ScienceMind Spring 2022 (Issue 11)

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Neural Circuits and Mapping the Brain part 1: From DEEP

DIVE

to Action Potential Neuron

n 2013, the US president Barack Obama made an amazing announcement He

WRITTEN

BY

ALEXI MERY

IEDITED AND DESIGNED BY WINNIE (WING YIN) LIU launched the BRAIN initiative, a plan whose goal was to map the brain This project was given a budget of 300 million dollars in funding. This goes to show the importance that neuroscience research has taken in the twenty-first century In this series of articles, we shall explore how research in neuroscience has grown to the point where such an initiative is now possible In this first part, we shall look at how our understanding of single neurons has grown We shall start by looking at the work of Santiago Ramón Y Cajal and Camillo Golgi and go all the way to the development of neurophysiology

Cellular neuroscience research only really started with the development of the field of histology in the late nineteenth century Histology is a field which studies anatomy of tissues, cells and organs under a microscope. Those of you that have used a microscope will know that the issue with cells and other structures is that they are often either transparent or all the structures have the same colour, making it very difficult to see The way around this is to use a stain, which is a chemical compound which colours a specific structure in a specific colour.

Our story starts with a certain Joseph von Gerlach He developed a hugely influential model for the organisation of the nervous system: the reticular theory, which he devised in 1871 This model suggested that the cells that comprised the nervous system were all connected without any gaps between them, in a sort of giant mesh which was named the ‘reticulum’. Gerlach came up with this theory based on a staining technique that he came up with What Gerlach saw with his stain was that nerve cells seemed to connect to each other in a continuous fashion Another scientist called Otto Friedrich Karl Deiters performed similar staining experiments to Gerlach and corroborated Gerlach’s model.

It was around this time that another scientist entered the stage: Camillo Golgi He was an Italian pathologist who worked at the university of Pavia In 1873, he made a remarkable discovery: he found that if you fix nervous tissue in potassium dichromate for several days, followed by immersion in silver nitrate,

one could obtain tremendously clear pictures of neurons, as seen in figure 1A The above reaction was named the ‘black reaction’ or ‘reazione nera’ This staining method was the histological breakthrough needed to start elucidating the microanatomy of the brain.

Unfortunately, in a turn of fate which shows that even nobel laureates can make mistakes, Golgi also believed in the reticular theory of neural organisation It would take a serendipitous encounter for the reticular theory to become disproven This was a meeting between Don Luis Simarro and Santiago Ramón Y Cajal, in 1887. Cajal was a young anatomist at this point and Simarro was a psychiatrist who had started using Golgi’s stain Cajal was instantly hooked He immediately decided to do his own work with this method and published drawings of the cerebellum and retina. It was Cajal who named the famous mossy and climbing fibres in the cerebellum and depicted how they were connected together

Unlike Golgi, Cajal believed that nerves were individual elements and did not touch each other, and this idea was actually synthesised by Waldeyer in 1891, a very influential German anatomist Waldeyer hypothesised that neural tissue was built from lots of individual neurons acting as functional units, working together Incredibly, Cajal also observed structures on dendrites that would become absolutely crucial later on in our story: dendritic spines (see figure 1B) In 1897, Charles Sherrington coined the term ‘ synapse’ and described it as the contact between neurons

Based on all the above work, Cajal developed his four principles of neural organisation from 1891 to 1906

Neurons are the basic building block of the nervous system Neurons communicate via synapses Neurons connect to other neurons in neural networks Information flows through neurons in a unidirectional way (dynamic polarisation)

Figure 1: Drawing of neurons by Cajal and Golgi (A) A drawing of the circuitry in the cerebellar cortex done by Golgi using Golgi staining and (B) is a drawing of pyramidal cortical neurons done by Cajal using methylene blue staining showing the neural spines (taken from Glickstein, 2006)

Paradoxically, Golgi and Cajal went on to share the Nobel Prize in Medicine in 1906 and that event rewarded the work of two men who really started the field of modern neuroscience However, there were still several more breakthroughs to go. In particular, now that we know what the anatomical building blocks are, it is time to understand how they work together to produce some of the functions of the nervous system Time to discuss the birth of the vast field of neurophysiology

This field really started with Luigi Galvani and the discovery of electricity in animals in 1780 In the eighteenth century, it was well known that a spark administered to an animal’s muscle causes a contraction, suggesting that electricity played a role in neural conduction. However many scientists still believed that it was the movement of fluids that allowed the nervous system to function Galvani chose to try to understand what importance electricity might have in living systems and he used the frog as his organism of choice What Galvani realised was that he did not need to stimulate the frog muscle to cause contraction.

Galvani realised this since if a frog muscle was suspended from an iron railing, just jostling it around could cause a contraction

This suggested to him that animals contained their own kind of electricity. In order to explain this finding, Galvani even went so far as to suggest that there were positive and negative charges on either side of the nerve fibre and that it was the movement of these charges across the nerve that caused the current he observed He also believed that channels would be needed to transport these charges, showing just how insightful he was.

Importantly, Galvani popularised a piece of equipment that would prove essential to understand the properties of the nervous system This was the ‘galvanometer’ , which could be used to detect the presence of an electric current. Although at the start it was very inaccurate, this did not stop the physiologist Carlo Matteucci from using it to measure a current between a nerve fibre and an injured muscle (injury current)

Figure 2: Image of a galvanometer from about the time of Luigi Galvani It works by deflecting a pointer when a current passes through a magnetic field In other words, the galvanometer uses the motor effect (taken from the Wellcome collection gallery)

IIn 1841, Matteucci’s experiment inspired Johannes Mueller, one of the most prominent physiologists of his time, to reproduce his findings He got a student of his, Emil Du BoisRaymond, to do it Du Bois-Raymond succeeded, in particular by developing a more accurate galvanometer, and he also extended some of Matteucci’s findings to nerves He realised that nerve current decreases with prolonged stimulation or tetany Du Bois-Raymond called this the ‘negative variation’

Du Bois-Raymond believed that it was this negative variation that was transmitted through nerves and he wanted to prove it by measuring its conduction velocity He also wanted to see whether the negative variation was larger than the resting potential or not and if there was a polarity reversal. The measurement of conduction velocity in nerves had actually already been made by another student of Johannes Mueller, Hermann von Helmholtz in 1850 He did this by creating an accurate measuring device that would measure the difference between the moment a stimulus was applied and the muscle twitch

This problem of measuring negative variation would eventually be passed on to one of his students: Julius Bernstein Using some extremely clever methods and by devising a series of new measuring techniques, he managed to record the first image of an action potential, as shown in figure 3 Bernstein’s great breakthrough was his ability to very finely change the timing of nerve current measurements He did this by using a device he invented called the ‘rheotome’

Figure 2: The first image of an action potential captured by Julius Bernstein in 1865 The more familiar representation would be an upside-down version of this image (taken from Schuetze, 1983)

35 years after measuring the above trace, Bernstein was finally in a position to publish a model for resting and action potentials He used studies done by scientists such as the chemist Walther Nernst to suggest that it was potassium ions (K+) that generate the resting potential gradient What is fascinating is that Bernstein did notice that there was an overshoot after the repolarisation, what we call hyperpolarisation However, he failed to include it in his own model Moreover, the importance of sodium ions (Na+) was also neglected, even though a scientist called Ernest Overton had already discovered that sodium ions were essential for proper nerve conduction It would take 40 years and a whole new generation of neurophysiologists for this mistake to finally be corrected However, that is a story for part 2

To be continued…

References

Bentivoglio, M , Cotrufo, T , Ferrari, S , Tesoriero, C , Mariotto, S , Bertini, G , Berzero A & Mazzarello P (2019) The Original Histological Slides of Camillo Golgi and His Discoveries on Neuronal Structure Frontiers in Neuroanatomy 13

https://doi org/10 3389/fnana 2019 00003

Connelly C & Chang H (2019) Galvanometers and the Many Lives of Scientific Instruments (F Willmoth J Nall & L Taub Eds ) Cambridge University Press; Cambridge University Press

https://www cambridge org/core/books/whi pple-museum-of-the-history-ofscience/galvanometers-and-the-manylives-of-scientificinstruments/2C8AA0727D87A087BC5AF3FC 907C2DC0

Delgado-García, J M (2015) Cajal and the Conceptual Weakness of Neural Sciences

Frontiers in Neuroanatomy 9

https://doi org/10 3389/fnana 2015 00128

Finkelstein G (2015) Mechanical neuroscience: Emil du Bois-Reymond’s innovations in theory and practice

Frontiers in Systems Neuroscience 9

https://doi org/10 3389/fnsys 2015 00133

Glickstein M (2006) Golgi and Cajal: The neuron doctrine and the 100th anniversary of the 1906 Nobel Prize Current Biology

16(5) R147–R151

https://doi org/10 1016/j cub 2006 02 053

Kandel E R (2006) In search of memory : the emergence of a new science of mind W W Norton & Co

Mishqat, I (2017, June 19) The Formation of Reticular Theory | The Embryo Project Encyclopedia Embryo asu edu

https://embryo asu edu/pages/formationreticular-theory

Scheuerlein, H , Henschke, F , & Köckerling, F (2017) Wilhelm von Waldeyer-Hartz A

Great Forefather: His Contributions to Anatomy with Particular Attention to “His” Fascia Frontiers in Surgery 4

https://doi org/10 3389/fsurg 2017 00074

Schuetze S M (1983) The discovery of the action potential Trends in Neurosciences, 6, 164–168 https://doi org/10 1016/01662236(83)90078-4

Verkhratsky A & Parpura V (2014) History of Electrophysiology and the Patch Clamp Methods in Molecular Biology, 1–19

https://doi org/10 1007/978-1-4939-1096-0 1

Nicotine’s Role in Preventing Parkinson’s Disease: A good excuse for smokers?

WRITTEN BY IRIS ZIELER

EDITED BY MAHTA HAGHIGHAT GHAHFAROKHI DESIGNED BY ZAHRAA BHATTI

Parkinson’s Disease (PD) is the most common movement disorder and the second most

common neurodegenerative disorder worldwide It is characterised by motor dysfunction such as bradykinesia, resting tremor, and muscular rigidity, as well as nonmotor symptoms, including olfactory dysfunction, constipation, disordered sleep, and disordered mood Importantly, these latter symptoms often precede motor dysfunction by up to a decade.

There are many factors contributing to the development of PD, and the exact mechanisms are still not fully

understood However, it has been demonstrated to involve the formation of Lewy bodies and loss of dopaminergic neurons in the substantia nigra region of basal ganglia More specifically, work done by Quik et al have identified the striatal 6ß2-nAChRs (nicotinic Acetylcholine Receptors) as being particularly susceptible to damage, with a decline in levels being closely associated with decrease in striatal dopamine levels (Figure 1)

While no modifiable risk factors for PD have been identified, head injury and pesticide exposure seem to correlate positively with the onset of PD.

Figure 1 Decrease in dopaminergic activity in the brain, visualised by FPCIT/PET neuroimaging A significant decrease and asymmetry can be seen as the condition worsens

On the other hand, caffeine, physical activity, ibuprofen, and tobacco smoking have shown an inverse relationship with the disorder In fact, Mappin-Kasirer et al draw our attention to a recent meta-analysis of observational studies, which reported a 60% decrease in PD risk associated with smoking.

The action of nicotine in the body

Nicotinic receptors are found throughout the entire body, such that nicotine, and other agonists acting on these receptors, tend to have widespread effects nAChRs are depolarising receptors, meaning that their activation causes the propagation of action potentials – the necessary response for signal transduction, cell-to-cell communication, and muscle contraction They are found in the central and peripheral nervous systems, as well as in skeletal muscle, enabling their actions in physiological, as well as psychological activity

Of particular interest with regards to PD are the cholinergic interneurons located in the striatum When activated, they interact with nAChRs on striatal cells to induce dopamine release Under normal physiological conditions, these interneurons are tonically active In PD patients, however, neuronal loss and downregulation of nAChRs in the striatum result in decreased signalling to the substantia nigra (SN) and hence reduced stimulation of dopamine release (Figure 2)

1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) is a dopaminergic neurotoxin used to induce PD in animal models When treating these animals with nicotine, studies have shown a reduction in MPTP-induced dopaminergic toxicity. One proposed mechanism is nicotine’s ability to promote the expression of neurotrophic factors involved in the survival of dopaminergic neurons Another putative mechanism is that the stimulation of nAChRs, prevents their disease-induced down-regulation, such that more receptors ‘survive’

The role of nicotine in Parkinson’s Disease

Downregulation of nicotinic receptors in the brain is one of the leading putative causes of degenerative diseases.

Figure 2. nAChR localisation within the nigrostriatal pathway. Lesion studies have indicated that the 64ß2ß3 (bold black) pathway is particularly vulnerable to damage, and hence has the biggest effect on DA release

Ritz et al underline that postmortem studies of PD patients have shown a significant loss of nicotinic receptors in the parietal frontal and temporal cortices, hippocampus, striatum, thalamus, substantia nigra pars compacta, and laterodorsal tegmental nucleus

The reason the involvement of the nicotinic cholinergic system is being investigated as a potential pharmaceutical target is that:

a) it has been shown to have a close association with the nigrostriatal dopaminergic system, suggesting that nAChR agonists could play a role in modulating the motor symptoms of PD

b) stimulation of this system has been shown to improve cognitive declines in PD patients, of which over 50% have been diagnosed with dementia.

c) Quik et al. draw our attention to the epidemiological evidence illustrating an inverse relationship between smoking and PD, attributing these effects to the nicotine in tobacco

More specifically, studies carried out on MPTP-treated non-human primates showed improvements in various neurochemical markers when chronically treated with nicotine, including tyrosine hydroxylase, dopamine and vesicular monoamine transporters, dopamine levels, and nAChR expression What all these markers have in common is that they are all involved in the dopaminergic pathway, playing a role in the synthesis, storage, or action

While animal models helped establish the prophylactic role of nicotine, further studies have

provided some evidence for nicotine’s efficacy in treating symptoms once PD has developed nAChR stimulation has been shown to ameliorate locomotor activity in un-lesioned animals, as well as improve motor behaviours in models of nigrostriatal damage Results are also pointing towards enhanced outcomes of pharmaceutical interventions for treatment of PD, for example, with levodopa

A meta-analysis of human trials looking at the treatment of parkinsonian symptoms with nicotinic agonists concluded that improvements in bradykinesia, tremor, rigidity, and gait disturbances were apparent Quik et al specify, however, that these results could only be attributed to chronic nAChR stimulation, as acute administration of nicotine had no significant effects on the patients’ conditions

But, smoking will never be prescribed by clinicians for PD treatment!

While an increasing number of studies demonstrate the persisting positive correlation between smoking and decreased risk of PD, an important point to remember is that we cannot be certain that the relationship doesn’t work in reverse, a concept coined “ reverse causation ” That is, it may be the susceptibility to the disease that causes behaviours such as abstinence from smoking, rather than not smoking increasing the risk of developing the disease In fact, Ritz et al demonstrated that patients with PD were less likely to become chronic smokers, having comparatively less reward-system activation in response to nicotine

than non-PD individuals.

Furthermore, nicotine receptors are located throughout the body, meaning that nicotine administration cannot be localised to the specific areas in the brain, causing neurodegeneration This will entrain a multitude of side effects, making its therapeutic use difficult and potentially dangerous.

Finally, “The risks of almost all noncommunicable diseases (eg ischemic heart disease, cerebrovascular disease, diabetes, chronic lung disease, and cancers) were found to be higher in current smokers than in nonsmokers” (Mappin-Kasirer et al , 2020), with the main exception being PD. Despite evidence linking smoking to the decreased onset of PD, this therapeutic effect is negligible when considering the vast array of adverse consequences of smoking As with all medical treatments, the benefits have to be compared with the disadvantages In this case, the latter by far outweigh the benefits

References

Benjamin Mappin-Kasirer Hongchao Pan Sarah Lewington, Jennifer Kizza, Richard Gray, Robert Clarke, Richard Peto Tobacco smoking and the risk of Parkinson disease

A 65-year follow-up of 30,000 male British doctors Neurology May 2020, 94 (20)

Domínguez-Baleón C Ong JS Scherzer

C R et al Understanding the effect of smoking and drinking behavior on Parkinson's disease risk: a Mendelian randomization study Sci Rep 11 13980 (2021)

Fahn, S (2007, October 1) Parkinson disease [Video file] In The Biomedical & Life Sciences Collection Henry Stewart Talks

Gale C & Martyn C (2003) Tobacco coffee and Parkinson's disease BMJ (Clinical research ed ), 326(7389), 561–562

Nicholatos, J W , Francisco, A B , Bender, C A et al Nicotine promotes neuron survival and partially protects from Parkinson’s disease by suppressing SIRT6 acta neuropathol commun 6, 120 (2018)

Ritz B Lee P C Lassen C F & Arah O

A (2014) Parkinson disease and smoking revisited: ease of quitting is an early sign of the disease Neurology 83(16) 1396–1402

Quik, M , Bordia, T , & O'Leary, K (2007) Nicotinic receptors as CNS targets for Parkinson's disease Biochemical pharmacology, 74(8), 1224–1234

iPSCs and CRISPR/Cas9

Genome Editing in Prospects for the Treatment of Neurodegenerative Disorders

WRITTEN BY EMMA VON SETH

EDITED BY ANDREA MAZGALEVA

DESIGNED BY ZAHRAA BHATTI

The continuous advancements in genome editing techniques have helped to provide

more comprehensive models and potential innovative therapeutic approaches for diseases that enable precise genome engineering, where the disease-associated gene has been identified as the best therapeutic target, even without a complete understanding of its biological mechanisms.

Yamanaka et al (2007) introduced the concept of reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) which share morphological features similar to those of embryonic stem cells (ESCs), having the potential to differentiate into advanced derivatives of all three germ layers. iPSCs maintain the genetic mutations found in the somatic cells, allowing them to generate the specific impacted cell types and providing patient specific cells in studying various diseases (1)

The clustered regularly interspersed short palindromic repeats (CRISPR) associated nuclease Cas9, is the most sequence specific gene engineering tool, which induces double-stranded

breaks (DSBs) at selected genomic loci using a single guide RNA (gRNA) that anneals to the target genomic sequence (2) The protospaceradjacent motif (PAM), a 3-bp DNA sequence, allows for Cas9 binding and cleavage when found next to the targeted DNA sequence Cellular repair mechanisms then repair the DSBs via non-homologous end joining (NHEJ) or homology-directed repair (HDR), replacing the target DNA sequence (Figure 1) NHEJ is an error-prone process that induces insertions and deletions (indels) at the site of the break and is thus used to obtain permanent gene inactivation (2) HDR is a more precise and error-free mechanism, operating with homologous DNA as a repair template.

The combination of iPSCs with CRISPR/Cas9 technology has proven to be more competent than classical gene transfer methods, providing prospects of alternative gene therapy,

where genes could be rescued or regulated in vitro and defective cells could be replaced with engineeredcorrected iPSCs in patients (3). Patient-derived iPSCs with the application of the CRISPR/Cas9 mechanism can be gene edited to obtain isogenic controls, permitting to focus on the specific diseasecausing mutations and demonstrating a correction of the disease-associated cellular phenotype (4).

Huntington's Disease (HD) is a lateonset neurodegenerative disorder that results from a polymorphic CAG trinucleotide repeat expansion mutation in the huntingtin gene (HTT) through gain-of-function mechanisms (2) The HTT CAG repeat was corrected in HD patient-derived iPSCs using CRISPR/Cas9 in combination with a piggyBac transposon-based selection system through HDR (5) The corrected isogenic iPSC lines maintained pluripotency and were able to be

differentiated into excitable and active neurons The HD iPSC-derived neural cells were observed to reverse a number of phenotypic abnormalities in isogenic controls, such as impaired neural rosette formation, increased cell death in response to growth factor withdrawal, and deficits in mitochondrial respiration (5) CRISPR/Cas9 gene editing strategies were also used to target mutant HTT DNA to generate pre-specified inactivating deletion mutations on the mutant allele alone6 The simultaneous use of two mutant allele-specific gRNAs eliminated a ~44 Kb DNA segment, which included the promoter region, transcription start site and expanded CAG repeats from the mutant allele without impacting the normal HTT allele, in patient-derived fibroblasts, iPSCs and neural progenitor cells (NPCs) This deletion prevented the generation of mutant HTT mRNA and protein, indicating complete and permanent mutant allele-specific

Figure 1 The CRISPR/Cas9 mechanism requires a target DNA strand, gRNA, Cas9 endonuclease, and a PAM site The induced double-stranded break is processed either via non-homologous end joining or homology directed repair, leading to gene inactivation and gene correction, respectively (adapted from Shin et al., 2018).

inactivation of the HD chromosome (6)

The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expansion of hexanucleotide (G4C2) repeats in the C9orf72 gene In the presence of a plasmid DNA donor template and with the use of CRISPR/Cas9-mediated HDR, an expanded G4C2 repeat in a patientderived iPSC line containing the C9orf72 mutation was successfully corrected (7) Disease-associated cellular phenotypes, including abnormal protein aggregation and stress granule formation, improved in motor neurons differentiated from gene-corrected iPSC lines

Making iPSCs immune compatible is one challenge for cell transplantation therapy In a successful study, human leukocyte antigen (HLA) genes, which are elemental in the major histocompatibility complex that accounts for cellular immune response, were manipulated to silence the cytotoxic immune rejection response using CRISPR/Cas9 technology (8) The tumorigenicity potential in transplanted iPSCs is another impeding component and the process to eliminate it is ongoing The research concerning this complication includes developing assays for screening carcinogenic mutations to distinguish and deplete cancer-related mutations in iPSC products before transplantation (RNA-sequencing analysis in vitro), and the quantification of the minimum number of transplanted cells that does not cause teratoma formation (3) The generation of

permanent changes to DNA with CRISPR/Cas9 gene editing in clinical applications, emphasises the concern of off-targeting (2) Methods such as optimising gRNA design techniques, using low levels of Cas9 or controllable Cas9, suggest possible reductions to this error The determination of an optimal treatment window is also essential in CRISPR/Cas9 therapeutics for progressive diseases of the nervous system, where if treatment is delivered too late damaged neurons might not be able to restore their functions and cellular integrity, while if treatments are applied too early adverse consequences due to compromised gene functions could arise (2). The development of animal models containing human genetic mutations that permit testing of allele-specific CRISPR/Cas strategies is thus important

The use of iPSCs with CRISPR/Cas9 gene engineering as a therapeutic means for confronting neurodegenerative diseases has been addressed in preclinical studies, and the potential of integrative methods has demonstrated to be successful (2) The progress of successful treatments is still, however, obstructed due to the ambiguous pathophysiology of the individual disorders Personal genomics is becoming more affordable, easing the detection of disease-associated genes and providing targets for CRISPR/Cas9 The potential to replace damaged cells with engineered patient-specific iPSCs that are capable of permanent integration and reversing neurological disease phenotypes, is a strong future prospect (Figure 2),

5 Xu X Tay Y Sim B et al (2017) Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells Stem Cell Reports, 8(3), 619-633 https://doi org/10 1016/j stemcr 2017 01 022

6 Shin J W Kim K -H Chao M J et al (2016) Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9 Human Molecular Genetics 25(20) 45664576 https://doi org/10 1093/hmg/ddw286

Figure

Patient-derived

cells reprogrammed to iPSCs that are corrected or modified with CRISPR/Cas9 gene editing mechanisms, before being used for autologous or allogeneic transplantation in treatment of monogenic diseases (adapted from Sguazzi et al , 2021)

References

1 Takahashi K Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126(4) 663-676

https://doi org/10 1016/j cell 2006 07 024

2 Shin J W , Lee J M (2018) The prospects of CRISPR-based genome engineering in the treatment of Neurodegenerative Disorders Therapeutic Advances in Neurological Disorders, 11 https://doi org/10 1177/1756285617741837

3 Sguazzi G P , Muto V , Tartaglia M , et al (2021) Induced Pluripotent Stem Cells (iPSCs) and Gene Therapy: A New Era for the Treatment of Neurological Diseases International Journal of Molecular Sciences, 22(24), 13674

https://doi org/10 3390/ijms222413674

4 McTague A , Rossignoli G , Ferrini A , et al (2021) Genome editing in IPSC-based neural systems: From disease models to future therapeutic strategies Frontiers in Genome Editing, 3

https://doi org/10 3389/fgeed 2021 630600

7 Mutihac R , Ababneh N , Scaber J , et al (2015) Modelling amyotrophic lateral sclerosis (ALS) using mutant and Cas9/CRISPR-corrected motor neurons from patients with C9ORF72 mutations reveals disease-specific cellular phenotypes Journal of the Neurological Sciences, 357 https://doi org/10 1016/j jns 2015 08 198

8 Xu H Wang B Ono M et al (2019) Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates IPSCs with Enhanced Immune Compatibility Cell Stem Cell 24(4) https://doi org/10 1016/j stem 2019 02 005

nonetheless, the precise mechanisms still need to overcome a number of challenges (3)

2.

somatic

THE EMERGING APPLICATION OF IMMUNOTHERAPY IN CANCER TREATMENT

WRITTEN BY SHREYA KAUSHIK | EDITED BY EMMA VON SETH | DESIGNED BY TAMARA YAP

Cancers are a group of diseases caused by the uncontrolled division of abnormal c-

ell growth, which lead to the formation of tumours, either localised to one area or with the potential to spread to other parts of the body The tumour forms a mass or lump in one's body as illustrated in an example of breast cancer in figure 1

Although cancer therapy has seen some progress, there is still a lot of ambiguity concerning the disease and its treatments, hence extensive oncological research to determine more successful treatment pathways is ongoing Methods of treatment depend on the type and stage of cancer an individual is diagnosed with, where current treatment options include Biomarker testing, Chemotherapy, Hormone therapy, Hyperthermia, Immunotherapy, Photodynamic therapy, Radiation therapy, Stem cell transplant, Surgery and Targeted therapy

This article will focus specifically on and discuss the use of immunotherapy and its alleviating impact in the treatment of cancer.

Figure 1 The progression in accumulation of cancer cells in a localised area in a breast showcasing breast cancer in a female patient

IMMUNOTHERAPY AND ITS APPLICATION IN CANCER TREATMENT

Immuotherapy is an emerging application that uses drugs, vitamins, minerals, transplantation,

and biological molecules to achieve a therapeutic effect in diseases, such as immunodeficiencies, autoimmune diseases, hypersensitivity reactions and most notably, cancer This was first applied by William B. Coley in 1891 when his treatment of a patient's inoperable sarcoma, with the introduction of a mixture of live and inactivated Streptococcus pyogenes and Serratia marcescens, showed to cause regression of the tumour This research helped identify immunotherapy as a mediator of immune responses that works to downregulate or upregulate the activity of the immune system. Since then, immunotherapy has emerged as a major topic of interest to pharmaceutical companies, researchers, and scientists in the treatment of cancer

The immune system safeguards humans from the development of cancer through the elimination of harmful cells in our body, however, it may fail to do so if the cancer cells escape the immune system or if they release signals that prevent the immune system from attacking them Immunotherapy treatments help our bodies to recognise and fight these cancer cells more effectively It is already the primary treatment for some types of cancers and is undergoing clinical trials for others. The different immunotherapies that oncologists use to treat patients are:

Monoclonal antibodies

Checkpoint inhibitors

Vaccines

Cytokines

CAR-T cell therapy

MONOCLONAL ANTIBODIES

Over the past 30 years, intense clinical assessment research and development has proven the wide potential that monoclonal antibodies have in cancer Monoclonal antibodies are synthetic antibodies produced in the laboratory that mimic natural antibodies to fight infection It involves producing numerous copies of one antibody that may then target cancerous cells, by engaging surface antigens differentially expressed in cancers. Examples of the use of monoclonal antibodies in targeted cancer therapy to induce cell death by blocking survival pathways and ligand-receptor growth would include: Rituximab targeting CD20 in the treatment of non-Hodgkin B cell lymphoma, Trastuzumab targeting HER2 in the treatment of breast cancer Cetuximab targeting EGFR in the treatment of colorectal cancer.

CHECKPOINT INHIBITORS

In 2018, James P Allison and Tasuku Honjo won the Nobel Prize in Physiology or Medicine for their discoveries in basic science allowing checkpoint inhibitor therapies This therapy targets key regulators of the immune system called immune checkpoints. To protect themselves, some cancers can trigger or stimulate immune checkpoint targets, which then reduces the immune response to the cancer stimulus

Checkpoint inhibitor therapy blocks these inhibitory checkpoints, which helps resto-

re immune function The first drug approved to target the immune checkpoint CTLA4 was ipilimumab Other examples of drugs that function as checkpoint inhibitors include: Nivolumab- it targets PD-1 to help fight cancers such as colorectal cancer, metastatic melanoma, renal cell carcinoma etc, Avelumab- it targets PD-L1 to help fight cancers such as renal cell carcinoma, merkel cell carcinoma, urothelial carcinoma etc

VACCINES

Therapeutic cancer vaccines help the immune system to identify proteins found on cancer cells and mount an attack against them Oncoviruses are the cause for some types of cancer including liver and cervical cancer, against which vaccines such as the HPV and hepatitis B vaccine have been developed A suitable approach to cancer vaccination is to separate the cancer cells and proteins, immunising patients against those proteins (those proteins act as antigens) in the hope of stimulating an immune response against the cancer cells. There is still ongoing research on immunisation for breast, lung, colon and various other types of cancer

CYTOKINES

Naturally found in the body, interferon and interleukin cytokines help boost our immune system and synthetic cytokines are used as a treatment for cancer These cytokines are produced by cells found within a tumour Interleukin-2 and interferonα can enhance anti-tumour activity allowing them to be used as therapeutic agents for cancer. Type I interferon-α has been proven clinically effective in the treatment of melanoma, chronic myeloid leukaemia, hairy-cell leukaemia, follicular lymphoma and AIDSrelated Kaposi’s sarcoma Interleukin2, on the other hand, is used to treat renal cell carcinoma and malignant melanoma

CAR-T cell therapy is a major area of interest for many oncologists due to its widespread clinical applications in cancer and autoimmune diseases. Cancer cells are killed through the genetic engineering of T cells to express chimeric antigen receptors that are produced in a manner that directs them towards antigens found on the tumour cells These T cells are then infused back into the patient where apoptosis of cancer cells is induced An example where CAR-T cell therapy has been used, is where CARs targeted the antigen CD19 which was present in acute

CAR-T CELL THERAPY

CONCLUSION

The application of immunotherapy in the treatment of cancer has emerged as an ext-

remely useful tool in improving the quality of life and survival in cancer patients Modulating the immune system and discovering its oncological impact on cancer patients has required many years of scientific research and its clinical translation The innovative use of antibodies, cytokines, chimeric antigen receptors, checkpoint inhibitors and vaccines in treating cancer has helped build upon a very foundation of science and how these basics can be further applied to treat one of the most common and feared diseases of humanity

References

Figure 2 Chimeric antigen receptor (CAR) T-cell therapy, specifically illustrating newly modified T cells harvested in the lab with CARs

The efficiency of a cancer treatment depends directly upon how efficiently a can-

drug can be delivered to its target site and indirectly upon how much damage is caused to the surrounding healthy tissues In a study conducted by a team of scientists from the University of Zurich, the effects of paracrine delivery of a drug rather than systemic administration was studied. This led to the discovery of the SHREAD gene therapy technique

Shielded Retargeted Adenovirus (SHREAD) is a gene therapy platform that utilizes non-replicative adenovirus serotype 5 for the paracrine delivery of cancer-drugs

The fundamental concept of its mechanism is that the nonreplicative adenoviral particles are equipped with targeting adapters and a reversible shield The targeting adapters recognize certain surface markers on tumor cells, in this case, human epidermal growth factor receptor 2 (HER2), and the function of the reversible shield is to de-target them from the liver and protect them from immune-based clearance mechanisms. To increase efficiency, monoclonal antibodies (mAb) are encoded into the DNA of the adenoviral genome to affect neighbouring tumour cells through paracrine mechanisms and these transformed tumour cells are converted into biofactories that secrete therapeutics

In the study BT-474 a human mammary ductal carcinoma cell line, was used as a HER2- positive tumour model HER2 is a protein that helps breast cancer cells grow quickly BT474 was selected as it is highly sensitive to anti-HER2 therapy

SHREAD: ADENOVIRAL PARACRINE DELIVERY SYSTEM FOR CANCER DRUGS

WRITTEN BY ASTRITI LAKSHMI ADITYA EDITED BY MAYA ROWLEY DESIGNED BY TAMARA YAP

Clinically approved anti-HER2 therapeutic antibody trastuzumab (TZB) was used as a model mAb. Precise biodistribution was determined using tissue clearing with passive CLARTY technique (PACT) This involved reconstructing the tumour microenvironment in a three dimensional structure and making tumours transparent

DID THE TRANSFORMED CELLS PRODUCE FUNCTIONAL ANTIBODIES?

Apanel of cells were transduced by the adenovirus encoding Ad-TZB and each virus

with the same multiplicity of infection To determine whether the cells produced functional antibodies, the cell supernatants were analysed after 72 hours through antigen capture enzyme-linked immunosorbent assay (ELISA) The TZB purified from Ad-TZB infected BT474 cell supernatants showed similar mass spectra as well as growth inhibitions as the TZB purified from Chinese hamster ovary cells transduced with the same Ad5 construct when applied to the same BT474 cells

COMPARISON STUDIES: GENERAL RESULTS AND TRANSDUCTION PERCENTAGES

The study consisted of fox chase severe combined immunodeficiency (SCID) beige mice bearing BT474 orthotopic xenografts being treated with phosphate-buffer saline (PBS), recombinant Herceptin (200 micrograms per dose), Ad-TZB and Ad-D1 3 (1 x 10^8 ivp Ad-TZB) for 3 doses. Tumour volumes were monitored for 60 days posttreatment initiation or until mice reached euthanasia criteria The fastest tumour outgrowth was exhibited by control Ad-D1 3 and PBS On the other hand, Ad-TZB mice showed delayed tumour outgrowth and Herceptin treated mice showed tumour regression

Figure

mice bearing orthotopic BT474 xenografts (A) Female Fox Chase SCID beige mice aged 7 to 8 weeks were given a subcutaneous 0 36-mg 17β-estradiol implant and then engrafted 2 days later with 4 × 106 BT474 cells in Matrigel in the fourth mammary fat pad Once tumors were established (50 to 250 mm^3), mice were treated intratumorally with 1 × 108 plaque-forming units (PFU) Ad-TZB or control Ad-D1 3, 200 μg recombinant Herceptin, or PBS every other day for a total of three doses (i e , on days 0, 2, and 4) Blood was collected on days 6, 11, 16 and at study termination (days 60 to 61) An additional cohort of mice was harvested at an interim time point on day 11 (B) Average tumor outgrowth measured for 60 days after initiation of treatment for Ad-TZB (n = 6), Ad-D1 3 (n = 7 until day 16, then n = 6 until day 48, when mice reached euthanasia criteria), PBS (n = 7), and Herceptin (n = 7) Error bars represent SE. The time point boxed in a dotted line represents the time point chosen for interim analysis (i e , day 11) (Smith et al , 2021)

In a quest to understand how specific the transduction of cells was, the following data was obtained: of the Ad-D1 3-transduced cells in the tumour, 93 7% were BT474 tumour cells and the remaining 6 33% were mouse-derived stromal cells This suggested good-specificity of the HER2-retargeted, shielded adenoviruses.

1. Efficacy of Ad-TZB treatment of Fox Chase SCID beige

COMPARISON STUDIES: LEVELS OF TZB IN THE TUMOR AND LIVER

To study effects of paracrine delivery of TZB in comparison to direct administration

of recombinant Herceptin on systemic exposure and pore formation within the tumour, visualization of the tumours were done at two time points: early time point (at day 11) and end point (day 61) The intact tumour tissue was prepared as 500 micrometre slices and the slices representing the centre of the tissue were stained with anti-human antibody conjugated with alexa 594 and imaged in 3-dimension. The amount of antibodies per a specified volume was analysed

At both time points, transduced tumour cells were well distributed through the 3D section and actively produced TZB mAb that was diffused significantly throughout the tumour The main site for the clearance of mAb is the liver Thus measuring the TZB levels in the liver gave an insight into the systemic clearance of circulating mAb Determining the levels of mAb in tissues with paracrine delivery gave these values: at day 11, there is 21-fold increase in TZB levels in the tumour, 89-fold decrease TZB in the plasma, and 2 2fold decrease TZB in the liver with paracrine delivery in comparison to direct administration of herceptin protein. This corresponds to an 1,800fold increase in the mAb concentration gradient from tumour to plasma and a 48-fold increase in the mAb concentration gradient from tumour to liver

from tumor to plasma and a 48-fold increase in the mAb concentration gradient from tumor to liver

Figure 2. Paracrine delivery leads to high intratumoral levels of antibody and lower drug systemic levels compared to direct administration of the protein Relative levels of TZB (yellow spheres) within the tumor (A) and in the liver (B) following treatment with Ad-TZB versus direct administration of Herceptin Images are shown from tissues of one of n = 3 mice (C) Quantification of fluorescent intensity (occupied volume, μm3) of tumor (n = 3, Left) and liver (n = 3, Middle) tissues stained for TZB with an antihuman antibody 11 d posttreatment with Ad-TZB or Herceptin The ratio of tumor over the liver signal in each mouse is also shown (n = 3, Right) The images shown in A and B are indicated in plots with red squares (Herceptin) or red triangles (Ad-TZB) All values were normalized to background signal obtained from PBS mouse tissues The solid black line represents the mean across the group; n s indicates P > 0 05, *P ≤ 0 05, and **P ≤ 0 01 from an unpaired t test performed using GraphPad Prism (D) Average serum concentrations of systemic TZB 6, 11, 16, and 60 d after initiation of treatment determined by a HER2-capture ELISA and standard curve interpolation Box plots represent five number summaries (min/Q1/med/Q3/max) for the antibody concentration for each mouse in the treatment group (n = 6 to 7) Data were analyzed using a two-way ANOVA with multiple comparisons using the Sidak method; P values for comparisons of Ad-TZB and Herceptin were <0 0001 (****) at day 6 and 11, <0 001 (***) at day 16, and not significant at day 60 (n s ; P > 0 05) from an unpaired t test performed using GraphPad Prism (E) Summary data comparing the amount of antibody reached in tumor, plasma, and liver with the paracrine-delivery strategy (i e , Ad-TZB) compared with direct administration of the antibody (i e , Herceptin) at day 11 Paracrine delivery leads to 21-fold higher antibody reached in the tumor with 89-fold less antibody in the plasma, thus increasing the overall concentration gradient from tumor to bloodstream 1,800-fold For the liver, paracrine delivery led to 2 2-fold less antibody in the liver, corresponding to a gradient effect of 48-fold from tumor to liver (Smith et al , 2021)

COMPARISON STUDIES: PORE FORMATION AND VASCULAR DISRUPTURE

Samples treated with Ad-TZB showed increased pore-formation and vascular disrupture

At day 11, tumours treated with Herceptin and Ad-TZB showed a four- and six-fold increase respectively in pore volume compared to PBS tumours However, at day 61, the average pore volume did not increase for Herceptintreated mice Whereas, Ad-TZB–treated mice showed a 16-fold increase

The porosity index was determined for both groups at day 11 and day 61 to determine the extent of angiogenic effects At day 11, tumours treated with Herceptin showed a two-fold increase whereas Ad-TZB–treated tumours showed a sevenfold increase in the porosity index compared to PBS tumours, respectively At day 61, the porosity index increased to fourfold and 16fold in Herceptin- and Ad-TZB–treated tumours, respectively. The combination of increased pore volume and porosity index in the tumours treated with Ad-TZB as compared to the other groups indicate despite the fact that herceptin showed a lower response in terms of overall tumour outgrowth, Ad-TZB shows greater therapeutic effects from within the tumour

In conclusion, SHREAD can be used for the successful and sustained production of antibodies within the tumour microenvironment with much lower amounts of the therapeutic diffusing into surrounding healthy tissue

References

Smith S Schubert R Simic B Brücher D Schmid M Kirk N Plückthun A (2021, May 17) The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody Retrieved from https://www pnas org/content/118/21/e2017 925118#abstract-2

MAJOR HISTOCOMPATIBILITY COMPLEX IN THE IMMUNE SYSTEM AND CANCER CELL ESCAPE MECHANISM

WRITTEN BY HARMONY, YUEN HAY YEE

EDITED BY ANOUCHKA AZRIA

DESIGNED BY TAMARA

YAP

In the immune system, many molecules are involved in different complex cellular processes The

Major Histocompatibility Complex (MHC) is a type of glycoprotein molecule involved in some key immune response mechanisms. MHC Class I (MHC-I) and MHC Class II (MHC-II) are two types of MHC molecules Both share similar functions in antigen recognition of T cells. The processed antigen displayed on the cell surface via MHC-I and MHC-II is recognised by CD8 (cytotoxic T cells) and CD4 (helper T cells) T cells respectively. The shape of each MHC is complementary to specific T cell receptors (TCR) on T cells Although identical TCR genes are used to synthesize TCRs in CD4 and CD8 T cells, the shapes of the binding site still vary due to different amino acid sequences within the variable region (V/V in figure 1)

This indicates that high specificities are required for TCR in T cells to recognise antigen peptides in different types of MHC molecules This is known as MHC-restricted recognition of antigen. When the shape of the TCR and the MHC with the antigen peptide is complementary, an immune response will be initiated

Figure 1 Interaction between the T cell and peptidepresenting cell. The TCR of the T cell binds to the MHC-I complex of the peptide-presenting cell. The antigen peptide shown in the figure was processed before it is presented on the MHC-I on the presenting cell

must be processed Similarly, both cells with MHC-I and MHC-II involve the breakdown of the original antigen into small sections of peptides However, they have contrasting intracellular interaction mechanisms in expressing processed antigens on the cell surface. The MHC-I is responsible for endogenous antigens, which could be antigens from viral proteins (Figure 2) On the other hand, MHC-II processes exogenous antigens that originate from the outside of the cell (Figure 3)

These mechanisms of MHC play a critical role in cell-mediated immune responses Nevertheless MHC-I might be a contributing molecule to the escape mechanism of cancer cells from T cells or NK Originally, CD8 T cells could potentially recognise cancer cells via the antigenpresenting pathway This pathway is similar to the path in figure 2, where TAP is involved in the translocation of the antigen to the rER, the endoplasmic reticulum aminopeptidase associated with antigen processing is responsible for the final trimming of the antigen into antigen peptides, and tapasin involves in the editing of the peptide antigens After editing peptide antigens, the peptide antigen binds to MHC-I and is transported onto the cell surface. However, cancer cells can avoid recognition by CD8 T cells

Figure 2. Antigen processing mechanism of MHC-I. The antigen in the cytoplasm is intracellular for the MHC-I mechanism That antigen is broken down by proteosomes and transported into the rough endoplasmic reticulum via the transporter associated with antigen processing (TAP) p88 is a type of protein involved in this mechanism The antigen peptide and MHC-I is transferred onto the cell surface after being transported to the Golgi

Figure 3. Antigen processing mechanism of MHC-II. The antigen is taken up by the cell by endocytosis and fuses with lysosomes to form antigen peptides Within the rER, MHC-II is attached to a protein known as the invariant chain The invariant chain will be degraded after the antigen peptide has bound to MHC-II The MHC-II and antigen peptide complex is then transported onto the cell surface

The 'missing self' hypothesis suggests that T cells and NK identify and destroy tumou-

rs lacking MHC-I Additionally, tumours could also be killed when the T cell receptor (TCR) of cytotoxic T lymphocytes (CTLs) recognises MHC-I molecules that comprise the tumour’s antigen A conclusion could be extrapolated that MHC could be involved in the ability of cancer cells to escape from the lines of defence in the immune system

Indeed, MHC is involved in the escape mechanism The escape mechanism is one of the processes cancer cells undergo to prevent recognition by the immune system This mechanism consists of the downregulation of the expression of MHC-I (HLA class I) on the cell surface using various regulatory mechanisms Hence, TCR will not be binding to the respective MHC-I molecules This indicates that CD8 T cells (cytotoxic T cells) will not recognise the antigen peptides Thus, the MHC-I antigen-presenting machinery is lost, and the cancer cells will not be recognised.

This escape mechanism of cancer cells from the immune system is one of the key processes of cancer development and needs to be further investigated. There is the clinical importance of understanding these mechanisms to improve cancer treatments to improve humans’ quality of life.

References

Cornel A M Mimpen I L & Nierkens S (2020) MHC Class I Downregulation in Cancer: Underlying Mechanisms and Potential Targets for Cancer Immunotherapy Cancers 12(7) 1760 https://doi org/10 3390/cancers12071760

Demanet, C , Mulder, A , Deneys, V , Worsham M J Maes P Claas F H & Ferrone S (2004) Down-regulation of HLA-A and HLA-Bw6, but not HLA-Bw4, allospecificities in leukemic cells: an escape mechanism from CTL and NK attack? Blood 103(8) 3122–3130 https://doi org/10 1182/blood-2003-072500

Dhatchinamoorthy, K , Colbert, J D , & Rock K L (2021) Cancer immune evasion through loss of MHC class I antigen presentation Frontiers in Immunology, 12 https://doi org/10 3389/fimmu 2021 636568

Fruci D Benevolo M Cifaldi L Lorenzi S Lo Monaco E Tremante E & Giacomini, P (2012) Major histocompatibility complex class I and tumour immuno-evasion: how to fool T cells and natural killer cells at one time Current oncology (Toronto, Ont ), 19(1), 39–41 https://doi org/10 3747/co 19 945

Ljunggren H G & Kärre K (1990) In search of the 'missing self': MHC molecules and NK cell recognition Immunology Today, 11(7), 237–244 https://doi org/10 1016/01675699(90)90097-s

Wood P (2011) Understanding immunology (3rd ed ) Pearson Higher Ed

Women's History Month

Professor Karen Uhlenbeck

In 2019, she is the first female winner of the Abel Prize, which is awarded by the King of Norway for outstanding mathematicians. She won for her pioneering work on gauge theory and minimal surfaces. The chair of the prize committee commented that her work had "dramatically changed the mathematical landscape."

Incretin Hormones: The Non-surgical Answer to Obesity

WRITTEN BY ADELINA KRUSTEVA | EDITED BY RHIANA CASTILLO | DESIGNED BY TAMARA YAP

One of the major public health challenges in the 21st century is caused by the prev-

alence of obesity, posing serious consequences for the economy, society and to the individuals. Obese subjects hold a vital risk for serious diet-related noncommunicable diseases such as non-alcoholic fatty liver disease, cardiovascular disease and type 2 diabetes mellitus For the development of effective treatment strategies, it is vital to understand the underlying mechanism of obesity pathogenesis

A recent discovery shows that the dysregulation of incretin hormone secretion and action has been observed in metabolic disorders and obesity Consequently, incretin-based therapies have been developed to help the fight with overweight issues and give more therapeutic options on the market (1) However, incretin mimetics present various chemical properties, giving them the advantage to be more potent as well as cope with the complications resulting from obesity Accordingly, could incretin hormones be the new non-surgical answer to obesity?

GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1), also characterised as gut peptides are the two known incretin hormones.

They are secreted after nutrient intake from K cells, found in the upper gut (GIP) and L cells, located in the lower gut (GLP-1) Together they are responsible for an “incretin effect”, which results in upraised insulin secretion (an anabolic hormone which modulates different metabolites like glucose, fatty acids, amino acids in our body) after oral glucose intake Incretin hormones are responsible not only for regulation of glucose levels, but also for influencing gastrointestinal tract, appetite, lipid metabolism and body weight (2) Recent studies have shown that administration of GLP-1 mimetics both into the CNS and general circulation results in reduced food intake due to decreased appetite and increased satiety (1) This effect is further enhanced as hedonic control of food intake is also regulated by GLP-1 (3) as, a decreased anticipatory food reward is associated with GLP-1 receptor activation (1) However, more research on the topic is still needed In addition, GLP-1 has been identified to play a vital role in fat metabolism

The hormone reduces white adipose tissue thickness converting it into thermogen-

ically active (brown) adipocytes and induces adipocyte hyperplasia (increase of lipid storage in order circulating blood glucose and fatty acids to be kept below toxic levels)

(4) Further research was done on the topic leading to the conclusion that exogenous activation of brain GLP-1 receptor signaling increases energy expenditure in mice (5) Additional analyses were done on humans, but more data on the study is yet to be gathered.

The second part of the article will focus on Incretin hormones in Obesity-Related Metabolic Diseases

(Figure 1) Despite the fact that diabetic patients are preserved from GIP and GLP-1, the loss of the incretin effect is observed Due to the apoptosis of β-cells causing diabetes, the pancreatic islets in subjects with T2DM (type two diabetes mellitus) are no longer responsive to GIP and have partial responsiveness to GLP-1

(6) When administered in pharmacological doses, GLP-1 is associated with increased insulin secretion, lowered blood glucose levels and weight-reducing actions (1)

Another obesity-related-metabolic disease is non-alcoholic fatty liver disease (NAFLD), characterised with excessive hepatic fat accumulation

Excessive body weight seems to lead to the progression of the disease and development of steatosis (extra fat accumulation in liver) A study with mice shows that elevated plasma DPP-4 activity is associated with lowered glucose induced active GLP1 levels

There is a lack of effective solution for NAFLD, however GLP-1 associated treatments result in reduction of body weight, therefore improvement of insulin sensitivity and reduction in hepatic triglyceride content (1)

Excessive visceral fat accumulation and insulin resistance is associated with the development of cardiovascular disease, which itself originates from obesity Several studies with human and animal models exhibit that GLP-1 receptors reduce blood pressure by body weight reduction, increasing urine excretion and vasodilation GLP-1 receptor agonists also diminish platelet aggregation and increase plaque stability On the other hand, GIP induces circulating lipids due to its effect on adipocyte related metabolism and regulates vascular inflammation. Due to these results GIP and GLP-1 hormones are suggested to be a potent medical treatment for cardiovascular disease However, further research is needed on the topic (7)

It is proven that incretin hormones reduce food intake and BMI significantly, consequently hav-

ing a positive effect on complications and diseases associated with excessive body weight Yet, is it possible for incretin hormones to replace the surgical solution to obesity? The half-life of both incretins is only a few minutes after which they are inactivated by the enzyme dipeptidyl peptidase 4 (DPP4) Therefore, DPP-4 inhibitors were clinically used for the prolonged effect of GLP-1 and GIP. (9) However, morbidly obese patients still need surgery for lowering their BMI

Incretin hormones’ effect is enhanced when bariatric surgery is performed and additional GLP-1 and GIP supplements are needed Incretin hormones can have a positive physiological effect on obesity, but more data analysis needs to be done concerning incretin treatment for patients with BMI of 35 and higher Regardless, GLP-1 and GIP are actively used for weight loss and complications resulting from obesity. The two hormones are yet to be further investigated and processed for permanent treatment for overweight patients

Figure 1. The image shows GLP-1 and GIP secreting cells and the hormones’ potential therapeutic effects on obese patients (1, 8)

References

[1] Michałowska, J , Miller-Kasprzak, E and Bogdański P 2022 Incretin Hormones in Obesity and Related Cardiometabolic Disorders: The Clinical Perspective

[2] Nauck M and Meier J 2022 Incretin hormones: Their role in health and disease

[3] Daniele, G , Iozzo, P , Molina-Carrion, M , Lancaster J Ciociaro D Cersosimo E Tripathy D Triplitt C Fox P Musi N DeFronzo, R and Gastaldelli, A , 2015 Exenatide Regulates Cerebral Glucose Metabolism in Brain Areas Associated With Glucose Homeostasis and Reward System Diabetes, 64(10), pp 3406-3412

[4] González, N , Moreno-Villegas, Z , González-Bris A Egido J and Lorenzo Ó 2017 Regulation of visceral and epicardial adipose tissue for preventing cardiovascular injuries associated to obesity and diabetes Cardiovascular Diabetology 16(1)

[5] Lockie, S , Heppner, K , Chaudhary, N , Chabenne J Morgan D Veyrat-Durebex C Ananthakrishnan G RohnerJeanrenaud, F , Drucker, D , DiMarchi, R , Rahmouni, K , Oldfield, B , Tschöp, M and Perez-Tilve D 2012 Direct Control of Brown Adipose Tissue Thermogenesis by Central Nervous System Glucagon-Like Peptide-1 Receptor Signaling Diabetes, 61(11), pp 2753-2762

[6] Opinto, G , Natalicchio, A and Marchetti, P , 2013 Physiology of incretins and loss of incretin effect in type 2 diabetes and obesity Archives of Physiology and Biochemistry 119(4) pp 170-178

[7] Jujić A Atabaki-Pasdar N Nilsson P Almgren, P , Hakaste, L , Tuomi, T , Berglund, L , Franks, P , Holst, J , Prasad, R , Torekov, S , Ravassa, S , Díez, J , Persson, M , Melander O Gomez M Groop L Ahlqvist, E and Magnusson, M , 2020 Glucose-dependent insulinotropic peptide and risk of cardiovascular events and mortality: a prospective study Diabetologia 63(5) pp 1043-1054

[8] Created with BioRender com

[9] Laferrère B 2016 Bariatric surgery and obesity: influence on the incretins International Journal of Obesity Supplements 6(S1) pp S32-S36

LIQUID GOLD: The Science Behind Blood Transfusions

WRITTEN BY REBECCA HAMMERSLEY EDITED BY KIRA LINKE DESIGNED BY TAMARA YAP

The history of the blood transfusion started in 1665 when the British physician Richard Lower revived a dog that had almost bled to

death via transfusing blood from another dog into a tied artery Yet the first human blood transfusion wasn’t performed until 1795 by Dr Philip Syng Physick The blood transfusions that we know today, however, were made feasible by the discovery of the ABO blood groups This landmark identification was made by Karl Landsteiner in 1901 Adding to the blood transfusion progression was the discovery of Decastello and Sturli, who identified the fourth blood group – AB These discoveries all led to the identification of blood type cross matching by Reuben Ottenberg who coined universal blood donor as a term to describe the blood type O

Our blood groups are categorised into four groups A, B, AB, and O Phenotypically there are only four different outcomes but these are created by six different genotypes The three different alleles are IA which deposits the A blood group antigen, IB which deposits the B blood group antigen, and i which makes a non-functional glycosyltransferase and deposits neither A or B antigens on the surface of the red blood cell The alleles IA and IB are codominant, meaning that when both alleles are present, they are expressed at the same level creating the AB blood type group The allele 'i' is recessive to all of the other blood groups, meaning to create the blood group O, two copies of the recessive allele are required This makes the blood type O and AB the rarest out of all of the blood groups, as out of all of the six possible genotypes, only one will produce the desired phenotype.

This is not the case for the blood groups A and B as there are two possible genotypes for both of the blood groups that will create the desired phenotype

These blood groups are determined by the formation of glycosylated side chains in the Golgi apparatus These side chains are known as the blood group antigens, and they depend on the specific glycoprotein patterns found on the surface of the red blood cells The different blood group antigens are created by differences in the glycosylation patterns

The blood group O builds the basic sugar skeleton is comprised of two galactose molecules, one fructose and one N acetyl-glucosamine. To create the A blood group the enzyme glycosyltransferase adds a N acetylgalactosamine to the basic sugar skeleton

For the creation of the B blood group antigen, instead of a N acetylgalactosamine, another galactose molecule is added to the sugar side chain Individuals who have the blood group AB have both the A blood group antigen and the B blood group antigen present on the surface of their erythrocytes

Our human immune system can detect and destroy nonself antigens and this is true for the antigens present in the blood groups Individuals of one blood group will have antibodies to target the antigen of the opposing group present in their plasma, meaning that an individual with blood type A will have antibodies for the B antigen and vice versa. Therefore, individuals who are of the blood group AB will have no antibodies against any of the blood group antigens making them the universal blood recipient

Figure 1. This image is a visual depiction of the blood group antigens that are present on the surface of the erythrocytes.

as they can receive any blood type in a transfusion, and they will have no immune response This immune response will cause agglutination of the red blood cells which is a problem as these block blood flow through small blood vessels such as capillaries Agglutination is when the specific antibodies will bind to the antigens on the surface of the cells in this case it is the antibodies binding the specific blood group antigens that are expressed on the surface of the red blood cells The antibodies will bind all of the red blood cells expressing the specific antigen together causing the formation of clumps of red blood cells

However, those who have the blood group O will have antibodies to all of the blood group antigens, meaning that they can only safely receive the blood group O via transfusion All of the blood groups have the same basic sugar skeleton - this sugar skeleton is the only glycosylated side chain on the erythrocytes of individuals who have blood type O Therefore blood type O can be put into any individual without the initiation of an immune response making it the universal blood donor group, making it liquid gold in the medical field

To add to all of the different blood classifications, there is also one more surface protein that can change the classification of individuals and this is the Rhesus (Rh) factor If an individual has the Rhesus factor present on the surface of their blood cells then the individual is Rh positive, and if this protein is lacking, the individual is Rh negative

Rh positive is the most common type of blood group The logic behind the Rhesus protein is very similar to the blood group antigens An individual with Rh positive can receive blood that is both Rh positive and Rh negative However, individuals who are Rh negative should only receive Rh negative blood as the transfusion of Rh positive blood can create antibodies against the Rhesus protein causing a transfusion reaction

The discovery of the universal blood donor has proved vital in medical and surgical advancement. This discovery has proved useful in emergency circumstances when the blood group is unknown, but it is also very useful in the treatment of blood disorders such as sickle cell disease. In conclusion, the most useful blood group is that of O negative as this can be received by anyone There is a demand for O negative blood from hospitals that cannot be met as only 8% of individuals have this blood type

References

Maerz, L L (2020) Transfusion and Autotransfusion: Overview Indications Component Transfusion eMedicine [online]

Mayo Clinic (2018) Rh factor blood testMayo Clinic [online] Mayoclinic org

News-Medical (2009) History of Blood Transfusion [online] News-Medical net

NHS Choices (2019) Blood transfusion [online] NHS

Women's History Month

Sarah Gilbert

Vaccinologist from The University of Oxford who played a pivotal role in developing the Oxford/AstraZeneca COVID-19 vaccine, of which 2 billion doses have been given.

Potential Drugs Switching On the Dormant Ability for Human Regenerative Medicine

WRITTEN BY VENUS HO

EDITED BY DRSHIKA MEHTANI

DESIGNED BY TAMARA YAP

Many of those who have lost a limb either from traumatic accidents or via amput-

ation from disease have dreamt of someday re-growing them Despite the installation of a prosthetic alternative, the functional properties of the lost limb are not fully recovered Scientists have been considering the possibilities of human limb regeneration for decades, and a recent breakthrough in drug treatment on the regrowth of an amputated frog leg has brought us one step closer to achieving this goal Regenerative capabilities can be observed throughout the human body yet such processes are carried out in a superficial manner. Examples include wound healing in skin cells and liver regeneration

POWER OF REGENERATION IN HUMANS

Both processes heavily rely on growth factors and cell signalling molecules The former depends on growth factors to initiate the healing process (Reinke, J M, Sorg, H 2012) Macrophages are then triggered to facilitate the process of angiogenesis for the production of tissue and blood vessels Finally, collagen is continuously formed and broken down to fine-tune the balance of toughness and re-modelling of the new tissue In the case of liver regeneration, partial liver regrowth occurs to restore its original mass after damage (Tao, Y , Wang, M et al , 2017). It first utilises cytokines to increase the sensitivity of hepatocytes to growth factors, promoting the re-entry of liver cells back to G1-phase for DNA replication and protein production This is followed by cell division for liver growth back to its initial size

Despite the fact that humans are capable of regenerating tissues and even organs, this begs the question of why can’t humans grow a new limb?

Figure 1 This diagram outlines the process involved in limb regeneration in a salamander To commence with, wound healing and closure occur after amputation with the absence of scar formation. Simultaneously, the debrisclearing process is then carried out by macrophages. A blastema is then formed by dedifferentiation Positional memory is also possessed by the cells, enabling them to arrange themselves into the correct for the regeneration of the limb (Joven, A , Elewa, A et al , 2019)

WHY CAN'T WE REGROW LIMBS NOW?

When limb loss occurs in humans, scarred tissue - a fibrous protein collagen

would grow around the wound along with a new skin layer, hence impeding the regrowth of a limb While organisms such as salamanders can achieve this regeneration due to the substitution of wound epidermis instead of scar tissue at the amputated site, sending signaling pathways to the site of injury(Joven, A , Elewa, A et al , 2019) This induces the formation of a blastema of progenitor cells which reverts cells to a less-specialised state, thus initiating the regrowth of muscle and tissue cells Studies have also revealed the significance of macrophages not only in the immune system but also in limb regeneration, as its depletion at a salamander wound causes failure of the regenerative process and closure instead (Godwin, J, W , et al 2013)

In addition, the complexity of limb regrowth poses a challenge in this therapeutic approach, as it involves the replacement and correct arrangement of tissues structures such as the skin, cartilage, bone, vessel, nerve, tendon, ligament, and muscle

From an evolutionary perspective a study suggests that the regenerative potential of human cells is suppressed by an anti-cancer mechanism Rapid cell division constitutes tissue regeneration, yet can dangerously lead to cancer Therefore, across of evolution of mankind, the importance of cancer prevention outweighs that of limb regeneration

A RECENT BREAKTHROUGH IN REGENERATIVE MEDICINE

Exciting news has been brought to the science community by a recent study on the

application of a drug cocktail on frogs with an amputated limb, which was followed by significant regrowth (Murugan N. J. Vigran H. J. et al. 2022). The majority of studies targeting regenerative medicine utilise naturally regenerative model organisms, most notably axolotls Nevertheless, Muragan and his colleagues experimented on organisms that did not possess such ability, which certainly brings a new perspective and direction to this field

African clawed frog – the species involved in the experiment, like humans, do not possess the capability of limb regeneration The organisms first had their hindleg amputated after being anaesthetized, ensued by the attachment of a BioDome – a 3Dprinted silicon cap, to the drugcoated limb stump of the frogs. The BioDome was intended to mimic the amniotic environment. While the cocktail of drugs contained 5 components that were designated for the termination of scar tissue growth, by alleviating inflammation and collagen production It was also purposed to trigger the process of outgrowth of desired tissue, patterning, and the development of sensorimotor function. After 24 hours, the BioDome was removed, and tissue development of the organisms was observed for the next 18 months

APPLICATION IN REGENERATIVE MEDICINE

Cell transplantation and bioelectric means are also concurrently explored, yet the drug delivery approach may be more direct compared to its counterparts This activation of an endogenous pathway leading to limb regeneration in frogs represents the prospect of setting this dormant ability in humans in motion The fact that the exposure to the drugs in a mere 24 hours induced such an extraordinary rate of lasting regrowth shines new hope to this approach in regenerative medicine However, Murugan and his team expressed their interest in testing this procedure on mammals and possibly on humans in the near future The regeneration of human limbs has always seemed to be a farfetched dream, yet this recent study has undoubtedly transformed this notion into an achievable goal. Much effort and thorough research on this therapeutic approach are still required before its introduction to human patients Perhaps the day when amputees no longer rely on any mechanical support might not be in the distant future

Figure 2. This image depicts the growth of soft tissue of three treatment of groups – treated with both BioDome and multidrug treatment (MDT), untreated with drug (BD), and received no treatment at all (ND) Out of the three, MDT displayed a significantly longer and more developed limb growth such as toe-like digits, which were absent in the other two (Murugan, N J , Vigran, H J et al , 2022)

References

Reinke J M & Sorg H (2012) Wound repair and regeneration European surgical research Europaische chirurgische Forschung Recherches chirurgicales europeennes, 49(1), 35–43

https://doi org/10 1159/000339613

Tao Y Wang M Chen E & Tang H (2017) Liver Regeneration: Analysis of the Main Relevant Signaling Molecules Mediators of inflammation, 2017, 4256352

https://doi org/10 1155/2017/4256352

Joven A Elewa A & Simon A (2019) Model systems for regeneration: salamanders Development (Cambridge, England), 146(14), dev167700

https://doi org/10 1242/dev 167700

Godwin J W Pinto A R & Rosenthal N A (2013) Macrophages are required for adult salamander limb regeneration

Proceedings of the National Academy of Sciences of the United States of America

110(23) 9415–9420

https://doi org/10 1073/pnas 1300290110

Murugan, N J , Vigran, H J , Miller, K A , Golding, A , Pham, Q L , Sperry, M M , Rasmussen-Ivey C Kane A W Kaplan D L & Levin M (2022) Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis Science advances 8(4) eabj2164

https://doi org/10 1126/sciadv abj2164

THE ROLE OF PROBIOTICS IN WOMEN'S HEALTH

Probiotic consumption has been one of the effective treatments for gastrointestinal

conditions ranging from acute infectious diarrhoea to irritable bowel syndrome The scientific interest in probiotics has grown beyond their role in the gastrointestinal system during the last decade.

Because of its expanding application, it is defined by World Health Organisation as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ (Probiotics in food Health and nutritional properties and guidelines for evaluation, n.d.). It is now also presented as nutritional supplements targeting women of reproductive age Due to a rise in the usage of probiotics amongst women, the time has come to examine if probiotics can prevent or treat the medical conditions related to women ’ s health

Probiotics promote vaginal health by maintaining the balance of the vaginal microbiome. Common vaginal disorders such as bacterial vaginosis could be one of the risk factors that contribute to pelvic inflammatory disease and HIV infection acterial vaginosis is characterised by the overgrowth of anaerobes without sufficient lactobacilli

WRITTEN BY NG WAI IN, JANINE EDITED BY ALEX EPSHTEIN DESIGNED BY TAMARA YAP

Lactobacilli establish vaginal eubiosis by creating an acidic environment of pH 4 5 by producing lactic and acetic acid and hydrogen peroxide (H2O2) (Buggio et al , 2019) It is thus unfavourable for the survival of pathogens and dysbiotic microbes.

The market viability of probiotics for pregnant women is extremely high as it is claimed to play a crucial role in preventing preterm delivery Bacterial vaginosis elevates the risk of spontaneous preterm delivery and neonatal complications Few studies have looked into the efficacy of probiotics, with one prospective cohort study showing the reduction of preterm delivery risk after the administration of probioticssupplemented milk during pregnancy (Myhre et al , 2010) A potential limitation is that the probiotic administration only explicitly reduces the vaginal pH in a randomised controlled trial, which may not be determinant evidence confirming its role in preventing preterm births (Hantoushzadeh et al., 2011). The role of probiotics in pregnancy is still unclear

as focused of lactobaiotics

Desirable criteria comprise the production of antimicrobial elements such as H2O2, the ability to adhere to vaginal epithelial cells and acidify the vagina The probiotics can be delivered orally or directly to the vaginal tract by capsules and tampons filled with freeze-dried lactobacilli (Tachedjian et al , 2017)

Although the media has put loads of emphasis on the vaginal health benefits brought by probiotics, some studies only demonstrate slight improvements in terms of shortterm clinical and mycological cures (Bradshaw et al , 2012) Strains of various members from the probiotic lactobacilli family are unable to survive in the lower female reproductive tract (FRT) in the long term. It is known that the natural microbiome balance in vaginas can be disrupted by sexual activities and the action of contraceptive pills In other words, the probiotic lactobacillus strains may be eliminated and fail to establish stable colonisation on the vaginal mucosa because of the external factors above. The inconsistent results among different studies could be affected by the clinical populations and trial methodologies Further research on the efficacy of probiotics is required

An old saying goes ‘beauty is only skin deep’, but this does not stop the overwhelming demand for antiageing strategies especially in recent years.

Probiotics may give some clues to the key to anti-ageing Factors contributing to ageing skin can be categorised as ‘extrinsic’ (i e environmental factors) and ‘intrinsic’ (i e hormonal and genetic influences)

UV light is perceived as one of the extrinsic ageing factors Since the link between sunlight exposure and photoaging has been recognised in recent years, interest in skin protection from UV light has surged. Probiotics are defined to be a possible therapy to tackle UV-induced skin damage

Probiotic metabolism produces a range of acidic molecules to restore the normal skin pH within the range of 4 2 - 5 6 For instance, lactobacilli produce free fatty acids and conjugated linoleic acids during fermentation Maintaining the acidic pH value may help prevent overstimulation of protease activities in the skin, further reducing the formation of wrinkles (Kober & Bowe, 2015)

Scientists have put in a lot of effort to substantiate the use of probiotics against photoaging, but some questions remain. In a human study, women received probiotic supplements with carotenoids in stimulated or natural sunlight The supplements speed up the recovery of immune system homeostasis after UV radiation exposure Yet, the sole effects of probiotics cannot be evaluated due to the absence of control groups. There were significant limitations in this study (BouillyGauthier et al , 2010)

Another study on skin conditions of healthy young women after probiotics intake

also suggests similar findings. The presence of wrinkles decreases more significantly after probiotic consumption in the intake group (MORI et al , 2016) It is believed that probiotics can act against harmful intestinal bacteria to improve skin conditions Yet, further experiments on the skin conditions of mature women are required to investigate whether probiotics can effectively mitigate skin conditions of different age groups

Based on the current understanding of probiotics, we can see that they hold the potential in vaginal health and slowing signs of ageing skin They also exhibit variations among individuals about clinical effects evident by numerous studies. It shows that we may need to develop personalised medicine approaches in future gut microbiome applications

References

Bradshaw, C S , Pirotta, M , De Guingand, D Hocking J S Morton A N Garland S M Fehler G Morrow A Walker S Vodstrcil, L A , & Fairley, C K (2012) Efficacy of Oral Metronidazole with Vaginal Clindamycin or Vaginal Probiotic for Bacterial Vaginosis: Randomised PlaceboControlled Double-Blind Trial PLoS ONE, 7(4), e34540

https://doi org/10 1371/journal pone 003454

Bouilly-Gauthier, D , Jeannes, C , Maubert, Y , Duteil, L , Queille-Roussel, C , Piccardi, N Montastier C Manissier P Piérard G & Ortonne, J -P (2010) Clinical evidence of benefits of a dietary supplement containing probiotic and carotenoids on ultraviolet-induced skin damage British

Journal of Dermatology 163(3) 536–543

https://doi org/10 1111/j 13652133 2010 09888 x

Buggio L Somigliana E Borghi A & Vercellini, P (2019) Probiotics and vaginal microecology: fact or fancy? BMC Women’s Health, 19(1) https://doi org/10 1186/s12905019-0723-4

Hantoushzadeh, S , Golshahi, F , Javadian, P , khazardoost, S , Aram, S , Hashemi, S , Mirarmandehi B & Borna S (2011) Comparative efficacy of probiotic yoghurt and clindamycin in treatment of bacterial vaginosis in pregnant women: A randomized clinical trial

The Journal of Maternal-Fetal & Neonatal Medicine 25(7) 1021–1024

https://doi org/10 3109/14767058 2011 61465

Kober, M -M , & Bowe, W P (2015) The effect of probiotics on immune regulation, acne, and photoaging International Journal of Women’s Dermatology 1(2) 85–89

https://doi org/10 1016/j ijwd 2015 02 001

MORI N KANO M MASUOKA N KONNO

T SUZUKI Y MIYAZAKI K & UEKI Y (2016) Effect of probiotic and prebiotic fermented milk on skin and intestinal conditions in healthy young female students Bioscience of Microbiota Food and Health, 35(3), 105–112

https://doi org/10 12938/bmfh 2015-022

Myhre R Brantsæter A L Myking S

Gjessing, H K , Sengpiel, V , Meltzer, H M , Haugen, M , & Jacobsson, B (2010) Intake of probiotic food and risk of spontaneous preterm delivery The American Journal of Clinical Nutrition, 93(1), 151–157

https://doi org/10 3945/ajcn 110 004085

Probiotics in food Health and nutritional properties and guidelines for evaluation (n d )

https://www fao org/3/a0512e/a0512e pdf

Tachedjian G Aldunate M Bradshaw C S , & Cone, R A (2017) The role of lactic acid production by probiotic Lactobacillus species in vaginal health Research in Microbiology 168(9-10) 782–792

https://doi org/10 1016/j resmic 2017 04 001

Women's History Month

Dr. Lam Sze Mun

One of three Malaysian Fellows selected for L'Oreal and UNESCO's Women In Science Fellowships in 2018 for her work on techniques to clean dirty water while also using it to create energy.

EXPLORING THE MULTIVERSE

WRITTEN BY ALEX EPSHTEIN

EDITED BY ASTRITI LAKSHMI ADITYA DESIGNED BY TAMARA YAP

The many worlds interpretation (MWI) has been a longstanding theory throughout

the history of quantum mechanics It is a collection of potentially observable alternate universes that share a universal hierarchy The reason it has not been proven, is because it goes against the quantum wave function: a mathematical description of the quantum state of an isolated quantum system i e an equation that proves that our universe is the only existing one, or is entirely separate to any other potential universes that may exist.

Although most physicists remember this theory first being explored by Everett in the 1950s from a mathematical perspective, Schrödinger pioneered the idea of the MWI half a decade earlier through a philosophical lens Although the scientists’ ideas differed in interpretation, both theories had the same premise: to disprove the “collapse of the wave function”

Diving deeper into Schrödinger’s most famous experiment, where he locked a cat in a box. This experiment had one outcome, but during its time in the box the cat

remained alive until a certain event triggered its inevitable death. Schrödinger explained that this experiment demonstrated a superposition of states, where there is one outcome, i e one solution to the wave function, albeit this does not mean that alternative solutions do not exist The experiment demonstrated two parallel universes: one where the cat lives and one where it dies You can open the box in one universe and reveal the cat’s corpse, while in another universe, a living cat The worlds are identical and occurring simultaneously, until something changes in one timeline, triggering a change in the fate of one of the cats, rather than there being a collapse in the wave function

Schrödinger pointed out the ridiculousness of expecting a quantum superposition to collapse just because we look at it It was, he wrote, “patently absurd” that the wave function should “be controlled in two entirely different ways, at times by the wave equation, but occasionally by direct interference of the observer, not controlled by the wave equation ” Everett expanded on this theory by introducing the idea of the universe ‘splitting’ when faced with quantum choices.

In Everett's version of the cat puzzle, there is a single cat up to the point where a drastic change occurs: then the universe splits in two one universe where the cat dies another where it lives, so the subsequent histories are correspondingly different But the parallel worlds can never communicate with one another

Let’s apply Schrödinger and Everett’s theory to the wave function, which describes the entire world as a superposition of states a Multiverse made up of a superposition of universes. The universal wave function indicates the position of every particle in the universe at a particular moment in time As the number of possibilities is restricted by the quantum nature of space and time, many versions in which stable stars and planets cannot exist However, there will be at least some universes resembling our own as often portrayed in science fiction. Provided it obeys the laws of fiction, there is no reason a particular fictional storyline cannot exist somewhere in the Multiverse

However, the wave function does not mention the change from one state to another, i e the flow of time Everett’s parameter includes a description of a world in which we exist, and all the records of that world’s history exist There will also be another identical universe, except the ‘time step’ has been advanced by, say, one second There is no suggestion that any universe moves along from one time step to another

Like with Schrödinger’s cat: there will be a ‘cat’ in another universe - as described by the universal wave function - who has all the memories

of the cat in our universe at this instant in time But it is impossible to say that the cat in our universe is the same cat as in the parallel universe. In Everett’s MWI, different time states can be ordered in terms of the events they remember, defining the difference between past and future, but remain unchanged from one state to another This proves that all states exist simultaneously, meaning our version of ‘time’, does not ‘flow’ as we know it to

References

The Many-Worlds Theory Explained (2020 May 20) The MIT Press Reader https://thereader mitpress mit edu/the-manyworlds-theory/

The parallel worlds of quantum mechanics (n d ) BBC Science Focus Magazine https://www sciencefocus com/science/theparallel-worlds-of-quantum-mechanics/

OntheRoadTowardsNovel TreatmentsforCOVID-19andCOPD

AN EXCLUSIVE INTERVIEW WITH PROFESSOR CLIVE PAGE WITH OLIVERA MITEVSKA | EDITED BY

Clive Page is a Professor of Pharmacology and the Director of the Sackler Institute of Pul

monary Pharmacology at King's College London His research focuses on inflammatory diseases of the lung

Throughout his 40-year career, he has made outstanding contributions in this area, for which he was awarded the Order of the British Empire in 2017 He is currently working on multiple projects designed to test the use of novel drug formulations, including two initiatives for the treatment of COPD and Covid-19

DRSHIKA MEHTANI

COPD is the third leading cause of death worldwide and at present there is no cure Those affected by the disease experience chest tightness, wheezing, and difficulty breathing due to bronchoconstriction Additional pain and discomfort are caused by lung inflammation. To ease symptoms, COPD patients are often treated with bronchodilators like β2-agonists and antimuscarinics, and mucolytic agents may also be prescribed to reduce symptom severity

To treat the underlying inflammatory response, patients are given steroids, which have

anti-inflammatory activity However, in recent years this standard course of treatment has been challenged by researchers like Professor Clive Page, who believe that there is something better on the horizon

“People with COPD have often smoked for decades before they see a doctor, and drugs like steroids are less effective,” he says “Another big issue about steroids is that they can interfere with steroid action in the endocrine system and elsewhere, so they are limited with health use One of the big revolutions in respiratory medicine over the last two decades has been the introduction of topically reactive or inhaled steroids which, if used properly, don’t get into the blood in any meaningful way, so they improve the anti-inflammatory activity whilst reducing side effects However, they can still have side effects, both locally and when the inhaler hits the back of the throat, which can cause opportunistic infections like Candidiasis There’s also a perception among some paediatricians that steroids have terrible effects, so we’d rather avoid them ” Much of Professor Page’s work has been devoted to finding molecules that are both nonsteroidal and anti-inflammatory which can treat a wide range of inflammatory lung diseases

One drug that could potentially satisfy all these criteria is ensifentrine - a dual inhibitor of the enzymes phosphodiesterase 3 and 4

Ensifentrine is currently being evaluated for use in COPD patients in a Phase III clinical trial organised by Verona Pharma, a company that the Professor co-founded Professor Page calls the molecule “bifunctional” as one side is bronchodilatory and the other anti-inflammatory “Formulating two separate drugs is not always straightforward – actually, one molecule is easier to formulateand we know from cancer and infectious diseases that doing this gives you much greater efficacy than you predict compared to treatment with two molecules. So we ’ re trying to make it easier for the patient and have both therapeutic actions in a single molecule,” he says

It's very rare for a drug to progress from the preclinical stage all the way to Phase III

trials “Developing medicines is a risky industry to be in,” Professor Page says “For every 50,000 molecules, maybe one will become an approved medicine Many fail because they are deemed unsafe, or not safe enough to ever be put into people.” Professor Page has been involved in the discovery and development of ensifentrine from its early stages throughout today, making him uniquely qualified to speak about the process

On the topic, he says, “First of all, we needed to show that ensifentrine is indeed a bronchodilator And we chose to use guinea pig trachea, which you can stimulate electrically to cause muscle contraction. We also used guinea pigs that we could then make allergic to see if inhaling ensifentrine stops lung inflammation

I chose this model because I knew it was useful in predicting what ultimately happened in the clinic We then looked at primates and got the same answers Once you ’ ve done that before you spend £1.5 million on your safety studies, it’s important to determine whether the drug binds the target in humans We do this on human bronchus that you can get from surgery and human inflammatory cells from blood volunteers Then you must screen for any potential drug-drug interactions, which is less of a problem for inhaled drugs because they’re unlikely to enter the liver and interfere with the metabolic processes there. To do these experiments under good laboratory practice, we outsourced the work to a specialist contract laboratory

regulators and seek permission to take that drug into a human being for the first time. We must be conservative with this process If you think you have a dose where the drug is going to work, you drop it 10fold or 100-fold when you first give it to humans Then we do a singleascending dose until you hit an adverse effect This could be a headache, nausea, or sometimes it’s more severe and you must stop. This gets you the dose that you put into your first patient studies, or Phase II clinical trials ”

Professor Page is confident that ensifentrine looks very promising “Ensifentrine is working,” he says “it’s a novel form of bronchodilation, it’s anti-inflammatory, and we’ll have to see how it pans out in its Phase III trial which I believe is due to read out later this year, ” the Professor adds He also notes that ensifentrine has been extremely well-tolerated in patients “because it’s designed by inhalation not to get into the blood ”

g interest glycosaminog produced by has a com profile. While known for properties, re that it can anti-inflamma Page explain highly anioni big polymer the molecule is anti-coagulant, but there are lots of other bits of the molecule that, by being anionic, can recognize cationic molecules, including various inflammatory mediators ” In the past 18 months, Professor Page has been working with an international consortium of scientists and clinicians to evaluate the use of off-label nebulized heparin on top of systemic heparin in Covid-19 patients

He says about his experience “When the lockdown first started, a group of us in the UK were charged with coming together to say if any of the drugs in development for different indications could provide benefit in the treatment of Covid, recognizing that the challenge was not to stop infection but to keep hospitalized patients out of ICU because we have a limited number of ICU beds I initially suggested we do heparin based on my own work because we knew there was an inflammatory response with a lot of cytokines being released, we knew that patients had coagulation - in particular alveolar coagulationwhich was probably causing the breathlessness

And systemic heparin has been very successful through Covid; many, if not all hospitalized patients have had it to treat cardiovascular abnormalities So, if you have a drug like heparin that’s anticoagulant and anti-inflammatory, and you nebulize it, it doesn’t get into the systemic circulation, meaning we wouldn’t put people at risk of bleeding and we’d still allow them to have the standard of care which was systemic heparin ”

The Professor notes that the process required tremendous cooperation and was largely built upon previous work.

Here, he's ails abou sk of get

he ground “We what’s called a brochure’ with all public domain to s to administer neb top of systemic hep not put anybody a decide what dos heparin to use, wha of that was based particularly like t Professor Jan S University of Portsmouth, we relied on a lot of her work on nebulized heparin ”

He also shares that the group initially faced some difficulties in convincing authorities that nebulized heparin was safe to administer via inhalation He says, “From day one, people were very concerned that if you inhale heparin patients are going to bleed to death. This was not news, in fact, in every trial with nebulized heparin

I’ve been involved in, going back almost 20 years, the ethics committee say, ‘heparin equals bleeding’ But there isn’t a single clinical trial that shows this – we made that part of our case And the regulators here in the UK could not have been more helpful in aiding this whole process, but if they had felt that the data said that nebulized heparin could pose serious adverse effects, they would not have allowed us to move forward Regulators are there to make sure that mad scientists can’t get away with doing anything just because they think they’ve got a good idea "

different health centres across 7 countries About his work, Professor Page says, “We showed that nebulized heparin had some benefit in keeping hospitalised patients away from intensive care. Nebulized heparin is anti-inflammatory, anticoagulant, and anti-viral, since in the meantime we showed that it binds the Spike protein of SARS-Cov-2 to stop infection We’re now getting data from our trial in Brazil that shows that the drug reduces death in people who have had it for a long enough time, which is obviously important This might have other implications for the treatment of other virus-induced pneumonias that exist and are yet to come, so that hopefully next time we are better prepared ”

Professor Page and colleagues are currently working towards an international meta-trial “We’re aiming to get around 700 people, and we ’ re up to around 450 to see whether nebulized heparin has any clinical benefit for Covid-19 in a broader population,” he says

We can expect to see more about nebulized heparin in the months and years

ahead In addition to the meta-trial, the Professor is also researching other ways that heparin can be used to treat other inflammatory lung diseases He says, “We’re now looking at ways to combine heparin with other drug classes to treat inflammatory conditions such as asthma and COPD. Though physicians were rightly concerned that heparin has anti-coagulant activity that you don’t need for these indications, my own research and others have shown that you can separate these two activities We are very interested in trying to understand how heparin is antiinflammatory, and whether you can find other molecules that are antiinflammatory without being anticoagulant, because outside of Covid19, having an anti-inflammatory drug based on an endogenous molecule that is not a steroid is actually something that I think is the holy grail of inflammation research,” he concludes

LIVING ON THE EDGE: HOW EXTREMOPHILES SURVIVE, THRIVE, AND DRIVE BIOTECHNOLOGICAL ADVANCES

WRITTEN BY ALICE KOSMIDER

EDITED BY DORIS YU

DESIGNED BY TAMARA YAP

The earth is far less hospitable than we think Over three quarters of our planet are

uninhabitable without technological assistance, encompassing deep oceans and arid deserts. But beyond our observed ecosystem are organisms which exceed these constraints, and flourish; the aptly named extremophiles Translated as ‘extreme loving’ they include all domains of life Extremophiles live and grow in numerous harsh environments which have selectively pressured their genomes, biochemistry and cellular physiology

THERMOPHILES

Thermophiles are organisms optimised for conditions above 40° and are usually found in areas of geothermal activity.

Certain species have been found above 120°C and many live in high pressure conditions which allows their water content to remain liquid. Their highly compact chains allows for tight and rigid packing to prevent melting GC rich regions in DNA and positive supercoiling further condenses chromatin while increasing the melting point Thermophilic proteins are reinforced via increased bonding networks: numerous hydrogen bonds, van der Waals forces and ionic interactions. A greater macromolecular packing density and high core hydrophobicity enhances rigidity of structures Charged surface residues inhibit nonspecific interactions caused by increased flexibility in elevated temperatures

PSYCHROPHILES

On the opposite side of the spectrum lie the cold inhabiting psychrophiles with optimal functionality below 20°C.

Psychrophiles often exploit high saline environments and intracellular concentrations

to lower their freezing point Low temperatures affect the fluidity of lipid membranes, decreasing permeability, which is regulated by membranes with branched chains and unsaturated fatty acids. Protein and enzyme thermodynamics must overcome energy barriers to fold or function Proteins contain additional flexible alpha helices and are protected from aggregation by coldshock proteins Enzymes maintain catalytic activity by solvent interaction and lower specificity Anti-freeze proteins maintain aqueous conditions and reduce mechanical damage from ice crystal formation by interfering with the order of water molecules

PIEZOPHILES

Piezophiles can withstand the force of intense pressures The physiological effects are comparable to freezing as cell membrane fluidity

and permeability is lost. To combat this, many piezophilic bacteria contain disordered fatty acids and specialised phosphate head groups in their membranes to reduce viscosity Cell structure and gene regulation adaptations include upregulation of chaperone-encoding genes, respiratory chain modifications and expression of different cell membrane porins. Low pressure adaptations need not be as specialised Many organisms can survive upper atmosphere or the vacuum of space; an external biolayer of microorganisms shields the organisms below the surface

HALOPHILES

Highly saline environments, such as deep hypersaline basins or anoxic lakes, harbour salt loving organisms, ranging from slight (200 to 500 mM NaCl) to extreme halophiles (2500 to 5200 mM NaCl)

Figure 2 Extremophile habitats are found all over the world in the most pressured of environments Image from https://doi org/10 3389/fmicb 2019 00780

Salt-in halophiles have highly concentrated intracellular saline environments They bala-

nce out the external osmotic pressure by accumulating ions within the cytoplasm Salt-in halophiles have evolved to express acidic residue coated proteins which trap water around them, preventing aggregation and denaturation Charged residues allow for stronger electrostatic interactions and salt bridges to maintain enzymatic stability Salt-out halophiles use energy to maintain near neutral salinity within the cytoplasm in comparison to external surroundings Turgor pressure must be maintained and osmoprotectants maintain osmotic balance and prevent salt influx which would interfere with metabolism

ACIDOPHILES

Most acidophiles maintain a near neutral cytoplasmic pH while surrounded by an extracellular environment of much greater hydrogen ion concentration, an influx of which can result in cell lysis. Evolutionary adaptations include membranes with near complete impermeability to protons Some acidophiles have smaller sized pores in membrane channels to prevent leakage and an internal net positive potential charge to offset the high external H+ concentration Many bacteria exploit active proton pumps and have organic acid degradation pathways to prevent proton uncoupling Thiosulphate and sulphur oxidisers secrete sulphate and protons, which significantly alter the surrounding acidity

ALKALIPHILES

Alkaliphiles maintain a near neutral intracellular pH, though slightly basic. They require hydrogen protons due to their bare proton desert-like surroundings Negatively charged acidic cell walls (often containing teichuronic acid or polyglutamic acid) lower the pH of the connecting environment which attract hydrogen protons and repel hydroxide ions, to generate enough proton force and synthesise ATP Many species generate proton force through Na+ or K+ antiporters which catalyse electrogenic exchange of outwardly moving ions while increasing proton intake These antiporters and acid production can lower overly high intracellular pH

RADIOPHILES BIOTECHNOLOGICAL APPLICATIONS

Radiophiles defy the hardiest of radioactive conditions which break DNA, damage p-

roteins, and cause oxidative stress

Deinococcus sp prokaryotes have thick cell walls, multiple membranes and are commonly arranged in a tetrad enclosed by a dense carbohydrate shell Multiple copies of highly condensed genomes link tightly to form ring like nuclei which hold DNA ends together and promote efficient and accurate repair Radiophiles reduce the effects of oxidative stress by purging reactive oxidative species (ROS), protecting proteins against oxidative damage, and producing antioxidants

UV protection mechanisms include photoprotective devices such as pigments, and a reduced number of bipyrimidine sequences to lower dimer lesion formation Manganese complexes are taken up on radiation exposure to prevent iron dependent ROS from accumulating and hydrolysis of damaged proteins promotes a cellular response to selectively remove potentially toxic proteins

The plasticity and environmental resistance of extremophiles makes them ideal for numerous potential biotechnological applications. In biofuel production, biomass is broken down by multiple extremophilic enzymes (extremozymes) under great temperatures, pressures and in the presence of organic solvents, which would denature other biocatalysts Extremozymes have also been sourced for industrial scale lipid hydrolysis, starch processing and fine chemical synthesis.

Environment-altering extremophiles have been used in bioleaching as they secrete metabolic reaction products that remove insoluble metal sulphides or oxides from ores Wastewater is then collected, and metal ions extracted for use in a cleaner, safer, and more economical approach to traditional mining. Bioremediation efforts utilise halophiles, acidophiles and radiophiles to clear up metal ion pollution from mines or industrial waste sites

Psychrophile-derived antifreeze proteins have been explored for use in agriculture Experimental research has demonstrated that spraying antifreeze proteins on seeds or used in biofertilizer improves plant resilience when planted in cold weather. Generating transgenic crops to withstand harsher winters or unexpected cold spells would undoubtedly increase product yield in an ever-demanding world.

The understanding of specialised protein domains not seen in non-extremophiles

has allowed for specialised protein engineering and design Extremozymes have been analysed as a basis for engineered biomimetics for coatings of aviation materials or equipment to withstand temperature fluctuations

Extremophile biochemistry has vastly influenced the field of molecular biology Most famous are the heat resistant Taq and Pfu polymerase used in PCR reactions (integral to covid-19 testing and sequencing techniques), derived from eubacterial microorganisms. Various modified enzymes which denature at different temperatures have allowed for more efficient experiments as solutions can be conveniently purified by thermal changes, leaving experimental products intact

Studies of the radiophile D. radiodurans have led to the discovery of novel DNA repair mechanisms and greater understanding of medical radiation treatment. Theoretical suggestions include the development of radioprotectants to incorporate into skincare products Anti-freeze proteins sourced from Arctic cod have been used for cryopreservation of tissues, cells, and embryos, and confer advantages over many other cryopreservation techniques Antifreeze proteins are easily available cost-effective and do not affect the preserved cells and tissue, whereas many popularised cryoprotective agents can penetrate cytoplasm and chromatin. Anti-freeze proteins have also been used as an experimental

adjuvant in tumour cryosurgery with increased ablation effects and antibacterial properties

The sheer diversity of life is yet to be fully discovered, let alone explained While the applicable potential of extremophiles is still in its infancy (restricted by current technology and exploration of tough ecosystems) research is working towards uncovering these wonders which redefine the parameters of life that can drive us beyond our limits

References

Coker, J A (2019) Recent advances in understanding extremophiles

F1000Research 8(F1000 Faculty Rev-1917)

https://doi org/10 12688/f1000research 207 65 1

Jin, M , Gai, Y , Guo, X , Hou, Y , & Zeng, R (2019) Properties and Applications of Extremozymes from Deep-Sea Extremophilic Microorganisms: A Mini Review Marine Drugs, 17(12), 656 https://doi org/10 3390/md17120656

Jin M Xiao A Zhu L Zhang Z Huang

H & Jiang L (2019) The diversity and commonalities of the radiation-resistance mechanisms of Deinococcus and its upto-date applications AMB Express, 9(1), 138 https://doi org/10 1186/s13568-0190862-x

Krüger, A , Schäfers, C , Schröder, C , & Antranikian, G (2018) Towards a sustainable biobased industry –

Highlighting the impact of extremophiles

New Biotechnology 40 144–153

https://doi org/10 1016/j nbt 2017 05 002

Merino, N , Aronson, H S , Bojanova, D P , Feyhl-Buska, J , Wong, M L , Zhang, S , & Giovannelli D (2019) Living at the Extremes: Extremophiles and the Limits of Life in a Planetary Context Frontiers in Microbiology, 10

https://doi org/10 3389/fmicb 2019 00780

Xiang, H , Yang, X , Ke, L , & Hu, Y (2020) The properties biotechnologies and applications of antifreeze proteins

International Journal of Biological Macromolecules, 153, 661–675

https://doi org/10 1016/j ijbiomac 2020 03 0

Women's History Month

American computer scientist who worked for IBM where she helped design supercomputers but was fired in 1968 after revealing she wanted to transition. She then joined Xerox PARC, where she co-authored 'Introduction to VSLI Systems' which is now a standard university textbook in computer science and later won 'Member of the National Academy of Engineering', the highest professional recognition for engineers.

Lynn Conway

A DEEP CLEAN: TACKLING THE ISSUE OF SINGLE-USE PLASTICS WITHIN DENTISTRY

WRITTEN BY AMY SCOTT

EDITED BY DRSHIKA MEHTANI DESIGNED BY TAMARA YAP

Sustainability is an issue that greatly underpins modern dental practice and is a chall-

enge yet to be tackled in-depth. In a clinical environment where sterility and patient safety are of number one concern, a lot of single use plastics are used to maintain these high standards of hygiene As a profession, dentistry consumes a high volume of single-use plastic that becomes clinical waste, making it exceedingly tricky to reuse or recycle. The use of PPE (Personal Protective Equipment) has increased exponentially during the COVID-19 pandemic, and currently contributes to the greatest volume of single-use plastic disposed of following a clinical procedure (Martin, 2021)

The need to maintain stringent hygiene standards and patient safety acts as the main challenge concerning the reduction of

single-use plastics within the clinical environment Single-Use plastics offer the guarantee of sterility, cleanliness, and convenience; all of which ensure the smooth running of clinical procedures and helps ensure quality of care received by the patient It is important to consider the factor of accessibility behind single-use plastics: for most practices they are cheap, easily available, and easy to use There is little, if any, maintenance associated with singleuse plastics for practices too Despite this, we cannot lose sight of the other, perhaps greater, side of this argument: do the environmental and public-health risks generated by excessive plastic waste outweigh their clinical benefits? (Duer, 2020).

On a daily basis within NHS dentistry alone, 300,000 single-use plastic cups are used and disposed of

This item alone accumulates to over 105 million pieces of plastic consumed per year,

which is a truly staggering volume of waste Plastic consumption within the dental practice is unavoidable; alongside plastic cups, it’s important to consider that plastic barrier wrap, and single-use instruments also need to be taken into account when considering the true extent of plastic consumption Despite this monumental amount of plastic consumption, it could be argued that the issue of single-use plastic is not exacerbated solely by increased demand in consumption, but also through the infrastructure that handles its production and disposal Plastic recycling rates in the UK have grown year-on-year, particularly through post-consumer recycling, which suggests not only an improved incentive to recycle, but also that we have the ability to The issue is- we can’t necessarily translate this to healthcare waste management. Dental waste, as per infection control policies, needs to be disposed of in orange clinical waste bins which are then incinerated Even though recycling can be an energy-intensive practice, the incineration process of clinical waste releases harmful toxins and carbon emissions from the plastic which then pollutes the air, posing a greater public health and environmental risk in comparison (Thomson, 2022) (British Plastics Federation, 2018)

Understandably, it’s an incredibly large issue to tackle, and it poses two main questions: How can I make a difference as an individual? How can Clinics shift to a more sustainable way of practice?

Answering the first question is, thankfully, getting easier Many more brands are waking up to the fact that consumers care more about sustainability and have since introduced many innovative products to the oral-care market to help consumers make more environmentally friendly choices A notable invention includes toothpaste tabs; small tablets in recyclable packaging designed to replace traditional toothpaste tubes, of which over 1 5 billion enter landfill or oceans per year. These tubes are often difficult, if not impossible, to recycle because the aluminium is layered between plastic film It’s also incredibly difficult to remove excess toothpaste residues after use to enable their recycling if it were possible (Parla, 2020) (LUSH, 2017)

Answering the second question, however, is considerably harder Not only would

it require an increase in budget, accessibility, and sustainable options, but also a complete overhaul of current infection-control guidance. As dental professionals, both in practice and in training, we can be the catalyst the NHS needs to tackle plastic pollution Given the negative public-health risks of single-use plastics, it could also be considered our duty of care to consider more sustainable alternatives Some emerging products include autoclavable plastics metal-based substitutes, and other possible uses of current equipment (For example, using the 3-in-1 and suction when encouraging a patient to rinse as opposed to offering a plastic cup)

It's difficult to predict how sustainability and clinical practice will merge in the future, but what is clear is that we can all take individual choices towards sustainability. It’s daunting at times to consider how we might tackle such widespread global issues, but individual choices all really do add up

References

Thompson, D (2022) Plastic Pollution & Dentistry Retrieved 13 February 2022, from https://www treetopsdentalsurgery co uk/ news/plastic-pollutiondentistry/#:~:text=Within%20the%20denta l%20practice%2C%20plastic,bags%2C%20 which%20are%20then%20incinerated

Duer, J (2020) The plastic pandemic is only getting worse during COVID-19 Retrieved 13 February 2022 from https://www weforum org/agenda/2020/07 /plastic-waste-management-covid19-ppe/

Macarthur E (2016) Plastics and the circular economy Retrieved 13 February 2022, from https://archive ellenmacarthurfoundation org/explore/plastics-and-the-circulareconomy#: :text=The%20use%20of%20pl astics%20has%20increased%20twentyfold %20in%20the%20past%2050%20years &t ext=In%202016%2C%20the%20Foundatio n%20published is%20lost%20to%20the% 20economy

Martin N (2021) Quantification of SingleUse plastics waste generated in clinical dental practice and hospital settings Retrieved 13 February 2022, from https://www sciencedirect com/science/ar ticle/pii/S0300571222000057

British Plastics Federation (2018) Plastic Recycling Retrieved 13 February 2022, from https://www bpf co uk/Sustainability/Plasti cs Recycling aspx

Parla Toothpaste Tabs (2020) Why Retrieved 13 February 2022 from https://parlatoothpastetabs com/pages/w hy

LUSH (2017) Introducing Lush’s new Toothpaste Tabs Retrieved 13 February 2022 from https://www lush com/uk/en/a/introducing -lushs-new-toothpaste-tablets

The progress of medicine is the continual tackling of ever more complex issues

Small molecule drugs are very well studied, and we are seeing their limitations. Progress in the field of biochemistry is providing more complex alternatives, with biologicals revolutionising treatments However, in this article we will be looking at a major breakthrough using the emerging field of self-assembling supramolecular chemistry

SPINAL CORD INJURIES AND SUPRAMOLECULESPROGRESS IN A PARALYSED FIELD

WRITTEN BY KIRA LINKE

EDITED BY YASH KHANNA DESIGNED BY TAMARA YAP

The concept of self-assembly refers to using small, simple components that are driven by their intermolecular forces to noncovalently bond into the more complex supramolecule Supramolecules can achieve complexity that can’t be generated by traditionally controlled multi-step covalent synthesis They are incredibly adjustable and biodegrade quickly in the host body. We've long observed this in nature through protein folding, virus mechanisms and DNA replication and expression But nature has had millions of years to develop these processes, and we are only just beginning to take notes

The self-assembly method introduces new variables of supramolecular dynamics, including plasticity and internal motion of components, which can be optimised for receptor binding

Confronted with this variability, scientists at Chicago’s Northwestern University began their mission to use a self-assembling system for a problem that currently has very limited treatment options - spinal cord injury.

CHAPTER ONE

Injuries of the central nervous system are notoriously difficult to heal and restore function to, as nerves seldom undergo mitosis Furthermore, astrocytes form glial scars, which inhibit axonal regeneration. The first selection barrier to overcome was to decide on the signalling pathways which should be activated to induce neuronal growth and repair, and how those signals should be delivered This was done using computational modelling

Two signalling factors were chosen for the task To encourage neural stem cells

into differentiating and extending axons, the laminin signal IKVAV was selected Laminin is a biologically active protein endogenously found in the basal lamina with influence on cell differentiation, migration, and adhesion It has been demonstrated to improve neural stem cell growth For the surrounding structural support needed in wound healing, the FGFR1 (fibroblast growth factor receptor 1) should be activated on the tissues localised around the wound This was done in the hopes of further promoting neuron differentiation and survival, as well as building extracellular support and angiogenesis An FGF2 mimetic peptide was concluded to be a suitable signal

These two signals need to be delivered in a way that ensures they’re still bioactive at the target tissue, and the supramolecular structure allows for the signalling molecules to activate the relevant receptors Here, fibril forming peptide amphiphiles (PAs) provide the solution They are easily modified in terms of size and bond rotation, form a supportive structure akin to the extracellular matrix, and rapidly biodegrade once the job is done. The peptides could be designed to express either signal, IKVAV or FGF2, at one terminus, while the alkyl tail could be modified to tune the mechanical properties of the subunit in the fibril, including non-covalent interactions and solubility The scientists calculated the degrees of motion for various PA structural analogues, which would be taken into the next phase of testing

CHAPTER TWO

In vitro testing is the most suitable method for observing the behaviour and activity of the chosen signals, IKVAV and FGF2, on analogous PAs

Human neural progenitor cells were treated with IKVAV-PA1 to IKVAVPA8 In solution, these form fibrils and associate closely with cells

Activation of the membrane receptor β-Integrin, an indicator of neuronal differentiation, was measured using antibodies, spectroscopy and western blot The greatest activation was produced by IKVAV-PA2 and IKVAV-5, which were predicted to be the most dynamic in silica. The amino acids included in the peptide sequence means they have the lowest hydrogen bond density, allowing internal motion and reducing adhesion to surrounding molecules

CHAPTER THREE

Knowing that higher internal motility increases the possibility of the signals reaching the target receptors and binding, in vivo testing started

Co-assemblies of IKVAV-PA2 with FGF2-PA1 or FGF-PA2 were carried through to this stage, which will hereby be referred to as PA2/PA1 and PA2/PA2 fibrils. The mixture of subunits was to be injected into murine models of

spinal cord injury, 24 hours after the injury was created. A new selection barrier was raised here, as the PAs had to assemble into fibres with similar mechanical properties as in vitro, even once in contact with the biological environment All PAs passed this barrier since they gelled at the injection site and localised around the damaged nerve They biodegraded gradually within 1-12 weeks, allowing sufficient time to initiate healing, but avoiding continued receptor activation Scientists labelled and observed regrowth in the corticospinal tract and serotonergic neurons, which mediate motor function

Surprisingly, PA2/PA1 fibrils significantly outperformed all other tested compounds and combinations in all measured variables, despite PA2 demonstrating significantly greater dynamics both in in silico and in vitro testing than PA1. Axon regeneration was seen from both PA2/PA1 and PA2/PA2, but the PA2/PA1 fibrils produced significantly more axon regrowth throughout the lesion, even completely spanning the lesion (Figure 1A). High levels of myelin basic protein were measured around the nerves after 3 months, a strong indication of re-myelination (Figure 1B) A glial scar was avoided and a fibrotic core was reduced, which strengthened the prospect of return of functionality

Both of these findings were likely a result of the low astrocyte density in the models injected with PAs, compared to sham Additionally, comparison with uninjured mice showed that the sham treatment led to tissue degeneration surrounding the nerve, and decreased vascular dimensions In contrast, all models treated with PAs avoided degeneration, and thePA2/PA1 co-assembly even increased vascular area fraction, length and branching, which provides better support for healing

Figure 1. Two chemically different PA scaffolds with two identical bioactive sequences reveal differences in growth of corticospinal axons after SCI (A) Scans of spinal cord sections GFAP indicates astrocytes (green), BDA indicates labelled axons (red); indicated are the lesion’s proximal border (PB), the distal border (DB), and the central part of the lesion (LC). (B) Three-dimensional scans of BDA-labelled axon regrowth (red) and myelin basic protein (MBP) (green) (left) and laminin (white) (right)

The success in improving the cellular variables was truly demonstrated by the beha-

vioural testing Within 1 week, all PA groups performed better than sham in footprint analysis and open field locomotor scores, in which the mice are allowed to freely roam around a large area Again, PA2/PA1 treated mice performed the best Their stride length and width was increased the most, returning almost to that of a healthy mouse (Figure 2) The cellular changes are directly reflected in the functional improvement

Protein analysis revealed that in the less bioactive PA2/PA1 fibrils, the FGF2 signal was almost completely inactive, unlike in the more therapeutic PA2/PA1 Why was this? Analysis of relaxation rates and anisotropy indicated that in PA2 fibrils, the FGF2 signal was greatly reduced in mobility Additionally, the co-assembly of two PA2-carried signals increased the intermolecular interactions between the same amino acid side chains PA1 and PA2 are differentiated by their tetrapeptide sequence in the motion-controlling region of the chain - PA1 consists of Val-Val-Ala-Ala, and PA2 is made up of Ala-Ala-Gly-Gly Even though PA1 is more disposed to forming structure beta sheets, which would greatly decrease fibril dynamics, the more heterogeneous amino acids in a PA1/PA2 combination significantly reduce intermolecular interaction, allowing for greater motility This study thereby provided a large step forward in spinal cord injury treatment

CHAPTER FOUR

The confidence gained from the results in the murine models prompted the scientists to take their PAs into differentiated human in vitro cells Human umbilical cord vein endothelial cells (HUVECs) were treated with the two established coassemblies. Within 48h, they observed that a branched network of capillaries was beginning to form

Treating paralysing spinal injuries remains an unsolved medical problem. It must be acknowledged that this animal model is only a budding solution for human patients The true value of this experiment lies elsewhere As the scientists themselves state, "The neuronal cell survival and functional recovery that we observed in dual-signal systems are unexpectedly linked to the differences in the chemical composition of their respective non-bioactive tetrapeptides"

Figure 2 Footprint analysis of animals injected with dual signal co-assemblies. (C) Representative footprints (D, E) Bar graphs representing (D) stride length in mm and (E) stride width in mm

They have demonstrated the therapeutic possibilities offered by self-assembling

systems and the importance of structural design, when modifying pharmacodynamics Nonetheless, progress remains the first step in the pursuit of a solution, especially in the ever-evolving field of regenerative biochemistry and supramolecular chemistry.

References

Álvarez Z Kolberg-Edelbrock A Sasselli I , Ortega, J , Qiu, R , & Syrgiannis, Z et al (2021) Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury Science

374(6569) 848-856 doi: 10 1126/science abh3602

WHY EPIGENETIC REPROGRAMMING MAY GIVE US SIGNIFICANTLY BETTER CONTROL OVER REGENERATION, AGEING, AND DISEASES

WRITTEN BY BJÖRN OLAISEN

EDITED BY ALEXI MERY

DESIGNED BY ZAHRAA BHATTI

Epigenetic reprogramming may be the most promising strategy to promote tissue regeneration

and extend the healthspan and lifespan of humans and other organisms. It has already been proven that this tool can turn dysfunctional tissues into functional ones and reverse the age in cells and tissues There have been huge advances in the field in recent years, a big increase in its popularity, and billions of dollars are being invested to improve our understanding of how epigenetic reprogramming works in order to create a therapy. Why does it seem that epigenetic reprogramming can make dramatic impacts on science, society, and human lives? And how can one single therapy affect regeneration, longevity, and diseases simultaneously?

What is the epigenome?

Although the many different cell types in our body can have vast differences in morphology and function, the genomes in the cells of an organism are identical, if changes due to genomic instability are not considered However, the epigenome varies between different cell types, enabling unique patterns of gene expression in different cells, and thus various characteristics and roles The epigenome is a set of chemical compounds and proteins that regulate which genes within the genome are transcribed and the level of transcription While the genome is relatively static, the epigenome is highly dynamic and is responsive to signals from within the cells other cells, or from the environment outside the body The epigenome does therefore play a central role during the whole course of the life of an organism, from enabling cell differentiation and tissue patterning during embryonic development to

responding to environmental stimuli and maintaining cell identities as we get older In the course of a lifetime, changes in the positions and nature of epigenetic marks on the DNA happen to all cells and tissues in the body (López-Otín et al., 2013). Epigenetic reprogramming is the reestablishment of these epigenetic marks, making the epigenome more similar, but not identical, to how it was early in the life of an organism This happens and is essential in the development of gametes and embryos (Jacob and Moley, 2005) Epigenetic alterations can be inherited and cause several common diseases, including cancers (Egger et al., 2004). Fortunately, changes in the epigenome can be reversed and promising therapies that target epigenetic regulatory enzymes are being developed

The discovery of cellular reprogramming

In 2012, Professor Shinya Yamanaka proved that mature, specialised cells could be brought back to an early stage of development by reprogramming He did so by identifying genes, called the Yamanaka factors, that were able to change a specific cell type such as a skin cell to an induced pluripotent stem cell (iPSC) An iPSC can be differentiated to any cell type in the body. The generation of iPSCs from specialised, adult cells is called cellular reprogramming The ability to generate iPSCs has been useful to make disease models and screen drugs, as well as in the development of personalised cell therapies and transplantation

A causative role of epigenetic alterations in ageing

Evidence suggests that epigenetic alterations are one of the primary causes of damage in cells, and this promotes aging (López-Otín et al., 2013). With age, there are reductions in the levels of enzymes that modify histones and DNA methylation, and also proteins that remodel chromatin Overexpression of several of these proteins, for example the sirtuins, improve aspects of aging and extend lifespan (López-Otín et al , 2013) Some of the consequences of aging on the epigenome are the movement of enzymes that regulate the epigenome, including the sirtuins, loss and redistribution of heterochromatin, hypomethylation across the DNA, and hypermethylation at CpG islands (Kane and Sinclair, 2019) As a consequence, genes that were turned off in cells get turned on and vice versa In this way, cells can lose their identity, meaning that a nerve cell for example loses its ability to do the functions associated with a nerve cell, and act and look more like another cell type Loss of cell identity may promote aging (Sinclair, D A , & LaPlante, 2019)

Tissue regeneration and age reversal using epigenetic reprogramming

Rejuvenation approaches that target aging are now being developed, based on the discovery made by Yamanaka A growing body of evidence suggests that epigenetic alterations are important drivers of the aging process and th

epigenome may be one of the m important targets when intervening with the aging process (López-Otín et al , 2013, Sinclair, D A , & LaPlante, 2019) The Sinclair Lab at Harvard Medical School and others published a ground-breaking discovery in 2020, based on the injection of the genes Oct4, Sox2, and Klf4 (OSK), which are three of the Yamanaka factors (Lu et al , 2020) Epigenetic reprogramming rejuvenated retinal ganglion cells (RGCs) in the eyes of mice, caused axons to regrow following injury and reversed vision in old mice and mouse models of glaucoma More specifically, the treatment increased survival in RGCs and changed DNA methylation patterns, and 90% of the mRNA levels altered due to aging, changed to a state associated with young cells The reprogramming was partial, which means that it did not rejuvenate the cells to such an extent that they became iPSCs OSK induced cellular processes that corrected the epigenetic marks so that the epigenome became more similar to how it was in an earlier stage in the life of the cells This remarkable finding indicates that cells contain a copy of the youthful state of the epigenome that can be accessed The beneficial effects on DNA methylation, vision, and axon regeneration required the two demethylases ten-eleven translocation methylcytosine dioxygenases TET1 and TET2. This suggests that DNA methylation plays a role in the aging process and is required to reverse it (Lu et al , 2020)

whether the age of a cell or an organism has changed?

Humans have two measurements of age: a chronological age, which is based on how many times the earth has circled the sun, and a biological age, which is based on health biomarkers and is influenced by lifestyle, aging, and health The reprogramming factors used in the Sinclair Lab partially reversed the Horvath Clock, which is a widely used biological clock that can predict your biological age with high accuracy, based on methylation patterns in the epigenome (Horvath, 2013).

Why do we age?

The Sinclair Lab is arguably a world leader in understanding the molecular causes of ageing and stopping and reversing it. The principal investigator of the Sinclair Lab, Professor David A Sinclair, has proposed the theory that a major cause of ageing may be that changes in the epigenome disrupt the pattern of gene expression that is associated with a youthful state (Sinclair, D A , & LaPlante, 2019) Furthermore, the changes in transcription can cause cellular dysfunction and senescence, and thus ageing and disease. This information theory of ageing is supported by epigenetic reprogramming and additional evidence in yeast and mammals For example, they developed the ICE mice (Inducible Changes to the Epigenome), which enabled them to

DNA breaks

s in the celerated the reason why nges in the epigenome may be due to the damage itself or the repair process, which would be an example of antagonistic pleiotropy

The proteins sirtuins 1, 6, and 7 have two main roles, which are to maintain cell identity by regulating the epigenome, but also to aid in DNA repair, by recruiting proteins and preventing damage of the surrounding epigenome When sirtuins have contributed to fixing the DNA damage, they may not find their way back to the place of the epigenome that they are supposed to regulate, which may cause aging

(Sinclair, D A , & LaPlante, 2019)

It seems that epigenetic alterations and DNA damage are upstream causes of aging, and that targeting these processes may ameliorate negative effects on health and longevity caused by other hallmarks of aging (Sinclair D. A. & LaPlante 2019). However, there are also other important characteristics of aging, some of which may be important drivers of the process (López-Otín et al , 2013) Evidence suggests that the mTOR pathway and the AMPK signaling pathway are particularly important in the molecular mechanisms of aging, and interventions targeting these pathways extend lifespan and healthspan in model organisms

(Sinclair, D A , & LaPlante, 2019) There are many questions that have to be answered before we precisely understand which hallmarks of aging are underlying causes and which are downstream consequences of aging In addition, the hallmarks are

interlinked, and it is not yet fully known which of them are the best drug targets

It was long thought that accumulation of mutations or free radicals were the principal causes of aging, but these hypotheses have been widely disproved (López-Otín et al. 2013). Organisms with accumulated mutations give rise to a healthy, young organism, and thus, mutations cannot be the main reason why we age (Sinclair, D A , & LaPlante, 2019) Also, antioxidants that reduce free radical activity have failed to extend lifespan, and in some instances even reduced it, which is thought to be due to the many beneficial physiological roles that free radicals have in the body (Sadowska-Bartosz and Bartosz, 2014 ) However, both mutations and excess free radicals can cause disease and aging in some scenarios

What is currently being done in the field of epigenetic reprogramming?

Numerous labs are trying to understand the mechanisms behind epigenetic reprogramming therapies, explore their applications and develop new reprogramming strategies The Sinclair Lab is for example trying to rejuvenate human senescent cells and brain organoids The discovery that epigenetic reprogramming can reverse the age of cells and tissues without promoting tumours has led to other scientists showing that the approach can successfully reverse the age of organs and aspects of aging of whole organisms (Chondronasiou et al , 2022) Altos Labs, a new biotechnology company, has gathered world-leading scientists, cians, and others to try to

develop cellular reprogramming therapies to reverse diseases. The Sinclair Lab is trying to understand where the backup copy containing information on what a youthful epigenome looks like is located, what it is, and how it is regulated. It is also possible that we will need to develop a reprogramming strategy that can target the methylation or packaging of mitochondrial DNA because mitochondria are central in the aging process and can affect the epigenome (López-Otín et al , 2013)

How can epigenetic reprogramming affect diseases, regeneration, and society?

Aging is the main risk factor for a range of diseases and is also associated with tissue decline and dysfunction in the body (López-Otín et al , 2013) Therefore, interventions that affect aging have a tendency to also slow, stop or reverse diseases and dysfunction This was for example the case when the mice with glaucoma got their sights back, and neuronal axons regenerated, in the experiments discussed (Lu et al , 2020) Epigenetic reprogramming may be the first treatment that can reverse vision loss due to glaucoma in humans. Current treatments prevent or slow down the pathological process (Sinclair, D A , & LaPlante, 2019) Additionally, if we better understand why we age, we may increase our understanding of age-related diseases and dysfunction, and thus be better able to treat those processes Understanding the mechanisms of epigenetic reprogramming will likely teach us more about the fundamental causes

of aging (Sinclair, D A , & LaPlante, 2019).

In the future, we may be able to rejuvenate our bodies periodically by combinations of drugs and injections of genes that initiate epigenetic reprogramming Hopefully, the epigenetic reprogramming treatment can be ingested in a pill Although we can likely reverse many diseases and aspects of aging in the future, we will have to continue to address diseases such as cancer which can occur sporadically due to replication errors or damage Extensions of healthspan and lifespan are likely to have a positive impact on the economy (Sinclair, D A , & LaPlante, 2019) The increased workforce and the money that is saved from healthcare costs could be used to try and fix societal problems such as inequality, global warming, conflicts, and suffering

References

Chondronasiou, D , Gill, D , Mosteiro, L , Urdinguio, R G , Berenguer, A , Aguilera, M Durand S Aprahamian F

Nirmalathasan N Abad M MartinHerranz, D E , Otto-Attolini, C S , Prats, N , Kroemer, G , Fraga, M F , Reik, W , & Serrano M (2022) Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming https://doi org/10 1101/2022 01 20 477063

Egger G Liang G Aparicio A & Jones P A (2004) Epigenetics in human disease and prospects for epigenetic therapy

Nature 429(6990) 457–463 https://doi org/10 1038/nature02625

Horvath S (2013) DNA methylation age of human tissues and cell types Genome biology, 14(10), R115 https://doi org/10 1186/gb-2013-14-10-r115

Jacob S & Moley K H (2005) Gametes and Embryo Epigenetic Reprogramming Affect Developmental Outcome: Implication for Assisted Reproductive

Technologies Pediatric Research 58(3)

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Kane, A E , & Sinclair, D A (2019)

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Serrano, M , & Kroemer, G (2013) The Hallmarks of Aging Cell, 153(6), 1194–1217

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Lu, Y , Brommer, B , Tian, X , Krishnan, A , Meer, M , Wang, C , Vera, D L , Zeng, Q , Yu, D , Bonkowski, M S , Yang, J , Zhou, S , Hoffmann E M Karg M M Schultz M B

Kane, A E , Davidsohn, N , Korobkina, E , Chwalek, K , Rajman, L A , Church, G M , Hochedlinger, K , Gladyshev, V N , Horvath, S Levine M E Gregory-Ksander M S

Ksander B R He Z Sinclair D A (2020)

Reprogramming to recover youthful epigenetic information and restore vision

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Sinclair D A & LaPlante M D (2019)

Lifespan: Why We Age-and Why We Don’t Have To Atria Books

ORGANISMS: What Weird and Wonderful Species Can Teach Us

WRITTEN BY EVA HAMRUD | EDITED BY KIRA LINKE DESIGNED BY TAMARA YAP

oo3 saw the completion of the biggest global initiative in biology – the Human Genome

project. Costing $2.7 billion and with worldwide collaboration, this initiative successfully sequenced the entire human genome Since then, other species have also had their genetic secrets revealed, and in 2018 a much more ambitious initiative was launched – the Earth BioGenome Project, which aims to sequence all taxonomically classified eukaryotes on earth This encompasses around 2 million species of plants, fungi and animals.

Sequencing the genomes of all complex life would greatly advance our understanding of ecosystems and help us conserve our increasingly threatened biodiversity With the growing pressures of global warming, deforestation and pollution, these reasons seem as good as any to head into the wilderness and start collecting species for sequencing. Yet a major goal of the Earth Biogenome Project is to not only protect biodiversity, but advance biomedical research Sequencing our own genomes makes sense when studying human health and disease, but the benefits of sequencing some of the more weird and wonderful species of our planet are less obvious.

Due to ethical reasons, experiments on humans are highly restricted and only used when absolutely necessary – for example to test new medications before they are released to a wider population Therefore, lots of biomedical research is carried out on model organisms – the lab rat (or mouse) Beyond mice and rats, we can thank yeast, fruit flies, nematode worms and other model organisms for a huge range of biological breakthroughs What these animals have in common is that they are extensively studied, each having a fully sequenced genome and wellestablished protocols for how to maintain and study them In order to branch out from these tried and trusted model organisms, it is necessary to sequence more species – but why do we need to do that?

Whilst we have learnt a lot from these common model organisms, some of the biggest biological breakthroughs have been possible thanks to more obscure species The Augustinian monk, Gregor Mendel, became the ‘father of modern genetics’ because of his experiments where he crossed different strains of pea plants in his monastery’s garden Charles Darwin developed the theory of evolution after carefully observing and documenting the adaptive

differences between species during his voyages, such as the varying beaks of the Galapagos finches

More recently, Elizabeth Blackburn, Carol Greider and Jack Szostak shared a 2009 Nobel Prize for their discovery of telomeres and telomerase, the essential anti-cancer mechanisms which protect the ends of chromosomes. This discovery was thanks to research using Tetrahymena, a single-cell organism more commonly known as pond scum

Future breakthroughs in biology will also require research in organisms other than the universally used lab mouse Popular model organisms like flies and mice are chosen because they reproduce quickly and are easy to maintain, but they can’t be used to study every biological process For example, ageing is one of the most important aspects of human health: as we get older the immune system weakens, cognitive abilities are reduced, and incidence of cancer and other diseases increases Laboratory mice typically live for 2-3 years, meaning that ageing research in this system is very slow. Alternative model species such as flies and nematode worms have much shorter lifespans, but as they are invertebrates they may age differently to humans In 2016, a small African freshwater fish began to emerge as an ideal vertebrate system to study ageing Turquoise killifish only live for 4 to 9 months and in this short lifetime display many hallmarks of ageing such as neurodegeneration and cancers

Since their identification, killifish have been used for ageing research in several labs around the world, for example this month a research group

from the Max Planck Institute in Germany were able to use killifish to characterise the agerelated loss of antibody diversity

Another unusual species, the axolotl, is being investigated to further our understanding of regeneration Regenerative medicine is an area of biology that seeks to develop therapies which can repair or regrow damaged tissues or organs Adult humans, along with mice and rats, have very limited regenerative ability Whilst humans can interestingly regenerate the tips of their fingertips after amputation, they cannot grow back entire limbs Axolotls, in contrast, can regrow not only amputated limbs, but severed spines and damaged brains By studying the genes and pathways involved in axolotl regeneration, research groups

that use this species hope to bring us closer to effective regenerative therapies for degenerative conditions

A final example is the tardigrade, also called water-bears Tardigrades are a family of 1mm long invertebrates that live all over the world in water and on land. Interestingly they can live almost anywhere – including at the extreme temperatures of -180C or 150C, and even in the vacuum of space Extensive research has been carried out to try to characterise the molecular mechanisms which allow tardigrades to be so hardy, and to see if any of these pathways could be stimulated in human cells

Interestingly, in 2016 a group of researchers from Tokyo University found that they were able to use a tardigrade protein to protect the DNA in human cells from X-ray radiation Understanding how genome integrity is maintained under high radiation levels is potentially interesting to the fields of cancer biology and radiotherapy

Historical breakthroughs in biology were not just made in the lab rat, but in a range of weird and wonderful species from around the world The enormous diversity between species continues to be a valuable resource that helps to further our understanding of human biology and develop new medical therapies In 2021 there were three times as many sequenced genomes as in 2018, and this number continues to rise. The completion of the Earth Biogenome Project will offer another 2 million species to the scientific community, some of which could hold the secrets to biology’s most important questions

References

Kim et al (2016) The short-lived African turquoise killifish: an emerging experimental model for ageing Disease Model and Mechanisms

Bradshaw et al (2022) Extensive agedependent loss of antibody diversity in naturally short-lived turquoise killifish eLife DOI: 10 7554/eLife 65117

Clarke et al (1998) Regeneration of descending axons in the spinal cord of the axolotl Neuroscience Letters

Maden et al (2013) Proliferation zones in the axolotl brain and regeneration of the telencephalon Neural Development

Jonsson et al (2008) Tardigrades survive exposure to space in low Earth orbit Current Biology

Hashimoto et al (2016) Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein Nature Communications

Lewin et al (2022) The Earth BioGenome Project 2020: Starting the clock PNAS

Women's History Month

British computer scientist who designed the Acorn Microcomputer, co-designing the BBC's one in 1998. In 1983, she designed the Acorn RISC Machine (ARM) which is an IP core found in most smartphones, laptops, and tablets today. In 2011, she was listed one of the top 15 most important women in tech history.

Sophie Wilson

Junk or Functional? Biological importance of the "underdog" non-coding RNAs

When it comes to RNA, the first thing that comes to mind for most people

would be the mRNA COVID vaccines. Even though it is more well-known and an essential part of the central dogma, only 3% of the human genome are protein-coding genes that code for mRNA.

WRITTEN AND DESIGNED BY WINNIE (WING YIN) LIU EDITED BY RHIANA CASTILLO

As sequencing technology advances, a new era of genetic research has brought about the discovery of more novel non-coding RNAs (ncRNAs), some with regulatory and pathological significance whereas many more with unknown functions Let's go over the known ncRNAs function and why ncRNAs are emerging as an important field

Types of mRNA

Function

Coding RNAs

Messenger RNAs (mRNAs)

Code for proteins

Ribosomal RNAs (rRNAs) Translation

Transfer RNAs (tRNAs) Translation

Small nuclear RNAs (snRNAs)

Splicing of pre-mRNA

Non-coding RNAs

Small nucleolar RNAs (snoRNAs)

MicroRNAs (miRNAs)

Small interfering RNAs (siRNAs)

Process and chemically modify rRNAs

Silence gene expression

Silence gene expression

Long noncoding RNAs (lncRNAs)

Act as scaffolding and regulate diverse cell processes including X-chromosome inactivation

Table 1. Types of RNAs produced by cells and their function Adapted from Krebs et al , 2017; Lewis et al , 2020

RNA interference

The first major discovery regarding ncRNAs was by Andrew Fire and Craig Mello in 1998, where they discovered gene silencing by doublestranded RNA This was later known as RNA interference (RNAi) and was awarded the 2006 Nobel Prize in Medicine for it. Both microRNAs (miRNAs) produced by the nucleus and synthetic small interfering RNAs (siRNAs) can act as guide strands in RNA induced silencing complex (RISC) Once recognized and bound to its mRNAs target, it silences gene expression by various mechanisms, such as cleavage of polyA tail leading to mRNA degradation and inhibition of translation The presence of the RNAi pathway allows a more finetuned regulation of gene expression

RNAi Therapeutics

Scientists sought to harness the RNAi pathway for clinical applications, with the major breakthrough of Patisiran, the first RNAi-based drug, approved by US Food and Drug Administration in 2018. Transthyretin (TTR) amyloidosis is an inherited and life-threatening disease caused by the deposition of TTR amyloids on various organs Patisiran is delivered into liver cells, where TTR is produced, as lipid nanoparticles encapsulating siRNA It silences both wild type and mutant TTR mRNA and downregulates TTR synthesis It is noteworthy that previous treatments for TTR amyloidosis only slow disease progression, thus patisiran turns a new page for patients suffering from this disease

Splicing of pre-mRNA

Pre-mRNAs, which are transcribed from DNA, have to be processed into mature mRNA before being exported out of the nucleus for translation. One of these modifications is the splicing of introns, which is carried out by spliceosomes consisting of small nuclear RNAs (snRNAs) in the form of ribonucleoprotein particles (snRNPs) and several other proteins snRNAs are a crucial part of the spliceosome assembly and splicing catalysis, enabling another level of gene expression regulation

Glossary

Central dogma: Proposed by Francis Crick in 1958, describes the flow of genetic information in which DNA is transcribed into messenger RNA (mRNA) and then translated into functional proteins.

Proto-oncogenes or tumour suppressor genes: mutation in these genes lead to cancer

Introns: segments of DNA that do not encode for proteins

Figure 1 Patisiran It is sold under the brand name Onpattro

Cancer Diagnosis

Another potential application of ncRNAs is as biomarkers for diagnosing cancer due to their highly tissue-specific expression patterns. NcRNAs that play a role in regulating gene expression of protooncogenes or tumour suppressor genes are often up or down regulated in tumour cells Furthermore, ncRNAs are relatively stable in bodily fluids, such as urine, serum and saliva, and thus less invasive to collect for diagnostic tests.

What is the takeaway?

It has become ever more evident that ncRNAs are key regulators of gene expression and protein synthesis, playing as, if not more significant role than coding RNAs An evergrowing number of novel RNA transcripts are identified by virtue of the development of high throughput RNA sequencing techniques

However, unravelling and understanding the mystery of their functions, on the other hand, would be a more challenging endeavour So next time you come across mRNA, remember its wonderful and fascinating "sister" non-coding RNAs

References

Fire A Xu S Montgomery M K Kostas S A Driver S E and Mello C C (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391 806–811

Krebs, J , Goldstein, E , and Kilpatrick, S (2017) Lewin’s GENES XII (Jones & Bartlett Learning)

Lewis, J , Morgan, D , Raff, M , Roberts, K , Walter, P , Alberts, B , and Johnson, A (2020) Molecular Biology of the Cell (W W Norton)

Pasquinelli, A E (2012) MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship Nature Reviews Genetics 13 271–282

Setten, R L , Rossi, J J , and Han, S (2019) The current state and future directions of RNAi-based therapeutics Nature Reviews Drug Discovery 18, 421–446

Sun Y -M and Chen Y -Q (2020) Principles and innovative technologies for decrypting noncoding RNAs: from discovery and functional prediction to clinical application Journal of Hematology & Oncology 13 109

Do you prefer video?

Watch this video by Henrik's Lab to learn more about ncRNAs

U ScienceMind U

HACKER DNA: HOW MALWARE CAN BE ENCODED INTO A GENE

WRITTEN BY MAHTA HAGHIGHAT GHAHFAROKHI

EDITED BY ANASTASIIA TARASENKO

DESIGNED BY TAMARA YAP

Science, at times, precedes fiction Domineering a computer with a simple touch in a world

animated by computers may well be one of this kind In 2015, a team of researchers from the University of Washington demonstrated the first successful DNA-based exploit of a computer system that executed the malicious code written into the artificial DNA strands while reading it To carry out the hack, researchers encoded malicious software into a small stretch of synthetic DNA Copies of the DNA were ordered online They then used it to take full control of a computer that attempted to process the genetic data after it had been converted into digital code by a DNA sequencing machine

To create this malware, the researchers translated a computer command into a stretch of 176 DNA letters The synthetic strands were passed through a sequencing machine, which converted the gene letters into digits 0s and 1s. DNA bases identifying as A, G, C, and T, each representing a binary pair (A=00, C=01, G=10, T=11)

The code infected the software and took control of the computer as this strand was sequenced and processed by the programme, the code infected the software and took control of the computer

This command was designed to target a particular flaw that the team had previously

discovered in the DNA processing programme The exploit took advantage of a basic buffer overflow attack, in which a software program executes the malicious command because it falls outside maximum length. The command then contacted a server controlled by the team, from where the researchers took control of a computer in the laboratory they were using to analyse the DNA file

“We analysed the security of 13 commonly used, open-source programs We selected these programs methodically, choosing ones written in C/C++," reads the research paper titled "Computer Security, Privacy, and DNA

Sequencing: Compromising Computers with Synthesized DNA, Privacy Leaks, and More "

"We found that existing biological analysis programs have a much higher frequency of insecure C runtime library function calls (e.g., strcpy). This suggests that DNA processing software has not incorporated modern software security best practices "

The researchers warn that hackers could one day use faked blood or spit samples to gain access to university computers, steal information from police forensics labs, or infect genome files shared by scientists.

References

https://www technologyreview com/2017/0 8/10/150013/scientists-hack-a-computerusing-dna/

After being banned for decades, scientists are now saying psychedelics could

become a valuable tool to relieve those suffering from anxiety, depression, and other severe mental disorders

WHAT ARE PSYCHEDELICS?

Remarkably, psychedelics have been used all throughout human history amongst various cultures The term ‘psychedelic’ is thought to have been coined in the 50’s by British psychiatrist Humphry Osmond, and derives from the Greek words psyche and delein, which translate to ‘mindmanifesting’ In the 60’s, they became widely used due to their heavy link to music and certain aesthetics, which was likely in response to American psychiatrist Timothy Leary’s strong advocacy towards psychedelics.

“Turn on, tune in and drop out.”

— Timothy Leary

A OR A IN SCIENCE? SHALLOW DIVE

In addition to the approximately 200 species of hallucinogenic mushrooms scattered all around the world, the ‘classical’ psychedelics impose a strong scientific and cultural impact on our society. These include psilocybin (the active compound found in magic mushrooms), lysergic acid diethylamide (LSD), dimethyltryptamine (DMT or ayahuasca, from a vine in the Amazon) and mescaline (extracted from mexican cacti)

In recent times, an increasing amount of research has also been conducted on ketamine and 3,4methylenedioxymethamphetamine (MDMA) Despite these drugs being termed dissociative anaesthetics rather than psychedelics, their effect on one ’ s consciousness is similar to that of the classical psychedelics

In high doses, ketamine causes dissociative effects, and has further shown to have an extremely rapid, sometimes immediate, antidepressant effect Meanwhile, MDMA can induce a state of empathy by turning off our brain’s “fear response ” , which is extremely important for those suffering with trauma-related disorders such as addictions or PTSD

WRITTEN BY CHELSEA BLAIR | EDITED BY NICOLA ALLEN | DESIGNED BY DORIS YU

These individuals often struggle with therapy due to an unwillingness to recall painful memories or the past Thus, MDMA can allow them a brief few hours to shut off their fear response and proceed with intense psychotherapy

Nonetheless, those suffering from depression generally do not have a pleasant psychedelic experience Integration psychotherapy sessions are therefore essential to ensure patients undergoing trips are speaking with a therapist who can help interpret their thoughts, visions and experience

UNDERSTANDING THE ACID TRIP EXPERIENCE…

Psychedelic intoxication provokes changes in an individual’s sensory and spatiotemporal perceptions, synthesia (where two or more senses become connected), and an alteration in our ways of thinking (e g adopting a more spiritual, intuitive and/or delusional mindset) These substances can also have an emotional impact, bringing certain core memories to the forefront of one ’ s mind

Scientists believe that psychedelics are not addictive in comparison to most dependence-forming substances such as alcohol or tobacco, which can cause a physical and mental dependency. Moreover, psychedelics are at a far lower risk in terms of toxicity, implying you would need an impractical amount in order to overdose However, the unpleasant effects that some experience are more likely to be mental rather than physical, hence why they are not necessarily classified as safe

THE PROPERTIES OF PSILOCYBIN

In 2016 a group at Imperial College London conducted a study showcasing that psychotherapy, alongside one or two doses of the psychedelic compound psilocybin, could help treat patients suffering with severe depression Numerous clinical trials have since confirmed this finding, and the US Food and Drug Administration (FDA) even granted psilocybin the title of "innovative treatment" at the end of 2019

Scientists from Western Connecticut State University have previously attempted to recreate the genes responsible for psilocybin, however due to certain regulations, the experiment was stopped prior to the creation of a ‘class 1 drug’ Nevertheless, another team independently found the psilocybin genes in a different genome of mushrooms, and were able to use these enzymes to produce the desired chemical compound

The team performed gene sequencing on three psilocybinproducing mushrooms and discovered a small cluster of genes responsible for creating the ‘magic mushroom’ composite They then proceeded to take the gene sequence of this cluster, extracting the protein and giving it tryptophan (the amino acid compound that makes you sleepy) and eventually created psilocybin.

This cluster of genes is thought to be associated with horizontal gene transfer, and is thought to have originated in dung and decaying wood environments Researcher Hannah Reynolds believes that these mushrooms benefit from producing psilocybin so they can grow and mature into adult fungi without being eaten by insect larvae The hallucinogenic effect of psilocybin can slow down the insects eating the decaying wood or dung (perhaps by creating olfactory confusion) allowing the mushroom to thrive and not get eaten

A MEDICAL RENAISSANCE

Around the world dozens of clinical trials are underway to assess the effectiveness of these psychoactive substances, both as tools for researching the inner workings of the brain and as ways to treat some of the most serious mental illnesses (e g depression anxiety, alcohol/tobacco addiction, obsessive-compulsive disorders, and eating disorders) In these illnesses, the brain is stuck in permanent rumination, where depressive or addictive thoughts are on a constant loop With newfound and regulated psychoactive treatments and therapies well underway, scientists are hopeful that psychedelics can break these patterns

Brain imaging techniques show that psychedelics have a profound disruptive effect on brain activity; this effect is mediated by targeting serotonin receptor 5HT2A. This receptor is peculiarly dense and is supposedly responsible for the rapid growth of the brain during recent human evolution

Furthermore, 5HT2A is located in the upper level parts of the brain cortexan area deemed responsible for our high level thinking, dreams, imaginations and new ideas. This area also integrates one ’ s hearing, thinking, smelling, vision, which are all disrupted when under the influence of psychedelics

David Nutt, head of Imperial College London’s Centre for Psychedelic Research, recently stated: “In simple terms, psychedelics put you back to what your brain was like as a child when all kinds of thinking was possible ”

Surprisingly, our neurodevelopment as human beings actually involves the shrinking rather than the growing of the brain. This is because as we age, our brains get rid of connections that are no longer needed, becoming more rigid and constrictive Nutt even described how “psychedelics disrupt this process and put you back into a state or child-like wonder. ”

Recent electrical physiological experiments have recently shown that these substances can also heavily affect your visual perception by inhibiting your visual cortex’s ability to reconstruct the signals transmitted by your eyes into images In other words, psychedelics induce the primary workings of your visual cortex, and so the images you see under these substances are similar to those you see at the very early stages of your life

If psychedelic research continues at its current rate, MDMA might be used to treat trauma-related disorders by 2023, and psilocybin a couple years after that. A radical ketamine-like drug, esketamine, has already been licensed for use in the UK to treat severe depression and has showcased prosperous results. Hopefully with more thorough research and detailed education on these topics, these drugs will be available through public health access, benefiting those especially in need

References

Lowe, H , Toyang, N , Steele, B , Valentine, H , Grant, J , Ali, A , Ngwa, W and Gordon, L (2021) The Therapeutic Potential of Psilocybin Molecules [online] 26(10) p 2948 Available at: https://www mdpi com/14203049/26/10/2948/htm#B13-molecules-2602948

Patra, S (2016) Return of the psychedelics: Psilocybin for treatment resistant depression Asian Journal of Psychiatry, 24 pp 51–52

Raemakers, J (2021) Interview with David Nutt and Jan Ramaekers - Drug Science [online] drugscience org uk Available at: https://www drugscience org uk/interviewwith-david-nutt-and-jan-ramaekers/

Reiff, C M , Richman, E E , Nemeroff, C B , Carpenter L L Widge A S Rodriguez C I Kalin, N H and McDonald, W M (2020) Psychedelics and Psychedelic-Assisted Psychotherapy American Journal of Psychiatry, 177(5), p appi ajp 2019 1

S M M A and C J (2019) Classical Psychedelics for the Treatment of Depression and Anxiety: A Systematic Review [online] Journal of affective disorders Available at: https://pubmed ncbi nlm nih gov/31382100/

Tupper, K W , Wood, E , Yensen, R and Johnson, M W (2015) Psychedelic medicine: a re-emerging therapeutic paradigm Canadian Medical Association Journal [online] 187(14) pp 1054–1059 Available at: https://www ncbi nlm nih gov/pmc/articles/ PMC4592297/

HEPARIN: A WONDERFUL BIOLOGIC

WRITTEN BY DRSHIKA MEHTANI

EDITED BY ANDREA MAZGALEVA

DESIGNED BY DORIS YU

Heparin has been widely known as a valuable anticoagulant and

antithrombotic agent for about a century now and is used in clinical practice for a variety of conditions. It is on the World Health Organisation’s List of Essential Medicines (Mulloy et al , 2015) Heparin belongs to a class of carbohydrates called glycosaminoglycans and has many closely related members heparan sulphate being the most significant of these (Atallah et al., 2020). Glycosaminoglycans are negativelycharged polysaccharides that are a part of every mammalian tissue They serve several purposes within the body depending on their molecular structure (Casale and Crane, 2021)

Heparin is most widely used for its actions as an anticoagulant but in recent times it has been realised that it has other beneficial pharmacological properties

These include but are not limited to its antiviral, anti-inflammatory, and antimetastatic actions (Mulloy et al , 2015). Heparin’s properties also led to it being a potential therapy for COVID-19 in early-stage research

(van Haren et al , 2020) This article aims to discuss the history of heparin, its beneficial pharmacological properties, its implications and the future prospects for its use in a variety of patients

Heparin is a biologic molecule and can be extracted from a myriad of animal sources The first preparations for use in the clinic were made from bovine lungs in the 1930s and then moved towards porcine mucosa due to the scarcity of bovine lungs in World War II It is known that heparin preparations differ based on species and the tissues they have been isolated from These can be structural or activity disparities Bovine heparin may not be as effective as porcine heparin in a clinical setting but reports have been inconclusive (Mulloy et al., 2015).

The history of heparin dates back to 1916 when it was first discovered by Jay McLean, a second-year medical student The name “heparin” was coined by Willian H Howell and L Emmett Holt, Jr at the Johns Hopkins Hospital in 1918 (Ong et al., 2019). The name “heparin” was given to reflect that the compound was found in a high abundance in the liver (Tyrell et al , 1999) Holt and Howell played a role in developing an isolation protocol for heparin because the methods used by McLean, Holt and Howell led to contaminated samples with other phosphatides.

Figure 1. (A) William Henry Howell (1860-1945). (B) Luther Emmett Holt, Jr (1895-1974) (C) Jay McLean (1890-1957) (Ong et al , 2019)

Holt also helped prove that heparin is not a phosphatide (after which it was named) but rather a carbohydrate as he tried to refine isolation protocols The history of the discovery of heparin is a complicated one and is conflicted on some accounts but is

nev credit Holt

Heparin tissues is (UH) and choice for use in several conditions such as surgical interventions

However, once the structure of heparin was devised, it led to the development of low molecular weight heparin (LMWH) and other heparin-based mimetics (Mulloy et al., 2015). There are efforts currently underway to develop recombinant heparin If successful, recombinant heparin would help eliminate the inconsistent heparin products and would help with the standardisation of the drug based on its activity profile (Thacker et al , 2022) The structure of heparin is described as a polysulphuric ester of a polysaccharide composed of glucosamine and ironic acid (Mulloy et al., 2015). Heparan sulphate on the other hand has the same polysaccharide backbone as heparin but the only difference between the two is the degree of sulphation, heparin being more highly sulphated and therefore the more potent of the two Heparin is mainly isolated from mast cell granules where it has effects on inflammatory mediators and in contrast heparan sulphate is virtually produced by all cells of the body

Highlighting the sources of both of these compounds is important when looking at ways to isolate them for clinical purposes (Hofmann et al , 2012) Figure 2 shows the structure of heparin compared to that of heparan sulphate, its closely related counterpart

Heparin is a drug that has a complex pharmacological profile and works through a range of different mechanisms Its most well-known action is that of an anticoagulant The mechanisms of its other actions as an anti-inflammatory agent or an antimetastatic drug are less clear (Mulloy et al , 2015) To produce its anticoagulatory effects, heparin binds to antithrombin, a glycoprotein that inhibits several components of the coagulation cascade of blood clotting Antithrombin inhibits the actions of another protein thrombin which is involved in the propagation of the coagulation cascade

Figure 2. Structure of heparin and heparan sulphate (Olczyk et al , 2015)

Heparin binding to thrombin causes a conformational change in its structure that increases the rate of inhibition of thrombin by antithrombin by up to 1,000 times, making heparin a potent antithrombotic agent Antithrombin is also involved in the inhibition of other proteases such as factor Xa, IXa and XIa which are also vital components of the coagulation cascade Inhibition of these proteases is also enhanced by the actions of heparin through antithrombin (Hofmann et al , 2012) To produce its effects as an anticancer drug, heparin works through several potential mechanisms involved in preventing the angiogenesis of cancer cells and their proliferation Heparin prevents the binding of fibroblast growth factor to fibroblast growth factor receptors on cancer cells. This downregulates the signalling of the receptor and therefore impacts cell growth and proliferation Heparan sulphate can have both upregulatory and downregulatory effects on cell proliferation (Atallah et al , 2020) Moreover heparin has antiinflammatory properties that can be exploited clinically to accelerate wound healing (Olczyk et al , 2015) and may help prevent and treat COVID-19 (van Haren et al., 2020)

while also acting as an antiinflammatory agent to help treat other inflammatory conditions (Mulloy et al , 2015) Heparin inhibits several enzymes and cytotoxic mediators that are released by proinflammatory cells to augment inflammation. Some include elastase, interleukin-8, cathepsin G and eosinophil peroxidase

Heparan sulphate also reduces neutrophil rolling at inflammatory sides by reducing cell to cell adhesion and therefore the propagation of the inflammatory response (Olczyk et al., 2015). To treat COVID-19, in particular, nebulised unfractionated heparin would be the treatment of choice as it would be able to mitigate local effects Unfractionated heparin would help in the prevention of Sars-CoV-2 binding to angiotensin-converting enzyme-2 (ACE-2) in lung tissue and therefore help stop the entry of the virus into cells and the progression of the disease

In addition, patients would greatly benefit from the use of unfractionated heparin to reduce the immune risks associated with COVID19 due to its anti-inflammatory and anti-mucolytic effects (van Haren et al , 2020)

While there are several benefits associated with the use of heparin and heparin-related products, there are also adverse effects that cannot be neglected Long-term heparin use is associated with the risk of heparin-induced thrombocytopenia, albeit this risk is reduced with the use of low molecular weight heparin products In addition to the risk of heparin-induced thrombocytopenia, there is a risk of excessive bleeding with overuse, osteoporosis, alopecia and an elevation of liver enzymes It is important to note that these risks are associated with long-term use and the incidence of these risks is based on several other factors as well

References

Atallah, J et al (2020) The use of heparin and heparin-like molecules in cancer treatment: a review Cancer Treatment and Research Communications 24

Casale J & Crane J S (2021) Biochemistry, Glycosaminoglycans StatPearls [online] Available from:

An antidote, protamine sulphate can be used to deal with heparin overdose Louis Barker Jaques declared in 1979 that heparin and related products should be called “linear anionic polyelectrolytes” rather than “anticoagulants” as they are involved in several other biochemical pathways that are unrelated to anticoagulation His concept has been supported by several studies and clinical trials that are currently underway to uncover its other effects (Mulloy et al , 2015) Over a century later, heparin has not failed to entice both pharmacologists and clinicians through its effects and only further research will be able to put a light on its full potential.

https://www ncbi nlm nih gov/ books/NBK544295/ (Accessed 22 February 2022)

van Haren, F M P et al (2020) Nebulised heparin as a treatment for COVID-19: Scientific rationale and a call for randomised evidence Critical Care 24 (1)

Hofmann, F B et al (2012) Handbook of Experimental Pharmacology Volume 207 Editorin-Chief [online] Available from: http://www springer com/series/164

Mulloy B et al (2015) Pharmacology of Heparin and Related Drugs Pharmacological Reviews [Online] 68 (1), 76–141

Olczyk, P et al (2015) Diverse Roles of Heparan Sulfate and Heparin in Wound Repair BioMed Research International 2015

Ong C S et al (2019) A Century of Heparin [Online] [online] Available from: https://doi org/10 1016/10 1016/j athoracsur

Thacker B E et al (2022) Multiplex genome editing of mammalian cells for producing recombinant heparin Metabolic Engineering [Online] 70155–165

Tyrrell D J et al (1999) Heparin in inflammation: potential therapeutic applications beyond anticoagulation Advances in pharmacology (San Diego Calif ) [Online] 46 (C) 151–208 [online] Available from: https://pubmed ncbi nlm nih gov/10332503/ (Accessed 22 February 2022)

STOP THE MUSIC! THE SCIENCE OF DRINK SPIKING

Drink spiking is at an epidemic level According to the home affairs select comittee,

up to 15% of women and 7% of men have been spiked before and this figure is set to increase Although drinking spiking carries a maximum sentence of up to 10 years if found guilty, it is difficult to catch offenders due to the fact that it can take up to 30-45 minutes for these drugs to have their desired effect, especially if they have already left the premises. The very nature of club nights exacerbates this as bar staff on club nights are exceptionally busy as there can be masses of people waiting for drinks and when you ’ re told to get your head down and get on with serving customers, it becomes more likely that they won’t be able to spot someone who’s just spiked someone At established bars and clubs, there are more staff around, but this isn’t always the case for student bars or clubs, which further adds to the problem This begs the question, is enough being done to prevent this on campus?

PREVENTION

A drink stopper is a device that is put over a glass to prevent someone from dropping stimulants into the drink. Several bars offer this with testing kits which can detect certain but not all types of drugs

WRITTEN BY THE FOUNDER EDITED BY WINNIE (WING YIN) LIU

DESIGNED BY TAMARA YAP

Speak to venue staff and the police as soon as you are suspicious of whether someone has been spiked A major red flag is strangers offering you drinks that you have not seen the bar staff make in front of you.

TOP 5 DRINK SPIKING AGENTS

Once mixed with alcohol, drugs' intended effects may be altered and since each drug has its own effect profile, this can be unpredictable and deadly This doesn't even begin to factor in how other drugs such as fentanyl can be laced with it This can cause nausea, vomiting, headaches, drowsiness, fainting, changes in blood pressure and loss of coordination, just to name a few.

ROHYPNOL (ROOFIE)

Drug type: Benzodiazepine

Use: Short-term insomnia, pre-medication in surgical procedures like anaesthesia

Effects: Sedation, muscle relaxation, anxiety reduction, prevention of convulsions, partial amnesia

Taken: Oral (whole tablet), crushed and snorted, dissolved in liquid

Onset: 10-15 minutes

Duration: 4-6 hours; residual effects found after 12 hours

GAMMA

HYDROXYBUTYRATE

(GHB)

Drug type: Depressant (Class C)

Use: Narcolepsy treatment

Effects: Euphoria, drowsiness, relaxation, ↑ libido

Taken: Oral (mixed into soft drink via pipette), colourless/odourless oily liquid - salty/soapy taste

Onset: 10 minutes – 1 hour

Duration: several hours; detectable in urine after 24 hours

ECSTASY (MDMA)

Drug type: Psychoactive (Class A)

Use: Recreational

Effects: Altered sensations, ↑ energy, ↑ empathy, ↑ pleasure, dilated pupils, tingling, tightening/movement of jaw muscles, ↑ Body temp, ↑ heart rate, nausea

Taken: Oral (pill; white/coloured or round/square/other shapes), Dabbed onto gums or snorted (powder; white/grey crystals) – bitter taste

Onset: 30-45 minutes (oral)

Duration: 3-6 hours; detectable in urine 1-4 days after

LYSERGIC ACID DIETHYLAMIDE (LSD)

Drug type: Psychedelic (Class A)

Use: Recreational Effects: Intensified thoughts, emotions, sensory perception (high doses: visual/auditory hallucinations)

Taken: Oral (odourless tab/pellet – micro dot), on tongue, onto food/into drinks

Onset: 30-40 minutes

Duration: 8-20 hours; detectable in urine 1-3 days after

KETAMINE

Drug type: Dissociative anaesthetic

Use: Induction/maintenance of anaesthesia

Effects: Anaesthesia, sedation, amnesia, trance-like state, pain relief

Taken: Oral (tablet), Nasal, intramuscular injection

Onset: 20 minutes – 1 hour (Oral), 15 minutes (Nasal)

Duration: Oral (30 minutes – 1 hour); detected in urine several days after

CALL TO ACTION

In October 2021, a campaign to raise awareness about drink-spiking was in full effect, however, what has happened since then? Dover Castle (DC) Bar was at the centre of attention, with multiple accounts of drinks being spiked On October 2nd, the Metropolitan Police said at the time that ‘ no arrests had been made’ and that ‘enquiries were ongoing’. In response to this, DC Bar claimed they have since started to provide drink stoppers upon request, put up posters about drink spiking and more cameras have been installed. Guy’s Bar and The Vault implemented similar measures, aiming to raise awareness after there were major boycotts of sports night which raises £18,000 per night. However, several reports of spiking are still being made at Guy's Bar

Thus, we've partnered with the 'Stop the Music!' campaign to tackle this

'STOP THE MUSIC' CAMPAIGN

The KCL Stop the Music Campaign aims to pressure university and student union decision makers into finally implementing a firm set of policies to combat spiking and sexual assault at KCLSU nightclubs

They include: Upon identifying a case of spiking, stop the music, turn on the lights, and announce to all attendees that there is someone in the event who is spiking drinks They also want to see the employment of medical professionals at KCLSU nightclub events and various other safeguarding measures to protect incapacitated attendees from sexual assault

References

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drinkaware (2021) Drinkaware Drinkaware co uk https://www drinkaware co uk/advice/stayingsafe-while-drinking/drink-spiking-and-date-rapedrugs#: :text=Recreational%20drugs%20like%20 Ecstasy%2C%20Lysergic

talktofrank (2022a) Ecstasy | FRANK Www talktofrank com

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talktofrank (2022b) GHB & GBL | FRANK Www talktofrank com

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Stop The Music Policies

1) Upon identification of a likely incident of spiking, stop the music, turn on the lights, and announce that someone has been spiked Advise attendees to check on friends and look out for suspicious behaviour before resuming the event

2) Employment of medical professionals at KCL nightclub events to administer to anyone targeted by spiking or dangerously over-intoxicated.

3) Establishment of safe zones at KCL nightclubs to offer a secure location to administer to victims of spiking and offer a broader welfare space for attendees

4) Any attendee carrying a clearly incapacitated person out of KCL nightclub venues must log in their name, phone number, and stand for a photograph for club administrators this will then be signed on by a medical professional and a security staff

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