A high-throughput screening system for SARS-CoV-2 entry inhibition, syncytia formation and cell toxicity.

BACKGROUND: The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is mediated through the binding of the SARS-CoV-2 Spike protein via the receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). Identifying compounds that inhibit Spike-ACE2 binding would be a promising and safe antiviral approach against COVID-19. METHODS: In this study, we used a BSL-2 compatible replication-competent vesicular stomatitis virus (VSV) expressing Spike protein of SARS-CoV-2 with eGFP reporter system (VSV-eGFP-SARS-CoV-2) in a recombinant permissive cell system for high-throughput screening of viral entry blockers. The SARS-CoV-2 permissive reporter system encompasses cells that stably express hACE2-tagged cerulean and H2B tagged with mCherry, as a marker of nuclear condensation, which also enables imaging of fused cells among infected EGFP positive cells and could provide real-time information on syncytia formation. RESULTS: A limited high-throughput screening identified six natural products that markedly inhibited VSV-eGFP-SARS-CoV-2 with minimum toxicity. Further studies of Spike-S1 binding using the permissive cells showed Scillaren A and 17-Aminodemethoxygeldanamycin could inhibit S1 binding to ACE2 among the six leads. A real-time imaging revealed delayed inhibition of syncytia by Scillaren A, Proscillaridin, Acetoxycycloheximide and complete inhibition by Didemnin B indicating that the assay is a reliable platform for any image-based drug screening. CONCLUSION: A BSL-2 compatible assay system that is equivalent to the infectious SARS-CoV-2 is a promising tool for high-throughput screening of large compound libraries for viral entry inhibitors against SARS-CoV-2 along with toxicity and effects on syncytia. Studies using clinical isolates of SARS-CoV-2 are warranted to confirm the antiviral potency of the leads and the utility of the screening system.

Origins of major archaeal clades correspond to gene acquisitions from bacteria.

The mechanisms that underlie the origin of major prokaryotic groups are poorly understood. In principle, the origin of both species and higher taxa among prokaryotes should entail similar mechanisms--ecological interactions with the environment paired with natural genetic variation involving lineage-specific gene innovations and lineage-specific gene acquisitions. To investigate the origin of higher taxa in archaea, we have determined gene distributions and gene phylogenies for the 267,568 protein-coding genes of 134 sequenced archaeal genomes in the context of their homologues from 1,847 reference bacterial genomes. Archaeal-specific gene families define 13 traditionally recognized archaeal higher taxa in our sample. Here we report that the origins of these 13 groups unexpectedly correspond to 2,264 group-specific gene acquisitions from bacteria. Interdomain gene transfer is highly asymmetric, transfers from bacteria to archaea are more than fivefold more frequent than vice versa. Gene transfers identified at major evolutionary transitions among prokaryotes specifically implicate gene acquisitions for metabolic functions from bacteria as key innovations in the origin of higher archaeal taxa.

Endosymbiotic origin and differential loss of eukaryotic genes.

Chloroplasts arose from cyanobacteria, mitochondria arose from proteobacteria. Both organelles have conserved their prokaryotic biochemistry, but their genomes are reduced, and most organelle proteins are encoded in the nucleus. Endosymbiotic theory posits that bacterial genes in eukaryotic genomes entered the eukaryotic lineage via organelle ancestors. It predicts episodic influx of prokaryotic genes into the eukaryotic lineage, with acquisition corresponding to endosymbiotic events. Eukaryotic genome sequences, however, increasingly implicate lateral gene transfer, both from prokaryotes to eukaryotes and among eukaryotes, as a source of gene content variation in eukaryotic genomes, which predicts continuous, lineage-specific acquisition of prokaryotic genes in divergent eukaryotic groups. Here we discriminate between these two alternatives by clustering and phylogenetic analysis of eukaryotic gene families having prokaryotic homologues. Our results indicate (1) that gene transfer from bacteria to eukaryotes is episodic, as revealed by gene distributions, and coincides with major evolutionary transitions at the origin of chloroplasts and mitochondria; (2) that gene inheritance in eukaryotes is vertical, as revealed by extensive topological comparison, sparse gene distributions stemming from differential loss; and (3) that continuous, lineage-specific lateral gene transfer, although it sometimes occurs, does not contribute to long-term gene content evolution in eukaryotic genomes.

Genomic perspective of triazole resistance in clinical and environmental Aspergillus fumigatus isolates without cyp51A mutations.

Aspergillus fumigatus is the most common etiologic agent of primarily all clinical manifestations of aspergillosis. A steady increase in the number of azole resistant A. fumigatus (ARAF) isolates from environment and clinical samples leading to therapeutic failures in clinical settings have alarmed the mycologists and clinicians worldwide. Although mutations in azole target cyp51A gene have been implicated in conferring azole resistance in A. fumigatus, recent studies have demonstrated occurrence of azole resistant strains without cyp51A mutations. In this study, next generation sequencing techniques and the expression profiling of transporter genes with single nucleotide polymorphisms (SNPs) in clinical and environmental ARAF isolates with (G54E) and without known cyp51A mutations was undertaken to understand the genetic background and role of transporters in azole resistance. The raw reads of four ARAF strains when mapped to Af293 reference genome (>100X depth) covered at least 93.1% of the reference genome. Among all four strains, a total of 212,711 SNPs was identified with 37,829 were common in at least two isolates. The expression analysis suggested the overexpression of MFS transporter, namely, mfsC in all ARAF isolates. None of the resistant strain showed significant upregulation of cyp51A and cyp51B gene. On the other hand, abcD was upregulated (5-fold) in the isolates with cyp 51A mutation (G54E). The whole genome sequence analysis showed the presence of two previously described amino acid substitutions S269F and F390Y in HMG1 gene in a clinical panazole resistant strain without cyp51A mutations. These mutations have been previously associated with azole resistance in A. fumigatus strains without cyp51A mutations. Further, several punctual mutations and a large-segment deletion among different strains were observed suggesting the involvement of resistance mechanisms other than cyp51A.

Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

One step beyond a ribosome: The ancient anaerobic core.

Life arose in a world without oxygen and the first organisms were anaerobes. Here we investigate the gene repertoire of the prokaryote common ancestor, estimating which genes it contained and to which lineages of modern prokaryotes it was most similar in terms of gene content. Using a phylogenetic approach we found that among trees for all 8779 protein families shared between 134 archaea and 1847 bacterial genomes, only 1045 have sequences from at least two bacterial and two archaeal groups and retain the ancestral archaeal-bacterial split. Among those, the genes shared by anaerobes were identified as candidate genes for the prokaryote common ancestor, which lived in anaerobic environments. We find that these anaerobic prokaryote common ancestor genes are today most frequently distributed among methanogens and clostridia, strict anaerobes that live from low free energy changes near the thermodynamic limit of life. The anaerobic families encompass genes for bifunctional acetyl-CoA-synthase/CO-dehydrogenase, heterodisulfide reductase subunits C and A, ferredoxins, and several subunits of the Mrp-antiporter/hydrogenase family, in addition to numerous S-adenosyl methionine (SAM) dependent methyltransferases. The data indicate a major role for methyl groups in the metabolism of the prokaryote common ancestor. The data furthermore indicate that the prokaryote ancestor possessed a rotor stator ATP synthase, but lacked cytochromes and quinones as well as identifiable redox-dependent ion pumping complexes. The prokaryote ancestor did possess, however, an Mrp-type H(+)/Na(+) antiporter complex, capable of transducing geochemical pH gradients into biologically more stable Na(+)-gradients. The findings implicate a hydrothermal, autotrophic, and methyl-dependent origin of life. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Networks of lexical borrowing and lateral gene transfer in language and genome evolution.

Like biological species, languages change over time. As noted by Darwin, there are many parallels between language evolution and biological evolution. Insights into these parallels have also undergone change in the past 150 years. Just like genes, words change over time, and language evolution can be likened to genome evolution accordingly, but what kind of evolution? There are fundamental differences between eukaryotic and prokaryotic evolution. In the former, natural variation entails the gradual accumulation of minor mutations in alleles. In the latter, lateral gene transfer is an integral mechanism of natural variation. The study of language evolution using biological methods has attracted much interest of late, most approaches focusing on language tree construction. These approaches may underestimate the important role that borrowing plays in language evolution. Network approaches that were originally designed to study lateral gene transfer may provide more realistic insights into the complexities of language evolution.

Acquisition of 1,000 eubacterial genes physiologically transformed a methanogen at the origin of Haloarchaea.

Archaebacterial halophiles (Haloarchaea) are oxygen-respiring heterotrophs that derive from methanogens--strictly anaerobic, hydrogen-dependent autotrophs. Haloarchaeal genomes are known to have acquired, via lateral gene transfer (LGT), several genes from eubacteria, but it is yet unknown how many genes the Haloarchaea acquired in total and, more importantly, whether independent haloarchaeal lineages acquired their genes in parallel, or as a single acquisition at the origin of the group. Here we have studied 10 haloarchaeal and 1,143 reference genomes and have identified 1,089 haloarchaeal gene families that were acquired by a methanogenic recipient from eubacteria. The data suggest that these genes were acquired in the haloarchaeal common ancestor, not in parallel in independent haloarchaeal lineages, nor in the common ancestor of haloarchaeans and methanosarcinales. The 1,089 acquisitions include genes for catabolic carbon metabolism, membrane transporters, menaquinone biosynthesis, and complexes I-IV of the eubacterial respiratory chain that functions in the haloarchaeal membrane consisting of diphytanyl isoprene ether lipids. LGT on a massive scale transformed a strictly anaerobic, chemolithoautotrophic methanogen into the heterotrophic, oxygen-respiring, and bacteriorhodopsin-photosynthetic haloarchaeal common ancestor.

Discovery of small molecule entry inhibitors targeting the linoleic acid binding pocket of SARS-CoV-2 spike protein.

Spike glycoprotein has a significant role in the entry of SARS-CoV-2 to host cells, which makes it a potential drug target. Continued accumulation of non-synonymous mutations in the receptor binding domain of spike protein poses great challenges in identifying antiviral drugs targeting this protein. This study aims to identify potential entry inhibitors of SARS-CoV-2 using virtual screening and molecular dynamics (MD) simulations from three distinct chemical libraries including Pandemic Response Box, Drugbank and DrugCentral, comprising 6971 small molecules. The molecules were screened against a binding pocket identified in the receptor-binding domain (RBD) region of the spike protein which is known as the linoleic acid binding pocket, a highly conserved motif among several SARS-CoV-2 variants. Through virtual screening and binding free energy calculations, we identified four top-scoring compounds, MMV1579787 ([2-Oxo-2-[2-(3-phenoxyphenyl)ethylamino]ethyl]phosphonic acid), Tretinoin, MMV1633963 ((2E,4E)-5-[3-(3,5-dichlorophenoxy)phenyl]penta-2,4-dienoic acid) and Polydatin, which were previously reported to have antibacterial, antifungal or antiviral properties. These molecules showed stable binding on MD simulations over 100 ns and maintained stable interactions with TYR365, PHE338, PHE342, PHE377, TYR369, PHE374 and LEU368 of the spike protein RBD that are found to be conserved among SARS-CoV-2 variants. Our findings were further validated with free energy landscape, principal component analysis and dynamic cross-correlation analysis. Our in silico analysis of binding mode and MD simulation analyses suggest that the identified compounds may impede viral entrance by interacting with the linoleic acid binding site of the spike protein of SARS-CoV-2 regardless of its variants, and they thus demand for further in vitro and in vivo research.Communicated by Ramaswamy H. Sarma.

Hybrid genome assembly and annotation of multidrug-resistant Staphylococcus aureus genotype ST672-SCCmec type IVd (2B).

OBJECTIVES: The emergence of multidrug-resistant Staphylococcus aureus strains is mainly mediated by mobile genetic elements, such as Staphylococcal Cassette Chromosome mec (SCCmec). Currently, SCCmec elements in S. aureus are classified into 15 types, with type IV being the most common in hospital and community-associated methicillin-resistant S. aureus. Among different subtypes of SCCmec type IV strains (IVa-IVn), the complete genome sequence of the SCCmec IVd (2B) subtype is still lacking. Here, we report the complete genome sequence of multidrug-resistant S. aureus SCCmec typeIVd (2B) isolate, S. aureus S145. METHODS: Staphylococcus aureus S145 was subjected to phenotypic and genotypic characterization. The whole-genome sequencing of S145 was performed using a hybrid-genome approach. The antibiotic-resistance genes were detected and compared with 112 publicly available S. aureus genomes. RESULTS: We obtained a complete genome of S145 with 2.7 Mbp length, Guanine-Cytosine (GC) content of 32.8%, and 2,548 protein-coding regions with 79 virulence factors and 90 antibiotic resistance genes. The S145 has ∼17-kb SCCmec, which encodes genes such as mecA, mecR1, ccrA2B2, and SCCmec IVd (2B) subtype gene CG002. We detected a ∼30-kb multidrug-resistant plasmid with eight antibiotic-resistant genes forming three clusters. Cluster1 encoded for penicillin (blaI-blaZ-blaR1), Cluster2 for aminoglycoside-streptothricin (aph(3')-IIIa-sat4-ΔANT(6)-Ia), and Cluster3 for macrolides (msr(A)-mph(C)) resistance genes. Comparative analysis of Cluster1-Cluster3 revealed that the genetic organization of these clusters resembles resistance genes present in plasmids of USA300 S. aureus SCCmec type IVa strains. CONCLUSION: Here, we report the complete genome sequence of S. aureus SCCmec IVd (2B) that can be used as a reference genome for further comparative genomic analysis.

In silico study identifies peptide inhibitors that negate the effect of non-synonymous mutations in major drug targets of SARS-CoV-2 variants.

Since its advent in December 2019, SARS-CoV-2 has diverged into multiple variants with differing levels of virulence owing to the accumulation of mutations in its genome. The structural changes induced by non-synonymous mutations in major drug targets of the virus are known to alter the binding of potential antagonistic inhibitors. Here, we analyzed the effects of non-synonymous mutations in major targets of SARS-CoV-2 in response to potential peptide inhibitors. We screened 12 peptides reported to have anti-viral properties against RBD and 5 peptides against Mpro of SARS-CoV-2 variants using molecular docking and simulation approaches. The mutational landscape of RBD among SARS-CoV-2 variants had 21 non-synonymous mutations across 18 distinct sites. Among these, 14 mutations were present in the RBM region directly interacting with the hACE2 receptor. However, Only 3 non-synonymous mutations were observed in Mpro. We found that LCB1 - a de novo-synthesized peptide has the highest binding affinity to RBD despite non-synonymous mutations in variants and engages key residues of RBD-hACE2 interaction such as K417, E484, N487, and N501. Similarly, an antimicrobial peptide; 2JOS, was identified against Mpro with high binding affinity as it interacts with key residues in dimerization sites such as E166 and F140 crucial for viral replication. MD simulations affirm the stability of RBD-LCB1 and Mpro-2JOS complexes with an average RMSD of 1.902 and 2.476 respectively. We ascertain that LCB1 and 2JOS peptides are promising inhibitors to combat emerging variants of SARS-CoV-2 and thus warrant further investigations using in-vitro and in-vivo analysis.Communicated by Ramaswamy H. Sarma.

Search for novel Plasmodium falciparum PfATP4 inhibitors from the MMV Pandemic Response Box through a virtual screening approach.

Owing to its life cycle involving multiple hosts and species-specific biological complexities, a vaccine against Plasmodium, the causative agent of Malaria remains elusive. This makes chemotherapy the only viable means to address the clinical manifestations and spread of this deadly disease. However, rapid surge in antimalarial resistance poses significant challenges to our efforts to eliminate Malaria since the best drug available to-date; Artemisinin and its combinations are also rapidly losing efficacy. Sodium ATPase (PfATP4) of Plasmodium has been recently explored as a suitable target for new antimalarials such as Cipargamin. Prior studies showed that multiple compounds from the Medicines for Malaria Venture (MMV) chemical libraries were efficient PfATP4 inhibitors. In this context, we undertook a structure- based virtual screening approach combined to Molecular Dynamic (MD) simulations to evaluate whether new molecules with binding affinity towards PfATP4 could be identified from the Pandemic Response Box (PRB), a 400-compound library of small molecules launched in 2019 by MMV. Our analysis identified new molecules from the PRB library that showed affinity for distinct binding sites including the previously known G358 site, several of which are clinically used anti-bacterial (MMV1634383, MMV1634402), antiviral (MMV010036, MMV394033) or antifungal (MMV1634494) agents. Therefore, this study highlights the possibility of exploiting PRB molecules against Malaria through abrogation of PfATP4 activity.Communicated by Ramaswamy H. Sarma.

In vitro antibacterial effects of combination of ciprofloxacin with compounds isolated from Streptomyces luteireticuli NIIST-D75.

Three phenazines, 1-methoxyphenazine (1), methyl-6-methoxyphenazine-1-carboxylate (2), 1,6-dimethoxyphenazine (4), and a 2,3-dimethoxy benzamide (3) were isolated from the Streptomyces luteireticuli NIIST-D75, and the antibacterial effects of compounds 1-3, each in combination with ciprofloxacin, were investigated. The in vitro antibacterial activity was assessed by microdilution, checkerboard, and time-kill assay against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi. According to the checkerboard assay results, each combination of compounds 1, 2 and 3 with ciprofloxacin resulted in a significantly lower minimum inhibitory concentrations (MICs) of 0.02-1.37 µg ml-1, suggesting synergistic combinations by fractional inhibitory concentration index, and displayed bactericidal activity in time-kill kinetics within 48 h. SEM analysis was carried out to determine the changes in morphology in S. aureus and E. coli during treatment with individual combination of ciprofloxacin and compounds (1-3), which revealed drastic changes in the cells such as dent formation, biofilm disruption, cell bursting, and doughnut-like formation, change in surface morphology in S. aureus, and cell elongation, cell burst with ruptured cell, and change in surface morphology in E. coli. Hep G2 cell viability was not affected by the compounds (1-3) that were tested for cytotoxicity up to 250 µM.

A rapid bead-based assay for screening of SARS-CoV-2 neutralizing antibodies.

Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. We describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies/metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2-Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2-GFPSpark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening.

Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction.

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. RESULTS: Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. CONCLUSION: Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

Networks uncover hidden lexical borrowing in Indo-European language evolution.

Language evolution is traditionally described in terms of family trees with ancestral languages splitting into descendent languages. However, it has long been recognized that language evolution also entails horizontal components, most commonly through lexical borrowing. For example, the English language was heavily influenced by Old Norse and Old French; eight per cent of its basic vocabulary is borrowed. Borrowing is a distinctly non-tree-like process--akin to horizontal gene transfer in genome evolution--that cannot be recovered by phylogenetic trees. Here, we infer the frequency of hidden borrowing among 2346 cognates (etymologically related words) of basic vocabulary distributed across 84 Indo-European languages. The dataset includes 124 (5%) known borrowings. Applying the uniformitarian principle to inventory dynamics in past and present basic vocabularies, we find that 1373 (61%) of the cognates have been affected by borrowing during their history. Our approach correctly identified 117 (94%) known borrowings. Reconstructed phylogenetic networks that capture both vertical and horizontal components of evolutionary history reveal that, on average, eight per cent of the words of basic vocabulary in each Indo-European language were involved in borrowing during evolution. Basic vocabulary is often assumed to be relatively resistant to borrowing. Our results indicate that the impact of borrowing is far more widespread than previously thought.

Anomalous Phylogenetic Behavior of Ribosomal Proteins in Metagenome-Assembled Asgard Archaea.

Metagenomic studies permit the exploration of microbial diversity in a defined habitat, and binning procedures enable phylogenomic analyses, taxon description, and even phenotypic characterizations in the absence of morphological evidence. Such lineages include asgard archaea, which were initially reported to represent archaea with eukaryotic cell complexity, although the first images of such an archaeon show simple cells with prokaryotic characteristics. However, these metagenome-assembled genomes (MAGs) might suffer from data quality problems not encountered in sequences from cultured organisms due to two common analytical procedures of bioinformatics: assembly of metagenomic sequences and binning of assembled sequences on the basis of innate sequence properties and abundance across samples. Consequently, genomic sequences of distantly related taxa, or domains, can in principle be assigned to the same MAG and result in chimeric sequences. The impacts of low-quality or chimeric MAGs on phylogenomic and metabolic prediction remain unknown. Debates that asgard archaeal data are contaminated with eukaryotic sequences are overshadowed by the lack of evidence indicating that individual asgard MAGs stem from the same chromosome. Here, we show that universal proteins including ribosomal proteins of asgard archaeal MAGs fail to meet the basic phylogenetic criterion fulfilled by genome sequences of cultured archaea investigated to date: These proteins do not share common evolutionary histories to the same extent as pure culture genomes do, pointing to a chimeric nature of asgard archaeal MAGs. Our analysis suggests that some asgard archaeal MAGs represent unnatural constructs, genome-like patchworks of genes resulting from assembly and/or the binning process.

Pax6 modulates intra-retinal axon guidance and fasciculation of retinal ganglion cells during retinogenesis.

Intra-retinal axon guidance involves a coordinated expression of transcription factors, axon guidance genes, and secretory molecules within the retina. Pax6, the master regulator gene, has a spatio-temporal expression typically restricted till neurogenesis and fate-specification. However, our observation of persistent expression of Pax6 in mature RGCs led us to hypothesize that Pax6 could play a major role in axon guidance after fate specification. Here, we found significant alteration in intra-retinal axon guidance and fasciculation upon knocking out of Pax6 in E15.5 retina. Through unbiased transcriptome profiling between Pax6fl/fl and Pax6-/- retinas, we revealed the mechanistic insight of its role in axon guidance. Our results showed a significant increase in the expression of extracellular matrix molecules and decreased expression of retinal fate specification and neuron projection guidance molecules. Additionally, we found that EphB1 and Sema5B are directly regulated by Pax6 owing to the guidance defects and improper fasciculation of axons. We conclude that Pax6 expression post fate specification of RGCs is necessary for regulating the expression of axon guidance genes and most importantly for maintaining a conducive ECM through which the nascent axons get guided and fasciculate to reach the optic disc.

Phylogenomic Analysis Reveals the Evolutionary Route of Resistant Genes in Staphylococcus aureus.

Multidrug-resistant Staphylococcus aureus is a leading concern worldwide. Coagulase-Negative Staphylococci are claimed to be the reservoir and source of important resistant elements in S. aureus. However, the origin and evolutionary route of resistant genes in S. aureus are still remaining unknown. Here, we performed a detailed phylogenomic analysis of 152 completely sequenced S. aureus strains in comparison with 7,529 non-Staphylococcus aureus reference bacterial genomes. Our results reveal that S. aureus has a large open pan-genome where 97 (55%) of its known resistant-related genes belonging to its accessory genome. Among these genes, 47 (27%) were located within the Staphylococcal Cassette Chromosome mec (SCCmec), a transposable element responsible for resistance against major classes of antibiotics including beta-lactams, macrolides, and aminoglycosides. However, the physically linked mec-box genes (MecA-MecR-MecI) that are responsible for the maintenance of SCCmec elements is not unique to S. aureus, instead it is widely distributed within Staphylococcaceae family. The phyletic patterns of SCCmec-encoded resistant genes in Staphylococcus species are significantly different from that of its core genes indicating frequent exchange of these genes between Staphylococcus species. Our in-depth analysis of SCCmec-resistant gene phylogenies reveals that genes such as blaZ, ble, kmA, and tetK that are responsible for beta-lactam, bleomycin, kanamycin, and tetracycline resistance in S. aureus were laterally transferred from non-Staphylococcus sources. In addition, at least 11 non-SCCmec-encoded resistant genes in S. aureus, were laterally acquired from distantly related species. Our study evidently shows that gene transfers played a crucial role in shaping the evolution of antibiotic resistance in S. aureus.

Insights into the Bacterial Profiles and Resistome Structures Following the Severe 2018 Flood in Kerala, South India.

Extreme flooding is one of the major risk factors for human health, and it can significantly influence the microbial communities and enhance the mobility of infectious disease agents within the affected areas. The flood crisis in 2018 was one of the severe natural calamities recorded in the southern state of India (Kerala) that significantly affected its economy and ecological habitat. We utilized a combination of shotgun metagenomics and bioinformatics approaches to understand the bacterial profile and the abundance of pathogenic and antibiotic-resistant bacteria in extremely flooded areas of Kuttanad, Kerala (4-10 feet below sea level). Here we report the bacterial profiles of flooded sites that are abundant with virulent and resistant bacteria. The flooded sites were heavily contaminated with faecal contamination indicators such as Escherichia coli and Enterococcus faecalis and multidrug-resistant strains of Pseudomonas aeruginosa, Salmonella typhi/typhimurium, Klebsiella pneumoniae, Vibrio cholerae. The resistome of the flooded sites contains 103 known resistant genes, of which 38% are plasmid-encoded, where most of them are known to be associated with pathogenic bacteria. Our results reveal an overall picture of the bacterial profile and resistome of sites following a devastating flood event, which might increase the levels of pathogens and its associated risks.