i = 1, 2,...,I

• Hr : the operating hour of OR r, r = 1, 2,...,R • zr : the total idle time for OR r, r = 1, 2,...,R • zmax : the max idle time among all the ORs • xir : decision parameter, if case i is operated in OR r ,xir = 1, Else xir = 0 The optimization model is as follows:

removes the cases operated during overtime from the list of cases to be scheduled on that day or extends OR operating hours to prevent delays on cases with short Case Windows.

These adjustments help determine a final one-day schedule of

EQUATIONS 6-12:

where constraint (6) ensures that each case is scheduled exactly once; constraint (7) is placed to limit the total surgery time of all cases in the specified day to be less than or equal to the total operating hours of all ORs; constraint (8) ensures that one OR’s to-be-scheduled case cannot be scheduled in other ORs; constraint (9) sets the value of the idle time for each OR; constraint (10) set the maximum OR idle time variable and finally, constraint (11) ensures non-negativity of the idle time variables. This model provides a solution for case schedules that minimized the maximum idle time of all of the ORs.

4.3 Phase 3: Further Case Adjustment and Resource Update

If the above optimization model is feasible, the optimal solution will minimize the maximum idle time between ORs. However, it is possible that some cases are not scheduled at all, which means case i can’t fit in any OR during that day. In this case, a further approach repeatedly adjusts the parameters of total operating hours (Hr) to be longer and run the optimization model again until there is a specific assigned OR for each operation. Then, this approach

cases and their corresponding ORs. As a result, the resource availability information can be updated based on the results.

The updated resource availability information can be used in Phase 1 for case scheduling the upcoming day. Iterating this process will yield a schedule for all business days.

5. R E S U L T S

The results of applying the proposed methodology to the existing data are provided in this section. Section 5.1 provides a detailed description of utilization for each room in each site. Section 5.2 compares the in-block and out-block utilization for each room to see how they follow block schedules. Also, this section presents utilization rates for each service in each room for each site. Section 5.3 applies the case scheduling model to a subset of historical case data to test the performance of our model against existing schedules.

5.1 Room Utilization

Room utilization results are provided for each room. As stated earlier, higher room utilization is desirable in our setting. The average room utilization of all five sites is

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STEM & TECHNOLOGY

desirable in our setting. The average room utilization of all five sites is 72.2%. Row 2 of Table 4 details results for each site. Site 1, 3, 4, 5 have lower utilization than Site 2. However, higher utilization for all 5 sites is expected. Figure 2 provides more granular analysis of the utilization in each room in Site 3 and Site 5. Figure 2 shows the average utilization of all rooms (except R07 for Site 5 and R15 for Site 3 since these two rooms are only used for special cases) for each site. Since R12 in Site 3 is used for emergency cases its utilization is low compared with other rooms.

In conclusion, ORs that are designed for emergency use are more likely to have significantly lower utilization. The next section analyzes whether the five sites are coherent with the block schedule.

5.2 Block Utilization

Section 5.2.1 provides detailed results on in-block and out-block utilization for each room on each site. Section 5.2.2 shows results for the in-block and total utilization for each service. Ideally, in-block utilization should be as close to total utilization as possible (the service follows the block schedule well), and both should be 100% (the service does not waste any assigned time, neither does it occupy time not assigned for it).

5.2.1 Block Utilization of Rooms

Table 5 shows the block

utilization results for each site.

Site 1 and Site 2 do not follow the block schedule well, with 42% and 39% out-block utilization, respectively, which are even greater than their in-block utilization. Site 3, Site 4, and Site 5 have in-block utilization greater than out block utilization but do not adhere to the block schedule well either, with around 20% out-block utilization.

Figure 3 provides a visual indication of block utilization for 12

each room in Site 3 and Site 5. In this figure, in-block utilization, out-block utilization, overtime percentages (the percent of time for the cases that begin during the operational hours, and run over), and average in-block and out-block utilization are presented for each room.

Figure 3 shows that Site 3 and Site 5 have significant outblock utilization, even though their in-block utilization is higher than their out-block utilization. R03 in Site 5 has a lower in-block utilization and higher out-block utilization than the averages. This is possibly due to the fact that the schedule for R03 is more complicated than other rooms. For example, R01 is scheduled for one service for all working days, while R03 is scheduled for different services every weekday.

This means that rooms dealing with one specific service are not likely to be scheduled for other services.

This adversely affects utilization

“Our methods and definitions could serve as a reference for future studies in this field.” because other rooms may be used for multiple types of services. A similar argument can be stated for the block utilization results of Site 3.

As expected, the ORs that are designed for urgent cases or have a complicated schedule would have significantly low inblock and high out-block utilization.

In the first case, not much can be done since there needs to be space for urgent use. In the second case, the complexity of the master block schedule for these specific

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rooms draws our attention. An optimization model will help better schedule surgical cases for sites that exhibit complicated schedules.

5.2.2 Block Utilization for Services

The main goal block utilization for services considers is whether low in-block utilization is caused by specific services that do not follow the schedule well, or by poor in-block utilization of all services. In addition, this study aims to distinguish whether each service needs more time than they are assigned to or if they actually require less scheduled time. In summary, this section explores in block and out-block utilization for each service, and the time each service needs compared with the average assigned for it.

Figure 6 is a breakdown of block utilization for all services in Site 3 and Site 5 and represents in-block utilization, good utilization, and total utilization for each service.

This figure shows the total number of hours assigned for each service and comparing in and out block utilization of each service. Also, it is useful to look at the percentages listed at the right of each bar, which indicates the percentage of time each service needs based on the time that it is scheduled for, to check whether more than sucient time is allocated for a particular service. If the percentage is greater than 100% (red vertical line in graph), it means that the service needs more time than scheduled. If the percentage is lower than 100%, it means it wastes some of its assigned time.

Figure 4 shows that services 12, 16, 34, 29 have significantly low in-block utilization in Site 3. This is a result of using the rooms assigned for these services for shared operations with other services.

In other words, the rooms are scheduled for these services a few weekdays during the time frame and are assigned for other services for the remainder of the time services. This

results in a more complicated scenario for the rooms and contributes to causing lower inblock utilization. On the other hand, most services in Site 5 have higher in-block utilization compared with Site 3. Lower in-block utilization is expected for Service 35 (Trauma).

Looking at the block schedule for Site 5, all rooms except R03 are assigned for one service only during all weekdays. This, and the point that Site 5 handles fewer services than other sites, causes less confusion while scheduling for Site 5 and results in higher in-block utilization.

Services in Site 1 and Site 2 have in-block utilization of around 20% to 30%, and some urgent services have lower in-block utilization. In summary, the results show that sites with fewer types of services have higher in-block utilization than sites with various types of services, and urgent services usually have lower in-block utilization.

5.3 Scheduling Model Validation

This section tests the scheduling model on historical cases in a two-week timeframe in one site in order to validate the model.

Cases that took place in Site 5 during the time-frame of 05/13/2019 to 05/24/2019 (10 operating days in total) were chosen. In Figure 5, the left diagram is the room utilization analysis of the original schedule and the right diagram is the room utilization analysis of the new schedule resulting from the model. Average In-block Utilization increased by 11%, from 57% to 68%. Average Out-block Utilization decreased by 5%, from 17% to 12%. Average Over time percentage decreased 9%, from 12% to 3%, and Average Room Utilization increased 6%, from 74% to 80%.

Moreover, the Total Utilization rate in each OR is more comparable, without rooms having extremely long/short idle time in two weeks.

In addition, the service utilization of the new schedule and of the original schedule was compared.

Figure 6 shows that the average Service In-block Utilization increased 7%, from 70% to 77%; the average Service Good Utilization increased 8%, from 77% to 85%. The goals of the model do not include improving service utilization, but the result actually reveals an improvement.

This is because the service Inblock Utilization is correlated to the room In-block Utilization.

Thus, this scheduling model has good applicability in achieving multiple optimization goals.

This utilization comparison analysis demonstrates that this case scheduling model is effective and achieves the three goals mentioned in Section 4.1. However, this analysis only tests a small portion of the total cases in one site. There is a need to further test the model robustness with the whole threemonth case data in each site. In addition, there are some inherent limitations to the scheduling model, and the model testing process does not comprehensively simulate the case scheduling process in real life.

6. D I S C U S S I O N & CONCLUSIONS

This section points out some limitations of the scheduling model in Section 4 and summarizes the work on operating room planning and scheduling.

The model testing results show an improvement of each utilization metric, but do not fully reflect the real-life scheduling by medical coordinators based on two reasons. First, the resource (PPEs, ICUs, and beds) capacity and the consumption of resources for each case in the model construction are considered, but not in model testing. Thus, the next immediate step of the research is incorporating the resource parameter into model

testing once the resource data for each case is available. Second, In Section 4, the Case Window for elective cases is calculated based on the actual case operating date, assuming that each elective case operates in accordance with the schedule. However, the actual case operating date is not necessarily consistent with the initial scheduled date, since sometimes elective cases get postponed when urgent cases come up in the last minute and take their blocks. Thus, there is a need to further revise the model testing method that can simulate the real-life situation by considering limited case information.

This paper exhibits an overall picture of the contemporary operating room planning and scheduling research. By summarizing prior work both in the medical community and in academia, widely used concepts and methodologies into a large academic medical center in the US are applied. The research presented operating room utilization analysis from multiple perspectives, including operating room, service, and the adherence of each service to the master block schedule. It also introduced various utilization metrics, which enables a more comprehensive interpretation of operating room utilization. The methods and definitions could serve as a reference for future studies in this field. After the utilization metrics were defined, a case scheduling model that can e ciently schedule elective cases to days and ORs in a mid-range time frame was developed. The scheduling model also takes PPEs and downstream resources into consideration, which will assist hospitals better manage medical resources, especially during this critical time, when the World Health Organization officially declared a public health emergency of international concern. officially declared a public health emergency of international concern.

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REFERENCES

1. Al-Jabir, A., Kerwan, A., Nicola, M., Alsafi, Z., Khan, M., Sohrabi, C., O’Neill, N., Iosifidis, C., Griffin, M., Mathew, G., & Agha, R. (2020). Impact of the Coronavirus (COVID-19) pandemic on surgical practice - Part 2 (surgical prioritisation). International journal of surgery (London, England), 79, 233–248. https://doi.org/10.1016/j. ijsu.2020.05.002 2. Institute for Health Metrics and Evaluation. IHME: COVID-19 Projections.

COVID-19. https://covid19.healthdata. org/united-states-of-america?view=socialdistancing&tab=trend.

3. Murray, C. J. L. (2020). Forecasting COVID-19 impact on hospital bed-days, ICU-days, ventilator-days, and deaths by US state in the next 4 months. MedRxiv. https://doi.org/10.1101/2020.03.27.20043 752

TABLES

Table 1: Number of operating rooms per site Site Number of ORs

1 8

2 16

3 15

4 16

5 7

Table 2: Population distribution among the five considered sites before and after exclusion of weekends and holidays.

Site Number of Cases Number of Cases after Exclusion

1 1,554 1,554

2 2,205 2,201

3 2,116 1,919

4 2,364 2,241

5 753 729

Total 8,992 8,644

Table 3: Distribution of case classification in the five considered sites.

Site Number of Elective Cases Number of Urgent Cases Total Cases Total Inpatient Cases Total Outpatient Cases

1 1,552

2 2,140

3 1,520

4 1,880

5 638

2

61

399

361

91

1,554 16 1,538

2,201 978 1,233

1,919 1,468 429

2,241 787 1,423

729 612 117

Table 4: Room Utilization Site Room Utilization

1 69%

2 80%

3 74%

4 68%

5 70%

Table 5: Room Utilization and in-block, out-block utilization Site In-block Out-block

1 30% 39%

2 38% 42%

3 53% 21%

4 43% 25%

5 51% 19%

SHENGWEI ZHANG

Shengwei is currently a third-year undergraduate studying Applied Mathematics and Computer Science. With an academic interest in data science, he is experienced in applying data science techniques to solve healthcare analytics problems, including optimizing the operating room schedule at Johns Hopkins Hospital and using Reinforcement Learning to predict COVID patient trajectory. Shengwei is passionate about undergraduate research at Hopkins because he values the opportunity to engage in a real-world project and see how his effort will make a difference in the organization.

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STEM & TECHNOLOGY

Figure 1: An illustration of a sample schematic of OR activities during operating hours

Figure 2: Room Utilization for Site 3 and Site 5

Figure 3: Block Utilization for Site 3 and Site 5

Figure 4: Block utilization for services at Site 3 and Site 5

Figure 5: Comparison of Room Utilization in Site 5 (date: 05/13/2019–05/24/2019).

Figure 6: Comparison of Service Utilization in Site 5 (date: 05/13/2019–05/24/2019).

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Correlation Between Fragile X Mental Retardation Protein and Metabotropic Glutamate Receptor Subtype 5 in Fragile X Syndrome S a m u e l D. M a r t i n

A B S T R A C T:

BACKGROUND: Quantifying the relationship between Fragile X Mental Retardation Protein (FMRP) and metabotropic glutamate receptor subtype 5 (mGluR5) expression in the living human brain of people with fragile X syndrome (FXS) is essential for understanding past failed results of clinical trials in FXS. METHODS: Eight participants, including six adult men with the full mutation of the FMR1 gene (FXS), one man with mosaicism of the FMR1 gene (FXS-M), and one woman with typical development and a premutation of the FMR1 gene (TDPM), underwent quantitative measurements of FMRP by polymerase chain reaction (PCR)/Southern Blot testing and mGluR5 by positron emission tomography (PET) with 3-[18F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB).

RESULTS: FMRP levels appear to be positively correlated to mGluR5 expression in the living human brain, and this pattern appears to be consistent across all measured brain areas.

LIMITATIONS: FXS affects a small portion of the population, and so recruiting participants presents financial and logistical constrictions. The small sample size provides a basis from which future studies may build; however, external validity is limited for the findings of this study.

CONCLUSIONS: The reductions of FMRP and mGluR5 for FXS subjects in key cortical and subcortical regions provides a basis for the neurobehavioral phenotype of FXS. Understanding this relationship could provide the basis for the development of novel interventions for FXS.

I N T R O D U C T I O N

The neurobehavioral phenotype of fragile X syndrome (FXS), which includes hyperactivity, impulsivity, anxiety, poor language development, and seizures, (Budimirovic & Kaufmann, 2011, Budimirovic & Kaufmann, 2016) has been reliably linked to a mutation of the fragile x mental retardation 1 (FMR1) gene, which results in an increase in CGG repeats in the promoter region of that gene (Bardoni, 2001). Individuals with typical development have between 16

5 and 45 CGG repeats, and people who have between 60 and 199 CGG repeats are classified as having a premutation of the mutation of the FMR1 gene. The presence of at least 200 CGG repeats is characterized as a full mutation of the gene and results in epigenetic silencing of the FMR1 gene via hypermethylation (Bardoni et al., 2001).

Silencing the FMR1 gene leads to a pronounced reduction of the main product of the gene, fragile X mental retardation protein (FMRP) (Brasic, et al., 2020). FMRP

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is an RNA binding protein with critical roles in the maturation of dendritic spines as well as protein synthesis at those dendritic spines. In fulfilling these roles, FMRP has been demonstrated to control around four percent of mRNA translation in the human brain (Bardoni et al., 2001). Reducing or eliminating FMRP is believed to result in overactive glutamatergic signaling, which affects the activation of downstream targets and results in excess protein synthesis (Bagni & Zukin, 2019, Soong et al., 2008). This hypothesis

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is well tested in FRM1 knockout (KO) of mouse models with FXS and is the substance behind the “mGluR theory” of neuronal dysfunction in FXS. This theory suggests that there is a strong connection between irregular protein synthesis at metabotropic glutamate receptors in FXS and the neurobehavioral deficits of FXS (Bear et al., 2004).

Because of its apparent control over such irregularities, FMRP expression in the brain is believed to be the most influential factor in determining the severity of cognitive deficit and problem behaviors associated with FXS (Budimirovic & Kaufmann, 2011, Budimirovic & Kaufmann, 2016).

However, many questions remain about the expression of the glutamate receptor associated with FXS: metabotropic glutamate receptor subtype 5 (mGluR5). For example, human autopsy studies and animal imaging studies have reported inconsistent values for mGluR5 expression, with some reporting increased signaling and others reporting reductions in activity (Budimirovic et al., 2017). In studies of KO mouse models in particular, many of which have been utilized to guide the design of clinical trials, mGluR5 expression was reportedly increased (Dahlhaus, 2018). However, in a PET imaging study designed to assess cerebral protein synthesis in the human brain, researchers found that subjects with FXS had decreased neuronal activity associated with application of the sedative propofol, which resulted in decreased protein synthesis (Qin et al., 2013). Therefore, it is possible that depressed mGluR5 expression is responsible for the symptoms observed in FXS.

To determine the true nature of neuronal signaling in the brains of people with FXS, unsedated individuals with both typical development (TD) and FXS must undergo in vivo quantification of mGluR5 expression using PET imaging techniques.

Furthermore, it is vital to obtain this measurement in the context of FMRP concentration to identify a possible relationship between these consequential variables.

M E T H Participants:

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Six adult men aged 21-35 years (mean 27.1, SD 4.7) with the FM of the FMR1 gene (FXS), one male aged 57 with a mosaicism of the mutation (FXS-M), and one female aged 56 with typical development (TD) were recruited for this study.

All participants were recruited and tested at the Institute for Neurodegenerative Disorders (IND) under the guidance of Dr. David Russell.

Testing:

PCR/Southern Blot (Cai et al., 2019) testing was utilized to quantify the level of mutation and confirm the characterization of each participant’s condition. FMRP values were obtained for each participant through venous blood sampling (LaFauci, 2016).

Participants underwent positron emission tomography (PET) with an mGluR5-specific radioligand, 3-[18F]fluoro-5-(2pyridinylethynyl)benzonitrile ([18F] FPEB). This radioligand is a highly potent and selective antagonist of mGluR5 receptors, and it has been demonstrated effective for use in quantifying mGluR5 expression in humans (Wong, 2013).

PET imaging measurements with FPEB indicate mGluR5 density, which serves as a representation of mGluR5 expression in key cortical and subcortical brain regions.

Participants with FXS were prepared for PET scans with mock scanner training performed by behavioral psychologists. The participants were also stabilized on the scanner bed with gauze bands, which were applied by nuclear medical technologists and

designed to hold their heads in the same position throughout the scan.

All participants then underwent MRI imaging, during which caregivers and parents of FXS subjects were allowed to stand by the scanner bed for support. Following this, participants were positioned appropriately on the PET scanner bed and received 185 megabequerels (MBqs) [5 millicuries (mCis)] of [18F]FPEB through intravenous bolus injections (Wong et al., 2013). Participants underwent PET imaging for 30 minutes after radiotracer injection on an ECAT EXACT scanner (Rahmim, 2005).

Concentration values were quantified in terms of binding potential (BPND) (Figure 1); however, scans at IND were initially measured in terms of standardized uptake value ratio (SUVR). Conversions of the SUVR values to BPND approximations were made by subtracting one from the SUVR values (SUVR-1) (Carson, 2003).

Data Analysis:

The [18F]FPEB uptake values were plotted against the FMRP concentration for all brain areas on one plot. This served to outline the general relationship between FMRP and mGluR5 expression across all brain regions.

To measure how consistent this pattern was throughout the brain, the slope of plots representing each of the individual brain areas were calculated and plotted with the uncertainty of each slope calculation represented by an error bar. The slope for each brain area was compared to the average slope across all brain areas, and the results were represented in Figure 3.

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PCR/Southern Blot testing confirmed FM (≥200 CGGs) for the six adults males with FXS, with a range of 600-1600 repeats. The adult male with mosaicism of the mutation (FXS-M) was confirmed to

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have 181 CGG repeats, which results in 20% methylation of the FMR1 gene. The adult female with typical development of the FMR1 gene (TD) was found to have 35 CGG repeats.

FMRP values for the six adult males with the FM of the FMR1 gene were 0.00-0.12 (mean .047, SD 0.04) ng/mcg of total protein.

The FMRP value for the FXS-M participant was .24ng/mcg of total protein and .87ng/mcg of total protein for the TDPM participant.

MRI imaging was completed with no major issues. The use of the mock scanner to prepare FXS participants for the loud noises of the MRI scanner was justified by the observed comfort of the participants during the scans. PET imaging was successfully carried out for the FXS participants with the proper precautions and preparations taken to ensure as comfortable an environment as possible during the scans. Vocal encouragement from parents, caregivers, and researchers was instrumental in ensuring this comfort.

A plot containing FMRP values on the x-axis and [18F] FPEB uptake on the y-axis reveals a possible positive relationship between FMRP and mGluR5 expression in various brain regions.

A linear fit to the data suggests a slope of around 1.05 ± .05 (SEM), with an R2 value of approximately .25 (p-value = 0.21) (Figure 2). Analyses of each individual brain area was conducted by calculating the linear fit of [18F]FPEB uptake and FMRP concentration, and the resulting slopes for each brain region are plotted in Figure 3. The calculated slope ± the uncertainty of our slope calculation for each brain area overlaps with the average slope across all brain areas included in this study, which suggests some uniformity in the relationship between FMRP and mGluR5 expression across the brain.

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D I S C U S S I O N

This study, along with related studies conducted at IND and Johns Hopkins University (JHU), (Brasic et al., 2020) marks the first time that mGluR5 expression has been quantified in the living human brain with FXS, demonstrating that MRI/PET imaging techniques are feasible methods of measurement in individuals with FXS (Cox et al., 2017).

This study reinforces the finding that

the proposed overactivation of downstream, glutamatergic signaling. These trials suggested an increased level of mGluR5 expression in patients with FXS. In these trials, the use of negative allosteric modulators (NAMs) of mGluR5s was employed with successful behavioral results for FMR1 KO mouse models of FXS. However, the application of NAMs to downregulate mGluR5s in the human brain with FXS did not achieve the

“Because of its apparent control over such irregularities, FMRP expression in the brain is believed to be the most influential factor in determining the severity of cognitive deficit and problem behaviors associated with FXS.” reduction in mGluR5 expression, as observed in the IND/JHU study, leads to lower levels of FMRP in FXS subjects, as levels of FMRP were approximately a standard deviation lower in the men with FXS in contrast to the healthy controls for key cortical and subcortical regions (Brasic et al., 2020). This contradicts the results of previous studies in mouse models of FXS (Bear et al., 2004).

A novel finding in this study is the possible relationship between FMRP and mGluR5 expression. The ubiquity of this apparent relationship in all brain regions measured suggests a large role for both FMRP and mGluR5 in FXS. Although not statistically significant in this study, future studies with larger sample sizes can be conducted to confirm and further explore this relationship to better understand how these variables interact with one another.

Clinical trials guided by the results of animal studies targeted

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same success, and the results of our study provide a possible explanation as to why that may be (Jong et al., 2019). The mechanism by which these NAMs operate is by binding allosterically to mGluR5s as noncompetitive antagonists, reducing the likelihood of post-synaptic excitation (Emmitte, 2013). Therefore, it is unsurprising that decreasing the already depressed mGluR5 expression in the brain of humans with FXS does not result in positive behavioral or cognitive results.

Another possible mechanism by which mGluR5 expression may be reduced in individuals with FXS involves the functional structure of mGluR5s and their supporting protein structures. A study analyzing the nature of mGluR5s in FXS suggested that the disorder can be characterized by a decrease in connectivity between mGluR5s and Homer proteins, which are

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the primary members of the postsynaptic density (PSD) that connect mGluR5s to their signaling complexes (Guiffrida, 2005). In fact, deleting the gene responsible for the irregular mGluR5-Homer scaffold interactions results in some positive behavioral effects (Ronesi et al., 2012). Regardless of the mechanism, it appears that FXS is better characterized by decreased neuronal activity rather than overactive signaling, as previously believed.

Given this, it makes sense to revisit a study examining the nature of cerebral protein synthesis (rCPS) in FXS that suggests decreased neuronal activity may be responsible for decreased protein synthesis. In this study, subjects were sedated with propofol and underwent PET imaging with L-[1-(11)C]leucine.

The researchers found that rCPS was reduced throughout the brain; however, the cause of this was uncertain. It was suggested that decreased neuronal activity, which resulted from sedation with propofol, could be responsible for alterations in rCPS. In turn, this could affect synaptic plasticity.

These all represent areas of research that warrant further investigation, as they suggest promising paths to clinical intervention in FXS. Larger studies are needed to strengthen confidence in the conclusions of this study. However, the findings presented here, in particular the reduced mGluR5 expression and its correlation with FMRP values, provide a great foundation for future studies and clinical trials.

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[18F]FPEB 3-[18F]fluoro-5-(2- pyridinylethynyl) benzonitrile ASD Autism spectrum disorder BPND Non-displaceable binding potential ERK Extracellular signal-regulated kinase FMR1 Fragile X mental retardation 1 FMRP Fragile X Mental Retardation Protein FXS Fragile X syndrome ID Intellectual disability LTD Long-term depression

MAPK Mitogen-activated protein kinase MBq Megabecquerel mCi Millicurie mGluR5 Metabotropic glutamate receptor subtype 5 MRI Magnetic resonance imaging mTOR Mammalian target of rapamycin (mTOR) NAM Negative allosteric modulator PET Positron emission tomography PET/MRI Positron emission tomography/ magnetic resonance imaging TACs Time activity curves TD Typical development

R E F E R E N C E S

1. Budimirovic, D. B. & Kaufmann, W. E. (2011). What can we learn about autism from studying fragile X syndrome? Dev. Neurosci. 33, 379–394.

2. Budimirovic, D. B. & Subramanian, M. (2016).

Neurobiology of Autism and Intellectual Disability: Fragile X Syndrome. in Neurobiology of Disease 375–384 (Oxford University Press).

3. Bardoni, B., Schenck, A. & Mandel, J. L. (2001). The Fragile X mental retardation protein. Brain Res. Bull. 56, 375–382.

4. Brasic, J. R. et al. (2020). Reduced Expression of Cerebral Metabotropic Glutamate Receptor Subtype 5 in Men with Fragile X Syndrome. Brain Sci. 10, 899.

5. Bagni, C. & Zukin, R. S. (2019). A Synaptic Perspective of Fragile X Syndrome and Autism Spectrum Disorders. Neuron 101, 1070–1088.

6. Soong, H. K., Markham, J. A., Weiler, I. V. & Greenough, W. T. (2008). Aberrant early-phase ERK inactivation impedes neuronal function in fragile X syndrome.

Proc Natl Acad Sci USA 105, 4429–4434.

7. Bear, M. K., Huber, K. M. & Warren, S. T. (2004). The mGluR theory of fragile X mental retardation. Trends Neurosci. 27, 370–377.

8. Budimirovic, D. B. et al. (2017). Updated report on tools to measure outcomes of clinical trials in fragile X syndrome. J. Neurodev. Disord. 9.

9.Dahlhaus, R. (2018).

Of Men and Mice: Modeling the Fragile X Syndrome. Front Mol. Neurosci. 11.

10. Qin, M. et al. (2013). Altered cerebral protein synthesis in fragile X syndrome: studies in human subjects and knockout mice.

J. Cerbral Blood Flow Metab. 33, 499–507.

11. Cai, X., Arif, M., Wan, H., Kornreich, R. & Edelman, L. J. (2019). Clinical Genetic Testing for Fragile X Syndrome by Polymerase Chain Reaction Amplification and Southern Blot Analyses. Methods Mol Biol 1942, 11–27.

12. LaFauci, G., Adayev, T., Kascsak, R. & Brown, W. T. (2016). Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP). Genes Basel 7, 121.

13. Wong, D. F. et al. (2013). 18F-FPEB, a PET radiopharmaceutical for quantifying metabotropic glutamate 5 receptors: a first-in-human study of radiochemical safety, biokinetics, and radiation dosimetry. J Nuc Med 54, 388–396.

14. Rahmim, A., Cheng, J. C., Blinder, S., Camborde, M. L. & Sossi, V. (2005).

Statistical dynamic image reconstruction in state-of-the-art high-resolution PET. Phys. Med. Biol. 50, 4887–4912.

15. Carson, R. E. (2003). Tracer Kinetic Modeling in PET. in Positron Emission Tomography: Basic Science and Clinical Practice 147–179 (Springer-Verlag).

16. Cox, A. D., Virues-Ortega, J., Julio, F. & Martin, T. L. (2017). Establishing motion control in children with autism and intellectual disability: Applications for anatomical and functional MRI. J. Appl. Behav. Anal. 50, 8–26.

17. Jong, Y. J. I., Harmon, S. K. & O’Malley, K. L. (2019). Location and Cell-Type-Specific Bias of Metabotropic Glutamate Receptor, mGlu 5, Negative Allosteric Modulators.

ASC Chem. Neurosci 10, 4558–4570.

18. Emmitte, K. A. (2013). mGlu5 negative allosteric modulators: a patent review (20102012). Expert Opin. Ther. Pat. 23, 393–408.

19. Guiffrida, R. et al. (2005). A reduced number of metabotropic glutamate subtype 5 receptors are associated with constitutive homer proteins in a mouse model of fragile X syndrome. J. Neurosci. 25, 8909–8916.

20. Ronesi, J. A. et al. (2012). Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome. Nat Neurosci 15, 431–440.

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TABLE

SAMUEL D. MARTIN

Samuel is from West Chester, Pennsylvania and is pursuing a BS/MS degree in neuroscience, with an additional major in psychology. He is a member of the class of 2023, and upon finishing his degrees at Hopkins, he hopes to pursue an MD/PhD. He enjoys completing research because it represents an opportunity for him to take what he has learned in the classroom and apply it to advance the study of his field.

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FIGURES Figure 1 This image depicts the results of PET imaging (top) and MRI imaging that were utilized to determine the density and the distribution of mGluR5s throughout the brain. Nondisplaceable binding potential (BPND) estimates the attachment of [18F]FPEB to the targeted receptors.This research was originally published in JNM. Wong et al. 18FFPEB, a PET radiopharmaceutical (2013).

Figure 2. This plot depicts the relationship between FMRP and [18F]FPEB uptake throughout various regions of the brain, as outlined in the legend. The relationship appears to be positive and linear. The p-value for this slope is .21, which indicates insignificance; however, the low n of this study merits acknowledgement.

Figure 3. This plot depicts the slope for the plot of [18F]FPEB uptake as a function of FMRP concentration for all 20 individual brain areas measured in this study. All slopes overlap with the average slope value obtained across all brain areas, which was 1.05

± .05 (SEM).

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T h e Tw o - W a y H i g h w a y Between the Gut and the Brain: How Microbiota Shape Neural Function Sid Kavuturu INTRODUCTION

More bacteria inhabit our gut microbiome than there are cells that constitute our bodies.

These bacteria play a crucial role in host physiological functions, aiding in digestion and absorption of macromolecules; moreover, by blocking gut attachment sites, they serve as a barrier to gut pathogens.

While it is well established that gut bacteria are beneficial to host physiological functions, little research has been done in studying whether these bacteria can potentially affect the development of neural systems. The following discussion sheds light on how microbiota can shape physiological responses and influence the immune response through altering development patterns in microglia, while also discussing the underlying mechanism by which it does so.

MICROGLIAL MATURATION IS PROMOTED BY CONSTANT INPUT F ROM HOST MICROBIOTA

Microglia are the resident macrophages of the central nervous system (CNS). They play a critical role 22

in maintaining tissue homeostasis, protection against pathogens, and brain development (Erny et al., 2015).

Also, numerous microglia-related genes have been implicated in various neuropsychiatric neurodegenerative disorders in humans (Erny et al., 2015). Although much is known about the effects that microglia have on the CNS and its relationship to disease pathology, very little is known about what controls their maturation and activation under homeostatic conditions (Erny et al., 2015). With this in mind, this paper

by Erny et al. (2015) specifically examines the effects of the gut microbiome on the maturation and maintenance of microglia in nondiseased brains (Erny et al., 2015).

Erny et al. (2015) began by examining global defects in microglia in Germ Free (GF) mice compared to Specific Pathogen Free (SPF) mice. As a consequence of their depleted gut microbiome, GF mice exhibited enlarged, heavier caeca and dark-colored cecal contents (Erny et al., 2015).

Quantitative deep sequencing was then conducted to determine wide mRNA expression profiles in both GF and SPF mice (Erny et al., 2015).

Marked differences in mRNA profiles of microglia genes between GF mice and SPF mice were observed (Erny

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et al., 2015). Compared to SPF mice, hundreds of genes were significantly downregulated and upregulated in microglia genes of GF mice (Erny et al., 2015). Among the genes that were downregulated were genes encoding for cell activation, transcription activation, and proteins involved in pathogen recognition (signs of premature microglia function) (Erny et al., 2015). Upregulated genes in GF mice included those encoding for inhibitors of transcription, and central microglia transcription and survival factors (normally downregulated in mature microglia) (Erny et al., 2015).

Ddit4, a gene regulating cell growth, proliferation, and survival, was found to be most amplified in GF mice (Erny et al., 2015). Furthermore, flow cytometry analysis showed an increase in expression of maturation markers that are normally decreased as maturation progresses, indicating that GF mice display phenotypically immature microglia (Erny et al., 2015). Overall, mRNA profiling and flow cytometry results show that commensal microbiota plays a crucial role in the development and maturation of microglia in steady state conditions (Erny et al., 2015).

It is known that microglia can attain certain polarized states, characterized by the terms M0, M1, and M2The differences between

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these states are defined by their specific genetic signatures (Erny et al., 2015). In GF mice, most genes that had been differentially regulated were in the M0-state whereas a limited bias was shown for genes in the M1 and M2 clusters, indicating a severe alteration in microglia steadystate conditions in the absence of gut microbiota (Erny et al., 2015).

Moreover, to elucidate the mechanism by which gut microbiota can affect microglia maturity, histopathological and morphological analyses of various brain regions were conducted across GF mice and SPF mice (Erny et al., 2015). GF mice failed to exhibit any notable histopathological changes in gross anatomy across the CNS (Erny et al., 2015). However, an increased number of microglia were found in all of the investigated brain regions (Erny et al., 2015). This increased number of microglia was present in gray matter as well as in subcortical white matter (Erny et al., 2015). No increase in the number of neurons, astrocytes, and oligodendrocytes was observed across brain regions in GF mice (Erny et al., 2015). Also, the morphological analysis revealed marked differences in microglia structure (Erny et al., 2015). In the absence of microbiota, microglia displayed longer processes, and an increased number of segments, branching, and terminal points (Erny et al., 2015). Microglia under GF conditions, but not in SPF conditions, also showed an overlap of processes with adjacent microglia (Erny et al., 2015). Since it has been shown that microglia development is dependent upon various survival factors, Erny et al. (2015) conducted a quantitative Polymerase Chain Reaction (qPCR) analysis to measure their expression across brain regions. GF mice failed to display a decreased expression of these survival factors and exhibited a normal level of cytokine and growth factor levels (Erny et al., 2015). This suggests that microglia irregularities

were not a result of altered survival factor levels (Erny et al., 2015).

Two experiments were conducted on GF mice and SPF mice to investigate the functional aspect of microglial immune response (Erny et al., 2015). In the first experiment, a lipopolysaccharide (LPS) protocol was employed, activating an innate CNS immune response (Erny et al., 2015). A qPCR analysis of LPS-

"More bacteria inhabit our gut microbiome than there are cells that constitute our bodies.” induced genes showed an increase in genes encoding for pro-inflammatory molecules as well as affected cytokine and chemokine pathways, eventually leading to a suppressed immune response in GF mice (Erny et al., 2015). A morphological investigation following the LPS introduction revealed malformed microglia in GF mice compared to SPF mice (Erny et al., 2015). Next, Erny et al. (2015) introduced the lymphocytic choriomeningitis virus (LCMV) and measured microglia expansion. SPF mice showed an increase in cell density after viral exposure compared to GF mice (Erny et al., 2015). An mRNA-profiling analysis of gene expression showed an alteration in the expression of genes responsible for microglial cell proliferation, differentiation, activation, transformation, and pro-inflammatory molecules (Erny et al., 2015). This diminished immune response in GF mice was accompanied by robust morphological changes to microglia

(Erny et al., 2015). Overall, these two experiments suggest that microbiota are necessary to encourage resistance against viral and bacterial exposure (Erny et al., 2015).

Erny et al. (2015) had only measured microglia alterations in the absence of gut microbiota. In order to see if microglia require constant input from host microbiota, they examined the effects on microglia after depletion of intestinal bacteria. SPF mice that were treated with broad-spectrum antibiotics caused depletion of intestinal microbiota, evidenced by their enlarged caeca (similar to GF mice) (Erny et al., 2015). Following antibiotic treatment, microglia exhibited an immature phenotype, as well as significant morphological changes similar to those in GF mice (Erny et al., 2015). Microglial cell density was not altered (Erny et al., 2015). Moreover, flow cytometry revealed an upregulation of genes encoding for survival factors, which are normally downregulated as maturation progresses (Erny et al., 2015). In aggregate, these data suggest that microglia require continuous input from host microbiota to properly develop and mature (Erny et al., 2015). To test whether the complexity of host-microbiota affected microglia, GF mice were tri-colonized (ASF-mice) with Bacteroides distasonis, Lactobacillus salivarius, and Clostridium cluster XI (Erny et al., 2015). ASF-mice showed an increase in the number of microglia and an increase in Ddit4 mRNA levels similar to GF conditions (Erny et al., 2015). ASFmice also displayed phenotypically immature microglia with significant alterations to cell morphology (Erny et al., 2015). Collectively, these results suggest a reduced host microbiota is not sufficient to foster proper microglia development and maturation (Erny et al., 2015).

Lastly, Erny et al. (2015) reported that defects in microglia

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activity can be partly corrected following exposure to gut microbiota or by way of short-chain fatty acids (SCFA). ASF-mice were co-housed with SPF mice, as a means to expose them to a complex microbiota (Erny et al., 2015).

ASF-mice exhibited cecal properties similar to those in SPF mice, indicating that colonization had occurred (Erny et al., 2015). Furthermore, ASF-mice post-exposure displayed a normal microglia cell count, Ddit4 levels, as well as a partly restored microglia morphology (Erny et al., 2015). The bacterial biomass was quantified, showing almost no detectable bacteria in GF mice and antibiotictreated SPF mice (Erny et al., 2015).

A high bacterial load was noted in SPF mice and co-housed ASF mice (Erny et al., 2015). Targeted qPCR showed enrichment of a greater number of bacterial clades in SPF mice and co-housed ASF-mice compared to tri-colonized ASF-mice (Erny et al., 2015). Together, these results indicate that it is bacterial complexity, not bacterial biomass, driving microglia development and maturation (Erny et al., 2015).

Recently, it has been shown that SCFAs may play a critical role in immune cell homeostasis in the colon (Erny et al., 2015). To this end, Erny et al. (2015) examined the effects of SCFAs microglia development and maturation by incorporating them into the drinking water of GF mice. Cecal properties were not restored to those in SPF conditions (Erny et al., 2015).

However, a normalized microglia density was observed, Ddit4 levels reduced to levels similar to those in SPF mice, and morphological defects were greatly restored (Erny et al., 2015). Microglial immaturity was also reversed (Erny et al., 2015).

It is known that SCFAs mediate innate immune response through interaction with their receptor FFAR2 (Erny et al., 2015). With this in mind, Erny et al. (2015) examined the effects of removing the SCFA 24

receptor. FFAR2 -/- mice displayed malformed microglia as well as severe morphological defects but retained microglial cell density (Erny et al., 2015). Immunofluorescence staining for FFAR2 revealed no receptors in the CNS, but receptors present in peripheral myeloid cells, specifically in the spleen (Erny et al., 2015). However, it has been previously shown before that receptors are not necessary for the entry of SCFAs into the brain (Erny et al., 2015). Accordingly, these results suggest that microbiome presence and complexity tightly regulate proper microglia development and maturation and that any defects in microglia development may be reconstituted with a re-introduction to a diverse microbiome or via supplementation of SCFAs (Erny et al., 2015).

THE INFLUENCE OF MATERNAL GUT MICROBIOTA PRODUCES SEXUALLY DIMORPHIC MICROGLIA IN PRENATAL AND ADULT OFFSPRING

Extrinsic factors such as environmental signals alter microglial properties via an array of receptors and signaling molecules, termed the sensome (Thion et al., 2018). However, little research has been done on the effects caused by intrinsic signals such as sexual identity (Thion et al., 2018). Recently, literature has shown that microglia may exhibit sexually dimorphic properties such as pain perception and differences in colonization between males and females (Thion et al., 2018). Moreover, some disorders display a sexual bias, such as Autism Spectrum Disorder (ASD) (affects a greater proportion of males), and various auto-immune diseases (affecting more females)

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(Thion et al., 2018). Finally, a link has been shown between microglial transcriptomic differences by sex along postnatal development (Thion et al., 2018). To this end, Thion et al.

(2018) examined how sexual identity may affect the microbiome, and subsequently how these changes can alter the development and maturation of microglia prenatally and postnatally. A transcriptomic and chromatin accessibility analysis during various stages of development reveals that microglia undergo stages of differentiation and that sensome genes start to be expressed in utero (Thion et al., 2018). In contrast, sexually dimorphic transcriptomic profiles develop postnatally (Thion et al., 2018). More interestingly, altering microbiome conditions affects males and females differentially at different time points of development: GF conditions affect embryonic microglia in males whereas notable changes in females were observed in adults (Thion et al., 2018). Also, depletion of microbiota produces acute and long-term sexually dimorphic alterations in microglia (Thion et al., 2018). Lastly, Thion et al. (2018) observe an overlap between human fetal microglia and the murine microglial transcriptome, paralleled by a reduced microglial sexual dimorphism during the mid-gestation period.

First, Thion et al. (2018) observed sexually dimorphic transcriptomic signatures in adult and embryonic mice, using RNASeq analysis. During the embryonic stage, few genes were expressed differentially across males and females. However, more genes were sexually dimorphic during the adult stage. In concordance with previous literature, adult female mice expressed genes associated with inflammatory responses, and apoptotic responses, displaying a more evoked immune state (Thion et al., 2018). Thion et al. (2018) also examined sexually biased microglial

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colonization patterns, if any, in embryonic and adult mice. Despite a small increase in microglial cell density in embryonic female mice, cell density was largely similar by sex. Therefore, sexually dimorphic genes are expressed differentially as development progresses, but microglial cell density remains relatively unaltered (Thion et al., 2018).

Since the microbiome has been shown to affect the development pattern of microglia, Thion et al. (2018) asked if prenatal maternal microbial exposure can potentially alter microglia development in dams and if these effects differed by sex. RNA-Seq was performed on microglia from the brains of embryonic and adult mice from GF and SPF dams (Thion et al., 2018). During the embryonic stage, maternal microbiota played a subtle role in shaping gene expression. However, as the time of birth approached, the absence of microbiota in GF dams played a more substantial role in both gene expression and sexually biased transcriptomic profiles. Embryonic male microglia expressed thousands of differentially expressed genes (DEGs), however, most of these genes were expressed at a lower level. In contrast, in adult male microglia, only twenty-six genes were deregulated (Thion et al., 2018). Further analysis showed that the genes deregulated in male embryos were associated with translation and metabolism.

An opposite effect was observed in females (Thion et al., 2018). During the embryonic stage, only twenty genes were differentially expressed.

However, in female adults, almost twenty times more genes were differentially regulated.

Genes deregulated in female adults were linked to inflammatory responses and genes upregulated were linked to regulation of transcription (Thion et al., 2018). Therefore, there is a clear time- and sex-specific relationship where DEGs arise, guided by

maternal microbial exposure (Thion et al., 2018). In males, this difference is more clearly noted during the embryonic stage, as opposed to females, where DEGs are more highly observed in adults (Thion et al., 2018).

As discussed in the report above, Erny et al. (2015) reported morphological alterations in microglia under GF conditions.

However, whether this difference is sexually dimorphic was not tested (Thion et al., 2018). In this paper, Thion et al. (2018) analyze microglial colonization patterns at different time points of development by sex (Thion et al., 2018). It was first established that embryonic GF mice do not exhibit any major histopathological changes in forebrain patterning, axonal tract development, or blood vessel formation compared to embryonic SPF mice (Thion et al., 2018).

However, an increased thickness of cortical layer V was observed in GF mice compared to SPF mice (Thion et al., 2018). Early in the embryonic stage, no differences in microglial density were observed in various brain regions in either sex (Thion et al., 2018). During the later stages of embryonic development, GF males expressed an increased microglial density compared to females (Thion et al., 2018). However, the opposite was shown in adult mice.

GF female adults showed a higher microglial density in the observed brain regions (Thion et al., 2018).

Throughout the embryonic stage, microglia mature to prime the fetus for postnatal challenges by regulating genes. An ATACSeq analysis revealed temporal changes in chromatin accessibility in GF mice and SPF mice (Thion et al., 2018). During early embryonic stages, chromatin accessibility of several regions increased in SPF mice, but not in GF mice (Thion et al., 2018). Differentially accessible regions (DARs) were also found to be sexually biased throughout

development (Thion et al., 2018).

Sexually dimorphic DARs became more prevalent postnatally.

Chromatin accessibility was similar in adult GF mice and SPF mice, whereas adult females displayed a sexually biased modification (Thion et al., 2018). Since accessibility in DEGs remained unaffected, in silico analysis was performed to determine transcriptional networks in these specific regions (Thion et al., 2018). Accessibility networks in DEG regions were prevalent in embryonic GF and SPF mice, whereas in females, they were more prominent in adult SPF and GF mice (Thion et al., 2018). Collectively, these results indicate that altered microbiota conditions can differentially affect chromatin accessibility and thus gene regulation. These effects are more prevalent in embryonic males and adult females (Thion et al., 2018).

Sexually dimorphic differences in microglia may be due to the absence of maternal microbiota, or due to the lack of microbiota in adults (Thion et al., 2018). To this end, Thion et al. (2018) studied the effects on transcriptomic modifications following antibiotic treatment in SPF mice. This antibiotic treatment produced DEGs in microglia of males and females, similar to SPF mice that hadn’t received the antibiotic treatment (Thion et al., 2018). In antibiotictreated (ABX) males, DEGs were associated with immune response, whereas in females, they were linked to regulation of transcription (Thion et al., 2018). The morphological and cell-density analysis was also performed on ABX males and females (Thion et al., 2018). However, no difference in morphology or microglial density was found in either males or females (Thion et al., 2018).

Thus, in adult mice, transcriptomic modifications in microglia result from long-term effects guided by microbiota (Thion et al., 2018).

As a further analysis, Thion

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et al. (2018) observed microglial gene expression in terminated human male and female fetuses during mid-gestation, attempting to draw a parallel consistent with their mice data (Thion et al., 2018).

Transcriptomic analysis showed that human fetal microglia do not exhibit sexually dimorphic gene expression during in-utero but express sensome genes, rendering them capable of detecting environmental stimuli, similar to mice (Thion et al., 2018).

A ONE-SYNAPSE NEURAL CIRCUIT BETWEEN THE GUT AND BRAIN

The papers discussed above provide evidence for gutbrain communication. Specifically, they corroborate modulations of the hypothalamic-pituitary (HP) axis and alterations in microglia function with regards to microbiome diversity (Erny et al., 2015; Thion et al., 2018). Even though communication between the gut and the brain has been established, few studies have questioned the underlying mechanism by which this communication occurs. Kaelberer et al. (2018) identifies “neuropod” cells: enteroendocrine cells in the intestinal lumen which form a one-synapse connection with vagal nodose neurons in the brainstem, using glutamate as the neurotransmitter.

First, mass spectroscopy revealed the expression of neuropeptides, CCK and PYY, in enteroendocrine cells (Kaelberer et al., 2018). These cells were also found to form connections with GFP expressing sensory nerve fibers – termed CCKGFP cells (Kaelberer et al., 2018). Using single-cell western blotting, synapsin-1, along with other pre-synaptic adhesion molecules, were found in about twothirds of CCKGFP cells, suggesting that these cells have the synaptic means necessary to form functional 26

connections (Kaelberer et al., 2018).

Once synaptic machinery was identified, Kaelberer et al.

(2018) used monosynaptic rabies tracing in vivo to see with which neurons the enteroendocrine cells formed synapses. The injected rabies virus differentially infects distinct enteroendocrine cells, spreading to their direct synaptic connection (Kaelberer, et al., 2018). These synapses were subsequently traced back to vagal nodose neurons, projecting into the nucleus solitarius in the brainstem (Kaelberer et al., 2018). This synaptic connection was also found in vitro; vagal nodose neurons expressing the appropriate receptors were infected with the rabies virus before being cocultured with organoids (Kaelberer et al., 2018).

Monosynaptic tracing, following an incubation period, revealed synaptic connections between the vagal nodose neurons and the organoids’ enteroendocrine cells (Kaelberer et al., 2018).

Luminal stimuli in vivo and whole nerve-electrophysiology in vitro were used to measure the transduction of stimuli from gut to brain (Kaelberer et al., 2018). Luminal stimuli included Ensure, sucrose, sucrose with a glucose transporter blocker (phloridzin), D-glucose, and fructose (Kaelberer et al., 2018). In vitro, electrophysiology experiments were conducted with vagal nodose neurons, before and after coculture with CCKGFP cells (Kaelberer et al., 2018). Ensure, sucrose, and D-glucose were found to increase vagal firing rate (Kaelberer et al., 2018). This increased response was not found when fructose was presented, when sucrose was perfused with phloridzin or intraperitoneally, or when the vagus nerve was severed (Kaelberer et al., 2018). A measure of glucose transporter levels showed the presence in intestinal epithelial cells, but not in vagal neurons. This suggests that stimuli from the gut are transduced by the intestinal

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epithelial cells and not directly by the vagal nodose neurons (Kaelberer et al., 2018). In vitro electrophysiology showed excitatory postsynaptic currents in vagal nodose neurons in response to D-glucose only following coculture with CCKGFP cells, but not prior (Kaelberer et al., 2018).

An optogenetic approach was used to test synaptic speed and specificity (Kaelberer et al., 2018).

Mice were bred with either the excitatory channelrhodopsin or with the inhibitory channel halorhodopsin (Kaelberer et al., 2018). Vagal firing rate responses were recorded in vivo as well as in vitro (Kaelberer et al., 2018). In vivo, light stimulation of channelrhodopsin increased the firing rate of vagal neurons to that similar to sucrose (Kaelberer et al., 2018). In the absence of sucrose, light stimulation was sufficient to increase the vagal firing rate (Kaelberer et al., 2018). Mice bred with channel halorhodopsin show an abolished response to sucrose when paired with light stimulation (Kaelberer et al., 2018).

In vitro, vagal nodose neurons before coculture with enteroendocrine cells with channelrhodopsin showed no response to light stimulation (Kaelberer, et al., 2018). After a synaptic connection was established between vagal nodose neurons and enteroendocrine cells, light stimulation elicited a postsynaptic current in the neuron within milliseconds (Kaelberer, et al., 2018).

Since a multitude of sensory-epithelial systems utilizes glutamate as the transduction signal, Kaelberer et al. (2018) hypothesized that the synapse between vagal nodose neurons and enteroendocrine cells was mediated by the neurotransmitter glutamate. qPCR and confocal microscopy revealed vesicular glutamate genes in intestinal enteroendocrine cells (Kaelberer et al., 2018). Furthermore, vagal nodose neurons expressed glutamate receptors (Kaelberer et

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al., 2018). A glutamate sniffer protein, which fluoresces in the presence of glutamate, was used to test whether enteroendocrine cells release this neurotransmitter (Kaelberer et al., 2018). After coculturing enteroendocrine cells with cells that express the glutamate sniffer protein, D-glucose was presented as the stimulus (Kaelberer et al., 2018). Fluorescence was observed, indicating that enteroendocrine cells release glutamate (Kaelberer et al., 2018). Next, enteroendocrine cells expressing channelrhodopsin were cocultured with vagal nodose neurons (Kaelberer et al., 2018). Light stimulation following the addition of an ionotropic glutamate receptor blocker abolished excitatory postsynaptic currents in the vagal nodose neurons (Kaelberer et al., 2018). The response was reinstated once the blocker was washed away (Kaelberer et al., 2018).

Previously, hormones were thought to be the primary signaling molecule to relay information between the gut and the brain (Kaelberer et al., 2018). However, the release of hormones is passive and often delayed relative to the stimulus (Kaelberer et al., 2018). To further understand the underlying mechanism, either a CCK receptor blocker (devazepide) or an ionotropic glutamate receptor blocker (kynurenic acid) was given to mice (Kaelberer et al., 2018). When devazepide was administered, a peak response from the vagal nerve was still recorded within milliseconds (Kaelberer et al., 2018). However, delayed activity by the neuron was abolished, suggesting that the delayed activity is being mediated

by CCK (Kaelberer et al., 2018).

The opposite effect was apparent following the application of kynurenic acid: the quick rapid peak response was abolished but failed to affect the delayed response of the vagal nerve (Kaelberer et al., 2018).

Here, vagal nerve recordings show

two phases of vagal nerve firing: one mediated by the rapid action of glutamate, and the other mediated by the delayed hormonal release (Kaelberer et al., 2018). Using vagal nerve recordings, Kaelberer et al.

(2018) were able to show that stimuli from the gut are rapidly transduced, by the termed neuropod cells, using glutamate as the neurotransmitter (Kaelberer et al., 2018).

CONCLUSIONS

The research thus discussed attempts to demystify the network between cells in our intestinal lumen and our brain, by evidencing that neural behavior depends on the composition of gut bacteria.

Furthermore, by showing that this communication occurs in two phases: rapid transduction using neurotransmitter, and a more delayed hormonally mediated route. However, in the future, it will be important to study the effects of varied microbiome diversity in diseased brains, since microbiota-modified neural systems have been shown to lead to agerelated neuropathology as well as a decreased immune function.

Furthermore, as important as studying diseased models is, future studies will need to be conducted in humans as well. Longitudinal studies, although being correlational, serve as an excellent starting point for bettering clinical interventions in patients.

There has been a large number of studies showing a clear link between alterations in the gut microbiota and abnormal morphological changes in immune functions in mice. In fact, a recent ancillary study conducted

by Sordillo et al. (2019) correlates alterations in gut microbiota to ASDlike behavioral patterns (Sordillo, J.E. et al.). If findings such as these can be validated in diseased humans, it will be increasingly important that large institutions, such as the FDA, start promoting the use of probiotic treatment as

a preventative means, potentially decelerating disease pathogenesis.

REFERENCES 1. Erny, et al. (2015). Host microbiota constantly control maturation and function of microglia in the CNS. Nature Neuroscience, 18(7), 965– 977. doi:10.1038/nn.4030 2. Thion, et al. (2018). Microbiome Influences Prenatal and Adult Microglia in a SexSpecifwic Manner. Cell, 172(3), 500–516.e16. doi:10.1016/j.cell.2017.11.042 3. Kaelberer, et al. (2018). A gutbrain neural circuit for nutrient sensory transduction.

Science (New York, N.Y.), 361(6408), eaat5236. doi:10.1126/ science.aat5236 4. Sordillo, J. E., et al. Association of the Infant Gut Microbiome with Early Childhood Neurodevelopmental Outcomes: An Ancillary Study to the VDAART Randomized Clinical Trial. JAMA network open, 2(3), e190905. doi:10.1001/jamanetworkopen.2019.0905

Sid Kavuturu

Sid is a Neuroscience major in the Class of 2021. After graduating, he plans to spend two years in the Boston area, pursuing research opportunities before he begins the application process for MD/PhD programs. His current research background lies in systems neuroscience, but in the future, he hopes to broaden his understanding of neurodegenerative disorders from both a clinical and translational point of view.

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Assessment of Biodegradation of Tr i m e t h o p r i m a n d Sulfamethoxazole in Anaerobic Digestion of Animal Manure Melody Mutra ABSTRACT

Livestock farms use significant quantities of veterinary drugs such as antibiotics. When ingested by livestock, many of these compounds are only partially metabolized or not metabolized at all and are excreted via urine or feces. Anaerobic digestion is a potential way to mitigate the release of these chemicals into the environment. The present study examined the biotransformation of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), during the anaerobic digestion of animal manure. Samples from lab-scale batch reactors containing microbial inoculum, dairy manure, nutrients, and the target antibiotics were collected and analyzed using liquid chromatography coupled with highresolution mass spectrometry (LC-HRMS). Both antibiotics degraded completely, with no impact on biogas production by the anaerobic microorganisms. Two transformation products (TPs) were detected for TMP and SMX each. TMP TPs resulted from demethylation degradation mechanisms, and SMX TPs were formed from the cleavage of the nitrogen-oxygen bond of the isoxazole ring. For SMX, the first-order degradation rate was 0.69 day-1, with a half-life of 1.00 days. For TMP, the first-order degradation rate was 0.24 day-1, with a half-life of 2.84 days. SMX, TMP, and their TPs did not exhibit toxic effects towards the anaerobic microorganisms. Some TPs exhibit greater toxicity and antibacterial activity than their parent compounds.

However, for the TPs identified in the present study, further research is needed to assess their impacts on aquatic organisms.

KEYWORDS

Trimethoprim (TMP), Sulfamethoxazole (SMX), Anaerobic, Biodegradation, Transformation Products (TPs)

INTRODUCTION

Livestock farms use significant quantities of veterinary drugs (including antibiotics and growth promoters), feed additives, feedstuff contaminants, disinfectants, pest control agents, and other chemicals.

When ingested by livestock, many of these compounds are only partially 28

metabolized or not metabolized at all and are excreted via urine or feces. Thus, these compounds have the potential to enter the environment, for example, entry into soil when manure is used as fertilizer.

As such, treatment technologies to eliminate the release of these compounds into the environment are of increasing interest.

Pharmaceuticals can degrade into transformation products (TPs) through processes such as anaerobic digestion, photolysis, oxidation, and other environmental reactions in surface bodies of water (Chibwe

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et al., 2017). Of particular interest is anaerobic digestion, a process in which bacteria break down organic matter in the absence of oxygen.

Anaerobic digestion converts animal waste into biogas, which can be utilized as a renewable energy source. It is largely unknown to what extent anthropogenic chemicals are removed during the anaerobic digestion of animal manure.

However, recent advancements in analytical instrumentation of liquid chromatography and highresolution mass spectrometry (LC-HRMS) allow for structures of

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previously undetected TPs to be elucidated with high confidence.

Liquid chromatography (LC) is a technique used to separate chemicals in a sample. A gradient consisting of organic and aqueous mobile phases are passed through a chromatography column loaded with the sample. After separation, analytes are directly analyzed by a high-resolution mass spectrometer (HRMS), which measures the massto-charge ratio (m/z-ratio) of ions with a very high accuracy. The mass spectrometer uses electrospray ionization (ESI) to produce ions, which are injected into a quadrupole to filter and guide the ions towards the orbitrap detector. Ions can also be fragmented in a collision chamber prior to orbitrap analysis.

The LC and HRMS system can thus be coupled for the identification and characterization of TPs. Trimethoprim (TMP) and sulfamethoxazole (SMX), shown in Table 1, were chosen as target veterinary antibiotics to analyze due to being commonly administered to farm livestock such as pigs (Lekagul et al., 2019) and frequently used in combination as cotrimoxazole (Batt et al., 2006). TMP and sulfonamides are known to elicit toxic effects in aquatic systems by blocking the formation of folic acid in bacteria.

The lack of folic acid leads to inhibitory activity in organisms such as most gram-positive aerobic cocci and some gram-negative aerobic bacilli (Gleckman et al., 1981). The TPs of SMX and TMP are of concern, as TPs have the potential to exhibit greater toxicity than their parent compounds (Gómez et al., 2010). In the environment, TPs can also exist in higher concentrations than their parent compounds (Gómez et al., 2010).

Anaerobic biodegradation is a major removal mechanism for TMP and SMX. Feng et al. (2017) observed the rapid anaerobic degradation of TMP and SMX in thermophilic (hot)

and psychrophilic (cold) conditions.

Although SMX degrades rapidly in

"When ingested by livestock, many of these compounds are only partially metabolized or not metabolized at all and are excreted via urine or feces. Thus, these compounds have the potential to enter the environment” anaerobic conditions (Carballa et al., 2007; Feng et al., 2017; Cheng et al., 2020), no TPs have been reported in methanogenic conditions, conditions characterized by the production of methane by bacteria. Ghattas et al. (2017) reported cleavage of the isoxazole ring of SMX under iron and sulphate-reducing anaerobic conditions. Additional alterations of the isoxazole ring have since been found, such as the reduction of the nitrogen-oxygen bond (Jia et al., 2017; Gonzalez-Gil et al., 2019).

Mohatt et al. (2011) identified four SMX TPs that result from an iron (II) redox-initiated reaction with the isoxazole ring under abiotic ironreducing conditions. Ilyas and van Hullebusch (2020) identified one TP, N-acetyl sulfamethoxazole, resulting from the anaerobic biodegradation of SMX. However, Ilyas and van

Hullebusch (2020) did not report any TPs for TMP. Trenholm et al.

(2020) observed TMP degradation in anoxic activated sludge but also identified no TPs. Liang et al.

(2019) found that TMP underwent O-demethylation transformation under anaerobic conditions, and Gonzalez-Gil et al. (2019) further identified the degrading enzymes as O-methyltransferase.

The purpose of the present study is to characterize the degradation of TMP and SMX under anaerobic methanogenic conditions and to use LC-HRMS and a non-target analytical workflow to identify any TPs formed.

MATERIALS AND METHODS Experimental design and sample collection

The experimental design consisted of batch reactor degradation experiments, in which TMP and SMX were spiked into bottles containing anaerobic seed bacteria, dairy manure, and microbial media.

The starting concentrations of TMP and SMX were each 1.0 mg/L. Liquid samples were collected from the batch reactors at time points of 0, 1, 2, 3, 7, 14, 21, 28, 35, 42, and 137 days.

Seed bacteria was collected from the Back River Wastewater Treatment Plant in Baltimore, Maryland. Dairy manure was acquired from Virginia Polytechnic Institute and State University. The media consisted of the following components per liter (Owen et al., 1979): 1 g resazurin, 26.7 g (NH4)2HPO4, 16.7 g CaCl­ •2H2O, 2 26.6 g NH4CI, 120 g MgCl2•6H2O, 86.7 g KCl, 1.33 g MnCl2•4H2O, 2 g CoCl2•6H2O, 0.38 g H3BO3, 0.18 g CuCl2•2H2O, 0.17 g Na2MoO4•2H2O, 0.14 g ZnCl2, 370 g FeCl2•4H2O, 500 g Na2S•9H2O, 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine hydrochloride, 5 mg riboflavin, 5 mg thiamine, 5 mg nicotinic acid, 5 mg pantothenic acid, 0.1 mg B12, 5 mg p-aminobenzoic

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acid, and 5 mg thioctic acid.

Experimental controls included positive and negative controls.

Positive controls did not contain TMP or SMX. Negative controls were autoclaved twice for 30 minutes at 15 psi and 121 °C in order to eliminate microbial activity. All conditions were tested in biological duplicates.

Additionally, a replicate experiment was prepared so that liquid sampling would not interfere with the biogas measurements. Bottles were capped with rubber stoppers and aluminum seal crimp caps and incubated at 35 °C in the dark.

Before any sampling, bottles were allowed to cool for 60 minutes to room temperature. All liquid sampling was conducted aseptically in an anaerobic glove box filled with N2 and H2. Separate liquid samples were collected for QuEChERS (quick, easy, cheap, effective, rugged, and safe) extractions, aqueous phase extractions, and pH measurements. Cumulative biogas was measured by puncturing the septum with a syringe and measuring the volume after equilibration with atmospheric pressure. Periodically, a 250 L gas sample from the reactor headspace was injected into a gas chromatograph (GC-TCD) to measure the percentage of CO2 and CH4.

QuEChERS and aqueous extractions

TMP, SMX, and their potential TPs were extracted from the liquid samples collected from the batch reactors using (1) a modified QuEChERS method and (2) an aqueous phase extraction method.

For the QuEChERS extraction, the samples were mixed with an extraction buffer (acetonitrile, methanol, and acetic acid), deionized water, and salts (magnesium sulfate and sodium acetate). The samples were then shaken, sonicated, vortexed, dried down, reconstituted, and filtered to isolate the organic contaminants into the liquid phase 30

prior to analysis by LC-HRMS. For the aqueous phase extractions, 1.0 mL of each sample was centrifuged and the supernatant was filtered prior to analysis by LC-HRMS.

Analytical methods

QuEChERS and aqueous phase extract samples were analyzed by LC-HRMS. The LC system consisted of an RSLC3000 nano highperformance liquid chromatography (HPLC) system equipped with an autosampler, pump, and column.

The Thermo Scientific Hypersil GOLD (150 mm × 1 mm, 3 μm particles) column was used to perform chromatographic separation of all samples.

Chromatographic separation was performed at a temperature of 30 °C and a flow rate of 75 μL/min. The mobile phase consisted of two components, termed A and B. Mobile phase A consisted of 0.1% formic acid in Milli-Q water, and mobile phase B consisted of methanol. A gradient was set up as follows: 5% B at 0 min, 50% B at 3 min, 95% B at 6.5 min, and 100% B at 8 min. Gradient elution was followed by a sawtooth method, in which the percentage of B in the mobile phase was dropped to 5% B at 10.1 min, then raised to 100% B at 10.6 min, then dropped to 5% B again at 11 min, where it remained until the end of the run. The total run time of the chromatographic run was 18 minutes.

The LC system was coupled to a Q Exactive HF Hybrid QuadrupoleOrbitrap Mass Spectrometer. The MS was run with ESI and used full MS/ddMS2 data acquisition in polarity switching mode.

Data analysis and TP identification

Compound Discover 3.0 (Thermo Scientific) software was used to identify TPs using a nontarget environmental research workflow with statistics. Differential analysis, retention time alignment, unknown compound detection,

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predicted elemental composition, and compound grouping were applied across all samples.

Compounds were identified using local and online database matchings, including mzCloud (with ddMS2) and ChemSpider. To identify matches, a mass tolerance of 5 parts per million (ppm) and minimum chromatographic peak intensity of 500,000 was used along with blank samples to subtract the chemical background.

After processing, compounds of interest were identified by manually analyzing the time series of compounds that accumulated in SMX or TMP samples but not in the controls.

Lastly, the structural similarity between the identified TPs and the parent compounds was analyzed by comparing fragmentation spectra.

Calibration and quantification

Quantification was performed by external calibration to obtain eight different concentration levels of each compound (1, 3, 10, 30, 100, 300, 1000 and 3000 ng/mL).

Calibration curves, plots of analytical response vs. analyte concentration, were generated using a 1/X weighted quadratic regression. All correlation coefficients were greater than 0.99. Between samples containing different parent compounds, blank samples of 50% methanol and 50% water were included to flush out contamination in the column.

Degradation kinetics

Kinetics were calculated for the QuEChERS extraction samples, accounting for the total compound present in the sample (liquid and solid phases). To calculate the degradation rates of TMP and SMX, a linear regression was conducted using the linearized first-order kinetics equation (1), where t is time (days), [c] is the concentration of the reactant (mg/L), k is the degradation rate constant (day-1),

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and [c]0 is the initial concentration of the reactant (mg/L). ln[c] = -kt + ln[c]­­0 ( 1 ) The experimental half-life of the target compounds was calculated using equation (2), where t1/2 is the half-life (days). This is derived from equation (1) when [c] equals one half the value of [c]0. t1/2 = ln(2)/k ( 2 )

RESULTS AND DISCUSSION Degradation kinetics

By 14 days of incubation, SMX degraded to completion and TMP degraded to near completion.

High correlation coefficients for both parent compounds confirmed the applicability of a first-order degradation model. Figure 1a displays the linearized regression fit for the degradation of TMP detected in the QuEChERS extraction samples. The graph shows a firstorder degradation rate of 0.24 day-1, a half-life of 2.84 days, and a correlation coefficient of 0.97.

Figure 1b displays the linearized regression fit for the degradation of SMX detected in the QuEChERS extraction samples. The graph shows a first-order degradation rate of 0.69 (day-1), a half-life of 1.00 days, and a correlation coefficient of 0.99.

All sets are displayed as averages of biological duplicates, and standard deviations are shown as error bars.

SMX TPs

Figure 2a shows a plot of SMX degradation and SMX TP formation. The abundance of the parent compound is shown in terms of normalized concentration (C/ C0; left y-axis), and the abundances of TPs are shown in terms of peak area (right y-axis). LC-HRMS analysis revealed the formation and accumulation of two TPs: TP253 and TP257. Both TPs were detected at increasing peak areas after day 0, with TP257 initially forming at

a faster rate. For each SMX TP, Table 2 shows the exact mass, MS2 fragments, molecular formula, mass error (in ppm), extraction method used, and proposed structure.

Figure 2b illustrates a proposed degradation pathway for SMX. Previous studies have also reported TP253 and TP257 as TPs of SMX, formed through cleavage of the nitrogen-oxygen bond of the isoxazole ring under anaerobic ironreducing conditions (Ghattas et al., 2017; Mohatt et al., 2011). The proposed mechanism for TP257 formation first involves the formation of TP253 through the rearrangement and reformation of the isoxazole ring by microbial iron (III) reduction (Mohatt et al., 2011). This reaction is followed by an aziridine ring-opening reaction (Mohatt et al., 2011). Figure 2c shows a comparison of the MS2 data of TP257 (top of figure) and parent compound SMX (bottom of figure). Shared mass peaks with blue arrows represent shared mass fragments between TP257 and SMX, and suspected structures matching these molecular masses are shown above the arrows. These shared mass fragments further confirm that TP257 is a TP of SMX, and the suspected structures illustrate which functional groups of SMX might be degrading further. The orange arrow points to a mass fragment of the MS2 of TP257 that is absent from the MS2 of the parent compound SMX, elucidating a portion of the chemical structure that is unique to the transformation product. These structural differences can lead to decreases or increases in ecotoxicity. Majewsky et al.

(2014), for example, found that TPs such as 4-NO2- and 4-OHSMX modified at the para-amino group exhibited greater inhibitory and antibacterial effects than the parent compound SMX, whereas TPs resulting from the breakdown of SMX retained less than 10% of SMX’s inhibitory effects on growth.

Additionally, although the acute

toxicity of TMP and SMX TPs decreases upon photochemical transformation, some TPs are highly toxic to daphnid green algae (Yang et al., 2020).

TMP TPs

Figure 3a shows a plot of TMP degradation and TMP TP formation. The abundance of the parent compound is shown in terms of normalized concentration (left y-axis), and the abundances of TPs are shown in terms of peak areas (right y-axis). Two TMP TPs were observed: TP276 and TP232. The concentration of TP276 increased initially and then decreased after day 2, indicating that TP276 is an intermediate of TP232. TP232 was detected at increasing peak areas starting at day 7. MS2 comparisons were performed for TMP TP confirmation and to better understand the structural differences between the compounds (data not shown). For each TMP TP, Table 3 lists the exact mass, identified MS2 fragments, molecular formula, mass error (in ppm), extraction method used, and proposed structure.

Figure 3b illustrates a proposed degradation pathway for TMP. TP276 forms through the removal of a methoxy group from the phenyl-methyl ether of TMP. The demethylation of the methoxy groups on the phenyl is potentially expected. This reaction has been observed in the removal of methyl ethers from benzenes by an O-demethylase enzyme system under acetogenic and fermentative conditions (Ghattas et al., 2017; Schilhabel et al., 2009). In the present study, the MS2 comparisons could not discern which methoxy groups were transformed. TP276 formation has been observed under aerobic conditions (Trenholm et al., 2020) and anaerobic conditions (Liang et al., 2019). However, the present study marks the first time TP276 formation was observed under anaerobic methanogenic conditions.

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Additionally, TP232 formation has not been reported in literature. After the formation of TP276, TP232 might be formed by a mechanism involving an enzyme. This mechanism results in the removal of an ether group from the phenyl-methyl ether group.

Biogas and pH

After 137 days of incubation, the cumulative liters of biogas per gram of volatile solids (L/gVS) was 0.31 +/- 0.02 in the bottles spiked with antibiotics and 0.33 +/- 0.03 in the bottles without antibiotics.

The addition of SMX and TMP did not affect the biogas production, indicating that these antibiotics and their TPs did not negatively impact the biogas production capacity of the anaerobic microbial community.

SMX and TMP degraded rapidly following apparent first-order degradation kinetics. The main metabolic pathways consisted of isoxazole ring cleavage for SMX and O-demethylation for TMP. Two TPs were identified for SMX and TMP each. These TPs were previously unreported under anaerobic methanogenic conditions. No toxic effects were observed from SMX, TMP, or their TPs, as biogas production and pH were consistent between the positive control samples and the samples spiked with TMP and SMX. However, future research needs to confirm the TPs identified in the present study and also assess their potentially toxic effects to aquatic organisms. The results of the present study will potentially provide a better

“The results of the present study will potentially provide a better understanding of agrochemical degradation in farm-based anaerobic digesters as a way to more effectively treat manure and remove anthropogenic contaminants.”

The pH levels in all samples remained consistent at 7.30 +/- 0.10 from day 0 to day 7 and increased slightly to 8.36 at day 14, indicating that the pH remained optimal for anaerobic digestion microorganisms.

The increase in pH also occurred in the positive control samples, suggesting that the antibiotics did not cause the pH change.

CONCLUSION

The primary goals of the present study were to characterize the degradation of TMP and SMX under anaerobic methanogenic conditions and to identify the TPs of these parent compounds. Both 32

understanding of agrochemical degradation in farm-based anaerobic digesters as a way to more effectively treat manure and remove anthropogenic contaminants.

REFERENCES

1. Batt, A. L., Bruce, I. B., & Aga, D. S. (2006). Evaluating the vulnerability of surface waters to antibiotic contamination from varying wastewater treatment plant discharges.

Environmental Pollution, 142(2), 295-302. https:// doi.org/10.1016/j.envpol.2005.10.010 2. Carballa, M., Omil, F., Ternes, T., & Lema, J. M. (2007). Fate of pharmaceutical and personal care products (PPCPs) during anaerobic digestion of sewage sludge.

Water Research, 41(10), 2139-2150. https:// doi.org/10.1016/j.watres.2007.02.012

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3. Cheng, D., Ngo, H. H., Guo, W., Chang, S. W., Nguyen, D. D., Liu, Y., Shan, X., Nghiem, L. D., & Nguyen, L. N. (2020). Removal process of antibiotics during anaerobic treatment of swine wastewater. Bioresource Technology, 300, 122707. https://doi. org/10.1016/j.biortech.2019.122707 4. Chibwe, L., Titaley, I. A., Hoh, E., & Simonich, S. L. M. (2017). Integrated framework for identifying toxic transformation products in complex environmental mixtures.Environmental Science & Technology Letters, 4(2), 32-43.https:// doi.org/10.1021/acs.estlett.6b00455 5. Feng, L., Casas, M. E., Ottosen, L. D. M., Møller, H. B., & Bester, K. (2017). Removal of antibiotics during the anaerobic digestion of pig manure. Science of the Total Environment, 603–604, 219–225. https:// doi.org/10.1016/j.scitotenv.2017.05.280 6. Ghattas, A. -K., Fischer, F., Wick, A., & Ternes, T. A. (2017). Anaerobic biodegradation of (emerging) organic contaminants in the aquatic environment.

Water Research, 116, 268-295, https:// doi.org/10.1016/j.watres.2017.02.001 7. Gleckman, R., Blagg, N., & Joubert, D. W. (1981). Trimethoprim: mechanisms of action, antimicrobial activity, bacterial resistance, pharmacokinetics, adverse reactions, and therapeutic indications.

Pharmacotherapy, 1(1), 14-20. https://doi. org/10.1002/j.1875-9114.1981.tb03548.x 8. Gómez, M. J., Gómez-Ramos, M. M., Malato, O., Mezcua, M., & FérnandezAlba, A. R. (2010). Rapid automated screening, identification and quantification of organic micro-contaminants and their main transformation products in wastewater and river waters using liquid chromatography-quadrupole-time-of-flight mass spectrometry with an accurate-mass database. Journal of Chromatography A, 1217(45), 7038-7054. http://doi. org/10.1016/j.chroma.2010.08.070 9. Gonzalez-Gil, L., Krah, D., Ghattas, A. -K., Carballa, M., Wick, A., Helmholz, L., Lema, J. M., & Ternes, T. A. (2019).

Biotransformation of organic micropollutants by anaerobic sludge enzymes. Water Research, 152, 202-214. https:// doi.org/10.1016/j.watres.2018.12.064 10. Ilyas, H., & van Hullebusch, E. D. (2020).

Performance comparison of different types of constructed wetlands for the removal of pharmaceuticals and their transformation products: a review. Environmental Science and Pollution Research, 27, 14342–

STEM & TECHNOLOGY 14364. https://doi-org.proxy1.library. jhu.edu/10.1007/s11356-020-08165-w 11. Jia, Y., Khanal, S. K., Zhang, H., Chen, G., & Lu, H. (2017). Sulfamethoxazole degradation in anaerobic sulfate-reducing bacteria sludge system. Water Research, 119, 12-20. https:// doi.org/10.1016/j.watres.2017.04.040 12. Lekagul, A., Tangcharoensathien, V., & Yeung, S. (2019). Patterns of antibiotic use in global pig production: A systematic review.

Veterinary and Animal Science, 7, 100058. https://doi.org/10.1016/j.vas.2019.100058

of sulfamethoxazole and trimethoprim by hydroxyl radicals in aquatic environment: mechanisms, transformation products and eco-toxicity evaluation. International Journal of Molecular Sciences, 21(17), 6276.

FIGURES Table 1. Veterinary target chemicals under study (Log P is the octanol-water partition coefficient).

A

13. Liang, B., Kong, D., Qi, M., Yun, H., Li, Z., Shi, K., Chen, E., Vangnai, A. S., & Wang, A. (2019).

Anaerobic biodegradation of trimethoprim with sulfate as an electron acceptor. Frontiers of Environmental Science Engineering, 13, 84. https://doi.org/10.1007/s11783-019-1168-6 14. Majewsky, M., Wagner, D., Delay, M., Bräse, S., Yargeau, V., & Horn, H. (2014).

Antibacterial activity of sulfamethoxazole transformation products (TPs): general relevance for sulfonamide TPs modified at the para position. Chemical Research in Toxicology, 27(10), 1821-1828. https://doi.org/10.1021/tx500267x

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Table 2. Transformation products of SMX detected by LC-HRMS. *MS2 column listing fragment ions in spectrum of TPs overlapping with abundant fragments of parent compound

15. Mohatt, J. L., Hu, L., Finneran, K. T, & Strathmann T. J. (2011). Microbially mediated abiotic transformation of the antimicrobial agent sulfamethoxazole under ironreducing soil conditions. Environmental Science & Technology, 45(11), 47934801. https://doi.org/10.1021/es200413g 16. Owen, W. F., Stuckey, D. C., Healy, J. B., Young, L. Y., & McCarty, P. L. (1979).

Bioassay for monitoring biochemical methane potential and anaerobic toxicity.

Water Research, 13(6), 485-492. https:// doi.org/10.1016/0043-1354(79)90043-5

Table 3. Transformation products of TMP detected by LC-HRMS. *MS2 column listing fragment ions in spectrum of TPs overlapping with abundant fragments of parent compound

17. Schilhabel, A., Studenik, S., Vödisch, M., Kreher, S., Schlott, B., Pierik, A. J., & Diekert. G. (2009). The ether-cleaving methyltransferase system of the strict anaerobe Acetobacterium dehalogenans: analysis and expression of the encoding genes. Journal of Bacteriology, 191(2), 588599. https://doi.org/10.1128/JB.01104-08 18. Trenholm, R. A., Vanderford, B. J., Lakshminarasimman, N., McAvoy, McAvoy, D. C., & Dickenson, E. R. V. (2020). Identification of transformation products for benzotriazole, triclosan, and trimethoprim by aerobic and anoxicactivated sludge. Journal of Environmental Engineering, 146(9), 04020094. https://doi. org/10.1061/(ASCE)EE.1943-7870.0001691 19. Yang, J., Lv, G., Zhang, C., Wang, Z., & Sun, X. (2020). Indirect photodegradation Hopkins Undergraduate Research Journal

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A

B

A

C

A

Figure 1. 1st order kinetic graphs with normalized linear fits for TMP on top in (A) and SMX below in (B) as detected in the QuEChERS extraction samples. ln[C] is plotted on the y-axis and time (days) on the x-axis. SMX was completely removed by 14 days.

Error bars show standard deviation from averaged

Figure 2.

biological duplicates.

A) Degradation plot of SMX in normalized concentration overlaid with SMX transformation product formation in peak area. Error bars show standard deviation.

B) Reaction pathways and transformation mechanisms of SMX. C) MS2 spectra comparison for SMXsulfamethoxazole (bottom) and TP 253 (top) with key matching (blue arrows) and unmatching (orange arrow) fragments identified and proposed structures drawn out

A

B

Melody Multra

Figure 3.

A) Degradation plot of TMP in normalized concentration overlaid with TMP TP formation in peak area. Error bars show standard deviation.

B) Reaction pathway and transformation mechanisms of TMP.

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Melody is a junior majoring in Chemical and Biomolecular Engineering. She is originally from Kaohsiung, Taiwan but is currently from Lake Zurich, Illinois.

Her research interests are in water/wastewater treatment and remediation, and she hopes at some point in the future to pursue a masters in environmental engineering. She is also involved in Hopkins Engineers Without Borders and the Intervarsity community.

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Subcellular Fractionation of Immune Cell Organelles for Synthetic Cellular Biology J o d y M o u , J o s h u a D e v i e r , J o s h u a C. D o l o f f A B S T R A C T: BACKGROUND: Immune cells consist of various subcomponents with unique and interdependent functions. Here, we aim to develop a novel synthetic biology platform where organelles such as nuclei, mitochondria, Golgi, and membranes can be separated and transferred between different cell types. This approach allows us to study roles of organelles in regards to cell function or observe changes in immune cell behaviors that may pave the way for new cellular engineering platforms.

METHODS: Our initial methods development consisted of an enucleation approach that allowed for nuclear transfer between two cell types.

We examined two potential methods, adapted from pre-existing protocols in chromosomal transfer in cell biology literature. We first investigated drug-induced cell fission techniques using demecolcine, which depolymerizes microtubules. In a second method, we utilized cytochalasin B for cytoskeletal disassembly followed by high-speed centrifugation for nuclear separation.

RESULTS: Using fluorescent microscopy, we showed drug-induced phenotypic changes resulting from demecolcine. We found cell fission to be unsuccessful as an approach due to variability in drug response, leading to lack of controlled fission. However, our second method demonstrated over 90% successful enucleation by visual inspection of fluorescent microscopy images in both adherent and non-adherent cell types.

LIMITATIONS: We developed our methods on immortal cell lines that may have differences in structural biology compared to primary cell types, which we wish to eventually study and utilize. In addition, the downstream processing steps including organelle separation and fusion remain to be explored.

CONCLUSION: We evaluated two potential enucleation protocols and introduced a novel paradigm for organelle-directed synthetic cellular biology.

INTRODUCTION

One of the defining characteristics of eukaryotic cells is the presence of organelles with diverse and interdependent functions. The importance of organelle distribution and function has been extensively studied (Marshall, 2012; Chang, 2017; Mukherji, 2014; Göpfrich, 2014).

As with many membrane-bound sub-compartments within the cell, most organelles contain specific biochemical pathways and functions that contribute to overall cell function

and phenotype (Tan, 2019). For instance, high mitochondrial density results in increased respiratory states for energy-intensive cells (Marshall, 2012), over-proliferation of endoplasmic reticulum and Golgi apparatuses are linked to heightened protein secretion (Marshall, 2012), and an abundance of lysosomes and peroxisomes indicates phagocytic activity in cells such as macrophages (Chang, 2017).

A previous study demonstrated that many cancer cells have abnormal organelle biology, including Golgilinked hyper-glycosylation, improper

cell surface marker expression, and aberrant nuclear structure (Knoblach, 2015).

Our focus on immune cells takes advantage of the complexity of organelles in immune cell subtypes (Figure 1). Thus far, there has been few published efforts to directly isolate and repurpose individual organelle components, with a majority of past work focusing on the replication of cell and organellespecific functions. For instance, artificial antigen presenting cells use beads conjugated with receptors

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to allow for T-cell expansion in adoptive immunotherapy (Neal, 2017; Sunshine, 2014). Biomimetic drug delivery carriers have been equipped with receptors such as CD47, CD44, and LFA1 to increase specificity and circulation times (Li, 2018; Anselmo, 2014). Disrupting human platelets with detergent created platelet ‘decoys’ which altered intracellular signaling pathways’ activation upon stimulation, leading to antithrombotic properties (Batrakova, 2011; Papa, 2019).

Rather than mimicking particular organelle functions, the main goal of the study was to develop methods to separate and transfer immune cell organelles between cell types. To do this, we aimed to engineer techniques for organelle-level manipulation by repurposing enucleation and cell fission techniques from cell biology literature. Our methods were based on microcell-mediated chromosome transfer and wholecell fusion methods previously used to study aneuploidy, epigenetics, and differentiation (Prescott, 1972; Goldman, 1973; Shay, 1975; He, 2007; Liskovykh, 2016). While these methods for whole-cell recombination result in non-dividing multinucleated cells, our methods are developed with the goal of maintaining longterm viability of cells in culture.

This may allow for stoichiometric recombination of desired organelles and subsequent characterization of cellular functionalities like proliferation, adhesion, migration, and secretory pathways.

MATERI AL S AND

These cells were selected for their differential cell surface expression and differences in adhesive ability (adherent versus suspension cell lines). Jurkat cells were cultured in RPMI-1640 Medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (Gibco) in T75 flasks. HEK cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS and 1% Penicillin/Streptomycin in both T75 and T150 flasks. Cells were maintained at 5% CO2 as per cell culture recommendations.

For routine passaging, cells were washed in phosphate buffered saline (PBS), treated with 10% Trypsin solution, and split at a 1:4 ratio.

Cellular Enucleation

Cytochalasin B is an agent that dissociates the actin skeleton in cells, reducing the structural integrity of the cytoskeleton and allowing for enucleation without significant disruption of the cellular membrane. Cytochalasin B in powder form (Cayman Chemicals, Cat# 1490-96-2) was resuspended in 50uL dimethyl sulfoxide (DMSO)

“… we aimed to engineer

techniques

for

organelle-level manipulation by repurposing enucleation and cell fission techniques from cell biology literature.”

METHODS Cell Culture Two immortalized immune cell lines were used: Jurkat cells (ATCC, Cat# TIB-152), a human cytotoxic T cell line, and human embryonic kidney (HEK) cells (ATCC, Cat# CRL-1573).

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and added to 50mL of cell culture media to make a final concentration of 20ug/mL. A Percoll solution was made using 20mL of Percoll (Sigma Aldrich, Cat# P1644), 1mL of 3M NaCl solution for hypotonicity correction, and 1mL of HEPES as a pH buffer

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(Gibco). Cells were counted using an automated cell counter and 70 million cells were harvested from growing cultures. Adherent HEK cells were washed with PBS and trypsinized for 5 minutes at 37˚C with 10% trypsin, recovered with media, counted, and washed in PBS. Jurkat cells were harvested directly from the media and washed in PBS. The cells were resuspended in 20mL of the cytochalasin B solution and added to a 70mL centrifuge tube. 20mL of the Percoll solution was added to the tube, and the solution was mixed via pipetting and inversion. The cells were then spun in a Beckman Coulter L-90K ultracentrifuge with a Ti-45 fixed angle rotor at 39,000 x g for 70 minutes at 37˚C. As a negative control, cells were resuspended in cytochalasin B solution at the same number and density but were not centrifuged. This was to confirm that cytochalasin B alone could not cause enucleation of cells without centrifugation with Percoll.

After centrifugation, a silica gel pellet was visible at the bottom, as well as one distinct band threequarters toward the bottom of the centrifuge tube. In the initial run with only Jurkat cells, fractions were pipetted out using a 1mL Pasteur pipette and separated into top, medium, and bottom fractions for further analysis. The bottom fraction was further centrifuged in a 15mL falcon tube on a benchtop centrifuge at 4,000 x g for 10 minutes. The additional band was analyzed, but it was found that all layers from the bottom fraction had viable and enucleated cells.

Microscopy

Fractions that had cytoplasts were stained with Trypan blue exclusion dye using 10uL of sample and 10uL of stain. They were counted on a Countess II Automated Cell Counter. Before or after counting, images were also taken on an EVOS

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inverted microscope under the 20/40 PH filter in a glass hemocytometer.

Viability, average cell size for live and dead cells, and a representative bright field photo were taken using the machine’s data export option.

For fluorescence microscopy, a Hoescht stain (Thermo, Cat# 62249) was used, as it is a livecell permeable dye that stains DNA with high specificity. Cells were washed in PBS and incubated in Hoescht solution (1uL per mL of PBS) for 15 minutes in the dark. For slide mounting, cells were resuspended in 20uL of 50% glycerol solution and placed on a slide with a coverslip.

For longer microscopy sessions, the coverslip was sealed with clear nail polish.

Imaging was performed on a ZEISS Apotome 2 with a DAPI filter set with exposure at 365 nm and Emission 445/50. Samples were imaged at 10x and 20x, using the DAPI filter set to locate the cells. A constant exposure was always kept between control and enucleated samples. Phase contrast images were also taken and the fluorescence images were overlaid.

Sample Storage

Samples from each layer and both control and treatment groups were resuspended in 5% v/v DMSO in cell culture media, pipetted into cryovials, and placed into a CoolCell freezing container at -80˚C prior to transfer to liquid nitrogen for longterm storage.

RESULTS Experimental Design

This study evaluated several potential routes for organelle fractionation. Multiple kits for isolating organelles have already been developed for commonly studied organelles such as mitochondria, lysosomes, and nuclei.

However, because these kits were developed for proteomic studies,

most of these reagents did not preserve the viability of the organelles as required for our goals. Organelles can be upwards of 2um in diameter, which is too large for conventional intra-membrane delivery systems such as electroporation.

As a result, the chosen strategy consisted of three primary steps followed by functional characterization (Figure 2). Firstly, we propose the separation of cells into subcellular vesicles to serve as liposome-like carriers of smaller organelle fractions.

Secondly, separation methods such as density centrifugation can be used to enable isolation of individual organelles still wrapped in lipid bilayer vesicles.

Lastly, using established cellular fusion techniques, vesicles can be fused back into intact recipient cells or combined with other vesicles (Yang, 2006).

Creating Subcellular Vesicles Approach #1: Cell Fission

We selected HEK cells and Jurkat cells as the starting cell lines due to their robust growth and phenotypic differences, namely being adherent and non-adherent cell lines, respectively. Our first strategy was to utilize drug-induced cell fission techniques (He, 2007; Yang, 2006) using demecolcine, a chemical that depolymerizes microtubules. When placed in demecolcine-containing solution, cells undergo swelling and loss of cytoskeletal form (Figure 3A). Because microtubules are essential for spindle fiber formation during mitosis, their inactivation results in micro-nucleation and cell fission when the cells attempt to divide. However, this first technique encountered several issues.

Under incubation of 20ug/mL demecolcine for 24 hours, HEK cells showed considerable differences in morphology (Figure 3B), with clumped appearances and loss of adhesions to coverslips (Figures 3C & D). Actin and DNA staining showed

that cells with various morphologies and differently sized nuclei were apparent, with a greater size distribution than in control samples.

The treatment was also extremely sensitive to variables such as confluency, culture volume, and the culture surface (for example: plastic, glass, or treated glass).

Creating Subcellular Vesicles Approach #2: Cell Enucleation The second approach was to focus solely on enucleating cells using a more controlled and reproducible fractionation method. To do this, we adapted historical methods of cell enucleation first developed in the 1970s by Prescott et al. and refined throughout the years by various cell biology groups (Prescott, 1972; Goldman, 1973; Shay, 1975; Liskovykh, 2016; Graham, 2018). Firstly, cells were incubated with cytochalasin B. The cells were then put under high-speed centrifugation through a density gradient of Percoll. Because actin is a key cytoskeletal structure, use of high-speed centrifugation in density gradient mediums leads to the disruption of cellular integrity and pelleting of highly dense organelles such as the nucleus. The nucleus detaches from the cell body in a stalk-like formation, eventually separating and forming the karyoplast (containing the nucleus) and cytoplast (containing all other cytoskeletal components).

Following centrifugation, a band of cellular material that was determined to be the cytoplasts was observed (Figures 4A & B). As a control, cells were incubated with cytochalasin B without centrifugation.

The cytoplast layer was removed and stained with a Hoescht DNA stain. Samples were observed using phase contrast microscopy overlaid with fluorescence microscopy. A 90% enucleation efficiency was observed in both HEK and Jurkat cells (Figures 4C-F). In addition, a trypan blue exclusion assay performed

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immediately after centrifugation showed that in various fractions, enucleated cells were intact (Figure 5). Viabilities ranged from 57% to 80%. Samples were then frozen at -60°C for further analysis.

DISCUSSION Proposed Future Work Improvements to Cellular Enucleation There are several aspects of the enucleation protocol that require additional optimization.

Firstly, additional Percoll density gradients, such as layered gradients, have been shown to better separate cytoplasts and nuclei-carrying karyoplasts.

Thus far, we have not been able to identify the karyoplasts in the centrifuged product, although they are likely present at the bottom of the tube. In addition, the time and speed of centrifugation could be further optimized for each cell type to achieve higher purity.

Vesicle Content Characterization and Separation

Past studies have demonstrated that cytoplasts retained endoplasmic reticulum, Golgi, mitochondria, and centrosomes, as well as cytoskeletal structures such as actin, vimentin, and microtubules.

Fluorescent microscopy will be used for a smallscale study of organelle distribution in various collected fractions, and to visualize cytoskeletal changes.

Additional centrifugation without chemical additives will be performed to separate by density. We expect that cytoplasts with different intracellular compositions can be separated by these methods, as previous proteomics techniques have often used density centrifugation as the basis for organelle separation (Gasser, 1988).

Vesicle Fusion

containing different organelles have been separated and characterized, we will use cell fusion mediated by polyethylene glycol (PEG). There are various combinations for potential fusion partners, comprising of cytoplasts or nuclei from either Jurkat or HEK cells. We propose to begin fusion experiments with HEK cell cytoplasts and Jurkat cell nuclei.

To visualize and differentiate cellular fusions between cytoplasts and nuclei, we will use differential cell adhesion as a simple way to separate fused and unfused cells. Since HEK cells are adherent, we expect that we can remove unfused Jurkat cell nuclei by simply washing away nonadherent cells and debris (Figure 6).

Studies have shown that enucleated cells can survive 24 to 48 hours; following 2 days, we expect that HEK cells without nuclei will disintegrate, leaving only nucleated HEK cells that may begin to divide.

Functional Assays

We envision utilizing assays for cell metabolic activity to evaluate enucleated cells, such as the MTT assay to measure cellular metabolism, RT-qPCR to measure gene expression, or Coomassie staining to look at protein content.

We may measure growth speed using scratch assays or migration potential using trans-well assays.

Lastly, we may also assay functional abilities that may be relevant to future therapeutic uses, such as cell mobility, infiltration, and cytotoxic ability.

Potential Applications

From a synthetic biology perspective, this work is novel, as previous studies have relied heavily on genomic or proteomic manipulation rather than larger scale manipulation at the organelle level.

The development of freezing and storage might allow for the formation of organelle recombination “toolkits” that could be used to explore

Once subcellular fractions

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questions in basic biology and produce new therapeutics (Figure 7A). Another intriguing question is the elucidation of cell subcomponent contributions to cell behavior. For instance, our enucleation protocols could be used to study nuclear and membrane dominance (Figure 7B).

We aim to move this research towards translational applications by using therapeutically relevant immune cell lines like Jurkats and later, natural killer cells, macrophages, and stem cells. Previous studies have demonstrated that enucleated T cells have the ability to bind target cells in vitro. One particular study of interest is testing the binding and cytotoxic potential of enucleated Jurkat cells, which can kill cancer cells in vitro under typical conditions.

Depending on other properties that enucleated cells possess, such as non-replication, enucleated cells could also serve to function as inert decoys when injected. Previous studies have shown that disrupted platelets without aggregation ability could prevent metastatic tumor formation in mouse models.

C O N C L U S I O N S This study has introduced a recombination strategy that focuses on separating cells into membrane-bound vesicles that contain organelles with three steps: (1) creating subcellular vesicles, (2) separation and characterization of vesicles, and (3) cellular fusion of vesicles using PEG. This study evaluated two different methods of cell separation: cell fission and enucleation.

Enucleation was ultimately selected for its reproducibility and potential for further development. HEK and Jurkat cells were both successfully enucleated using a cytoskeletal drug combined with gradient centrifugation. Lastly, this study presented several exciting future steps that focus on refining existing techniques and using previously

STEM & TECHNOLOGY

established assays to test and compare these constructs with whole cells.

While much of the above work is preliminary, it opens the possibility for the development of a new organelle-level cellular engineering platform, and demonstrates the huge potential in adapting techniques found in basic biology research for subsequent translational applications.

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Neal, L. R. et al. (2017). The Basics of Artificial Antigen Presenting Cells in T Cell-Based Cancer Immunotherapies. J. Immunol. Res. Ther. 2, 68–79.

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Li, R., He, Y., Zhang, S., Qin, J. & Wang, J. (2018). Cell membranebased nanoparticles: a new biomimetic platform for tumor diagnosis and treatment. Acta Pharm. Sin. B 8, 14–22.

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Marshall, W. F. (2012). Organelle size control systems: From cell geometry to organelle-directed medicine. BioEssays 34, 721–724.

Anselmo, A. C. & Mitragotri, S. (2014). Cell-mediated delivery of nanoparticles: taking advantage of circulatory cells to target nanoparticles. J. Control. Release 190, 531–41.

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Chang, A. Y. & Marshall, W. F. (2017). Organelles - Understanding noise and heterogeneity in cell biology at an intermediate scale. J. Cell Sci. 130, 819–826.

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Göpfrich, K., Platzman, I. & Spatz, J. P. (2018). Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells. Trends Biotechnol. 36, 938–951.

Goldman, R. D., Pollack, R. & Hopkins, N. H. (1973). Preservation of normal behavior by enucleated cells in culture. Proc. Natl. Acad.

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Shay, J. W., Gershenbaum, M. R. & Porter, K. R. (1975). Enucleation of CHO cells by means of cytochalasin B and centrifugation: The topography of enucleation. Exp. Cell Res. 94, 47–55.

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AC K N O W L E D G E M E N T S

We would like to acknowledge Dr. Doloff for his tremendous encouragement and support of this project, as well as other members of the Doloff Lab and TTEC for their support and guidance.

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FIGURES Figure 1. (left) Examples of important immune cell organelles. The cell membrane carries various receptors that are critical to cell

A

function, such as CD45, a common lymphocyte marker. Lysosomes allow for phagocytic ability in macrophages; chimeric antigen receptors are responsible for the efficacy of CAR-T cell therapy; and the Golgi apparatus glycosylates antibodies produced

by B cells.

Figure 2. (below) Proposed cellular fusion and separation protocol. (1) Subcellular vesicles are created that keep organelles

A

such as the nucleus separated in vesicles surrounded by a lipid bilayer. Karyoplasts and cytoplasts are vesicles containing the nucleus and other organelles, respectively. (2) Density centrifugation and microscopy are used to characterize the vesicles. (3) PEG mediated fusion combines vesicles into new cell constructs. (4) Cells are subjected to functional tests such as metabolism and mobility.

Figure 3. (above) Approach #1: Cell Fission using Demecolcine shows drug response variability. (A) Phenotypic changes seen with Demecolcine, including swelling and micronucleation. (B) Trypsinized cells following Demecolcine treatment. Various nuclei (stained blue) and cell sizes are visible. (C, D) Cells showed phenotypic changes with (C) and

µg/mL of

without (D) the addition of 20

Demecolcine. Actin (green) and DNA (blue) shows loss of planar adhesion to coverslips, and formation of cell clumps. Fluorescence overlaid with phase contrast microscopy. All images shown were taken at 20x.

Figure 4. (above and right) Approach #2: Cell Enucleation is successful using cytochalasin B and centrifugation. (B) Bands of cellular material after initial high-speed centrifugation. (C) The bottom portion (“bot”) was centrifuged for further separation.

(D-G) Over 90% enucleation was seen by visual inspection in centrifuged (L) versus control (R). All images shown at 20X magnification with DAPI and brightfield overlay.

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STEM & TECHNOLOGY Figure 5. (left) Trypan blue exclusion assay shows intact enucleates. Four additional fractions, 1mL each, were taken from the bottom band (Fig. 4C. Enucleated cells were found throughout all the bands. All fractions showed the ability to exclude trypan blue, although the bottom fraction (B4) had low cell counts and low viability. Images at 20X.

Figure 6. (above) Proposed fusion methods involving Jurkat karyoplasts (red) and adherent HEK cell cytoplasts (green). To select for fused cells (brown), karyoplasts can simply be washed away, and the enucleated cytoplasts are expected to disintegrate after 48h or more.

A

B

Figure 7. Potential next steps and applications of the presented method. (A) A cellular fractionation “toolkit”, where frozen but viable components can be mixed and matched. (B) A potential application of cell enucleation is studying membranenuclear exchange, which could answer basic biology questions about membrane or nuclear dominance in combining differentiated cell types.

JODY MOU

Jody is a Biomedical Engineering senior from Toronto, Canada. Her research interests within the Doloff Lab center around the development of novel methods for synthetic cellular biology. Following her time at JHU, she plans to pursue a PhD in protein design and molecular immunology.

JOSHUA M. DEVIER

Joshua is a Biomedical Engineering major in the Class of 2023 based out of New Orleans, Louisiana. He is involved in synthetic and computational biology research at the Doloff lab, and also works in medical device development as part of the design team HydroGene. He plans to pursue a combined MD/PhD degree with an interest in the clinical applications of gene circuits and synthetic biology.

PROF. JOSHUA C. DOLOFF

Professor Doloff is a PI and Assistant Professor in Biomedical Engineering. The Doloff Lab focuses on Immunoengineering and Regenerative Medicine, innovative therapies for cancer, the autoimmune system, and implant/transplant systems.

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African Americans in Pharmacogenomics: Opportunities for Care Improvement A B S T R A C T:

Elise Moore

There is a long history of mistreatment and neglect of African Americans in the medical field. It is even

more apparent when focusing specifically on the mental health outcomes of African Americans. In the current era of progressive action in favor of equity for African Americans, there is an opportunity to examine and improve treatments for their mental health.

Pharmacogenetics is the field of pharmacology that examines the genetic relationship with how drugs work in the human body. As the field has only recently begun to be studied, there is a unique opportunity to make a conscious effort in making sure that African Americans receive fair and equal treatment. To do this there must be an in-depth examination of current and past practices and how those have impacted the relationship between race and medical accessibility in America.

The focus of this paper will be on the role that research and testing in pharmacogenetics have in mental health diagnosis and the necessity to acknowledge the toxicity of underrepresentation and bias. Lastly, there will be an examination of how the knowledge and practices presented can be put into tangible action items.

I N T R O D U C T I O N Mental Health in African Ancestry, Not Race Americans

It is important to make the clarifying distinction that the contents of this paper will examine the effects of different ancestry, not race. The wide array of genetic differences in the human body are attributed to the differences in ancestry. Race, being a social construct, is not the way that humans are different biologically, and it can be harmful to label these differences as such (Fujimura, 2010). In the past, there has been caution to not research the genetic differences between races in fear of encouraging racial bias (Sankar, 2004). However, if we shy away from addressing these differences directly, we are committing a disservice to communities who have been clumped together and studied with less finite detail in the name of their similarities, the most prominent case of this being those falling under the label ‘black’.

In the current era of progressive action in favor of equity for African Americans, there is an opportunity to examine and improve treatments for their mental health.

Currently in the United States, of the African Americans who meet the criteria for receiving mental health resources, only 33% are treated (Delencour, 2016), and those who are treated are more likely to receive a lower quality of care compared to white patients (Wang, 2000). The inequalities African Americans face also contribute to adverse long-term consequences such as more serious mental disorder diagnosis (Bell, 2015; Schwartz, 2014) and higher rates of incarceration for mental illnesses (Hawthorne, 2012).

Pharmacogenetics is the relatively new field of pharmacology that examines the genetic relationship of how drugs work in the human body. Because of the newness of the

field, there is a unique opportunity to make a conscious effort in making sure that African Americans receive fair and equal representation.

PGx (pharmacogenomic) testing is designed to predict drug doses, physiological responses, and risk of toxicity, specific to the individual being tested (Daly, 2017). This accessibility to personalized medicine could be revolutionary but, as of now, personalized refers to the niche of those that are of European descent.

Antipsychotic Therapeutic Outcomes

Antipsychotics, used mainly to treat varying forms of mood and psychotic disorders, have a gaping history of harmful effects in the African American community.

Looking at the therapeutic outcomes of antipsychotic medication in African Americans exemplifies the consequences of providing care to people as an afterthought, where they are often put into

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dosage categories specific to those of European descent. The inappropriate generalization of drug interactions and genetic factors to include African Americans results in incorrect dosages, damaging their bodies (Shah 2005). One study found that African Americans were 1.8 times more likely than white patients to be overprescribed typical antipsychotics (Diaz, 2002). One study showed that patients with the candidate gene TCF7L2, commonly found in African Americans, who were overprescribed antipsychotics were significantly more likely to develop Type II diabetes (Irvin, 2009).

The adverse side effects that can result from these generalizations needs to be addressed.

The D2 dopamine receptor (DRD2), used as a pharmacological target for antipsychotics, is another example of the need for more detailed research on the genetic markers of African Americans. In one study, the minor allele 141C, used as a marker to predict the reaction the body will have to a specific drug, was present in only 10% of Caucasian participants while appearing in 50% of African Americans (Zhang, 2010). The risk allele is more common in African ancestry, yet it is consistently used as an indication of treatment response leading to the false conclusion that African Americans are less likely to respond to an antipsychotic drug that treats schizophrenia when consulting this marker compared to those of European descent. This false inference of drug response is a dangerous miscalculation that can affect someone’s response to treatment.

Multifactorial Reasoning for Mental Health Disparities

Blaming the problem of mental health disparities faced in the African American community solely on the shortcomings of pharmacogenetics is not the intention of this work. The role of 44

genetics is only a small part of the overall picture. By looking at the environmental factors that plague African Americans, the amount of environmental racism that they experience correlates directly with their mental health (Pinto, 2008).

In the United Kingdom, much like in the United States, minorities make up a smaller percentage of the overall population but still show a much higher incidence of schizophrenia (Boydell, 2001). In the past, schizophrenia, a mood disorder that causes disruption and dysfunction in the form of psychosis or delusions, was best predicted through genetics but recent data shows that there might be environmental effects to the presentation of the disorder (CantorGraae, 2005; Krabbendam, 2005).

A study observing black males with schizophrenia in the United Kingdom showed a strong correlation between the presentation of their illness and the isolation and exclusion they faced in their daily lives. The researchers were able to link the perception of social inequality as a major contributor to schizophrenia (Cantor-Graae, 2005). This study is reflective of the conditions and experiences that many African Americans face in their communities in the United States. Another study conducted in a similar population found that the link between poverty and presentation of schizophrenia was two times higher in urban communities than in rural, possibly due to the increased frequency of racial disparities experienced in this environment (Krabbendam, 2005).

And although the majority of African Americans live in the rural portions of the U.S, that number is assumed to be even higher here due to the sheer size of our urban cities, where there is an unequal distribution of impoverished African Americans, and as a consequence, the lower amount of mental health services available to the majority of the poorer population.

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U N D E R R E P R E S EN T A T I O N Current Research

As mentioned before, current methods for pharmacogenomics do not always take African American differences into account when making decisions regarding medication. It is common to use data collected from those of primarily European descent and to assume it is generalizable. This makes sense since historically, white Europeans have been the most researched population, which leads to scientists being more likely to include European genetic variations in subsequent studies, causing the omission of the genetic differences among minority groups (Quansah, 2018).

Genome-wide association studies (GWAS) are genetic tools used to identify genomic factors in mental health and neurological disorders (Quansah, 2018). It shows the molecular etiology of these disorders by highlighting genes that can potentially predict the presence or likelihood of the individual having a specific disease or trait. In other words, GWAS identifies genetic and environmental risk factors that are predictive of the onset of diseases within and across populations. GWAS has recently made the progressive step of accounting for admixture, gene mapping based on ancestry, which allows for more specified medical findings for individuals of different ancestral backgrounds, but there is still a lot of work to be done to expedite the process of making the resource more inclusive. One of the more recent analyses of the contents of GWAS showed that in 2016, 81% of the data in GWAS was from white participants while only 3% was from black research participants (Quansah, 2018). This study also examined the overall makeup of 569 neurogenomic studies and found that 89% of them exclusively focused on people of European descent. Only 1% of the studies focused exclusively

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on people of African descent.

This large gap in intentionally gathered data demonstrates the effort that must be put forth moving forward to achieve equity of data in the medical community.

The Clinical Pharmacogenetics Implementation Consortium (CPIC) is another tool for clinicians. It is a compilation of guidelines based on current data meant to bridge the gap between genetic lab testing and prescribing drugs to patients. Here, there is a lack of data available on African Americans and inconsistencies in the current guidelines addressing racespecific recommendations due to the lack of studies being conducted (Zhang, 2019). Of the 50 drugs listed on the CPIP website, only 32 contain guidelines specific to the ancestry of African Americans, and of those, some have barely any details on specific recommendations. Codeine and warfarin hold most of the attention of studies conducted across ethnicities. On the CPIC website, there is conclusive data on these two drugs on the effects and dosage amounts for African Americans. The solid foundation of research for these two drugs has encouraged subsequent studies investigating them. Antipsychotics, antidepressants, and anticonvulsants, three types of drugs used in mental health treatment, in most cases, have no present specific guidelines on medication recommendations for African Americans or most other non-European minorities.

On their website, looking at the guidelines for CYP2D6 and CYP2C19 genotypes, used for prescribing tricyclic antidepressants, yields low acknowledgement of African ancestry with only one mention with data collected based solely on Caucasians of European ancestry and those of Asian ancestry (Hicks, 2016).

Consumer Availability

For most people, consumer

tests such as Navigenics, 23andMe, and deCODEme are their only interactions with pharmacogenetics (Roberts, 2017). Consumer testing affects public interpretations of pharmacogenetics as a whole because of the labeled trendiness and publicity these tests have received from the media. However, the information presented in one of the tests showed that more than 11 of the 22 available disease reviews were only applicable to those of European descent. There was limited availability of data for African Americans, and in some cases less than 10% of variants offered were even applicable. For prostate cancer, 2 out of 13 of the listed variants applied to African Americans. Type II diabetes, a disease that disproportionately affects African Americans, had only 1 variant that was relevant out of 15 (Haga, 2010). There is danger that comes with having limited data on the population these tools are

PubMed, it is only within the last 20 years that the initiative to be more inclusive in pharmacogenomics has gained popularity. In order to begin incorporating more studies and testing opportunities for African Americans and other non-European groups, the question of why this was challenging to do in the past must be acknowledged in order to present withstanding solutions.

The need for homogeneous study participants which has led to all European studies is one of the most consistent arguments (Quansah, 2018; Perera, 2015).

Homogeneous populations easily avoid the problematic type I errors of finding data significant when it is inconclusive by studying differences in allelic frequencies within only one population. This was decided to be more reliable than missing differences that occur if there are different subpopulations being studied. This problem is showing itself to be miniscule in the current

“Though there are many projects based in Africa that have an emphasis on African ancestry, there is still a shortage of similar projects in the United States and other countries that claim to be at the forefront of medical advancement.” intended to support. Limited data leads to an increase in false positives in non-European groups, making the advantages of having advanced personalized treatment inaccessible.

Historical concerns

Although the importance of diversity in pharmacogenetics is widely accepted now, it is important to understand why, in the past, there was a precedent of single-grouped focused research and consumer limitations. Based on trends of the dates of articles published on

age of technological advances where principal component analysis can correct for stratification (Need, 2009). This is still difficult to do but it should no longer be valid to favor exclusivity for this reason. It will only improve with the advancement of computer software.

There are specific challenges that come with having an AfricanAmerican centered cohort. Due to the complexity of African ancestry, there is higher allelic diversity, creating difficulty to track subpopulations from the subset variability and

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the genetic differences between populations. In one study, 94% of the African American participants showed some degree of admixture of up to 19 different ancestral components (Akinyemi, 2016). This observation matters because, as seen in a study with Abacavir, a HIV/ AIDS drug, genetic variance causes different reactions and differences in adherence to medication (Rotini, 2017). This makes it hard for control groups to reflect the large diversity needed to make results marketable.

A current solution is to have larger cohorts. By testing a larger sample population the intention would be to increase the validity of the results (Quansah, 2018).

Again, additional software can address the confounding factors bred through this (Peprah, 2014).

The current screening methods make it hard to screen for diseases in non-European populations. There is a need for genetically matched controls since using European controls for African American studies can lead to a type II errors. The rare variants that African Americans possess are not reflected in European descent data because of the differences in allele frequencies (Need, 2009). This is because genetic data relies on linkage disequilibrium mapping, the correlation between variant regions found in the individual compared to the population, and tag SNPs, used to identify genetic variations at the nucleotide level (Perera, 2015). The length of linkage disequilibrium in African Americans is considerably shorter than European descents from more recombination as a result of being the more ancestral population.

However, there are advantages to these shorter segments. These shorter segments are good for fine mapping, where regions of interest are systematically sequenced. This has led to discoveries of disease susceptibility markers by looking at allele frequencies (Dalvie, 2015). This 46

has been groundbreaking in gaining knowledge for identifying markers for Type II diabetes. (Rotini, 2017).

N E X T S T E P S Promoting Current Efforts

The work to reach the African American community through pharmacogenomics is already underway. There needs to be a promotion of the current efforts in terms of time, money, and resources.

One of the most successful current projects is the 1000 Genome Project.

This initiative is focused on getting a more expansive log of homogeneous African ancestry established to encourage more research activity.

Human Health and Heredity in Africa (H3Africa) Initiative has a similar mission:to gain knowledge of the differences in genetic variation and tackle some of the diseases spreading through the continent (Dalvie, 2015). Another project, African Genome Variation Project (AGVP), has made great strides in these efforts, finding new loci associated with the susceptibility and pathogenesis of diseases that have killed Africans for centuries such as malaria, hypertension and Lassa fever (Peprah, 2014).

Developing this method has also benefited the Western world, introducing details crucial to further understanding asthma. However, in order to continue these efforts, there needs to be a stronger emphasis towards funding these projects. One study found that most funding for these projects comes from Iceland, where the main focus is on their own population, which is mostly of European descent (Need, 2009).

There needs to be a conscious effort to promote these initiatives with the message that this is beneficial to all. So many beneficial discoveries have come from these projects that have helped countless people in the short time they have been around.

More researchers and clinicians should be eager to continue them.

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Research Challenges

Though there are many projects based in Africa that have an emphasis on African ancestry, there is still a shortage of similar projects in the United States and other countries that claim to be at the forefront of medical advancement.

The reasons for this shortage must be addressed. Firstly, there is a lack of representation among the scientists who make these discoveries (Dalvie, 2015). Data released by the National Institute of Health (NIH) shows that the amount of African or African American scientists employed by the NIH is barely 20%. Although this is fairly representative of the overall African American population within the U.S, it has an effect on the direction of research focus and decision making. One study found that African American scientists at the NIH were 13% less likely to receive a grant when compared to their white coworker, the lowest of any ethnic group (Ginther, 2012).

The challenge of increasing representation presents itself again in the search for qualified candidates for African-American centered cohorts. It has already been shown that in order to validate and increase the significance of these cohorts, a larger sample size will be needed to account for the increased genetic variability. There needs to be a focus on creating the infrastructure to accommodate a larger size and the added changes that come with that (Peprah, 2014). Part of this design involves addressing consumerpatient concerns and the poor access to healthcare in AfricanAmerican communities (Need, 2009).

Both of these factors contribute to the struggle of recruitment.

A reason for hesitance from participants comes from the distrust of the health care system stemming from the fear of exploitation. This comes from centuries of racial discrimination in medicine. If moving

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forward, there is not an increased effort to address the history of relations between African Americans and medical advancement, there will always be an underlying barrier of fear (Bates, 2005). There is evidence, though, that suggests that unwillingness may contribute less to lower recruitment amounts than the lack of access that African Americans face (Haga, 2010).

Recruitment Challenges

Voluntary bias, only recruiting from the subset of participants that is readily available and who are, in many cases, seeking alternative treatment options, excludes a whole group of people who may not know of these opportunities or who are not comfortable with the medical community. Looking specifically at pharmacogenomics, part of this discomfort comes from the fear that racist informed medical discrimination is still influencing racial based genomics (Bates, 2004). One study observing the public’s opinion of PGx testing recorded a participant noting that he was not certain he trusted what they said they were doing. He and many others referenced the Tuskegee experiments, an example of the abuse of black men by the medical community that spanned 40 years, beginning in 1932. Data showed that people who share personal identities with victims of medical abuse, such as socioeconomic status and race, perceive higher risk of medical mistreatment for themselves (Alsan, 2016). This has led to the fear of race designed medicine, a perceived key problem with the individualization of pharmacogenomics (Condit, 2003).

In order to begin the healing necessary to mend these justifiable apprehensions, there needs to be more educational opportunities about clinical trials and current clinical practices (Scherr, 2017).

This point has been brought up in conversations among health

practitioners on how to improve racial health disparities to the extent that it is nearly moot. However, there needs to be a stronger focus on education based on the concerns of the audience that is trying to be reached (Need, 2009). However, there needs to be a stronger focus on education based on the concerns of the audience that is trying to be reached (Need, 2009). In terms of pharmacogenetics, there cannot be the assumption that simply teaching the basics of genetics and the process for how the tests work will improve people’s confidence in the health care system (Bates, 2005). Studies show that there is no correlation between the information being presented to patients and their understanding of genetics.

Additionally, the patient may consider the data to be irrelevant if that is not the basis of their concern.

C O N C L U S I O N

Normally, the protocol is that, when speaking to patients about pharmacogenetics, it is important to emphasize the benefits and to lead with addressing common attitudes and beliefs surrounding PGx testing (Scherr, 2017). While this is important, clinicians and researchers must also include the importance of addressing common attitudes and concerns that may arise in specific communities, including African Americans. In addition, it is important to honor the numerous factors related to disparities as it relates to pharmacogenetics by acknowledging the ways of current and past research design, the underrepresentation of African Americans in the medical field, and promoting the current efforts to improve these inequalities. This is the chance to strive towards equity in medicine, and pharmacogenetics is the place to do it.

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of Science, vol. 41, no. 1, 2010, pp. 5–30., doi:10.1177/0306312710379170.

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30. Scherr, Courtney L, et al. “A Review of African Americans’ Beliefs and Attitudes About Genomic Studies: Opportunities for Message Design.” Frontiers in Genetics, Frontiers Media S.A., 14 June 2019, 31. Schwartz, Robert C, and David M Blankenship. “Racial Disparities in Psychotic Disorder Diagnosis: A Review of Empirical Literature.” World Journal of Psychiatry, Baishideng Publishing Group Inc, 22 Dec. 2014.

32. Shah, Rashmi R. “Pharmacogenetics in Drug Regulation: Promise, Potential and Pitfalls.” Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 360, no. 1460, 2005, pp. 1617–1638., doi:10.1098/ rstb.2005.1693.

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“Inconsistency in Race and Ethnic Classification in Pharmacogenetics Studies and Its Potential Clinical Implications.” Pharmacogenomics and Personalized Medicine, Volume 12, 2019, pp. 107–123., doi:10.2147/pgpm. s207449 37. Zhang, Honghong, et al. “The Advantages and Challenges of Diversity in Pharmacogenomics: Can Minority Populations Bring

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Us Closer to Implementation?” Clinical Pharmacology & Therapeutics, vol. 106, no.

2, 30 Apr. 2019, pp. 338–349., doi:10.1002/ cpt.1491 38. Zhang, Jian-Ping, et al. “Association of a Schizophrenia Risk Variant at theDRD2Locus With Antipsychotic Treatment Response in First-Episode Psychosis.” Schizophrenia Bulletin, vol. 41, no. 6, 2015, pp. 1248–1255., doi:10.1093/schbul/sbv116.

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167, no. 7, 2010, pp. 763–772., doi:10.1176/ appi.ajp.2009.09040598.

ELISE MOORE

Elise is a December graduate from Hopkins with a degree in both Psychology and Natural Sciences Area, officially part of the Class of 2021. While at Hopkins, she enjoyed her time as a member of the JHU Women’s Basketball team, the Phi Mu Gamma Tau sorority, and an executive member of both the Student Athlete Advisory Committee and the Black Student Athlete Association. She is from Minneapolis, MN and is excited to be returning home next fall to attend pharmacy school at the University of Minnesota School of Pharmacy, where she conducted the research she has written about with help from their Experimental Pharmacology department. This is a pivotal time in the expansion of the field of pharmacology and personalized medicine where efforts should include communities suffering from health disparities.

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T H E V I L L A G E P A N C H AYA T A S A N I N S P I R AT I O N F O R T H E I N D I A - PA K I S TA N CONFLICT A B S T R A C T:

Riya Jain The formal legal system in India fails citizens on two levels: it operates on Western values that run adversarial to

Indian culture and is prohibitively slow and inaccessible. The panchayat system, however, has been resolving disputes in villages for centuries and in many ways is one of the most democratic institutions in the world. Similarly, the Lok Adalat system greatly improves access to and quality of justice and is an alternative dispute resolution mechanism inspired by local village panchayats. Traditional dispute resolution mechanisms are uniquely different from formal legal systems in a post-colonial society specifically because they are highly attuned to a society’s values, needs, beliefs, and culture. Interestingly, the panchayat system links India and Pakistan, along with other countries in South Asia, through centuries of conflict resolution at the village level. The cultural and psychological framework behind the system run native to both countries. Could a system designed to resolve hyper-local, uncomplicated, low-stakes disputes resolve one of the most complex, multi-faceted, traumatic conflicts in the world between two nuclear powers that share centuries of history? This paper advances the common understanding of intractable conflicts and proposes that international conciliators could incorporate its underlying framework into modern conflict resolution techniques, especially in the case of India and Pakistan.

I N T R O D U C T I O N

India’s legal system is responsible for providing justice to an incredibly diverse population of 1.3 billion people. Across just 1.3 million square miles of land (onethird the size of the United States), India’s population represents more than 2,000 ethnic groups, every major religion, and 120 languages.

Ensuring that everyone receives timely, equitable, and quality judicial outcomes is undoubtedly a formidable task–a task made even more difficult by decades of colonial rule and modern-day poverty and corruption.

Since achieving independence in 1947, India primarily relies on the common law legal system inherited from the British, as described in its constitution and administered by the Indian judicial system. The formal legal system is complex, modern, and governs constitutional, criminal, property, tax, and personal matters (among many others). The last area,

family law, consists of separate sets of laws that govern Hindus, Sikhs, Jains, Muslims, Christians, and other religious communities across the country. While the Gupta Empire and the Mughal Empire and their respective judicial traditions predate British colonialism, virtually no part of those customs have been incorporated within India’s present legal institutions either in form or in content. As a result, Indian institutions involve Western-based paradigms, philosophies, and values, key among them certainty and uniformity. On a conceptual level, this Britishimport runs adversarial to traditional dispute resolution mechanisms, and thus rustic norms and values as well. On a practical level, the formal legal system is prohibitively slow, marred by corruption, and requires money and expertise that most people simply do not have.

The primary criticism against the Indian court system is that it is painfully slow. As of 2016, more

than 22 million cases are pending in India’s district courts, another 4.5 million waiting to be heard in the high courts, and still another 60,000 in the Supreme Court (Doshi, 2016).

According to analysts, if the judges were to dispose of 100 cases every hour, it would take more than 35 years to fully catch up (Kant 2016).

Lawyers, who charge per hearing, routinely appeal decisions to drag cases on further. Another major reason for backlog is the paucity of judges. India has only 13 judges per one million people, one of the world’s lowest judge to population ratios, compared to 50 in developed nations (Doshi 2016). The impact of such a large backlog is manifold.

First, people wait for decades to receive justice. Victims of violent crimes such as rape or murder may be forced to relive the trauma for perhaps 60 years until their case is finally settled. Witnesses may die before they even have the chance to give their testimony in court. And

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people facing petty lawsuits have to live with the headache of litigation.

94% of cases in high courts have been pending for 5 to 15 years, and in most instances, justice delayed is justice denied. Second, Indians are being held in prison during or awaiting their trial, without having been convicted of a crime. Nearly 300,000 “undertrial” prisoners (two-thirds of all prisoners in India) languish in jail, many for longer than their maximum formal sentence if convicted (Amnesty International, 2017). Legal analyst Alok Prasanna summarizes that “Parties don’t see litigation as a way to resolve disputes. They get what they want by making the other party suffer and come to court repeatedly for years,” (Doshi, 2016). When people feel like the court system doesn’t reflect their culture and that it extorts their time and money, people don’t believe the system is just. Problematically, effective legal institutions require popular legitimacy.

Given that the Indian formal legal system does not adequately meet the needs of its citizens, either conceptually or practically, it is worth exploring whether an effective alternative exists. Part I of the paper addresses this question by tracking the evolution of legal pluralism in India. Beginning with South Asia’s local tribunals, I argue that the village Panchayat has been grounded in and accurately reflects the values, cultural norms, and legal philosophy of the subcontinent.

The paper then examines Lok Adalat courts, an alternative dispute resolution mechanism that effectively gives a statutory basis to the ancient concept of local tribunals. In highlighting the failures of India’s formal legal system, I will demonstrate that Lok Adalat is a valuable alternative and necessary to truly secure justice for all citizens.

Part II of the paper proposes a new way of conciliating interstate conflict through traditional legal 50

systems. The panchayat system links India and Pakistan, along with other countries in South Asia, through centuries of conflict resolution at the village level. This paper posits that international conciliators could incorporate the underlying framework of India’s customary legal systems and alternative dispute resolution mechanisms into modern conflict resolution techniques.

For clarification, I offer brief definitions of the following terms: litigation, arbitration, conciliation and mediation. The four terms can be placed on a scale (Table 1). Factors such as time, money, and education can be similarly placed on the scale (Table 1).

Mediation occurs when a neutral, third-party facilitates a conversation between the conflicting parties. Mediators simply allow discussion to take place. They do not take sides, offer their opinion, propose or influence a solution in any way. Rather, they guide the two parties towards reaching a solution themselves. Conciliation takes place when the neutral third-party adopts a more active role. They still facilitate conversation, but conciliators can also propose solutions and persuade parties to see an arrangement as mutually beneficial. Both mediation and conciliation are voluntary.

Arbitration occurs when the thirdparty takes the role of a decisionmaker. Upon hearing arguments from the conflicting parties and viewing evidence, the third-party gives his or her decision. The process in private and the decision is often binding and final. Litigation is a far more formal, complex, and rules-oriented process.

The third-party, similarly, is the decision-maker, but the conflicting parties are represented by legal counsel and do not communicate with the decision-maker directly. Both arbitration and litigation are courtlike processes, but litigation is public and strictly follows due process.

Furthermore, the decision is binding

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but not final and can be appealed.

This paper discusses justice systems ranging from the informal village panchayat to the Lok Adalat courts to India’s formal legal system. An understanding of these terms and how they are applied to the present context will be useful for understanding the functional differences between methods of dispute resolution at the local, national, and international levels.

I. LEGAL PLURALISM IN INDIA A. History of Panchayat

The panchayat system is the oldest form of justice provision and conflict resolution in the Asian subcontinent, specifically in rural India, Pakistan (known as jirga), Bangladesh (known as shalish), and Nepal. Literally translating to “assembly” (ayat) of “five” (panch), the system consisted of five, wellrespected and honorable village elders with one elected or informally acknowledged leader (sarpanch) that handled disputes between individuals and villages. Traditionally, the vast majority of cases fell into three broad categories: women (zan), gold (zar), and land (zamin), with land disputes forming the largest category of cases (Chaudhury, 1999).

While these may have been the objects of a dispute, most plaintiffs at their core were looking to restore or balance the principles of honor, modesty, and the defense of honor/ women. Few villagers would dare to disregard or appeal the panchayat’s decision, even though it was never binding in a legal sense. The sarpanch was a community leader, greatly respected, and often the oldest in the village. To disagree with him would be dishonorable and could ruin a family’s reputation. In these times, caste may have been a criterion for the upper-most village positions such as the sarpanch, but other positions were not–indicating that village republics have some

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but not ultimate social mobility.

Through centuries of various sultanates, empires, and British leaders, the panchayat system has undergone periods of increasing and diminishing influence to reach its present constitutional maturity and structure. The current iteration is a modernized, three-tiered system formalized with the passage of the 73rd amendment to the Indian constitution in 1992. The council consists of 5 representatives elected by the people of the village with elections held every 5 years. They have jurisdiction over both petty civil and criminal cases up to 1 crore or roughly 134,000 USD. The council has the powers for summoning witnesses, discovery and production of documents; the process is free and the decision is not binding.

In criminal cases, the Panchayat can only impose a fine up to 200 rupees and it cannot sentence anybody to imprisonment. Finally, lawyers cannot appear before panchayat in any of its proceedings.

This ancient, traditional system has since its inception been grounded in the cultural legal principles of South Asia. First and foremost, the panchayat system is rooted in villages. Mahatma Gandhi, inspired by the village economy, claimed that “the future of India lies in its village.” He recognized the value of decentralization which allowed villages to be in charge of their own affairs and responsive to the needs of a small group of people. Sir Charles Metcalf, Governor-General of India in 1835, wrote about India: “The village communities are little republics, having nearly everything they can want within themselves and almost independent of any foreign relations. They seem to last when nothing else lasts. Dynasty after dynasty tumbles down; revolution succeeds revolution but the village communities…has contributed more than any other cause to the preservation of the people of India.”

This preservation can be seen in the way that Panchayats are highly attuned to the needs, priorities, struggles, conflicts, and cohesion of their people. A fishing village in Andhra Pradesh, for example, will give rise to different types of disputes and accepted means of restoring harmony than handweavers in rural Rajasthan. The resilience of the panchayat as a legal system reflects the strength of village communities.

Second, the panchayat system protects the customs and traditions of the village. India is a highly hierarchal society, and the panchayat oversees and promotes harmony and by acting for the benefit of society as a whole. Furthermore, the council makes decisions based on values that matter to people, notions of justice such as duty, honor, community. Rather than strictly following the facts, the panchayat system proposes solutions that weigh the relative justice in order to save the honor and status of both parties. Knowing that people in the subcontinent can bear material loss but not the loss of honor, panchayat members provide “both parties with face-saving resolutions, offering them different doses of honor and material benefits. The weaker party is usually privileged in terms of material benefits while the powerful party is promised an elevation in status,” (Kadir, 2018).

Finally, fair gender representation is mandated by law. Women and other marginalized groups in India struggle to attain adequate representation in Parliament and state assemblies, however, they have successfully secured political power at the grassroots level. The 1992 Constitutional amendment guaranteed that one-third of the available panchayat seats would be reserved for members of scheduled castes or tribes and another onethird for women, barring otherwise adequate representation. As of 2020, 20 states have increased the

allotment of seats for women to a full 50% in their state constitutions.

There are currently more than 3 million elected representatives (of which more than 1 million are women) for panchayats at all levels (UN Women, 2019).

The panchayat system garners a lot of popular support in Indian and Pakistani villages, perhaps because they are more convenient or they provide better outcomes or there may be no alternative. It, of course, does not always lead to “ideal” outcomes by most reasonable measures. Decisions can be based on caste, religion, community, personal or political considerations, leading to a higher chance of injustice. For example, panchayats have ruled in several instances that a victim of rape must marry her rapist. One of the panchayats in the Western state of Maharashtra ordered a virginity test of a bride by putting a white bedsheet on the bed on her first night of marriage. As recently as February 2021, a village panchayat in UP announced a punishment of four slaps and a 1,000 lakh rupees (or 1,375 USD) for the rape of an 8 year old boy by his 16 year old cousin.

Pakistani panchayats or jirgas very often sanction violence against women. More women in Pakistan are murdered by close family members for immoral behavior and bringing dishonor to the family than Pakistani civilians are killed by terrorism (Mahmood, 2018). Virtually all of Pakistan’s jirgas are maledominated, except the first and only all-women council which was formed in 2013 (Inayat, 2016). Such abhorrent treatment of women has no place in any justice system and major reform is needed for traditional systems to be in accordance with internationally accepted standards of human rights. Still, 70% of cases in rural India today are redressed

by Indigenous methods of dispute resolution. Community participation in traditional mediation lends itself

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to decisions in which every member of that community feels obliged.

The village panchayat was in many ways one of the most democratic institutions in the world. When viewed holistically, it is the people’s system.

B. Rise and Evolution of Lok Adalat

The system of Lok Adalat, or the People’s Court, was created under the Legal Services Authorities Act in 1987 to provide Alternative Dispute Resolution (ADR) services.

Through arbitration and compromise, Lok Adalat draws inspiration from the traditional panchayat system.

During the time of its formation, politicians and government officials saw Lok Adalat courts as an opportunity to ease the burden of the formal court system and widely increase access to justice. Marred by corruption, poor infrastructure, awful processing times, and prohibitively high costs, formal Indian courts are often characterized as highly ineffective providers of justice. It is not uncommon for people to wait forty or fifty years before their case gets resolved, by which point the resolution may be obsolete or the disputants may have taken the issue into their own hands (sometimes leading to violent outcomes).

Those without money or with little education are left stranded with no chance at legal recourse because the court system can bureaucratic nightmare. The creation of Lok Adalat was intended to fix this.

When a case is pending trial or pre-litigation stage, the disputing parties have the option to reach an amicable settlement through Lok Adalat. It is an arbitration process where parties represent themselves and interact directly with the presiding officer. The presiding officer is a practicing or retired judge and joined by two other members, usually a lawyer and a social worker.

This ensures that decisions are made with significant legal understanding 52

but also informed by social and cultural conditions. Lok Adalats have jurisdiction over civil and minor criminal cases but are especially effective in the settlement of money claims, including partition suits, motor accident and insurance claims, and even matrimonial cases.

Disputes which require creative solutions or offenses that are so grave that the state (i.e. police) must be involved are rarely referred to Lok Adalat (Jephi, 2018). The process is free and proceedings are public. The judge’s decision is final and binding, but if a settlement isn’t reached, the case will back to state court.

This method of ADR radically expands access to justice across India. Free from the complexities of legal proceedings, Lok Adalat courts generally hear cases and deliver the decision on the same day. Lok Adalat requires no attorney’s fees and the presiding officers work on a volunteer basis affording even the poorest individuals a chance at legal recourse. The Lok Adalat serves to facilitate a between parties, so they are free to litigate and also free from complex terminology and official procedural rules.

Thus, the process is more easily understood by those with less formal education and can be conducted in local languages and for the illiterate. Because they require little infrastructure, Lok Adalat courts can be established in remote parts of the state where government services are infrequent or virtually nonexistent.

Finally, the process provides clashing parties the opportunity to “reduce hostility, regain a sense of control, gain acceptance of the outcome, and resolve the conflict in a peaceful manner,” (Singh, 2018).

Lok Adalat courts do face challenges, mainly those related to time constraints. These courts are regarded as a device in managing the judiciary’s caseload, so the focus has shifted from providing quality justice to resolving as many cases

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as possible. In other words, quality is often sacrificed for the purposes of efficiency. Lines outside Lok Adalat courts are long, the presence of lawyers is inconsistent, and parties are given very little time to present their case. The result is limited negotiation with little to no room for give and take, key components for a successful dispute resolution. Most Lok Adalat proceedings, therefore, are underlined with an element of coercion or perceived pressure to settle. In Bangalore, a coordinated statewide Lok Adalat program resolved over 20,000 cases in a single day (Relyea, 2009). Whether full justice was achieved in any of these cases is highly questionable.

However, for many filing minor suits, the benefit of a speedy resolution likely outweighs the inconvenience of being shuffled around a protracted maze of litigation, courts, and lawyers’ offices. For example, in another recent Bangalore mediation, a complex property dispute filed in the 1960s was settled completely by all interested parties after only 4 hours of mediation.

Though cases like this may seem extreme, they certainly aren’t uncommon, highlighting the immense benefit of the Lok Adalat courts.

Due to the failures of the modern legal system, Lok Adalat is a necessary alternative especially for those who could not access the formal legal system otherwise. By mid-2004, over 200,000 Lok Adalats have been held, more than 16 million cases have been settled, and nearly 6.7 million people have benefited through free legal aid and advice.

Statistics compiled by the Law ministry show that from 2015 to 2018, more than 5 million cases have been disposed of each year (Laskar, 2012).

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II. TOOL FOR INTER-STATE CONFLICT

Although they still face challenges, traditional ADR mechanisms work.

Village panchayats and Lok Adalat courts successfully resolve conflicts, can provide higher quality justice, and are timely and accessible to all individuals irrespective of castes, socio-economic status, and locality.

Thus far, we have seen how they resolve disputes in villages and towns. This section of this paper will explore the utility and applicability of customary legal systems at the macro-level. Could a system designed to resolve hyper-local, uncomplicated, low-stakes disputes resolve one of the most complex, multi-faceted, traumatic conflicts in the world between two nuclear powers that share centuries of history? The conflict between India and Pakistan is characterized by nearly seven decades of violence, so the potential benefit for utilizing traditional dispute resolution mechanisms at the international level, if successful, is enormous.

Such a breakthrough in international dispute resolution tactics need not be limited to South Asia. Many of today’s conflicts are between culturally and geographically close nation-states such as North-South Korea, Serbia-Croatia-Bosnia, and India-Pakistan. Further research into local justice systems in each of those regions could give rise to a similar analysis as presented here. Given that no method of reconciliation has been entirely successful in the past, the opportunity for traditional legal systems to make progress is, at the very least, worth considering.

A. The Micro-Level

Traditional dispute resolution mechanisms are uniquely different from formal legal systems in postcolonial society specifically because they are native to the people and not a foreign export. Case studies

from around the globe consistently prove that justice systems cannot be effectively transplanted to other cultures because the populace does not identify with it (Tamanaha, 2011).

For example, Native American tribes view land as communal and sacred, while Europeans see land as a commodity with rights to ownership.

Laws developed in connection with land use will naturally differ. As another example, Middle Eastern cultures believe in collective responsibility and collective punishment because familial ties are incredibly strong. A crime includes the perpetrator and also his family, the victim and the victim’s family, so justice is achieved only when all these parties are satisfied. In the West, justice is achieved when the right individual is caught and penalized–his or her family has no bearing on the outcome. Such fundamental differences make it clear that local populations will not see foreign legal codes as their law and serving their needs; Thus, it is the cultural insight and attention to shared history with which customary systems mediate that allow them to be successful at the micro-level.

Similarly, the psycho-social processes underlying the panchayat and Lok Adalat system make them uniquely better suited to achieving justice in South Asia. First, Indian and Pakistani life is highly interdependent and hierarchical. Family is the basic unit of social organization.

The individual is affected by family events and the individual’s preferences and choices are often subordinated to collective solidarity.

Prestige and position is higher for kin rather than a stranger, the older rather than the younger sibling, male rather than female, and a person belonging to the upper castes rather than the lower ones (Krishnan, 2018).

The hierarchy is pronounced and respected, so one might think it is a major source of division. While it certainly is for reasons related to

women’s rights and human rights, the hierarchal ordering of society, especially before the modern era, fostered cohesiveness and harmony– every individual had an established position, role, and duty to perform in society. Thus, people tend to have “collective behavior when it comes to solving issues and facing related problems. They work together in a hierarchical social structure that evolved at some point in their community. The group becomes the major element to fall back on rather than the state or its legal system,” (Mathew & Pellissery, 2009). Indian and Pakistani society is an intricate web of social relationships, and traditional legal systems accordingly deliver collective justice. Second, justice in South Asia is largely restorative, not punitive. In the Western conception of justice: “…every legal proceeding is an adversary proceeding. Someone is against someone else. The issue is who is guilty and who is innocent.

According to the principle of dharma, the purpose of law is to right the balance that has been put out of order or to make things harmonious again. It was not a question of who is guilty or innocent; it is a question of how can we restore the balance.”

Dharma is a concept ubiquitous in many Indian religions, most prominently in Hinduism, Buddhism, Jainism, Sikhism. While it is not an influential concept in Islamic tradition, local Pakistani panchayats place a similar emphasis on a restorative, peacemaking process. In other words, the Western framework puts the perpetrator on trial and assumes that consistent and equal application of law will satisfy the victim. The South Asian model seeks to provide restitution to the victim’s family and simultaneously punish the perpetrator.

B. The Macro-Level to

Using the term “intractable” describe conflicts like that

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between Israel-Palestine and IndiaPakistan tends to be controversial because it implies that resolution is impossible and undermines any attempt at peace. Parties engaged in an intractable conflict remain stuck at a dangerous impasse, resist any attempt at negotiation, and often maintain high-intensity relations for decades. Common to all intractable conflicts is a struggle that draws the conflicting parties’ fundamental moral values and identities into question. What seems like pettiness or stubbornness to an observer is actually a fight for survival–and so, as a matter of principle, negotiation or compromise is impossible. While a peaceful resolution to such conflicts is stubbornly elusive, they are certainly worth dealing with primarily because they are often the most destructive. They easily escalate and cyclical periods of violence can put the affected populations through extreme physical and emotional distress. If a conflict is perceived as intractable, disputants will resort to desperate measures like suicide bombings or terrorism, further increasing the intractability of the conflict. Left alone, the desire for self-preservation and consequent hatred of the other parties festers, causing immense bloodshed.

The enmity between India and Pakistan is said to be simultaneously over territory, national identity, and power position in the region. Each country’s history of involvement in the hostilities is highly contested and too long to restate in its entirety. Briefly, India is a secular country, with strong institutions, stable democracy, and genuine rule of law. Pakistan is a Muslim state with a long history of military autocracy, weak political and civil institutions, and a wellestablished track record of exporting violent extremism across its borders.

Both are nuclear powers. India’s population, its defense budget, and its economy are seven times as large as Pakistan’s, and as the regional 54

hegemon, India seeks to assert itself aggressively in Jammu and Kashmir by routinely deploying thousands of troops to the disputed region (Dalrymple, 2015). In response, Pakistan takes every measure of defense, including relying on jihadi proxies (i.e. terrorists according to India, freedom fighters to Pakistan) to carry out their security objectives.

The two countries disagree on a lot, but much of the attention centers around ownership over the highly disputed region of Kashmir.

There are three common causes of intractability of which some permutation have appeared in almost every conflict over the last century and all in India and Pakistan’s dispute over Kashmir (Burgess & Burgess, 1996). First is irreconcilable moral differences. Generally steeped in differing religious or cultural views, differences in beliefs and in what is fundamentally right or wrong are so personal that they leave no room for negotiation. For example, India insists, as a matter of security but also of principle, that Pakistan ceases all state-sponsored terrorism activities before any progress towards peace can be made.

Second, high-stakes distributional issues are over tangible assets, often land or natural resources, and intangible assets, such as opportunity or wealth. Inequity in distribution, especially of physical resources, could be because there physically is not enough of an asset to satisfy both parties or because each party holds a special claim over the asset. India and Pakistan claim historical and religious ties, respectively, to the entire region of Kashmir–an area that cannot be divided or shared without major concessions. Finally, domination conflicts are over power and status.

In the international context, countries engaging in negotiations are deeply concerned with their positioning in the global and regional hierarchy and how peace proceedings may

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impact their status as the stronger or weaker party. India, as the regional hegemon, is especially resistant to international mediation over the Kashmir issue because (among many other reasons) bilateralism with Pakistan guarantees that India maintains the dominant position.

Politically and religiously, India and Pakistan are two very different countries, and yet, “the India-Pakistan conflict owes more to historical and psychological “nearness” than to mere competition for resources or territory,” (Kadir, 2019). That similarity is enough to justify the potential for international conciliators to draw inspiration from Indian and Pakistani informal justice systems because the cultural framework for dispute resolution– owing to the preferences for collective and restorative justice–are very similar. Thus, an appreciation for shared culture and history is also a necessary aspect of international, not just local, mediation. Traditional conflict resolution often attempts to construct universal models and techniques that are applicable across all social and cultural domains of conflict. While conflict is universal, how it is perceived, expressed, and managed is not and is determined by local traditions and norms.

The panchayat system is definitely effective at resolving conflict at the village level. The possibility of effective international mediation between India and Pakistan is only a matter of “understanding the psycho-dynamics behind this micro institution of Panchayat and utilizing it at the macro level interstate conflict resolution mechanism,” (Kadir, 2019).

The suggestion here is not that international mediators replicate a village tribunal with India and Pakistan in front of the U.N. (or whoever the third-party may be) “elders”. Rather, the goal of mediation–and where a traditional framework can be especially powerful–is to facilitate

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the normalization of relations, not to immediately resolve the territorial dispute between India and Pakistan.

For as long as India sees Pakistan as the enemy, New Delhi has no reason to soften its intransigence stance and accept third-party intervention.

As long as Pakistan remains the weaker power, Islamabad will desperately continue to perpetuate violence in Indian-controlled Kashmir. Normalizing relations is a prerequisite to serious negotiations and permanent peace. It is also a task based on cultural understanding, perception, and emotion and a prerequisite to permanent peace.

There are two specific ways the international community can draw inspiration from the village panchayats. It is critical to understand that these options ought to influence the way that mediation is conducted, not the architecture of mediation. In other words, logistics such as a place for meeting, the rules for proceedings, and the writing of a treaty and can continue to follow internationally accepted norms and historical precedents. The first way is face-saving techniques. India and Pakistan are highly concerned with preserving their honor and not appearing weak in front of the other.

Similar to village disputes, third party mediation is necessary here because it is often easier to make concessions to a third-party than to the opposing party (Ting-Toomey, 2015). One can bear the material loss but not the loss of face. India’s insistence on bilateral negotiations in many ways is due to the desire to save face. India believes that it can “handle” Kashmir the ways the way it handled militancy in Punjab and that accepting international help would be a sign of weakness. India also fears that international powers will force it into and undesirable settlement and knows that bilateral negotiations ensure a dominant Indian position. Finally, India fears that losing Kashmir would severely

undermine its secular identity and also embolden other religious groups in other states. Pakistan fears that losing Kashmir would serve a blow to its Muslim identity and expose its militant efforts–both of which would be a major embarrassment on the world stage. A third-party that understands these fears and their repercussions can facilitate conversation that preserves each country’s honor. The second way is improving perception. A third-party that understands the tense and entrenched dynamic between India and Pakistan can persuade each country to believe in the urgency of peace. One aspect of managing conflict for village panchayats is understanding what is at stake.

The India-Pakistan conflict is not just about territorial, boundary, and sovereignty issues; it is also about acknowledgment, representation, and legitimization of different identities and ways of living, being, and making meaning (LeBaron, 2003).

Resolving the conflict on paper and signing a peace agreement may not actually satisfy the enmity built up over decades of fighting. For India and Pakistan, material designation is certainly at stake, but so is forgiveness for deeply emotional, traumatic experiences. Currently, however, the preponderance of a war narrative over the peace narrative by hawkish media breeds mutual mistrust. Lives are lost and ruined as a result every day that the conflict continues. An international conciliatory process sensitive to the native context is necessary for the countries and their people to reckon with their history of hostility.

The international community ought to be aware of their tendencies towards Western conceptions of justice, propose agreements that commit to collective and restorative justice, and mediate with awareness of the emotionally charged nature of every negotiation point. Following traditional norms also bridges the

trust gap between the third-party mediator and the conflicting parties.

In a village, the council elders are wellknown and well-revered individuals.

Implicit to the trust formed between the councilmen and villagers is shared history, values, and kinship.

C O N C L U S I O N Traditional alternative dispute resolution mechanisms like the village Panchayat and Lok Adalat are highly effective at resolving conflict at the local level. They are based in centuries of culture, custom, tradition, and norm and are highly attuned to the needs and values of the people they serve. For the most part, these informal systems operate alongside the formal justice system, “not bound to follow code of civil procedure or code of criminal procedure and are only guided by the principles of natural justice,” (Times of India, 2018). A panchayat or Lok Adalat court do not have the capacity to resolve important or complex cases, but significantly help ease the caseload of state courts by taking on petty civil and criminal cases. Three things are important to clarify. First, these systems vary across regions and boundaries, especially in regards to how progressive and compatible with international standards they are. This paper does not intend to suggest that they operate in the same manner across all villages in South Asia. Instead, the information presented attempts to draw broad strokes in generalizing common cultural characteristics that may allow for comparison with Britishexported conceptions of justice. It also hopes to discover commonalities across India and Pakistan about the system at its core. Second, urbanization and globalization have dramatically changed people’s relationship with and the relevance of local justice systems. People in New Delhi, Mumbai, Kolkata, Karachi, Lahore, and other cities are likely

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disconnected from rural forms of justice and solely rely on the state legal system. Despite massive urbanization during the last three decades, however, around 70% of people still live in villages in both India and Pakistan. Finally, despite the positive light with which this paper discusses village councils, significant reform is needed. Legal pluralism in India is fairly cohesive and inclusive, providing the poorest and least educated citizens with a chance for legal recourse. It also sanctions horrible violations against women’s rights and is susceptible to personal or political influence. Fortunately, meeting 21st-century international norms regarding human rights does not require panchayats or Lok Adalat courts to lose the spirit of collective and restorative conciliation.

The spirit of traditional legal systems in India and Pakistan, rooted in cultural and societal norms, is precisely what allows these systems to fill the gaps left by the formal legal system. Given their effectiveness, it is worth considering whether traditional knowledge can be useful beyond the local context. Indians and Pakistanis share centuries of history, are hierarchical and familyoriented, and prioritize harmony and societal stability. Thus, this paper proposes that the panchayat system can inspire international mediators to take a new approach towards resolving the India-Pakistan conflict. Such a suggestion is certainly ambitious considering how diametrically opposite India and Pakistan are religiously and politically. Yet, both countries use the same method of resolving conflicts in their villages. This method prioritizing collective and restorative justice by using face-saving techniques and improving perception between the disputing parties–techniques that could be greatly useful to international mediators. This is not to say that traditional knowledge ought to replace the institutional 56

layers of the modern international system. Rather, it can inform mediation practices in an effort to normalize relations between India and Pakistan. Undoubtedly, one of the biggest challenges will be convincing India to give up its longheld stance on bilateral negotiation and accept third-party intervention.

To borrow from rather than to ignore indigenous psychology could prove to be a breakthrough for international conciliation efforts.

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11. Kant A. (2017). How to speed up judiciary: Make India’s slow courts world class. New Delhi, India: The Economic Times. Retrieved from 12. Krishnan L. (2018). Culture and Distributive Justice: Some insights from the Indian Context.

New Delhi, India: Oxford University Press.

13. Laskar M. (2012). Lok Adalat System in India.

SSRN Electronic Journal.

14.

LeBaron M. (2003).

Culture and Conflict. Boulder, CO: Moving Beyond Intractability. Retrieved from 15. Mahmood B. (2018). Pakistan’s Jirgas and Women’s Rights.

Washington D.C.: The Diplomat.

16. Matthew L., Pellissery S. (2009). Enduring Local Justice in India: An Anomaly or Response to Diversity? Prayagraj, India: Psychology and Developing Societies.

17. (2018). More than 50 lakh cases disposed

by National Lok Adalats. Times of India.

18. Relyea G., Niranjan B. (2009).

Comparing Mediation And Lok Adalat: Toward An Integrated Approach To Dispute Resolution In India.

19. Singh A. (2016). ADR Mechanism in India: Achievements and Challenges. Gujarat, India: Paripex-Indian Journal of Research.

20. (2019). Take Five: “Elected Women Representatives are Key Agents. UN Women.

21. Tamanaha B. (2011). The Rule of Law and Legal Pluralism in Development. St. Louis, MO: Washington University in St. Louis School of Law’s Legal Studies Research Paper Series.

22. Ting-Toomey S. (2004). Translating Conflict Face-Negotiation Theory into Practice. Thousand Oaks, CA: Sage. .

Table 1: Definitions.

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RIYA JAIN

Riya is a junior at Johns Hopkins University where she double majors in Economics and Political Science. In 2019, she was awarded the prestigious David L. Boren Scholarship to study Arabic in Amman, Jordan and her experiences abroad inspired her research on informal legal systems and alternative dispute resolution mechanisms.

HUMANITIES & SOCIAL SCIENCES

The Authenticity of the Letter of Thomas Aquinas to Brother John:A Natural Language Processing Analysis

John Schindler ABSTRACT

For centuries, the authorship of many texts, from Shakespeare’s plays to the Federalist Papers, has been

disputed. In this paper, I use statistical techniques to attempt to determine the true authorship of a Medieval Latin text, which has traditionally been attributed to Thomas Aquinas. Using three different stylometric techniques programmed with Python, I show that there is significant statistical evidence that the Letter of St. Thomas Aquinas to Brother John differs from other letters known to have been authentically written by Thomas Aquinas. This represents the first statistical analysis of this much-debated text, and while the evidence is suggestive that Aquinas did not author the letter, further study is necessary to conclusively identify the true author.

I N T R O D U C T I O N For centuries, scholars have debated the authenticity of various works, from Shakespeare’s plays to the Federalist Papers. Analysis and comparison of works were done manually by experts who would compare subtle elements of a document such as writing style and vocabulary to determine the authorship. Today, computer programs can be used to detect elements of an author’s style, such as word frequencies, which would be nearly impossible to do manually.

Thomas Aquinas was a 13thcentury theologian and Catholic saint who wrote tremendous amounts, including many works that are still studied today. Among his minor works is a much-beloved letter known as “De Modo Studendi .” I will refer to the letter using its more informal title “The Letter to Brother

John”. The brief letter is supposedly advice from Thomas Aquinas to this unknown Brother John about the best way to study. As Aquinas is regarded as one of the great intellects of all time, having a letter with his advice to students regarding good study habits holds special significance to young scholars everywhere.

Unfortunately, while the letter was attributed to Aquinas as early as the middle of the 14th-century, no study of its authenticity has ever been conducted. This is probably due to the absence of evidence, its lack of theological significance, and its size – it is one of the briefest works attributed to Aquinas at just 183 words. While the authenticity was not debated for almost half a millennium, some scholars began to wonder about the authenticity of the text in the late 19th-century, when interest in Aquinas’s theology reemerged.1

This paper uses three statistical techniques for natural language processing to examine Thomas Aquinas’ Letter to Brother John. The evidence produced by this analysis suggests that there is a statistically significant difference between the known letters of Thomas Aquinas and the Letter to Brother John. However, two issues make a definitive conclusion more challenging. First, the Letter to Brother John is short, which means statistical analysis is harder to do with any level of confidence.

Second, most NLP techniques were developed for modern languages, which means that some aspects of the ancient Latin language may not be properly accounted for. Both of these challenges are areas I am working to improve.

Beyond the concerns about the statistical techniques

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used, statistical analysis of a text is just one piece of evidence that should be used in determining the authorship of a text, and it must be combined with other information to form a stronger determination of the letter’s author. Therefore, while this statistical analysis was necessary, further analysis should be done to reach more definitive conclusions.

For example, determining the exact age of the original manuscript would be very helpful in assessing the authenticity of the letter.

M E T H O D S Analysis Using Stylometric Techniques

Stylometry is the statistical analysis of texts in order to empirically measure characteristics, such as word frequencies. These techniques are often used to compare texts in order to help determine authorship.

In this paper, I use three stylometric techniques to analyze the Letter of St. Thomas Aquinas to Brother John: Mendenhall’s Characteristics Curves of Composition (MCCC), the ChiSquared Test, and the Delta Method.

"...suggests that there is a...difference between the known letters of Thomas Aquianas and the Letter to Brother John” Mendenhall’s Characteristic Curves of Composition

The first technique is known as Mendenhall’s Characteristic Curves of Composition (MCCC) .

This method uses the length of the author’s words to discern authorship.

According to Mendenhall’s theory, a plot of the word length usage of a given author’s works would produce a characteristic curve. This curve would look the same for any sufficiently large excerpt of that author’s writings. While this may be a crude method of authorship identi58

fication, this method can potentially identify an author whose style was mimicked, since the word length is a subtle characteristic of an author’s style that is not easily replicated.

Figure 1 shows lines representing the characteristic curves of several letters. The green line, labeled L1, shows the characteristic curve of Aquinas’ letter to Bernard, which is widely accepted as authentic. The other lines, labeled L2-L7, similarly show the characteristic curves of other letters widely regarded as authentic.

The blue line shows an average of all these letters, as well as a few of Aquinas’ other short works.

Finally, the orange line shows the characteristic curve of the disputed Letter to Brother John. The x-axis shows the length of words and the y-axis shows the proportion of all words of that length in the document. For example, for the L1 letter, words of length 4 represent 17.5 percent of the document.

Looking at the lines for L1-L7 we see that there is considerable variation between letters in terms of the frequency of use of words of certain length. Importantly for our purposes, however, the frequencies in the Letter to Brother John highlight key differences between this letter and the others. In particular, while words of length two range from 1014 percent of the authentic letters, in the Letter to Brother John, those words account for 20 percent of the letter, 6 percentage points higher than in any other letter. This comes at the expense of slightly longer words, including words of length 3, 4, and 5 letters. In each case, the Letter to Brother John has far fewer words of this length. We see that the Letter to Brother John sticks out for words of length 7, where it again has the highest proportion. Given 7 comparators, it seems odd that one letter should have the maximum or minimum usage for four of the seven most common word lengths.

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While this is not definitive proof, it is suggestive that the Letter to Brother John features a different style.

Chi-Squared Test

The second test utilized was the Chi-squared test. This test is a method of measuring the “distance” between the vocabulary in two bodies of text. A lower score means that the two texts use similar vocabulary, indicating that they may have the same author. In this method, we use the ‘n’ most common words in a corpus made by combining the works of the two authors. We then predict the expected number of times that each of the most common words would appear in the original two corpora if written by the same author. For example, if n is ten we would calculate the number of times we expect each of the ten most common words to appear in the disputed text, assuming it was written by the author of the known authentic works. We could then determine whether there is any significant difference between the number of times we expected each of the ten words to appear and the actual number of times they occurred. By finding the difference between the actual and expected number of tokens in each author’s corpus, we can calculate the chisquared statistic, which gives us a measure of the “distance” between two texts. Whereas with the characteristic curves, we get a visual representation, that representation does not give us a statistical test of whether the two items we are seeing are different. That assessment is left to our judgement based on the charts. With the Chi-squared test, we get a statistical measure that tells us just how far apart one measure of the style of authorship is from another. The chi-squared formula is shown in Equation 1.

For this test, I chose three authors to compare the disputed letter to. First, of course would be Thomas Aquinas, to whom

HUMANITIES & SOCIAL SCIENCES

the disputed letter is traditionally attributed. The second author, Thomas of Sutton, was a Dominican and an early defender of Aquinas. A few of his works were falsely ascribed to Aquinas.2 The third author, Francis of Assisi, was the founder of the Franciscan order and a contemporary of Aquinas. Both Thomas of Sutton and Francis lived at about the same time as Aquinas and wrote on similar subjects, making them good candidates for comparison.

The analysis was conducted using the 50 and 100 most common words found in the sets of texts. Table 1 shows the results obtained when we compare the corpuses of Aquinas, Sutton, and Francis to the disputed letter: Both values of n produce the same results. The disputed letter is more similar to Aquinas’ writings than to Sutton’s or Francis’ writings. These results, however, are not conclusive proof that the disputed letter was written by Aquinas, only that it was more likely to have been written by him than by either of the other two authors.

Delta Method

The final method I used was the delta method. This method measures how much the disputed document and the works of each author differs from the average statistics of all the texts put together. The big advantage of this is that each feature, in this case each of the most common words, is given equal weight, thus eliminating the issue of common words playing an outsized role in the results.

Mathematically, the delta method calculates the percentage of each author’s work that a given feature comprises. For example, the word “and” might make up 4% of the words in one author’s corpus. For each feature in each author’s corpus and the disputed letter, we compute the mean and standard deviation of the percentages as well as a z-score

(formula below), which measures how far the feature is from the mean.

Finally, we calculate the delta score by comparing the disputed letter to each of the authors. The lowest delta score indicates the author to whom the disputed letter is most similar.

The results from the delta method are in Table 2. Additionally, for reference, I calculated the delta scores for seven letters known to be written by Aquinas.

If you look in Table 2, you will note that using either 20 or 30 most common words, the Letter to Brother John has the lowest Delta score for Francis. That means that of the three authors, the Letter to Brother John is most similar to the writings of Francis, who certainly did not author the letter. However, Aquinas is more likely to have authored the letter than Sutton, which is also interesting.

This may suggest that the style of the letter is simpler and more akin to Francis’ style than it is to the style of typical Thomistic writings. As a test of the delta method, I selected a very short letter known to be written

by Francis, the “Epistola s. Clarae de ieiunio”, and compared that to the works of all three authors. The delta method correctly attributes the work to Francis, as shown in Table 4.

One possible explanation is that Francis had the simplest writing style of the three authors, and therefore any short and simple letter, such as the Letter to Brother John, would be attributed to Francis.

C O N C L U S I O N In conclusion, statistical analysis is mixed but seems to suggest that Aquinas is not likely to have been the author of the Letter to Brother John unless the letter is uncharacteristic of his authentic letters. The delta method correctly attributed the authentic letters of Aquinas to him, but did not attribute the disputed letter to him. This method is generally considered the most accurate, since it gives equal weight

to each feature, which prevents the most common words from having too much influence on the results.

The characteristic curves method also shows significant differences between the disputed letter and Aquinas’ other letters. Weighing against that is the fact that the chi-squared method attributed the disputed letter to him. However, one drawback of the chi-squared test is that with only a few comparators, as in our case, it is not a statistically powerful test. Future work could look at a broader body of texts to increase the power of the chi-squared test.

F O O T N O T E S 1. The letter was first attributed to Thomas Aquinas around the year 1330 by Venturino of Bergamo, O.P. Some authors, such as Echard (1719) and Grabmann (1928) accept the authenticity of the letter, although Grabmann states that the “historical proof could be set on firmer ground”. (Eschmann Catalogue) Others, however, such as Torrell (1993) consider the letter to be an unauthentic work.

2. On the Corpus Thomisticum website, there are 6 letters written by Thomas of Sutton, which are under the heading of works falsely ascribed to Thomas Aquinas.

R E F E R E N C E S 1. Alarcón, E. (n.d.). Corpus Thomisticum.

Retrieved December 1, 2020, from http:// www.corpusthomisticum.org/ 2. Bourke, V. J. (1965). Aquinas’ search for wisdom: Vernon J. Bourke. Milwaukee: Bruce Publishing Company.

3. Gilson, E. (1956). The Christian philosophy of St. Thomas Aquinas. With A catalog of St. Thomas’ works. New York: Random House.

4. Laramée, F. D. (2018, April 21). Introduction to stylometry with Python. Retrieved from https://programminghistorian.org/en/ lessons/introduction-to-stylometry-with-python 5. Mendenhall, T. C. (1887). The Characteristic Curves Of Composition. Science, 9 (214S), 237-246.

6. Morton, A. Q. (1978). Literary detection: How to prove authorship and fraud in literature and documents. New York: Scribner’s Sons.

7. Torrell, J. & Royal, R. (2005). Saint Thomas Aquinas. Washington, D.C.: Catholic University of America Press.‌

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F

I

G

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E Q U A T I O N S

Figure 1: Characteristic Curves of Aquinas and Disputed Letter

Equation 1: Chi-Squared Formula

Equation 2: Z-Score

Equation 3: Delta Score

TABLES Table 1: Chi-Squared test for the corpuses of Aquinas, Sutton, and Francis n

Aquinas

Sutton

Francis

100 61.08 82.62 161.45 50 41.9 52.66 123.12 Table 2: Delta Method z-scores for Aquinas, Francis, and Sutton with Letter to Brother John n

Aquinas

Francis

Sutton

20 1.81 1.58 2.01 30 2.04 1.73 2.25 Table 3: Delta Method z-scores for Aquinas, Francis, and Sutton with Known Letters of Aquinas Test Case (n=30) Aquinas Francis Sutton Letter 1 1.38 1.55 1.46 Letter 2 1.80 1.95 2.30 Letter 3 2.15 2.22 2.25 Letter 4 1.30 1.49 1.73 Letter 5 1.44 1.55 1.48 Letter 6 1.07 1.61 1.42 Letter 7 1.42 1.60 1.61 Table 4: Delta Method z-scores for Aquinas, Francis, and Sutton with Known Letter of Francis Aquinas 30 most common 2.71 60

Francis

Sutton

2.10

2.82

Hopkins Undergraduate Research Journal

JOHN SCHINDLER Class of 2022, John is a junior from Rockville, Maryland. He is majoring in Computer Science and Economics, and has studied Latin for several years. He is interested in research involving Natural Language Processing.

HUMANITIES & SOCIAL SCIENCES

Understanding the Drop Out Decision: A Dynamic Optimization Model ABSTRACT

Serena Goldberg

This paper presents a theoretical dynamic optimization model of the choice to enroll and attend college in the United States. In particular, this paper focuses on low-income and poor students, given their particularly low graduation rate . The ultimate goal of this paper is to explain why low-income students in the United States may start but fail to complete college, by specifically incorporating studies of stochastic shocks to the opportunity cost of attending college. This model suggests policies that lower the frequency and size of these shocks or, alternatively, reduce the impact of these shocks on one’s ability to graduate, can theoretically reduce dropout rates.

I N T R O D U C T I O N

parental income, student income, Student loan debt in the or loans. For low-income students, United States has greatly increased whose families, by definition, have the last several years as college little income or limited savings, some tuition and the wage premium of a combination of student work and college degree have respectively loans is often necessary (Singletary increased (Hamilton Project, 2017; 2019). Income from student work has Johnson 2019, Singletary 2012). the benefit of complete fungibility.

Even without considering grants and It may be used for tuition, but if a scholarships, the cost of attending student drops out, it can also be spent college for low-income students is on rent, food, or other purchases. often quite high (Goldrick-Rab and However, student work consumes Kendall 2016), and the number of a significant amount of time that students who drop out of college could be spent taking classes or with a large student loan debt studying. As students, many are only grows (Peterson and Robb only eligible for low-skill wage work, 2018). On average, individuals which often involve irregular work who never complete their degree shifts, leading students with a huge earn significantly less than those difficulty of selecting their course with a college degree, but both schedules (Golden 2015).Ultimately, must pay back their student loan students may struggle to take debt. Dropouts therefore have, on enough credits each semester or to average, much smaller disposable regularly attend required classes. incomes than college graduates. Additionally, students who work have Given this, policy makers may aim less time to study, and consequently, to increase the graduation rate of the probability that they pass their students who begin a college degree. classes may decrease as a function To pay for high tuition costs, of work. On the other hand, students students turn to family savings, can also get a loan to cover their

tuition. The benefit of loans is that borrowing does not require a time investment. Instead, the borrower can focus on their schoolwork and earning credits, allowing them to earn their degree most efficiently.

Given the high expected return of attending college, the question here is why low-income students are not getting more loans.

Personal financial volatility may be an important factor in students ultimately not graduating. There is a high probability that low-income students will experience a stochastic shock to opportunity cost during their college career. These shocks include bad luck like being sick before exams, a family or medical emergency, or losing parental support. Due to limited family savings (Singletary 2019), even small shocks can cause dramatic upheaval in students’ lives. Presuming that students earn utility from their family relationships, when a student’s family is financially stressed, students may fear that their family relationships will deteriorate if they choose not to work to

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supplement their family income.

Similarly, students will likely face some disutility from seeing their family struggle. Each of these struggles increases the opportunity cost of attending college by significantly increasing the utility of working. The opportunity cost of going to school may be so high that students decide to drop out of school. Given the non-zero probability of dropping out for many low-income students, students may borrow less than is otherwise considered ‘optimal.’

M

O

D

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L

To better understand the decision making framework, we use the model in Equation 1 (see equations at end) to describe the process students undergo to get a loan and then continuing school.

We can further simplify the function by making each variable a discrete binary choice, as in Equation 2.

As clarification, student loans are deferred while in school (US Department of Education 2019), and so the cost of having loans, δ, is only paid when the individual has borrowed in a previous period and is also no longer a student. Because debt ‘follows’ the student throughout their time in college, Lt-1=1 as long as the student has borrowed in any period before, or inclusive of, period (t-1). Additionally, the direct and indirect costs of attending school, X, is modeled as a normal random variable with mean μ and variance σ2, although the variance captures mostly changes in indirect costs, given little expected variance in direct costs. Our new simplified equation then becomes Equation 3.

We are most interested in the decision making of potential students, or those for whom H=0. For an individual who has just graduated from high school, Lt-1=0. To model this individual, we look at Equation 4.

I do not include the case of v(0,0,0,1) as we can assume that nonstudents are not eligible for student 62

loans. We can also say that if an individual does not attend college, there is an essentially zero probability that they will earn a college degree.

While there are some who go back to school in the future, we will consider these individuals beyond the scope of this paper, and a small enough population such that we can still yield some interesting results by making this simplifying assumption.

So, we can say that for an individual who has just graduated from high school, they will follow Equation 5.

in the max function. Otherwise, they evaluate against the first. If the high school graduate knows they will have to borrow, then they must ask themselves whether the direct and indirect costs are outweighed by the time-discounted future expected value of attending college over having never gone to college, using values based off of having borrowed.

The expected value, as discussed above, considers both the value of dropping out with debt, the value of earning a degree with debt, and the probability of earning a degree

In other words, the new high having borrowed. If the high school school graduate will first evaluate graduate knows they will not borrow, Equation 5.2 for whether they will then they must make a similar get a loan should they go to college, calculation, but they will instead which is determined entirely by the consider the choice using values expected long-term benefits of based on having not borrowed. borrowing, or the weighted average We can also treat the decision to continue college or to dropout as intermediary choices between ”Given the high the time a student graduates high school and earns their degree. expected return of Understanding the choice to drop attending college, out will give us a sense of how to estimate α10,1,L, how it may differ the question...is with changing μ and σ2, and how each may then impact borrowing. why low-income If a student has not borrowed, the decision to drop out is equivalent students are not to the choice of whether to go to college in the first place, as all getting more loans” values in v are still zero. If a student has borrowed before, we calculate between the value associated with Equation 6, assuming that the debt dropping out with or without debt cannot be paid off in one period. and graduating with or without debt, Note that Equation 6 where the probability with which suggests that the choice to drop out these weights are being calculated after borrowing is structured similarly are also determined by the choice to, though not exactly the same as, to borrow. As described above, we the choice to drop out having never can estimate that the probability of borrowed. The biggest difference graduating college increases with is that we now consider the cost more debt, or that α10,1,0 < α10,1,1. related to paying the interest and

Next, the high school balance of the debt borrowed, δ. We graduate works backwards and also consider the expected future considers Equation 5.1 and whether value of earning a degree having or not to attend college, given their borrowed, instead of compared borrowing choice. If they will borrow, to the future value of earning a then the high school graduate degree having never borrowed. evaluates against the second input No matter whether we

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HUMANITIES & SOCIAL SCIENCES

borrow or not, we can now calculate Equation 7, for those who are just starting college and are entering a four year program.

From this, we see that α10,1,Lt decreases as μ or σ2 increase, based on the shape of the normal cumulative distribution function, φ(z). Similarly, α10,1,Lt increases with ct, which changes based off of borrowing decisions, the discount factor, the return on a degree, and the cost of loans. It seems reasonable to assume that low-income and poor students will have both higher μ and σ2. So the probability of graduating is lower for low-income and poor students. This matches what we see in the data (Burns 2018).

Notably, α10,1,Lt changes depending on how many years remain in a particular program. For example, those who are in their second to last year and determining whether to enter their final year in the program instead calculate Equation 8.

If we assume Xt is independent from Xt+1, this is necessarily smaller than the previous value calculated, as any probability must be valued from zero to one. This explains why most drop outs occur in the first year and decline thereafter (Ishitani 2016). Looking at our original model, we can also conclude that is larger in the second-to-last year than at the beginning of the first year, as the benefits of earning a degree will be earned within a year compared to within four (or two) years. Thus, c3 is also likely higher than c0, c1, and c2, as more weight is placed on the benefits of a higher salary over the costs of another year, further increasing α10,1,Lt. A further extension of this may be applied to help explain why students opt for two year programs over four year programs, even though the former often have lower student outcomes overall. It is certainly reasonable to say that the probability of experiencing a shock large enough to drop out in a particular

year t will be lower if attending a four year institution compared to a two year institution, either because of a larger ct due to a higher value for a four year degree or lower μ and σ2 because of greater availability of support services at four year institutions, or both. However, if the former probability is not sufficiently higher, it is possible that the overall probability of graduating from a two year institution will still be higher than the overall probability of graduating from a four year institution, because there are fewer draws from the distribution. More research is still needed however to determine what other factors may contribute to the decision to attend two year programs over four year programs.

We next look at how a higher μ and σ2 impact the choice to borrow.

The new high school graduate starts with Equation 5.2. Notice that as α10,1,L decreases, more weight is placed on V(0,0) and V(0,1) on each side of the inequality. Given that we can be reasonably sure that V(0,0) > V(0,1), we can say that both larger and more volatile expected shocks should reduce borrowing. Now, certainly this does not guarantee that all of these individuals with high μ and σ2 will choose not to borrow. If the cost to borrow, δ, which impacts V(H,0)-V(H,1), is low enough, or the probability of graduating is high enough if L=1 (ie α10,1,1 >> α10,1,0 ) , we will still see students choose to borrow. Policies that also reduce μ or σ2 will also directly increase the likelihood of graduating, and therefore increase borrowing. However, some of these policies are probably better than others in addressing our original goal of reducing the number of dropouts. For example, considering Equation 6, reducing the cost of borrowing may also increase the probability of dropping out with debt, ceteris paribus. Evidence for policies that reduce search costs of borrowing have had mixed results,

with potentially slightly encouraged borrowing and modestly improved academic performance (Booji, Leuven, and Oosterbeek 2012; Stoddard, Urban, Schmeiser 2017; Marx and Turner 2019), but not by a large extent, suggesting that this policy may not be the best route to reducing dropouts. On the other hand, reducing both the direct and indirect costs of going to school directly increases the probability of graduating, and causes students to borrow with more focus on maximizing their chance of graduating. More research is needed, but some studies have shown promising results of stipends in addition to reduced tuition (Serino 2018), especially when also combined with additional support services (Weiss et al. 2019).

C O N C L U S I O N

On the surface level, it seems irrational to enroll in college only to drop out. However, many students struggle with circumstances out of their control. The dynamic optimization model presented in this paper demonstrates how these stochastic shocks to opportunity cost may impact a rational student’s choices. While an agent may be able to estimate the probability of a highor low-impact stochastic shock to opportunity cost, it is impossible to know with certainty whether such an event will occur. Given the high value of a college degree, it may be worth enrolling in college for the chance of earning a degree.

The model presented by this paper, given this context, suggests that policies that reduce the cost of college, provide support services or otherwise smoothen the opportunity cost for students will theoretically increase graduation rates.

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E Q U A T I O N S

Attainment.” American Economic Journal 11 (2): 108-141.

Equation 1:

10. Peterson, Christopher and Cliff Robb.

2018. “The Student Debt Crisis: Could it Slow the US Economy?” Knowledge at Wharton.

11. Serino, Louis. 2018. “As Free College Programs Move Forward, Experts Agree Design Matters.” Brookings Institution.

Equation 2:

12. Singletary, Michelle. 2019. “Does America have a savings crisis?” Washington Post.

13. Singletary, Michelle. 2012. “There Seems to Be No End to the Rise in Student Loan Debt.” Washington Post.

Equation 3: Equation 4:

14. Stoddard, Christina, Carly Urban, and Maximilian Schmeiser. 2017. “Does Salient Student Loan Information Affect College Students’ Academic and Borrowing Behavior?” Economics of Education Review 56: 95–109.

Equation 5.1 and 5.2:

15. US Department of Education. 2019. “Deferment vs. Forbearance.” Federal Student Aid.

Equation 6:

Equation 7:

Equation 8:

R E F E R E N C E S 1. Booij, Adam S., Edwin Leuven, and Hessel Oosterbeek. 2012. “The Role of Information in the

2. Take-Up of Student Loans.” Economics of Education Review 31 (1): 33–44.

Burns, Bridget. 2018. “College Rankings Need More Focus on Graduation Rates of Low-Income Students.” Washington Post.

3. Dynarski, Susan M. 2015. “An Economist’s Perspective on Student Loans in the United States”, CESifo Working Paper, No. 5579, Center for Economic Studies and Ifo Institute (CESifo), Munich 4. Golden, Lonnie. 2015. “Irregular Work Scheduling and Its Consequences.” Economic Policy Institute.

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5. Goldrick-Rab, Sara and Nancy Kendall.

2016. “The Real Price of College.” The Century Foundation.

6. Hamilton Project. 2017. “The Education Wage Premium Contributes to Wage Inequality.” Brookings Institution.

7. Ishitani, Terry. 2016. “Time-Varying Effects of Academic and Social Integration on Student Persistence in the First and Second Years of College: A National Data Approach.” Journal of College Student Retention. 18(3): 263-286.

8. Johnson, Daniel. 2019. “What Will it Take to Solve the Student Loan Crisis?” Harvard Business Review.

9. Marx, Benjamin and Lesley Turner. 2019.

“Student Loan Nudges: Experimental Evidence on Borrowing and Educational

Hopkins Undergraduate Research Journal

SERENA GOLDBERG

Class of 2021, Serena is a senior majoring in Economics, Mathematics, and Applied Mathematics and Statistics. She first became involved with research as a freshman through the Woodrow Wilson Undergraduate Research Fellowship because she wanted to better understand individual decision making and learn how to leverage research to inform policy. Outside of research, she is involved with the Economic Policy Issues Colloquium and Johns Hopkins Model UN Conference.

Next year, she will be pursuing a PhD in Economics at Yale.

HUMANITIES & SOCIAL SCIENCES

U.S.-India Maritime Strategic Cooperation A v i P. K i r p e k a r ABSTRACT

The United States and India are experiencing converging interests and a strengthening bilateral relationship across many domains. Since the 2005 landmark Civil Nuclear Deal, the U.S. and India have become closer partners and cooperation between the two nations has increased. The maritime domain receives relatively little attention despite being a critical arena in which the U.S. and India have developed robust coordination on military exercises, defense equipment trade, and cooperation agreements. Washington and New Delhi have similar strategic objectives in the Indo-Pacific, and the threat of a rising and revisionist China underpin the U.S.-India partnership. The Indian Ocean is a crucial region for all three countries, and as such, the United States and India have been and will continue to work together to preserve the balance of power. China’s increased presence in the Indian Ocean Region (IOR) through the Twenty-First Century Maritime Silk Road (MSR) is deeply concerning to the United States and regional nations due to the ambiguity of Beijing’s intentions. Maritime cooperation is and will continue to be a critical aspect of the broader United States-India relationship, and both nations will need to work closely together and with other nations to secure the region and prevent or neutralize any threats presented by increased Chinese presence. This article examines numerous studies, think tank publications, and government readouts to argue that in the maritime domain, the United States and India are experiencing a convergence of interests and will continue to cooperate to maintain the balance of power.

INTRODUCTION The United States and India possess converging interests as their bilateral relationship strengthens across many domains. Since the 2005 landmark Civil Nuclear Deal, the U.S. and India have become closer partners, and cooperation between the two nations has increased. The maritime domain receives relatively little attention despite being a critical arena in which the U.S. and India have developed robust coordination on military exercises, defense equipment trade, and cooperation agreements.

Washington and New Delhi have similar strategic objectives in the Indo-Pacific, and the threat of a rising and revisionist China underpin the U.S.-India partnership. The Indian Ocean is a crucial region for all three countries, and as such, the United States and

India have been and will continue to work together to preserve the balance of power.

China’s increased presence in the Indian Ocean Region (IOR) through the Twenty-First Century Maritime Silk Road (MSR) is deeply concerning to the United States and regional nations due to the ambiguity of Beijing’s intentions. China is currently involved in territorial disputes with various nations in both the East and South China Seas. In these areas, Beijing is building artificial islands and military installations to be used for naval operations while engaging in naval standoffs with surrounding countries (Cooper, 2017). China will likely pursue similar actions in the Indian Ocean, which would disrupt the current power system at the expense of Washington and New Delhi. India has historically been the

major IOR power, and with China now developing the connectivity and capabilities to challenge that status, the future of the Indo-Pacific is dependent on U.S. and Indian responses to the MSR. The United States has already committed to maintaining and promoting a free and open Indo-Pacific, stipulating that all nations be secure in their sovereignty and be able to pursue economic growth in line with international rules, norms, and principles of fair competition (DOD, 2019). Thus, maritime cooperation is and will continue to be a critical aspect of the broader United States-India relationship, and the two nations will need to work closely together along with other nations to secure the region and prevent or neutralize any future threats presented by increased Chinese

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presence.

This paper will argue that in light of growing Chinese actions and ambitions across the Indian Ocean Region and Western Pacific, the United States and India will need to increasingly work together in the maritime domain in order to prevent China from disrupting the balance of power in the Indo-Pacific and the world. Part I of this paper will provide a background of U.S.-India relations from the outbreak of the Sino-Indian War in 1962 to the present. Then, Part II will highlight several Chinese actions, both seemingly provocative and benign, in the Indian Ocean.

Part III will discuss instances of maritime be between Washington and New Delhi. Part IV will explain the future of the partnership between the United States and India. Part V will then conclude with some observations about the current security environment in the IOR and areas for future research.

HISTORYOF U. S. - I N D I A PARTNERSHIP FROM 1962-PRESENT The United States and India have not always been close partners.

A majority of the relationship was characterized by estrangement dictated by Cold War politics, and the two countries ended up on opposite sides as the latter half of the 20th century unfolded. During the Cold War, India was at the forefront of the Non-Aligned Movement (NAM). Also, India–Soviet Union relations and U.S.–Pakistan relations prevented a closer U.S.–India relationship. The United States found Pakistan to be a strategic asset that the U.S. could not afford to alienate, while Pakistan viewed the United States as a valuable partner in ensuring the survival of the young nation and 66

counterbalancing India (Coutto, 2015). This led to closer ties between Washington and Islamabad, at the same time as India was pursuing closer ties with the Soviet Union through military and economic assistance (Donaldson, 1972).

Despite occasional cooperation and assistance, Cold War politics kept the U.S. and India from establishing stronger ties during the latter half of the twentieth century.

When war erupted between India and China in 1962 over a border dispute, Prime Minister Jawaharlal Nehru appealed to the United States and President John F. Kennedy for support. The United States supported India against China by providing arms and air assistance and by recognizing the McMahon Line, the demarcation along the border of Northern India and Tibet set forth by colonial administrator Sir Henry McMahon in 1914 (COFR, 2020). After China unilaterally declared a ceasefire to avoid a conflict with major powers, the United States decided to increase aid to India to contain Chinese influence, demonstrating anxieties about China’s influence as early as 1962. Shortly after this, India returned to purchasing defense equipment from the Soviet Union.

China’s influence in the region also increased as Pakistan, angered over the United States’ support for India, turned more towards Beijing.

Despite U.S. support for India during this conflict due to concerns about growing Chinese influence, the overall dynamic of the bilateral relationship remained unchanged (Devereux 2009). For the next 30 years, U.S.-India relations were weak as Washington navigated numerous Indo-Pakistani conflicts and the proliferation of nuclear weapons on the subcontinent.

In 1998, however, after decades

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of tumultuous relations between the United States and India, New Delhi surprised U.S. intelligence by completing a series of underground nuclear tests close to the border with Pakistan. As the possibility of nuclear conflict between India and Pakistan became more likely, the United States criticized these tests and imposed economic sanctions, damaging the U.S.-India relationship.

Prime Minister Atal Bihari Vajpayee sent a letter to President Bill Clinton claiming China as the major nuclear threat to India (Burns, 1988). These tests awakened the United States to the fact that nuclear weapons were a fundamental barrier to partnership between the United States and India, and as such, Washington used this moment to encourage developing closer ties with New Delhi (Vickery, 2018). This was evidenced

by President Bill Clinton’s trip to India in 2000, the first U.S. presidential trip to India since 1978. The Bush administration continued cultivating a stronger relationship by lifting economic sanctions and pursuing a comprehensive defense agreement and a nuclear agreement (COFR).

Numerous high-level meetings and negotiations took place throughout 2005, a watershed year for the U.S.-India bilateral relationship. U.S. Secretary of State Condoleezza Rice and Indian Minister of External Affairs Natwar Singh established a dialogue on energy security in March, and in June, the two countries signed a new defense partnership framework.

Most importantly, on July 18, 2005, officials from both countries drafted the landmark civil nuclear deal, and the U.S. and India embarked on a strategic partnership that continues today. Indeed, China’s rise and growing power in Asia was a motivating factor for the deal, and the United States used it as a

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way to solidify relations with the world’s largest democracy in order to counterbalance China (Bajoria and Pan, 2010). The agreement represented the bridging of a chasm between the two countries that lasted throughout the Cold War and into the 1990s, and its legacy was a foundational step in the development of a geostrategically crucial 21stcentury partnership (Mukherjee and Sivaram, 2018). The rise of China and threats it posed to the U.S. and India remains one of the most pivotal factors in the U.S.-India relationship.

India continues to be an important part of preserving the balance of power in the Asia-Pacific, and one of the motivating factors behind a stronger bilateral relationship is that an economically and militarily strong India was in the best interest of the United States. Ties continued to strengthen throughout the next decade as bipartisan support for the U.S.-India relationship grew in the context of an increasingly threatening China.

Under Prime Minister Narendra Modi and Presidents Barack Obama and Donald Trump, U.S.India relations improved in almost every arena. In the defense domain specifically, in 2015, Obama and Modi signed the Joint Strategic Vision for the Asia-Pacific and Indian Ocean Region. In 2016, the United States elevated India to the status of a major defense partner, and in 2018, the Trump administration took another step forward in the defense partnership by negotiating and signing one in a series of defense cooperation agreements (COFR). In the maritime sphere, both nations regularly engage in naval exercises, information and equipment sharing, and strategic high-level dialogues.

Similar to 1962 and 2005, China will continue to be the focus of the U.S.-India relationship as the two

nations work together to secure their strategic goals.

CHINESE ACTIONS IN THE INDIAN OCEAN REGION China envisions the MSR as a vehicle to increase connectivity between China and Southeast Asia, the Indian Ocean, East Africa and the Middle East (Green, 2018). Taken at face value, this international development initiative seems to be relatively benign; but when taken with increasing Chinese antagonism in the East and South China Seas and more aggressive foreign policy overall, the MSR is concerning for IOR countries, especially India and the United States (Sahgal and Thakker, 2018). Beijing has been building artificial islands in the South China Sea with military installations capable of launching naval operations, in addition to stoking territorial disputes in both bodies of water (Cooper). Under the name of the Belt and Road Initiative, China has engaged in debt-trap schemes across the Indo-Pacific. One of the most striking examples of this was Sri Lanka’s Hambantota port, where Sri Lanka sold the port to China to pay off the debts accrued during the construction of the port (Ferchen, 2020). Indeed, the Chinese policy of building areas of influence across the Indian Ocean, known as China’s String of Pearls, will present security complications for India and the balance of power in the IOR (Khurana, 2008). Lastly, China’s more aggressive policies outside of the maritime domain, such as the border dispute with India in the Ladakh region, increasing restrictions on Hong Kong, and the treatment of Uyghur Muslims, likely points to more aggressive actions within the maritime sphere in the IOR. In the context of China’s actions around

the globe, presence in the Indian Ocean is worrying for surrounding nations and the United States, who has an active interest in maintaining the division of power in the region.

Although exact ambitions remain to be seen, China is undoubtedly actively pursuing the capabilities necessary to hold military operations and missions in the region. Indeed, China’s military, the People’s Liberation Army (PLA), is undergoing a broad modernization campaign, with the PLA Navy expanding its fleet of replenishment, auxiliary, and submarine vessels, building amphibious military installations, and developing major surface combatants (White, 2020).

Additionally, the establishment of China’s first overseas military base in Djibouti indicates that Beijing is attempting to establish a permanent PLA presence in the region, which can potentially undermine India’s and the U.S.’s geostrategic advantage in the IOR (Upadhyaya, 2017). This growth in military capabilities and resources are likely meant to promote and secure Chinese interests in the IOR with the possibility of military conflict becoming much more plausible.

The Indian Ocean is a critical region for China for a number of reasons related to the shipping routes through which China obtains raw materials, energy sources and natural resources. China is the world’s second largest economy and export country, and as such, continued growth is intrinsically linked to its ability to secure the Sea Lines of Communication (SLOCs) for uninterrupted supply of those vital goods and access to new global markets (Upadhyaya). Additionally, China faces a “Malacca dilemma” in that over 80% of maritime oil imports come through the Malacca Strait.

This presents a chokepoint that, if blocked, would be devastating

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for China (China Power, 2020).

Thus, China is seeking to diversify its oil import sources, avoiding the Malacca Strait where possible and establishing a network of pipelines via Myanmar, Pakistan, Russia and the central Asian republics, but with limited success (Upadhyaya).

Overall, China’s main interests in the Indian Ocean are to protect its SLOCs and create a pathway to

focused on managing the rise of China and ensuring no wrongdoing in the Indo-Pacific and the world writ large. Specifically, India has enjoyed naval preeminence in the Indian Ocean, aiming to be a net security provider in the region through a modern, robust, and balanced navy, capable of operating across the entire spectrum of naval operations from peacetime

“India has historically been the major power [in the Indian Ocean Region], and with China now developing the connectivity and capabilities to challenge that status, the future of the Indo-Pacific is dependent on U.S. and Indian responses to the [Twenty-First Century Maritime Silk Road].” secure its oil imports bypassing the Malacca Strait. In achieving these goals, China will look for ways to challenge the United States and India, and for this reason, Washington and New Delhi will cooperate in the maritime domain to prevent China from supplanting the balance of power.

U. S. - I N D I A MARITIME SECURITY COOPERATION Security cooperation between the United States and India manifests in many different ways, including combined military exercises, defense agreements, and high-level dialogues. All of these instances of collaboration are focused on achieving the U.S. and India’s common strategic goals and objectives, which currently are 68

to total war (Upadhyaya). While the United States has had a strong naval presence in Asia since World War II, the deepening partnership with India, exemplified by recent expansion of combined military exercises and signed cooperation agreements, will help with burden sharing and reduce the strain on both armed forces (Sahgal and Thakker).

This high level of cooperation also reassures allies and partners, deters potential adversaries and contributes to timely responses to a range of possible contingencies (Cancian, 2016). Both Washington and New Delhi are active in building maritime domain awareness (MDA), which is the functional understanding of anything associated with the maritime domain that could impact the security, safety, economy, or environment, in the Indian Ocean (Economic Times, 2018). It is

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imperative for the United States and India to have a high level of MDA in order to combat Chinese aggression in the IOR, and these instances of cooperation will effectively improve the nations’ contingency planning and knowledge of the region.

Furthermore, combined military exercises increase the interoperability of the two nations’ militaries, which is beneficial in the event of a contingency that requires U.S.-India cooperation. Three main exercises demonstrate heightened maritime security cooperation between the United States and India: Tiger Triumph, Rim-of-thePacific (RIMPAC), and Malabar.

In November 2019, the amphibious Tiger Triumph was the first-ever tri-service exercise between the two. During the weeklong exercise, U.S. Marines and sailors partnered with forces from India’s ground, naval and air armed services to rehearse humanitarian aid delivery and disaster response (DOS, 2020). This drill is a reflection of the need for developing amphibious capabilities in the IOR if a contingency necessitates a combined response from the United States and India, similar to the 2005 Indian Ocean Tsunami (Rajagopalan, 2019).

The RIMPAC exercise is the world’s largest international maritime exercise, designed to “foster and sustain cooperative relationships, critical to ensuring the safety of sea lanes and security in support of a free and open Indo-Pacific region” (U.S. Pacific Fleet Public Affairs, 2020). India, along with 24 other nations, was invited to participate in the 2020 iteration of this U.S.-led international exercise taking place off the coast of Hawaii from August 17 to August 30. These exercises demonstrate a commitment to pursuing the maritime goals of both

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the U.S. Department of Defense’s Free and Open Indo-Pacific and the U.S.-India Joint Strategic Vision for the Asia Pacific and Indian Ocean Region.

Finally, the Malabar exercises are of particular significance due to a partnership known as “The Quad,” comprising the United States, India, Japan and Australia.

This was popular in the beginning of this century but collapsed in 2008 due to skepticism and anxieties about China’s response. Almost 10 years later, in November of 2017, the Quad revived amid mounting shared concerns about China’s foreign policy, as well as growing trust, comfort, and compatibility among the four democracies. Despite the resurgence of strategic cooperation,

the U.S., Japan, Australia and the Philippines in sailing through and conducting a naval exercise in the South China Sea (Rajagopalan), and the July 2020 U.S.-India combined naval exercise off the coast of the Andaman and Nicobar islands near the Malacca Strait, which is a critical chokepoint for Chinese oil imports (Miglani, 2020). The increased cooperation between Washington and New Delhi, the revival of the Quad and India’s more aggressive posture in the greater Indo-Pacific all reflect shared anxieties about China’s growing power and presence in the Indian Ocean and concerns about China replacing India and the United States as regional and global hegemons respectively.

In addition to military exercises,

“China’s rise and revisionist ambitions are increasingly concerning for the U.S. and India, and as such, the countries will continue working together to preserve the balance of power in the Indo-Pacific.” meetings and interactions were kept at the working level because India was not yet comfortable enough to elevate this partnership (Smith, 2020). In 2019, however, India was warming to the Quad, and most significantly, New Delhi invited Australia to participate in the 2020 Malabar exercises. Typically, these exercises are trilateral among the U.S., India and Japan, but with the recent increase in Chinese activity in the Indian Ocean, the sense that New Delhi should increase its presence in the Western Pacific has materialized (Pant, 2020). This is also seen in India’s decision to join

the United States and India have signed a series of defense cooperation agreements that facilitate information and resource sharing, increase interoperability, and work towards common interests.

These agreements all serve as examples of the deepening U.S.India relationship and the increasing convergence of strategic interests for the two nations. Four such agreements stand out as significant: the U.S.-India Defense Technology and Trade Initiative (DTTI), the Logistics Exchange Memorandum of Agreement (LEMOA), the Communications Compatibility and

Security Agreement (COMCASA), and the Basic Exchange and Cooperation Agreement for GeoSpatial Cooperation (BECA). DTTI, LEMOA and COMCASA are all signed, while BECA continues to be negotiated by U.S. and Indian officials.

The DTTI began as a way to facilitate cooperation on defense technology and trade in 2012 and was affirmed by President Obama and Prime Minister Modi three years later. Since then, numerous meetings have taken place between high-level defense officials of both countries that have resulted in billions of dollars worth of defense trade (Indian Express, 2019). The technology trade under this initiative is paramount for developing interoperability capabilities in the Indian Ocean, and the co-production and codevelopment of defense equipment have positive implications on the two nations’ maritime cooperation.

LEMOA was signed in August 2016 and is an important piece in enhancing regional and extraregional naval and strategic outreach. The agreement enables the United States and India to use each other’s naval facilities for supplies and services formally and on a more regular basis, which will likely facilitate military exercises, training, and humanitarian missions.

Additionally, it is now more probable that the United States will add a military base in the Indian Ocean other than Diego Garcia (Mishra, 2018). Essentially, this agreement will again work to improve interoperability between the two navies in addition to operationalizing sharing the burden of ensuring the security of the Indian Ocean.

COMCASA, signed in September 2018, will assist the Indian military in getting better surveillance of the IOR, which is

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especially important in the context of growing Chinese presence in the region. The Indian military will gain access to high-end technologies from the United States, including communications system networks and reconnaissance aircraft, that will allow closer and more secure cooperation and communication between U.S. and Indian navies (Mishra). While LEMOA improves access to both nations’ resources, COMCASA improves the military capabilities and competencies for India and the United States, which will prove to be crucial in containing China’s revisionist ambitions in the Indian Ocean.

Lastly, BECA was signed in October 2020, as U.S. and Indian leaders recognize the importance of this agreement to easier facilitate MDA cooperation. It will enable the United States to share advanced satellite and topographical data for long-range navigation and missile-targeting with India (Pandit, 2020). This agreement will boost the navigation and tracking capabilities, again proving useful in responding to contingencies and monitoring Chinese movements in the Indian Ocean.

These combined military exercises, especially those involving the Quad countries, and foundational defense cooperation agreements represent a deepening partnership between the United States and India amid converging interests. Clearly, the rise of China on both the regional and global levels is causing anxieties in both Washington and New Delhi, and India’s renewed commitment to the Quad indicates a willingness to confront the threat posed by China.

Presence activities, like exercises and agreements, reassure allies and partners while also providing a deterrent to potential adversaries.

It is clear that the United States 70

and India have a common interest in maintaining the global and regional balance of power, so these and future actions by Washington and New Delhi will increasingly be focused on managing China’s rise, influence, and intentions.

FUTURE OF U. S. - I N D I A PARTNERSHIP For the foreseeable future, China will continue to threaten the balance of power in the Indo-Pacific and world writ large, and as such, United States-India relations will be focused on limiting Beijing’s power to disrupt the power structure.

While the Indian Ocean Region and the Western Pacific are two of the domains in which the U.S. and India cooperate and resist Chinese aggression, other arenas such as outer space, emerging technology and the economy will also be critical in countering China. U.S. strategists have been keeping a close eye on the development of India because an economically strong and rising India is in the best interest of the United States. An empowered New Delhi will frustrate Chinese regional and global hegemonic aspirations, reduce the costs to the U.S. associated with preventing those aspirations, and substantially offset Chinese influence in Central Asia, Southeast Asia and Africa (Mead, 2020). Thus, the United States should bolster India’s economy in order to ensure a wealthy, powerful and democratic India emerges as an ally in this new era of great power competition.

Despite potential points of divergence between the United States and India on issues of human rights or the Russia-India relationship, both nations will likely prioritize their converging interests over these points of contention.

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China’s rise and revisionist ambitions are increasingly concerning for the U.S. and India, and as such, the countries will continue working together to preserve the balance of power in the Indo-Pacific. This will require further levels of cooperation and collaboration between U.S. and Indian officials in other sectors of the government, further solidifying the partnership between the two nations.

The 2+2 dialogue, consisting of the U.S. Defense and State Departments and the Indian Defense and External Affairs Departments, has been highly effective in encouraging closer cooperation. Finding common interests in areas other than defense and security, such as energy and technology, will be important for future cooperation, and an open dialogue similar to the 2+2 dialogue can prove beneficial (Rossow, 2019).

While cooperation in other sectors may not address the China threat directly, increased cooperation is indicative of a strengthening partnership between the U.S. and India and a more robust response to increasing Chinese presence in the IOR.

CONCLUSION Due to China’s increasingly aggressive and powerful position on the world stage, the United States and India have converging interests and many instances of deep cooperation. China’s actions in the Indian Ocean Region and the South China Sea point to a more combative and expansive foreign policy, and its ambitions in engaging in these maritime regions are ambiguous, contributing to anxieties across the world about China’s rise.

While Beijing is challenging both the balance of power in the IOR and in the world, Washington and New Delhi have ignited a strategic partnership that is focused on containing

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Chinese ambitions in the IndoPacific. Through combined military exercises and defense cooperation agreements, the two nations are collaborating in order to maintain their respective regional and global power, and this partnership will continue as the threat of Chinese dominance continues for the foreseeable future.

AC K N O W L E D G E M E N T S I thank Ronak Desai for inspiring my passion for South Asian security issues and Professor Steven David for allowing me to pursue my passions, despite the lack of resources available to me. I would further like to thank both Mr. Desai and Professor David for all their help in numerous rounds of proposals, outlines, and edits.

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AVI P. KIRPEKAR Class of 2022, Avi is a junior studying International Studies and Political Science with a minor in Economics. He will attend JHU SAIS this Fall as part of the BA/MA program where he will study Conflict Management and South Asia Studies.

He is originally from Cleveland, Ohio, and his main interests lie in the intersection between Indian and American security and strategic interests in the global community.

He hopes to work for the State Department after graduation to help solve international crises using diplomacy and negotiation.

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The Hybridization of M o d e r n i t y a n d Tr a d i t i o n in the Age of Indian Matrimonial Websites J emina Opman-Auge ABSTRACT

Today, internet matchmaking services are replacing more traditional forms of marriage intermediation. With

this evolution comes the possibility of change and disruption of the status quo. In this paper, I will delve deeper into the processes behind modern Indian marriage, placing special emphasis on the evolving role of individual agency, premarital romance, and cultural rootedness.

How do modern couples balance a desire for greater participation in spouse selection, all while remaining within the boundaries of what society considered ‘acceptable’? What compromises are made when vying for both social legitimacy and individualism in marriage, and how might these compromises impact religious divides and social hierarchies? Matrimonial websites are only accessible to those with an internet connection and a valid phone number. As a result, this analysis will primarily serve as a window into the marital trends of the urban, literate class. In the subsequent paragraphs, I will suggest that urban Indian couples are today reshaping the social consensus on marriage toward greater agency and individual compatibility, all while maintaining an emphasis on strong kinship and community ties. This is achieved through a hybridization of Western modernity and Indian tradition, ultimately helping to maintain religious and caste divides.

INTRODUCTION Marriage in India implies arranged. Currently ninety percent of India’s marriages reportedly fall in the ‘arranged’, as opposed to ‘love’, category (Titzmann, 2013). To facilitate matchmaking based on traditional criteria (namely religion, education, horoscope, caste, and profession), arranged marriages have for decades relied on forms of intermediations. These include one’s own social circle (kinship and caste networks), as well as third-party marriage bureaus and matrimonial newspaper advertisements. Today, the most recent iteration of marriage intermediation, arising in conjunction with the widespread usage of internet technology: matrimonial websites (Mukhopadhyay, 2012; Seth and Patnayakuni, 2009).

First appearing in the late 1990s, Indian websites dedicated to matrimonial matchmaking have grown exponentially in the last two 72

decades (Seth and Patnayakuni, 2009). The subject of this paper, Shaadi.com, claims to be the world’s oldest and most successful matchmaking service, founded in 1996 and with over 35 million members in its database (Shaadi.com, 2019).

While matrimonial websites are also prevalent in the West, India-based portals such as Shaadi.com continue to incorporate distinctively South Asian characteristics. Upon logging into Shaadi.com, for instance, one must provide information regarding specific diets followed (with options including Jain and eggetarian), community and subcommunity, and who the account is being created for (self, child, sibling, friend). Further optional specifications include complexion, blood group, and astrological details.

Indian matchmaking websites, far from abandoning tradition in exchange for Western ‘modernity’, are evidently targeting a specifically South Asian audience.

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As a result, websites like Shaadi. com become especially useful for considering current trends taking place in this region of the world.

P A R T 1: E M E R G I N G TRENDS ON SHAADI.COM Arranged marriage, often referred to as a union between two families rather than two individuals, is a form of marital alliance orchestrated by family members with the goal of creating ‘suitable matches’. Parents typically initiate and mediate the selection of a spouse for their child by relying on social networks or third parties, granting little agency or say to the prospective bride and groom. The practice is geared around preserving traditional notions of compatibility and a sense of community obligation, with key criteria including community, religion, caste, education, and social status (Mody, 2002). Romantic love, however,

is not a necessary precondition to marriage. This selection of partners based on traditional criteria rather than individual compatibility implicitly serves to consolidate communities and maintain existing social hierarchies.

Prospective brides and grooms are given very little opportunity to get to know each-other in any meaningful way; instead, they are expected to remain consenting in a show of familial obligation and community loyalty, regardless of whether they are ‘in love’ or not (Mody, 2002).

Diametrically opposed to arranged marriage, love marriage is based on the Western notion of marriage in which two individuals choose for themselves to wed as a result of romantic involvement (Mody, 2002). This union is socially illegitimate, commonly viewed as a “most unholy” affront on Indian values, community obligations, and the ‘natural’ social hierarchy (Mody, 2002, p.225). In particular, love marriage is viewed as threatening and anti-social because of the risk it poses to the persistence of traditional social hierarchies and divides (Mody, 2002). Love and romance do not take into account caste, social status, or religion thus introducing the possibility of marriage outside one’s own group.

As a result, where arranged marriage is geared toward preserving traditional notions of compatibility, love marriage threatens to destroy long-standing community ties.

The rigid binary between ‘social choice’ and ‘individual choice’ often found in literature portrays love marriage and arranged marriage as largely mutually exclusive, with agency and premarital love both residing firmly outside the social pale.

And yet, given the rise of technology and novel digital match-making solutions, it is worth reexamining

the applicability of this dichotomy of terms in the modern urban Indian context. Do agency and premarital love remain socially illegitimate?

Or, conversely, has social legitimacy expanded beyond the restrictive confines of traditional ‘love’ and ‘arranged’ marriage definitions?

To gain insight on these questions, I use data from the Shaadi.com matrimonial website as a reflection of modern urban Indian attitudes on marriage, romance, and agency. I analyze four months of testimonials, posted from July to November 2019 and featured on the ‘Shaadi pride’ section of the website.

These testimonials, freely available to all internet users, were written by couples who met on Shaadi.com and are now endorsing the platform by sharing details of their ‘success stories’. One concern associated with self-written testimonials is that they may provide a skewed perspective of the actual premarital selection process. Couples, for instance, might exaggerate or embellish reality to paint their unions in a more favorable light (for instance, with ‘love at first’ cliches). This risk, however, is not of much practical concern for the conclusions of this paper. Here, the testimonials serve to gain insight on the values and characteristics that individuals consider important when choosing their partners. While perhaps not painting a perfect picture of reality, the testimonials, at the very least, accurately reflect how these ‘success-story’ couples would like their unions to be perceived by society.

In terms of methodology, I filter out any responses repeated verbatim over five times to remove autogenerated text from the analysis, for a total of fifty testimonials. Further, I view marriage on a spectrum, with traditional arranged marriage (no individual agency or romance before

unions) at one extreme and love marriage (no consideration given to family or tradition) at the other. This avoids categorizing the testimonials in the traditional rigid binary, allowing instead for the emergence of ‘gray zones’ in between the two extremes that might complicate the narrative of matrimony in India.

Upon analyzing the testimonials, just over half seem to lie somewhere in the middle of the spectrum, characterized by an emphasis on family approval on the one hand, and individual agency on the part of the prospective brides and grooms on the other (Table 1). These unions, referred to in Uberoi’s analysis of popular Indian cinema as ‘arranged love marriages’ (Uberoi, 2006), can be summarized in one of the analyzed testimonials: I got an interest from Deepak and I liked his profile too so interest was accepted. We exchanged our numbers and spoke to each other about our jobs, lifestyle and families and we found each other compatible so we decided to meet up as our families wanted us to meet first. In few days we decided to involve our parents and then everything fell in place by gods grace. We happily got engaged on 4th Oct 19!!!

Tulika and Deepak The general storyline that emerges from these testimonials, and in line with Uberoi’s arranged love marriage concept, is that a couple meets on Shaadi.com, has some degree of contact (in-person and/ or virtual communication), develops romantic feelings (often described as ‘meant to be’ or ‘love at first sight’), and seeks family approval.

Ultimately, once consent has been received, the family finalizes the

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engagement. Receiving the family’s blessing, however, is not always easy: Eventually we fell in love but families were not ready to accept our pair. Sanket had to convince his parents a lot and despite the struggle he had to go through, he never backed off.

Sneha & Sanket Sanket’s insistence on validating individual compatibility with social compatibility reinforces the importance accorded to family approval. Two central tendencies, mutually exclusive according to traditional definitions of love and arranged marriage, therefore surface. On the one hand, there is a direct articulation of agency and choice on the part of the marriageable youth themselves, highlighted by the omnipresent emphasis on communication and individual compatibility. The couples are clearly establishing themselves as key players in their own relationships, with strong romantic connections and love given center-stage in the selection process. On the other hand, however, family consent of the union remains indispensable for taking the next steps toward marriage: in each of the thirty cases, the authors felt compelled to specifically mention that family approval was received before proceeding on to legitimizing their relationships, indicating that the social legitimacy of their love at least partly relies on maintaining a facade of adherence to tradition.

The next group that emerges, with the shared characteristic that a family member, rather than ‘self’, was the first to initiate contact, lies nearer to arranged marriage on the metaphorical spectrum. Ten out of the fifty testimonials are included within this category, yet continue to reside in the gray zone as a result of 74

their deviation from the traditional arranged marriage definition: while the unions are arranged in the sense that a family member or friend made the initial selection, there remains an emphasis on individual agency and modern notions of premarital romance incompatible with traditional understandings of acceptable matrimony.

Our meeting was arranged by our parents, but on the first meeting itself we felt that spark between both of us and our journey started.

- Kawal preet & Gursimran

individual compatibility and agency, with the prospective brides and grooms being the key players in the marriage process. And yet, while no mention of family is made, this does not imply that seeking and receiving parental consent was actually absent. As such, the number presented here should not be considered equivalent to the number of socially illegitimate elopements undertaken without family approval.

Further, even within this sphere dominated by western notions of love and individual agency, I continue to find references to Indian tradition: in nine out of the ten testimonials, the attached photos depict the couples

“...it becomes essential that couples seeking social legitimacy demonstrate a certain degree of adherence to tradition.”

The mention of “spark” and “our journey” paint the portrait of couples who have regained agency over their relationships, who emphasize premarital romance and individual compatibility - rather than blind obedience to the dictates of parental authority - as reasons for marriage. Contrary to attempting to hide their romance or ‘spark’, these newly-weds and newly-engaged deliberately attempt to exhibit a certain degree of ‘modernity’.

Finally, ten out of the fifty testimonials, all of which do not include any mention of parental consent or family, lean closer to love marriage. For instance:

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Instinctively both of us felt that this was the right choice. After that we met [a] few more times and decided to take it further. We got engaged.

Shirkant & Priyanka The central focus is here on

in traditional South Asian clothing.

The saris, kurtas, bindis, and mehndi serve to represent an enduring identification with cultural roots, even in spite of a clear departure from traditional understandings of socially acceptable marriage.

To summarize, in each of the three cases, whether the union was closer to arranged, love, or arranged love marriage, I find a desire common among almost all testimonial couples to present themselves as both ‘traditional’ (adhering to Indian values) and ‘modern’ (valuing premarital compatibility, agency, and romance), to varying degrees.

This general trend is reflected in Shaadi.com advertisements, which emphasize both the role of family and the individual in finding suitable spouses. In a commercial titled “Like Nahin Like Like”, for instance, a father tirelessly attempts to find a match for his daughter, only to have her turn down the offers because she likes, but does not ‘like like’, the

suggested candidates. (“Select Shaadi : Like Nahin Like Like”, 2018).

This example solicits notions of romantic love and individual choice, pointing toward a hybridization of Indian tradition on the one hand and Western modernity on the other.

Matrimonial websites inherently facilitate greater involvement of prospective brides and grooms in the process of selecting a spouse. For one, internet-mediated matchmaking provides greater anonymity, thereby reducing the burden of social pressures and stigmas characteristic of reliance on familial social networks for intermediation (Seth and Patnayakuni, 2009; Agrawal, 2015). As it becomes possible to engage and disengage without fear of adverse social consequences, additional steps geared toward couples getting to know eachother are emerging without directly challenging social norms. Further, future husbands and wives are now selected among millions of strangers in a virtual database, unvetted by the social networks or marriage bureaus that once served to provide context and credibility. As a result, greater communication can even be considered a security imperative associated with the digital age (Agrawal, 2015). With messaging and calling bringing down all barriers to communication, the very concept of limited contact before marriage nearly seems to be incompatible with modern matrimonial websites in a technology age. These developments, in turn, create an opportunity for the role of parental intervention in intermediation to recede. As marriageable individuals become increasingly active in selecting their spouses, Western notions of love and compatibility dominate. In the testimonials, there is repeated mention of “soulmates”, “swept off my feet”, “love at first sight”

- which all could have easily been taken from the scripts of Hollywood or Bollywood movies. Couples therefore seem to be using this greater individual agency to ensure that romance exists prior to marriage.

And yet, even with this newfound ease in communication and facilitation of increased individualism, kinship and community ties remain strong. The couples in the testimonials, reluctant to marry for exclusively romantic reasons, blend traditional criteria with individualistic expectations and make independent decisions while staying culturally rooted. The validation provided by family approval and the upholding of traditional values continues to be considered vital, with romantic love alone perceived as insufficient to sanctify marriage. The importance of tempering the pursuit of Western notions of love and compatibility with an emphasis on cultural rootedness possibly stems from the very nationalism at the foundations of Indian identity: throughout the entire phase of the national struggle, “the crucial need was to protect, preserve and strengthen the inner core of the national culture, its spiritual essence” – a responsibility that fell especially on women (Chatterjee, 1989, p.624).

Failing to highlight tradition comes with the risk of being seen as having betrayed Indian culture. Balancing tradition with modernity, rather than embracing a love marriage extreme foregoing social obligations, may therefore be born out of a national imperative with origins in a struggle against colonialism, thereby remaining essential in the pursuit of social legitimacy.

As a result of this hybridization between Western and Indian norms, the results from the analysis of testimonials, lying somewhere in between the love and arranged marriage extremes, do not fit into a

rigid binary. This problematization of the existing age-old dichotomy can prove to be a source of confusion for couples, who struggle to find the proper label for their unions: The more we talked the more we found a perfect partner in each other. We talked to our families and they were too happy and agreed to accept […] So we call it an arranged marriage because we just choose each other but family approved our choices then it happened! - Daljeet & Chanchal One gains clarity, however, when appreciating the existence of ‘gray zones, characterized by the various compromises that individuals make between individual and social compatibility to reach a sphere of social legitimacy. The simplified polarization between love and arranged marriage, upheld by choice and consent defined in exclusive terms (either choice / consent of society or choice / consent of individual), therefore does not accurately reflect the reality on matrimonial websites where individualism does not preclude strong kinship and community ties.

As labels are increasingly difficult to apply, this suggests that the very concept of an Indian marriage implying ‘arranged’ deserves revision in an urban context. Hence the importance of recognizing a spectrum of possibilities, which allows for the emergence of nuances beneath the seemingly straightforward arranged versus love construct.

To conclude, in a telling sign of changing times, couples today post testimonials for the world to see in which notions of romance and agency take center stage. What was an excluded and socially illegitimate minority - couples seeking romantic

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love before marriage - has now integrated itself into the socially acceptable urban norm. These couples have gained social legitimacy through a compromised individualism oriented toward tradition and family. The emphasis on modernity in the testimonials suggests that, in an urban context, arranged marriages might appear ‘too traditional’, in a sense almost backwards relative to the emerging trend valuing agency. In a similar vein, however, the overwhelming insistence on family values and consent indicates that love without community backing might very well remain ‘un-Indian’ and outside the social pale. As individuals continue to seek family approval to validate their unions, what might the result be for society’s religious and caste divides?

Might an insistence on adherence to tradition imply a persistence of the social hierarchies? Or is the trend towards agency pointing to a reversal of existing community norms? I will address these questions in the following section.

P A R T 2: MATRIMONIAL WEBSITES AND SOCIAL HIERARCHIES Rigid religious and caste structures have long been used as means of division in Indian society, and have been perpetuated by the practice of arranged marriage (Mody, 2002). Social hierarchies are maintained by portraying endogamy (also referred to as in-marriage, or marriage within one’s own group) as an imperative to community integrity. Elders select spouses that fit within caste, class, and religious boundaries, ruling out the risk of outmarriage (marrying outside one’s group) (Mody, 2002). The same cannot be said for love marriage.

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Here, the focus on individual rather than social compatibility introduces the possibility of socially illegitimate inter-caste or inter-religious unions that stray from the endogamous norm (Mody, 2002). As a result, one of the greatest obstacles faced by love-marriage couples in their vying for social inclusion is the threat they pose to traditional social divides and community homogeneity.

And yet, as seen in the previous section, matrimonial websites are today blurring the love and arranged marriage extremes in favor of increased agency coupled with cultural rootedness. With this, it is worth asking: do internet matchmaking services threaten religious and caste divides, or promote endogamous marriage along community lines? In the analysis that follows, I pursue a more theoretical approach by delving into three primary factors that interact to maintain, and perhaps even strengthen, the social hierarchy: the promotion of social divides rooted in the functioning of matrimonial websites, the insistence on family approval that suggests the mimicking of traditional value by prospective brides and grooms themselves, and the substantially larger pool of available candidates satisfying specific sub-caste preferences.

Upon signing in to Shaadi. com, the influence of distinctively South Asian social characteristics directly comes to the fore. Before viewing any profiles, for instance, individuals are required to indicate their religion, caste, and subcommunity. Similarly, when selecting preferences that will filter out potential available spouses, key criteria include questions pertaining to one’s openness toward various specific communities and religions.

Especially telling, the default is set to ‘not open to inter-caste caste

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or inter-religious marriage’. These sign-up features reflect search options and filtering criteria that are unabashedly organized along caste, religious, and community lines, with an emphasis on favoring intracommunity marriage. The website’s structure alone, by facilitating and promoting endogamous unions mirroring traditional matchmaking considerations, seems to maintain caste and religious divides.

Further, as reviewed above, the analysis of success story testimonials points to an overwhelming majority of couples (forty out of fifty) placing strong emphasis on family, validating romantic love with parental approval. Saurabh and Deepika, for instance, “made sure everyone is happy in [their] families”. This almost omnipresent emphasis on family strongly suggests that traditional caste and religious criteria are being mimicked by the potential brides and grooms themselves, even when they exhibit substantial agency in the selection process. As couples vie for social legitimacy and parental consent, these young adults seem to be taking up the community’s original gatekeeping role, replicating traditional community values to gain family approval. In only one of the fifty success stories is there an explicit reference to marriage outside of the sub-caste divides: Suchma is a “from Garhwali Brahmin family” and Mukesh is “nonGarhwali Brahmin, still because of the qualities he had, all goes with flow.” Yet even here, this is an intersub-caste, rather than inter-religious or even inter-caste, marriage. Rather than an exception, this case serves to further reinforce the notion of persevering community divides: with the issue of sub-caste portrayed as a negative that has to be justified and outweighed by a positive (“qualities”), Suchma seems to

acknowledge the potentially socially controversial nature of the union (even when it remains intra-caste).

Lastly, matrimonial websites are removing the geographic and network limitations that would otherwise have made maintaining strict in-marriages more challenging.

Taken alone, urbanization has the potential to normalize heterogeneity and thereby threaten religious and caste divides perpetuated through arranged marriage. With urbanization comes increased geographic and social mobility, as individuals move from their traditional villages to seek economic opportunities in urban centers. As social and geographic mobility increase, the extended family structures and social networks decline, making it more difficult to identify suitable partners for marriageable offspring (Seth and Patnayakuni, 2009; Mukhopadhyay, 2012; Agrawal 2015). Social circles are also inherently limited, the ability to find a ‘perfect match’ largely dependent on, and restricted by, the reach of available contacts.

With matrimonial websites, these threats to in-marriage are essentially eliminated. There are now thousands of active users to choose from - lack of choice is certainly not a significant concern. No longer limited by social circles or geographic barriers, the availability of options capable of maintaining endogenous matrimonies increases exponentially.

This facilitates the unions of those who, without access to matrimonial websites, might otherwise have been left with little alternative than to marry outside their specific communities.

By both offering a far larger pool of potential candidates and encouraging social categorization based on community lines, internet matchmaking services are simultaneously able to satisfy the two seemingly contradictory priorities of

modern urban couples: an emphasis on individual compatibility (though greater agency and romance before marriage) and social compatibility (through family approval). As prospective brides and grooms replicate traditional preferences for community endogamy while bolstering their involvement in the selection process, matrimonial sites seem to facilitate the reinforcing of existing social hierarchies and divides associated with traditional arranged marriage. A comparison can here be drawn with Facebook.

While the breadth of information available on the platform is nearlimitless, “users tend to aggregate in communities of interest, which causes reinforcement and fosters confirmation bias, segregation and polarisation”, just as users of Shaadi. com tend to filter the profiles they see and receive invites from based on community lines (Vicario, et al, 2016).

An increase in choice therefore does not necessarily equate to greater diversity in either thought or marriage.

In the previous section, I examined how couples seeking romance and individual compatibility before marriage make compromises when vying for social legitimacy, namely by maintaining a degree of adherence to tradition. I now add a corollary that follows from the above analysis: an insistence on family approval, compounded by the matrimonial websites’ filtering criteria and abundance of available potential spouses, serves to promote matrimony along the lines of community endogamy. At the root of the public outcry accompanying the legalizing of love liaisons in India’s civil marriage law was an apprehension of the possibility of social mixing between communities and castes (Mody, 2002). With this in mind, to obtain social legitimacy, the modern urban couple must

balance their newfound agency with an upholding of religious and caste structures. By doing so, they reassure society that love need not destroy the ties of obligation between individuals and their communities. Marriage, as such, can continue to serve as a tool that consolidates communities in a modern urban setting. The traditional arranged marriage process may be undermined by the rise of matrimonial websites - but the same cannot be said for the social hierarchy.

CONCLUSION To summarize, this paper, through the lens of the Shaadi. com matchmaking service, has problematized the existing dichotomy between love and arranged marriage, indicating that the bifurcation between the two terms as mutually exclusive polar opposites is not applicable to the modern urban Indian setting. As fundamental principles of traditional arranged marriage gradually give way to the Western notions of romantic love, individual compatibility, and agency, it becomes essential that couples seeking social legitimacy demonstrate a certain degree of adherence to tradition in order to retain an appearance of ‘Indianness’. This is coherent with the analysis of testimonials: I find no mention of an unabashed embrace of ‘modernity’ or a revolt against age-old social divides, but rather a deliberate effort to temper increased agency with an emphasis on the seeking and receiving of family consent. While love alone therefore seems to remain insufficient to sanctify marriage, urban couples are gradually normalizing agency and romance before marriage, once deemed socially illegitimate, through the retention of Indian values.

This insistence on approval in turn suggests these couples are mimicking

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the traditional endogamous criteria as they select a spouse. In other words, the social legitimacy of a union rooted in individual compatibility seems to depend on a fundamental compromise: the taking up of the communitygatekeeping role by the couples themselves, ensuring the endurance of existing social homogeneity.

One can therefore speculate that marriages lying within the gray areas, characterized by greater agency and premarital romance, are not necessarily accepted because the social consensus has drastically liberalized; rather, they are accepted precisely because these unions, as a result of their emphasis on tradition and consequently endogamy, are seen as non-threatening to the persistence of long-standing social structures and hierarchies.

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11. Seth, Nainika and R. Patnayakuni. “Online Matrimonial Sites and the Transformation of Arranged Marriage in India.” Social Networking Communities and E-Dating Services: Concepts and Implications.

Ed. Celia Romm-Livermore and Kristina Setzekorn. New York: Information Science Reference, 2009: 329–352. Print.

12. Shaadi.com, www.shaadi.com, accessed 30 November 2019 13. Sharangpani, Mukta. “Browsing for Bridegrooms: Matchmaking and Modernity in Mumbai.” Indian Journal of Gender Studies 17.2 (2010): 249–276. Print.

14. Titzmann, F.-M. 2013. Changing patterns of matchmaking: The Indian online matrimonial market. Asian Journal of Women’s Studies 19(4): 64–94.

15. Titzmann, F.-M. 2015. Matchmaking 2.0. the representation of women and female

agency in the indian online matrimonial market. Internationales Asienforum 42(3-4): 239–256.

16. Titzmann, Fritzi-Marie. “Medialisation and Social Change: The Indian Online Matrimonial Market as a New Field of Research.” Social Dynamics 2.0: Researching Change in Times of Social Media and Convergence: Case Studies from Egypt, Lebanon, Iran, India, Malaysia and Indonesia. Ed. N. Schneider and B. Graf.

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17. Uberoi, Patricia. Freedom and Destiny: Gender, Family and Popular Culture in India.

Delhi: Oxford University Press, 2006. Print.

18. Vicario, Michela Del, et al. “The Spreading of Misinformation Online.” PNAS, National Academy of Sciences, 2 Jan. 2016, www.pnas.org/content/ early/2016/01/02/1517441113.abstract.

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Table 1. Summary of findings: number of testimonials relative to criteria

ACKNOWLEDGEMENTS

I thank Ghazal Asif for her guidance throughout writing this paper, and for introducing me to the conceptions of nation, community, tradition and belonging in the modern South Asian context.

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JEMINA OPMAN-AUGE Class of 2023, Jemina is a sophomore from Los Angeles majoring in Economics, with minors in Entrepreneurship and Management.

After graduating from high school, Jemina spent nine months on a gap year in Pune, India. There, she lived with a local family and volunteered as a teaching assistant with Teach for India. After watching her host sister find a husband on Shaadi.com, Jemina became fascinated with the dynamics between matrimonial websites and arranged marriage in the urban Indian context.

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