A Perspective on Protein Structure Prediction Using Quantum Computers.

Despite the recent advancements by deep learning methods such as AlphaFold2, in silico protein structure prediction remains a challenging problem in biomedical research. With the rapid evolution of quantum computing, it is natural to ask whether quantum computers can offer some meaningful benefits for approaching this problem. Yet, identifying specific problem instances amenable to quantum advantage and estimating the quantum resources required are equally challenging tasks. Here, we share our perspective on how to create a framework for systematically selecting protein structure prediction problems that are amenable for quantum advantage, and estimate quantum resources for such problems on a utility-scale quantum computer. As a proof-of-concept, we validate our problem selection framework by accurately predicting the structure of a catalytic loop of the Zika Virus NS3 Helicase, on quantum hardware.

mosGILT controls innate immunity and germ cell development in Anopheles gambiae.

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide a crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Anopheles gambiae mosGILT regulates innate immune genes and zpg expression.

Gene-edited mosquitoes lacking a g amma-interferon-inducible lysosomal thiol reductase-like protein, namely ( mosGILT null ) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILT null A. gambiae was therefore compared to wild type (WT) by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILT null A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg , an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILT null mosquitoes. These results provide the crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC.

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.

Design and Synthesis of Thiazole Scaffold-Based Small Molecules as Anticancer Agents Targeting the Human Lactate Dehydrogenase A Enzyme.

A new series of thiazole central scaffold-based small molecules of hLDHA inhibitors were designed using an in silico approach. Molecular docking analysis of designed molecules with hLDHA (PDB ID: 1I10) demonstrates that Ala 29, Val 30, Arg 98, Gln 99, Gly 96, and Thr 94 possessed strong interaction with the compounds. Compounds 8a, 8b, and 8d showed good binding affinity (-8.1 to -8.8 kcal/mol), whereas an additional interaction of NO2 at the ortho position in compounds 8c with Gln 99 through hydrogen bonding enhanced the affinity to -9.8 kcal/mol. Selected high-scored compounds were synthesized and screened for hLDHA inhibitory activities and in vitro anticancer activity in six cancer cell lines. Biochemical enzyme inhibition assays showed the highest hLDHA inhibitory activity observed with compounds 8b, 8c, and 8l. Compounds 8b, 8c, 8j, 8l, and 8m depicted significant anticancer activities, exhibiting IC50 values in the range of 1.65-8.60 µM in HeLa and SiHa cervical cancer cell lines. Compounds 8j and 8m exhibited notable anticancer activity with IC50 values of 7.90 and 5.15 µM, respectively, in liver cancer cells (HepG2). Interestingly, compounds 8j and 8m did not induce noticeable toxicity in the human embryonic kidney cells (HEK293). Insilico absorption, distribution, metabolism, and excretion profiling demonstrates that the compounds possess drug-likeness, and results may pave the way for the development of novel thiazole-based biologically active small molecules for therapeutics.

Genomics to Notebook (g2nb): extending the electronic notebook to address the challenges of bioinformatics analysis.

We present Genomics to Notebook (g2nb), an environment that combines the JupyterLab notebook system with widely-used bioinformatics platforms. Galaxy, GenePattern, and the JavaScript versions of IGV and Cytoscape are currently available within g2nb. The analyses and visualizations within those platforms are presented as cells in a notebook, making thousands of genomics methods available within the notebook metaphor and allowing notebooks to contain workflows utilizing multiple software packages on remote servers, all without the need for programming. The g2nb environment is, to our knowledge, the only notebook-based system that incorporates multiple bioinformatics analysis platforms into a notebook interface.

PDAUG: a Galaxy based toolset for peptide library analysis, visualization, and machine learning modeling.

BACKGROUND: Computational methods based on initial screening and prediction of peptides for desired functions have proven to be effective alternatives to lengthy and expensive biochemical experimental methods traditionally utilized in peptide research, thus saving time and effort. However, for many researchers, the lack of expertise in utilizing programming libraries, access to computational resources, and flexible pipelines are big hurdles to adopting these advanced methods. RESULTS: To address the above mentioned barriers, we have implemented the peptide design and analysis under Galaxy (PDAUG) package, a Galaxy-based Python powered collection of tools, workflows, and datasets for rapid in-silico peptide library analysis. In contrast to existing methods like standard programming libraries or rigid single-function web-based tools, PDAUG offers an integrated GUI-based toolset, providing flexibility to build and distribute reproducible pipelines and workflows without programming expertise. Finally, we demonstrate the usability of PDAUG in predicting anticancer properties of peptides using four different feature sets and assess the suitability of various ML algorithms. CONCLUSION: PDAUG offers tools for peptide library generation, data visualization, built-in and public database peptide sequence retrieval, peptide feature calculation, and machine learning (ML) modeling. Additionally, this toolset facilitates researchers to combine PDAUG with hundreds of compatible existing Galaxy tools for limitless analytic strategies.

Choline chloride attenuates the allergic airway disease by inhibiting the lysophosphatidylcholine induced response in mouse model.

Aims: Allergic airway disease manifestation is induced by lysophosphatidylcholine (LPC) through CD1d-restricted Natural killer T (NKT) cells. Choline chloride (ChCl) and LPC both have the "choline" moiety in their structure and this may interplay the effect in allergic airway disease pathway. Main methods: To test the hypothesis, mice were sensitized with cockroach extract (CE); challenged with CE or exposed to LPC and were given ChCl 1hr later. Key findings: A significant increase in Airway hyperresponsiveness (AHR), total and differential cell count, Th2 cytokines, 8-isoprostanes level in bronchoalveolar lavage fluid (BALF) and inflammation score based on lung histology were observed on challenge with CE or exposure to LPC (p â€‹< â€‹0.05) indicating LPC induced airway disease manifestation in mice. These parameters were reduced significantly after administering mice with ChCl (p â€‹< â€‹0.05). The inflammatory parameters were significantly increased in LPC exposed mice, not sensitized with CE, which were significantly decreased when mice were administered with ChCl demonstrating its role in the inhibition of LPC induced allergic airway disease manifestation. Docking of CD1d with LPC and ChCl indicated the competitive inhibition of LPC induced effect by ChCl. This was validated in vivo in the form of decreased CD1d-restricted NKT cells in BALF and lung of the immunized mice on ChCl administration. There was no effect of ChCl administration on CD1d expression in BALF and lung cells. Significance: This study shows that ChCl attenuates the allergic response by inhibiting the LPC induced- NKT cell mediated AHR, inflammation and oxidative stress by competitive inhibition to LPC in binding to CD1d.

Expanding the Galaxy's reference data.

Summary: Properly and effectively managing reference datasets is an important task for many bioinformatics analyses. Refgenie is a reference asset management system that allows users to easily organize, retrieve and share such datasets. Here, we describe the integration of refgenie into the Galaxy platform. Server administrators are able to configure Galaxy to make use of reference datasets made available on a refgenie instance. In addition, a Galaxy Data Manager tool has been developed to provide a graphical interface to refgenie's remote reference retrieval functionality. A large collection of reference datasets has also been made available using the CVMFS (CernVM File System) repository from GalaxyProject.org, with mirrors across the USA, Canada, Europe and Australia, enabling easy use outside of Galaxy. Availability and implementation: The ability of Galaxy to use refgenie assets was added to the core Galaxy framework in version 22.01, which is available from https://github.com/galaxyproject/galaxy under the Academic Free License version 3.0. The refgenie Data Manager tool can be installed via the Galaxy ToolShed, with source code managed at https://github.com/BlankenbergLab/galaxy-tools-blankenberg/tree/main/data_managers/data_manager_refgenie_pull and released using an MIT license. Access to existing data is also available through CVMFS, with instructions at https://galaxyproject.org/admin/reference-data-repo/. No new data were generated or analyzed in support of this research.

Evaluation of vertical and horizontal distribution of particulate matter near an urban roadway using an unmanned aerial vehicle.

Measurement of traffic emissions has gained a lot of interest in recent times due to its contribution to urban pollution. This paper reports the outcome from an unmanned aerial vehicle (UAV) based measurement of PM concentration near an urban roadway at Kolkata, India. A total of 54 flights were carried out for simultaneous measurements of PM1, PM2.5 and PM10 mass concentration and meteorological parameters in vertical as well as in horizontal direction. Results for the vertical flight up to 100 m showed that the PM1, PM2.5 and PM10 concentrations at higher altitudes are less (mean; 24.6, 39.9 and 103.8 µg m-3) compared to the respective ground level concentrations (mean; 26.3, 50.4 and 201.9 µg m-3). For all the three particle sizes, the majority of the cases of higher PM concentration at higher altitudes happened during the evening flight. Low mixing height and low wind speed are suggested to be the reasons for the poor dispersion of pollutants in the evening. While there was a 7-10% fall of fine particles (PM1 and PM2.5) mass concentrations up to 90 m away from the road, no trend could be seen for PM10. The random forest model to predict the UAV/Ground concentration ratio showed high accuracy (R2 = 0.82-0.95) for all three particle sizes. This is an important finding from this study, which shows how UAV measurement data can be used to generate models that can predict the higher altitude concentrations from the ground based measurements.

Re-Expression of ERα and AR in Receptor Negative Endocrine Cancers via GSK3 Inhibition.

DNA methylation, catalyzed by DNA methyltransferase (DNMT), is a well-characterized epigenetic modification in cancer cells. In particular, promoter hypermethylation of AR and ESR1 results in loss of expression on Androgen Receptor (AR) and Estrogen Receptor (ER), respectively, and is associated with a hormone refractory state. We now report that Glycogen Synthase Kinase 3 (GSK3) phosphorylates DNMT1 at S714, which is localized to a 62 amino acid region referred to as auto-inhibitory linker, which functions to occlude the DNA from the active site of DNMT1 to prevent the methylation of unmethylated DNA. Molecular Dynamics simulation indicates that phosphorylation at S714 resulted in conformational rearrangement of the autoinhibitory domain that inactivated its ability to block the methylation of unmethylated DNA and resulted in enhanced DNA binding. Treatment with a novel and more selective inhibitor of GSK3 resulted in decreased methylation of the promoter region of genes encoding the Androgen Receptor (AR) and Estrogen Receptor alpha (ERa) and re-expression of the AR and ERa in AR negative prostate cancer and ER negative breast cancer cells, respectively. As a result, concurrent treatment with the GSK3 inhibitor resulted in responsiveness of AR negative prostate cancer and ER negative breast cancer cells to inhibitors of the AR or ER, respectively, in in vitro and in vivo experimental models.

Artificial Intelligence in Surveillance, Diagnosis, Drug Discovery and Vaccine Development against COVID-19.

As of August 6th, 2021, the World Health Organization has notified 200.8 million laboratory-confirmed infections and 4.26 million deaths from COVID-19, making it the worst pandemic since the 1918 flu. The main challenges in mitigating COVID-19 are effective vaccination, treatment, and agile containment strategies. In this review, we focus on the potential of Artificial Intelligence (AI) in COVID-19 surveillance, diagnosis, outcome prediction, drug discovery and vaccine development. With the help of big data, AI tries to mimic the cognitive capabilities of a human brain, such as problem-solving and learning abilities. Machine Learning (ML), a subset of AI, holds special promise for solving problems based on experiences gained from the curated data. Advances in AI methods have created an unprecedented opportunity for building agile surveillance systems using the deluge of real-time data generated within a short span of time. During the COVID-19 pandemic, many reports have discussed the utility of AI approaches in prioritization, delivery, surveillance, and supply chain of drugs, vaccines, and non-pharmaceutical interventions. This review will discuss the clinical utility of AI-based models and will also discuss limitations and challenges faced by AI systems, such as model generalizability, explainability, and trust as pillars for real-life deployment in healthcare.

The Ser/Thr protein kinase PrkC imprints phenotypic memory in Bacillus anthracis spores by phosphorylating the glycolytic enzyme enolase.

Bacillus anthracis is the causative agent of anthrax in humans, bovine, and other animals. B. anthracis pathogenesis requires differentiation of dormant spores into vegetative cells. The spores inherit cellular components as phenotypic memory from the parent cell, and this memory plays a critical role in facilitating the spores' revival. Because metabolism initiates at the beginning of spore germination, here we metabolically reprogrammed B. anthracis cells to understand the role of glycolytic enzymes in this process. We show that increased expression of enolase (Eno) in the sporulating mother cell decreases germination efficiency. Eno is phosphorylated by the conserved Ser/Thr protein kinase PrkC which decreases the catalytic activity of Eno. We found that phosphorylation also regulates Eno expression and localization, thereby controlling the overall spore germination process. Using MS analysis, we identified the sites of phosphorylation in Eno, and substitution(s) of selected phosphorylation sites helped establish the functional correlation between phosphorylation and Eno activity. We propose that PrkC-mediated regulation of Eno may help sporulating B. anthracis cells in adapting to nutrient deprivation. In summary, to the best of our knowledge, our study provides the first evidence that in sporulating B. anthracis, PrkC imprints phenotypic memory that facilitates the germination process.

Pharmacologically induced reversible hypometabolic state mitigates radiation induced lethality in mice.

Therapeutic hypothermia has proven benefits in critical care of a number of diseased states, where inflammation and oxidative stress are the key players. Here, we report that adenosine monophosphate (AMP) triggered hypometabolic state (HMS), 1-3 hours after lethal total body irradiation (TBI) for a duration of 6 hours, rescue mice from radiation-induced lethality and this effect is mediated by the persistent hypothermia. Studies with caffeine and 6N-cyclohexyladenosine, a non-selective antagonist and a selective agonist of adenosine A1 receptor (A1AR) respectively, indicated the involvement of adenosine receptor (AR) signaling. Intracerebroventricular injection of AMP also suggested possible involvement of central activation of AR signaling. AMP, induced HMS in a strain and age independent fashion and did not affect the behavioural and reproductive capacities. AMP induced HMS, mitigated radiation-induced oxidative DNA damage and loss of HSPCs. The increase in IL-6 and IL-10 levels and a shift towards anti-inflammatory milieu during the first 3-4 hours seems to be responsible for the augmented survival of HSPCs. The syngeneic bone marrow transplantation (BMT) studies further supported the role of radiation-induced inflammation in loss of bone marrow cellularity after TBI. We also showed that the clinically plausible mild hypothermia effectively mitigates TBI induced lethality in mice.

Targeting DNA Repair through Podophyllotoxin and Rutin Formulation in Hematopoietic Radioprotection: An in Silico, in Vitro, and in Vivo Study.

Drug discovery field has tremendously progressed during last few decades, however, an effective radiation countermeasure agent for the safe administration to the victims of radiation exposure is still unavailable. This multi-model study is aimed at elucidating the mechanistic aspects of a novel podophyllotoxin and rutin combination (henceforth referred as G-003M) in the hematopoietic radioprotection and its involvement in the DNA damage and repair signaling pathways. Using in silico study, we identified the binding sites and structural components of G-003M and validated in vitro. We further studied various in vivo endpoints related to the DNA repair and cell death pathways in mice pre-administered with G-003M, irradiated and subsequently euthanized to collect blood and bone marrow cells. In silico study showed the binding of podophyllotoxin to ß-tubulin and presence of a functional hydroxyl group in the rutin, suggested their involvement in G2/M arrest and the free radical scavenging respectively. This experimentation was further validated through in vitro studies. In vivo mice studies confirmed that G-003M pre-administration attenuated DNA damage and enhanced repair after whole body exposure. We further noticed a decrease in the levels of γH2AX, p53BP1, and ATM kinase and an increase in the levels of DNA pk, Ku 80, Ligase IV, Mre 11, Rad 50 and NBS 1 in the blood and bone marrow cells of the G-003M pre-administered and irradiated mice. We noticed an overall increase in the pro-survival factors in the G-003M pre-treated and irradiated groups establishing the radioprotective efficacy of this formulation. The lead obtained from this study will certainly help in developing this formulation as a safe and effective radioprotector which could be used for humans against any planned or emergency exposure of radiation.

Rescuing Self: Transient Isolation and Autologous Transplantation of Bone Marrow Mitigates Radiation-Induced Hematopoietic Syndrome and Mortality in Mice.

The inflamed bone marrow niche shortly after total body irradiation (TBI) is known to contribute to loss of hematopoietic stem cells in terms of their number and function. In this study, autologous bone marrow transfer (AL-BMT) was evaluated as a strategy for mitigating hematopoietic form of the acute radiation syndrome by timing the collection phase (2 h after irradiation) and reinfusion (24 h after irradiation) using mice as a model system. Collection of bone marrow (BM) cells (0.5 × 106 total marrow cells) 2 h after lethal TBI rescued different subclasses of hematopoietic stem and progenitor cells (HSPCs) from the detrimental inflammatory and damaging milieu in vivo. Cryopreservation of collected graft and its reinfusion 24 h after TBI significantly rescued mice from lethal effects of irradiation (65% survival against 0% in TBI group on day 30th) and hematopoietic depression. Transient hypometabolic state (HMS) induced 2 h after TBI effectively preserved the functional status of HSPCs and improved hematopoietic recovery even when BM was collected 8 h after TBI. Homing studies suggested that AL-BMT yielded similar percentages for different subsets of HSPCs when compared to syngeneic bone marrow transfer. The results suggest that the timing of collection, and reinfusion of graft is crucial for the success of AL-BMT.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase.

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

Ligand and Structure Based Models for the Identification of Beta 2 Adrenergic Receptor Antagonists.

Ligand bound beta 2 adrenergic receptor (ß2AR) crystal structures are in use for screening of compound libraries for identifying inducers and blockers. However, in case of G protein coupled receptors (GPCR), docking and binding energy (BE) calculations are not enough to discriminate agonist and antagonists. Absence of a reliable model for discriminating ß2AR antagonist is still a major hurdle. Docking of known antagonists and agonists into activated and ground state ß2AR revealed several key intermolecular interactions and H-bonding patterns, which in combination, emerged as a model for prediction of antagonists. Present study identifies an alternative binding orientation, within the binding pocket, for blockers and a minimum grid size to lessen the false positive predictions. Cluster analysis revealed structural variability among the antagonists and a conserved pattern in case of agonists. A combination of docking and structure activity relationship (SAR) model reliably discriminated antagonists. Based on key intermolecular interactions, a set of agonists and antagonists useful to SAR, quantitative structure activity relationship (QSAR) and pharmacophore modeling, has also been proposed for identifying antagonists.

Prilocaine hydrochloride protects zebrafish from lethal effects of ionizing radiation: role of hematopoietic cell expansion.

Drug repositioning is an approach of significant translatability, and the present study was undertaken to screen a collection of FDA approved small-molecule clinical compounds for identification of novel radioprotective agents. Screening of JHCCL (Johns Hopkins Clinical Compound Library), a collection of 1,400 FDA approved small molecules, lead to identification of prilocaine hydrochloride, a local anesthetic used widely during dental procedures, as a potential radioprotector. Prilocaine, at a concentration of 20 µM, protected zebrafish from radiation induced (20 Gy) pericardial edema (PE), microphthalmia and rendered 60 % survival advantage over radiation exposed controls. While 40 % survival advantage over radiation exposed controls was achieved with 10 µM prilocaine. Prilocaine, in a dose-dependent manner, scavenged, radiation-induced hydroxyl radicals and maximally (43 %) at the highest concentration (1 mM) tried in this study. However, prilocaine exerted a mild superoxide anion scavenging potential (around 5 %) at all the concentrations used within this study. Prilocaine, at 20 µM concentration, significantly increased erythropoiesis, a marker for HSC function, in caudal hematopoietic tissue (CHT) in wild type and anemic zebrafish embryos (1.48 and 0.85 folds respectively) when compared to untreated (1) and phenylhydrazine (PHZ) (0.41 fold) treated control groups respectively. These results suggest that prilocaine is a radioprotective agent and free radical scavenging and HSC expanding potential seems to be contributing towards its radioprotective action.

Insights into the Origin of Clostridium botulinum Strains: Evolution of Distinct Restriction Endonuclease Sites in rrs (16S rRNA gene).

Diversity analysis of Clostridium botulinum strains is complicated by high microheterogeneity caused by the presence of 9-22 copies of rrs (16S rRNA gene). The need is to mine genetic markers to identify very closely related strains. Multiple alignments of the nucleotide sequences of the 212 rrs of 13 C. botulinum strains revealed intra- and inter-genomic heterogeneity. Low intragenomic heterogeneity in rrs was evident in strains 230613, Alaska E43, Okra, Eklund 17B, Langeland, 657, Kyoto, BKT015925, and Loch Maree. The most heterogenous rrs sequences were those of C. botulinum strains ATCC 19397, Hall, H04402065, and ATCC 3502. In silico restriction mapping of these rrs sequences was observable with 137 type II Restriction endonucleases (REs). Nucleotide changes (NC) at these RE sites resulted in appearance of distinct and additional sites, and loss in certain others. De novo appearances of RE sites due to NC were recorded at different positions in rrs gene. A nucleotide transition A>G in rrs of C. botulinum Loch Maree and 657 resulted in the generation of 4 and 10 distinct RE sites, respectively. Transitions A>G, G>A, and T>C led to the loss of RE sites. A perusal of the entire NC and in silico RE mapping of rrs of C. botulinum strains provided insights into their evolution. Segregation of strains on the basis of RE digestion patterns of rrs was validated by the cladistic analysis involving six house keeping genes: dnaN, gyrB, metG, prfA, pyrG, and Rho.