Highlights Thumyat Noe `23 Joyce Chen `23 Vignesh Subramanian `24 Peter Gillespie `25

Figure 1 Beyond upscale production, the barriers for microbial pigment implementation are low, as many experiments have demonstrated microbial pigments' capability to hold viability as substitute colorants for synthetics currently in use. Two values were determined based off of the experiment: minimum inhibitory concentration (MIC) of violacein (minimum concentration at which a degree of microbial growth inhibition occurs compared to negative controls), and minimum bactericidal concentration (MBC) of violacein (minimum concentration at which complete inhibition or killing of microbes is achieved). MIC for violacein and vancomycin were 3.9 μg/mL and 1.56 μg/mL, respectively. Similarly, MBC values for violacein and vancomycin were 15.6 μg/mL and 6.25 μg/mL, respectively. However, it was determined no killing activity was present for either, but rather, only complete growth inhibition. Although values for MIC and MBC were approximately 2.5 times higher in violacein than vancomycin, the results still show significant antibacterial activity of violacein. Currently, there is a desperate need for the discovery of safe and effective treatments to methicillin- and vancomycin-resistant strains of SA; as such, violacein may provide a solution to this problem (8). Microbial pigments have also been identified as possessing anticancer properties. Since the 1990s, prodigiosin, a red pigment derived from marine Vibrio sp, has been shown to possess antiproliferative properties, inhibiting the growth of cancer cell lines in vitro. Recent in vivo studies have also demonstrated its efficacy against a variety of cancers; however, the mechanisms behind its anticancer activity are unclear (9). In a recent study, researchers tested the efficacy of prodigiosin against glioblastoma, the most common and deadliest brain cancer, and sought to determine its mechanism of action. Glioblastoma cell lines were cultivated and then treated with prodigiosin at varying concentrations. Caspase 3 levels were then measured as a marker for cell death. To detect target proteins and measure the amount of target RNAs present in the sample, researchers conducted a western blot and polymerase chain reaction, respectively (9). Analysis found that caspase 3 levels were high in treated cultures, indicating activation of cell death pathways. Results from the western blot suggest that multiple proteins related to endoplasmic reticulum stress were also present. The endoplasmic reticulum is responsible for protein production and transport, and when it becomes overloaded, stress pathways are activated, which can lead to cell death. The PCR conducted on the prodigiosin-treated samples found the expression of RNA that encodes for these ER stress proteins was significantly higher than in untreated control samples. These results suggest prodigiosin kills glioblastoma cancer cells by increasing ER stress, sensitizing the cancer cells to pathways associated with protein overproduction induced death, as cancer cells already produce excessive amounts of protein (9).

Conclusion

Currently, microbial pigments are still in their infancy. Upscale production of microbial pigments for industry application relies heavily on the optimization of culturing methods and the maturation of bioreactor technology. However, progress continues in these areas, as seen by the advent of AI-based technologies that help determine optimal culture conditions and manage the precarious balancing act that giant bioreactors present. Beyond upscale production, the barriers for microbial pigment implementation are low, as many experiments have demonstrated microbial pigments' capability to be viable as substitute colorants for synthetics currently in use. Many parts of the world have also started shifting towards safer and more eco-friendly colorants, with Europe leading at 85% use of natural pigments (6). As it stands, future research into microbial pigments should aim to explore how production of pigments can be increased through the use of microbiological and genetic engineering techniques, as well as to elucidate the specific mechanisms that give microbial pigments their biotherapeutic properties. References

1. M. Narsing Rao, M. Xiao, and W. Li, Fungal and bacterial pigments: secondary metabolites with wide applications. Frontiers in Microbiology 8, 1-8 (2017). doi: 10.3389/fmicb.2017.01113. 2. L. Ardila-Leal, et al., Brief history of colour, the environmental impact of synthetic dyes and removal by using laccases. Molecules 26, 1-40 (2021). doi: 10.3390/molecules26133813. 3. Aman Mohammadi, et al., Microbial pigments as an alternative to synthetic dyes and food additives: a brief review of recent studies. Bioprocess and Biosystems Engineering 45, 1-12 (2021). doi: 10.1007/s00449-021-02621-8. 4. S. Mani and R. Bharagava, Exposure to crystal violet, its toxic, genotoxic and carcinogenic effects on environment and its degradation and detoxification for environmental safety. Reviews of Environmental Contamination and Toxicology 237, 71-104 (2016). doi: 10.1007/978-3-31923573-8_4. 5. M. Kanelli, et al., Microbial production of violacein and process optimization for dyeing polyamide fabrics with acquired antimicrobial properties. Frontiers in Microbiology 9, 1-53 (2018). doi: 10.3389/fmicb.2018.01495. 6. C. Venil, L. Dufossé, and P. Devi, Bacterial pigments: sustainable compounds with market potential for pharma and food industry. Frontiers in Sustainable Food Systems 4, 1-17 (2020). doi: 10.3389/fsufs.2020.00100. 7. O. Darwesh, et al., Isolation and optimization of Monascus ruber OMNRC45 for red pigment production and evaluation of the pigment as a food colorant. Applied Sciences 10, 1-15 (2020). doi:10.3390/app10248867. 8. C. Aruldass, et al., Antibacterial mode of action of violacein from Chromobacterium violaceum UTM5 against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). Environmental Science and Pollution Research 25, 5164-5180 (2018). doi: 10.1007/s11356-0178855-2. 9. S. Cheng, et al., Prodigiosin stimulates endoplasmic reticulum stress and induces autophagic cell death in glioblastoma cells. Apoptosis 23, 314-328 (2018). doi: 10.1007/s10495-018-1456-9.

Introduction

Writing is a practice so ingrained in modern human life that it can easily be taken for granted. Yet, there was a time when writing was a novel creation, and the idea of creating physical messages, records, or important cultural narratives was unknown. Early writing systems were largely lost to time, and only recently has it become possible to understand the role that writing played in ancient societies. While a human analysis of ancient archeological finds suffices for a discernment of the identity and significance of ancient scripts, anything more technical, such as an identification of authorship, requires more sophisticated techniques. A variety of algorithmic methods, including the creation of heatmaps and pixel patches, can allow for an analysis of ancient text on the level of individual letters or individual parts of letters. By utilizing a variety of algorithmic approaches that examine text with varying levels of detail, from the overall shape of the letters down to handfuls of pixels, it is possible to identify authorship with a degree of accuracy that human analysis alone could never achieve.

Human Analysis

When faced with a newly discovered ancient text, meaning is the most logical place to begin an analysis, and an understanding of meaning can be achieved with standard human knowledge of language. One newly discovered text of great importance was found on a piece of ceramic smaller than a four centimeter by four centimeter square. In the Shephelah region of modern-day Israel lies the Bronze/Iron Age site of Tel Lachish, where archeologists have unearthed a variety of ancient artifacts, including this piece of ceramic. On the fragment, there are six letters in two lines, which were derived from Egyptian hieroglyphics and appear to be primitive iterations of letters used in the various Semitic languages, such as Hebrew.

While a human analysis of ancient archeological finds suffices for a discernment of the identity and significance of ancient scripts...an identification of authorship, requires more sophisticated techniques.

Figure 2 Fragment of ceramic discovered in the Shephelah region (other side to the left).

Figure 3 Fragments of pottery such as this are known as ostraca, and are what comprise the Arad Corpus. This finding marks the earliest example of alphabetic script found in the southern Levant, with all earlier fragments being located in Egypt. Thus, this artifact represents a bridge between the earliest known alphabets from Egypt, and the more well-known alphabets of the Levant of later centuries, filling in a gap in the historical record. A simple examination of the writing was all that was necessary to understand the nature of the text. By comparing the primitive letters to extant Hebrew, they could be identified, and with the known age of the site, the writing’s place in history was established (1). While this not insignificant examination was achieved without the use of computers, it constitutes a relatively simple, surface-level analysis. An exploration of meaning does not involve an exploration of the details of how any given letter was written, only an identification of each letter present in a given text. Thus, for any analysis more sophisticated than an analysis of meaning, computer assistance is needed for a truly accurate understanding of ancient texts.

Heatmaps

Outside of meaning, authorship is perhaps the most important aspect of any text, and yet for many of the most ancient, culturally significant texts, authorship remains a great mystery. The Dead Sea Scrolls are one of the oldest collections of biblical manuscripts, and a careful analysis of their authorship could provide insights into the scribal culture prior to the creation of the proper Hebrew Bible. Human analysis of the scrolls has long struggled to parse out how many scribes composed the scrolls and which scrolls they may have worked on. The obstacle for researchers lies in the difficulties in determining which variations in the script are due to different authorship. In addition, since any given letter can appear on a scroll thousands of times, the idea of a human identifying the authorship of any one document becomes a Sisyphean task. Researchers from the University of Groningen attempted to circumvent the limits of human analysis in a 2020 study, wherein they applied a novel algorithmic approach to one of the most famous Dead Sea Scrolls, the Great Isaiah Scroll, in order to determine its authorship. To do this, the researchers took digital images of the scroll, rendered the text on them as simple black and white images, and removed artifacts such as stray ink marks so that the text could be read more clearly. From these cleaned up images, particular features of the text, such as varying degrees of bending when writing curved letters, were analyzed with an algorithmic feature extraction method known as Hinge. Hinge features indicate whenever an author subconsciously slowed down or sped up while writing, such as a particular bend in a character. By inputting Hinge features, the researchers provided their algorithm with hundreds of variables to compare to one another. In addition to algorithmic feature analysis, the researchers sought to synthesize the algorithmic analysis with traditional analysis through the creation of heatmaps. No letter is written the same way twice, with variations in line thickness, angle of pen strokes and length of strokes differing between each instance of a letter. Heatmaps are aggregated visualizations of every instance of each letter, showing the frequency of different variations across the whole of a given text. Researchers posit that these heatmaps could supersede traditional charts which do not aggregate each instance of a letter, as the heatmaps can demonstrate that what might otherwise be viewed as an insignificant deviation in writing style is actually indicative of a specific hand. The Great Isaiah Scroll notably has two distinct halves which were sewn together, with the second half’s Hebrew appearing distinct from the first’s. This distinction led to some scholars suggesting throughout history that two different scribes with similar writing styles composed the manuscript, but the belief that the scroll was composed by a single scribe was still deeply held. Thus, heatmaps were created for the entirety of the scroll as well as for each half, so that the frequency and types of variation might be compared between the different sections of the manuscript. The