Evolutionary Sequence Analysis Research Articles

Evolutionary Profile for (Host and Viral) MLKL Indicates Its Activities as a Battlefront for Extensive Counteradaptation.

Pathogen infection triggers host innate defenses which may result in the activation of regulated cell death (RCD) pathways such as apoptosis. Given a vital role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptosis. Yet, it is unclear whether components of this emerging RCD pathway display signatures associated with pathogen conflict that are rare in combination but common to key host defense factors, namely, rapid evolution, viral homolog (virolog), and cytokine induction. We leveraged evolutionary sequence analysis that examines rates of amino acid replacement, which revealed: 1) strong and recurrent signatures of positive selection for primate and bat RIPK3 and MLKL, and 2) elevated rates of amino acid substitution on multiple RIPK3/MLKL surfaces suggestive of past antagonism with multiple, distinct pathogen-encoded inhibitors. Furthermore, our phylogenomics analysis across poxvirus genomes illuminated volatile patterns of evolution for a recently described MLKL viral homolog. Specifically, poxviral MLKLs have undergone numerous gene replacements mediated by duplication and deletion events. In addition, MLKL protein expression is stimulated by interferons in human and mouse cells. Thus, MLKL displays all three hallmarks of pivotal immune factors of which only a handful of factors like OAS1 exhibit. These data support the hypothesis that over evolutionary time MLKL functions—which may include execution of necroptosis—have served as a major determinant of infection outcomes despite gene loss in some host genomes.

HIV-1 molecular transmission network among sexually transmitted populations in Liaoning Province, China.

Introduction:In recent years, with the development of molecular epidemiology, molecular transmission networks based on evolutionary theory and sequence analysis have been widely used in research on human immunodeficiency virus (HIV)-1 transmission dynamics and precise intervention for high-risk populations. The HIV-1 molecular transmission network is a new method to study the population's access to the network, the characteristics of clustering, and the characteristics of interconnection in the network. Here, we analyzed the characteristics of the HIV-1 molecular transmission network of sexually transmitted people in Liaoning Province.Methods:A study of HIV-infected persons who were sexually transmitted in Liaoning Province from 2003 to 2019. HIV-1 RNA was extracted, amplified and sequenced, and a phylogenetic tree was constructed to determine the subtype using the well matched pol gene region sequence. The gene distance between sequences was calculated, the threshold was determined, and the molecular transmission network was constructed.Results:109 samples of pol gene region were obtained. The main subtype of HIV-1 was CRF01_AE, followed by B, CRF07_BC, etc. 12.8% of them were resistant to HIV. At the threshold of 0.55 gene distance, 60.6% of them entered the HIV-1 molecular transmission network. Workers, sample source voluntary counseling and testing, other testing, subtype B and drug resistance are the factors influencing the access to HIV-1 molecular transmission network. The subtype of CRF01_AE formed 6 clusters in the molecular transmission network. In the network, the difference of connection degree between different subtypes was statistically significant.Discussion:The three subtypes CRF01_AE, CRF07_BC and B that enter the molecular transmission network do not have interconnections, and they form clusters with each other. It shows that the risk of transmission among the three subtypes is less than the risk of transmission within each subtype. The factors affecting HIV-1 entry into the molecular transmission network were occupation, sample source, genotype and drug resistance. The L33F mutation at the HIV-1 resistance mutation site constitutes the interconnection in the largest transmission cluster in the network. The epidemiological characteristics of HIV-infected persons in each molecular transmission cluster show that 97% of the study subjects come from the same area and have a certain spatial aggregation.Conclusion:Constructing a molecular transmission network and conducting long-term monitoring, while taking targeted measures to block the spread of HIV can achieve precise prevention and control.

Analysis of codon usage patterns and influencing factors in rice tungro bacilliform virus.

Rice tungro bacilliform virus (RTBV) belongs to genus Tungrovirus within the family Caulimoviridae harbors circular double-stranded DNA (dsDNA). Rice tungro disease (RTD) caused by RTBV, responsible for severe rice yield losses in South and Southeast Asia. Here, we performed a systematic evolutionary and codon usage bias (CUB) analysis of RTBV genome sequences. We analysed different bioinformatics techniques to calculate the nucleotide compositions, the relative synonymous codon usage (RSCU), and other indices. The results indicated slightly or low codon usage bias in RTBV isolates. Mutation and natural selection pressures have equally contributed to this low codon usage bias. Additionally, multiple factors such as host, geographical distribution also affect codon usage patterns in RTBV genomes. RSCU analysis revealed that RTBV shows mutation bias and prefers A and U ended codons to code amino acids. Codon usage patterns of RTBV were also found to be influenced by its host. This indicates that RTBV have evolved codon usage patterns that are specific to its host. The findings from this study are expected to increase our understanding of factors leading to viral evolution and fitness with respect to hosts and the environment.

Evolutionary Sequence Analysis and Visualization with Wasabi.

Wasabi is an open-source, web-based graphical environment for evolutionary sequence analysis and visualization, designed to work with multiple sequence alignments within their phylogenetic context. Its interactive user interface provides convenient access to external data sources and computational tools and is easily extendable with custom tools and pipelines using a plugin system. Wasabi stores intermediate editing and analysis steps as workflow histories and provides direct-access web links to datasets, allowing for reproducible, collaborative research, and easy dissemination of the results. In addition to shared analyses and installation-free usage, the web-based design allows Wasabi to be run as a cross-platform, stand-alone application and makes its integration to other web services straightforward.This chapter gives a detailed description and guidelines for the use of Wasabi's analysis environment. Example use cases will give step-by-step instructions for practical application of the public Wasabi, from quick data visualization to branched analysis pipelines and publishing of results. We end with a brief discussion of advanced usage of Wasabi, including command-line communication, interface extension, offline usage, and integration to local and public web services. The public Wasabi application, its source code, documentation, and other materials are available at http://wasabiapp.org.

Molecular evolutionary and structural analysis of human UCHL1 gene demonstrates the relevant role of intragenic epistasis in Parkinson\u2019s disease and other neurological disorders

BackgroundParkinson’s disease (PD) is the second most common neurodegenerative disorder. PD associated human UCHL1 (Ubiquitin C-terminal hydrolase L1) gene belongs to the family of deubiquitinases and is known to be highly expressed in neurons (1–2% in soluble form). Several functions of UCHL1 have been proposed including ubiquitin hydrolyze activity, ubiquitin ligase activity and stabilization of the mono-ubiquitin. Mutations in human UCHL1 gene have been associated with PD and other neurodegenerative disorders. The present study aims to decipher the sequence evolutionary pattern and structural dynamics of UCHL1. Furthermore, structural and interactional analysis of UCHL1 was performed to help elucidate the pathogenesis of PD.ResultsThe phylogenetic tree topology suggests that the UCHL1 gene had originated in early gnathostome evolutionary history. Evolutionary rate analysis of orthologous sequences reveals strong purifying selection on UCHL1. Comparative structural analysis of UCHL1 pinpoints an important protein segment spanning amino acid residues 32 to 39 within secretion site with crucial implications in evolution and PD pathogenesis through a well known phenomenon called intragenic epistasis. Identified critical protein segment appears to play an indispensable role in protein stability, proper protein conformation as well as harboring critical interaction sites.ConclusionsConclusively, the critical protein segment of UCHL1 identified in the present study not only demonstrates the relevant role of intraprotein conformational epistasis in the pathophysiology of PD but also offers a novel therapeutic target for the disease.

Self-compatibility in peach [Prunus persica (L.) Batsch]: patterns of diversity surrounding the S-locus and analysis of SFB alleles

Self-incompatibility (SI) to self-compatibility (SC) transition is one of the most frequent and prevalent evolutionary shifts in flowering plants. Prunus L. (Rosaceae) is a genus of over 200 species most of which exhibit a Gametophytic SI system. Peach [Prunus persica (L.) Batsch; 2n = 16] is one of the few exceptions in the genus known to be a fully self-compatible species. However, the evolutionary process of the complete and irreversible loss of SI in peach is not well understood and, in order to fill that gap, in this study 24 peach accessions were analyzed. Pollen tube growth was controlled in self-pollinated flowers to verify their self-compatible phenotypes. The linkage disequilibrium association between alleles at the S-locus and linked markers at the end of the sixth linkage group was not significant (P > 0.05), except with the closest markers suggesting the absence of a signature of negative frequency dependent selection at the S-locus. Analysis of SFB1 and SFB2 protein sequences allowed identifying the absence of some variable and hypervariable domains and the presence of additional α-helices at the C-termini. Molecular and evolutionary analysis of SFB nucleotide sequences showed a signature of purifying selection in SFB2, while the SFB1 seemed to evolve neutrally. Thus, our results show that the SFB2 allele diversified after P. persica and P. dulcis (almond) divergence, a period which is characterized by an important bottleneck, while SFB1 diversified at a transition time between the bottleneck and population expansion.

Antibiotic Substrate Selectivity of Pseudomonas aeruginosa MexY and MexB Efflux Systems Is Determined by a Goldilocks Affinity.

Resistance-nodulation-division (RND) efflux pumps are important contributors to bacterial antibiotic resistance. In this study, we combined evolutionary sequence analyses, computational structural modeling, and ligand docking to develop a framework that can explain the known antibiotic substrate selectivity differences between two Pseudomonas aeruginosa RND transporters, MexY and MexB. For efficient efflux, antibiotic substrates must possess a "Goldilocks affinity": binding strong enough to allow interaction with transporter but not so tight as to impede movement through the pump.

Zebrafish ubiquitin-specific peptidase 5 (USP5) activates interferon resistance to the virus by increase the expression of RIG-I.

The ubiquitin specific peptidase (USP) family is involved in many life processes, of which antiviral is also an important basic function. One of the more important ways is to activate interferon. In this study, we reported the antiviral function of the ubiquitin specific peptidase 5(USP5) gene in zebrafish. Evolutionary and comparative protein sequence analysis of the USP5 was performed. The localization of USP5 in FHM cells cytoplasm was determined. Overexpression of USP5 significantly evoked higher expression of mRNA that encode IFNφ1 and ISGs, the promoteractivities of IFNφ1 and IFNstimulated response element (ISRE) were augmented likewise. USP5 was also able to enhance the expression of RIG-I and activate higher levels of IFNφ1 stimulated by Poly (I: C). Viral infection and interference tests demonstrated that USP5 inhibited the replication of SVCV in vitro. In summary, this study reveals that USP5 is able to activate higher levels of interferon by increasing RIG-I protein levels, and thus implement antivirus functions.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

BackgroundStable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis. These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended, and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood.ResultsTo characterize this population of non-coding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~ 360 nt, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3′ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons.ConclusionIn total, our results indicate sisRNAs are selected intron regions with distinct properties and may play a role in gene expression regulation.

Noncoding regions underpin avian bill shape diversification at macroevolutionary scales.

Recent progress has been made in identifying genomic regions implicated in trait evolution on a microevolutionary scale in many species, but whether these are relevant over macroevolutionary time remains unclear. Here, we directly address this fundamental question using bird beak shape, a key evolutionary innovation linked to patterns of resource use, divergence, and speciation, as a model trait. We integrate class-wide geometric-morphometric analyses with evolutionary sequence analyses of 10,322 protein-coding genes as well as 229,001 genomic regions spanning 72 species. We identify 1434 protein-coding genes and 39,806 noncoding regions for which molecular rates were significantly related to rates of bill shape evolution. We show that homologs of the identified protein-coding genes as well as genes in close proximity to the identified noncoding regions are involved in craniofacial embryo development in mammals. They are associated with embryonic stem cell pathways, including BMP and Wnt signaling, both of which have repeatedly been implicated in the morphological development of avian beaks. This suggests that identifying genotype-phenotype association on a genome-wide scale over macroevolutionary time is feasible. Although the coding and noncoding gene sets are associated with similar pathways, the actual genes are highly distinct, with significantly reduced overlap between them and bill-related phenotype associations specific to noncoding loci. Evidence for signatures of recent diversifying selection on our identified noncoding loci in Darwin finch populations further suggests that regulatory rather than coding changes are major drivers of morphological diversification over macroevolutionary times.

Gene transfer and nucleotide sequence evolution by Gossypium cytoplasmic genomes indicates novel evolutionary characteristics.

The DNA fragments transferred among cotton cytoplasmic genomes are highly differentiated. The wild D group cotton species have undergone much greater evolution compared with cultivated AD group. Cotton (Gossypium spp.) is one of the most economically important fiber crops worldwide. Gene transfer, nucleotide evolution, and the codon usage preferences in cytoplasmic genomes are important evolutionary characteristics of high plants. In this study, we analyzed the nucleotide sequence evolution, codon usage, and transfer of cytoplasmic DNA fragments in Gossypium chloroplast (cp) and mitochondrial (mt) genomes, including the A genome group, wild D group, and cultivated AD group of cotton species. Our analyses indicated that the differences in the length of transferred cytoplasmic DNA fragments were not significant in mitochondrial and chloroplast sequences. Analysis of the transfer of tRNAs found that trnQ and nine other tRNA genes were commonly transferred between two different cytoplasmic genomes. The Codon Adaptation Index values showed that Gossypium cp genomes prefer A/T-ending codons. Codon preference selection was higher in the D group than the other two groups. Nucleotide sequence evolution analysis showed that intergenic spacer sequences were more variable than coding regions and nonsynonymous mutations were clearly more common in cp genomes than mt genomes. Evolutionary analysis showed that the substitution rate was much higher in cp genomes than mt genomes. Interestingly, the D group cotton species have undergone much faster evolution compared with cultivated AD groups, possibly due to the selection and domestication of diverse cotton species. Our results demonstrate that gene transfer and differential nucleotide sequence evolution have occurred frequently in cotton cytoplasmic genomes.

Domain-Dependent Force Selectivity in the Mechanosensitive Ion Channel Piezo1

Piezo1 is a mechanosensitive ion channel noted for its exceptional complexity and involvement in force sensing in a broad range of tissues, including cartilage, bone, skin, blood vessels and lymph nodes. While cell-specific splice variants have been reported in the closely related Piezo2 channel, and various membrane proteins and lipids have been demonstrated to modulate the mechanotransduction process, it is less clear how Piezo1 attunes itself to tissue microenvironments with greatly divergent force profiles. Here, we describe chimeric cpGFP/Piezo1 probes that selectively respond to shear stress with a strong and irreversible increase in fluorescence intensity. Probe sensitivity is correlated with the presence an actin cytoskeleton, the inhibition of which dampens but does not eliminate the signal. The reporters retain functional pores and allow cation entry in response to the Piezo1 agonist Yoda1, as well as poking with a glass probe and exposure to a hypotonic environment, but these stimuli do not trigger a fluorescent response. Similarly, no shear response is provided by cpGFP inserted into other regions of Piezo1, as well as non-mechanosensitive reporters (Lck-cpGFP and the voltage-sensitive reporter ASAP1). Evolutionary analysis of Piezo1 sequences show that different clusters of conserved and co-mutating residues exist in the blade region, suggesting that multiple allosteric pathways may be responsible for conveying different types of mechanical stress to the pore. We propose that other sets of Piezo1 insertions can be used to determine the function of these clusters.

Molecular diversity and antibiotic resistance gene profile of Salmonella enterica serovars isolated from humans and food animals in Lagos, Nigeria.

Outbreaks of Salmonellosis remain a major public health problem globally. This study determined the diversity and antibiotic resistance gene profile of Salmonella enterica serovars isolated from humans and food animals. Using standard methods, Salmonella spp. were isolated from fecal samples, profiled for antimicrobial susceptibility and resistance genes. Seventy-one Salmonella isolates were recovered from both humans and food animals comprising cattle, sheep, and chicken. Forty-four serovars were identified, with dominant Salmonella Budapest (31.8%). Rare serovars were present in chicken (S. Alfort, S. Wichita, S. Linton, S. Ealing, and S. Ebrie) and humans (S. Mowanjum, S. Huettwillen, S. Limete, and S. Chagoua). Sixty-eight percent of isolates were sensitive to all test antibiotics, while the highest rate of resistance was to nalidixic acid (16.9%; n = 12), followed by ciprofloxacin (11.3%; n = 8) and tetracycline (9.9%; n = 8). Five isolates (7%) were multidrug-resistant and antimicrobial resistance genes coding resistance to tetracycline (tetA), beta-lactam (blaTEM), and quinolone/fluoroquinolone (qnrB and qnrS) were detected. Evolutionary analysis of gyrA gene sequences of human and food animal Salmonella isolates revealed variations but are evolutionarily interconnected. Isolates were grouped into four clades with S. Budapest isolate from cattle clustering with S. Budapest isolated from chicken, whereas S. Essen isolated from sheep and chicken was grouped into a clade. Diverse S. enterica serovars with high antibiotic resistance profile are ubiquitous in food animals; hence, there is a need for surveillance and prudent use of antibiotics in human and veterinary medicine.

Structure of the intergenic spacers in chicken ribosomal DNA

BackgroundRibosomal DNA (rDNA) repeats are situated in the nucleolus organizer regions (NOR) of chromosomes and transcribed into rRNA for ribosome biogenesis. Thus, they are an essential component of eukaryotic genomes. rDNA repeat units consist of rRNA gene clusters that are transcribed into single pre-rRNA molecules, each separated by intergenic spacers (IGS) that contain regulatory elements for rRNA gene cluster transcription. Because of their high repeat content, rDNA sequences are usually absent from genome assemblies. In this work, we used the long-read sequencing technology to describe the chicken IGS and fill the knowledge gap on rDNA sequences of one of the key domesticated animals.MethodsWe used the long-read PacBio RSII technique to sequence the BAC clone WAG137G04 (Wageningen BAC library) known to contain chicken NOR elements and the HGAP workflow software suit to assemble the PacBio RSII reads. Whole-genome sequence contigs homologous to the chicken rDNA repetitive unit were identified based on the Gallus_gallus-5.0 assembly with BLAST. We used the Geneious 9.0.5 and Mega software, maximum likelihood method and Chickspress project for sequence evolution analysis, phylogenetic tree construction and analysis of the raw transcriptome data.ResultsThree complete IGS sequences in the White Leghorn chicken genome and one IGS sequence in the red junglefowl contig AADN04001305.1 (Gallus_gallus-5.0) were detected. They had various lengths and contained three groups of tandem repeats (some of them being very GC rich) that form highly organized arrays. Initiation and termination sites of rDNA transcription were located within small and large unique regions (SUR and LUR), respectively. No functionally significant sites were detected within the tandem repeat sequences.ConclusionsDue to the highly organized GC-rich repeats, the structure of the chicken IGS differs from that of IGS in human, apes, Xenopus or fish rDNA. However, the chicken IGS shares some molecular organization features with that of the turtles, which are other representatives of the Sauropsida clade that includes birds and reptiles. Our current results on the structure of chicken IGS together with the previously reported ribosomal gene cluster sequence provide sufficient data to consider that the complete chicken rDNA sequence is assembled with confidence in terms of molecular DNA organization.

Phase separation-deficient TDP43 remains functional in splicing

Intrinsically disordered regions (IDRs) are often fast-evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. Here, we combine systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allows customization of the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, even a mutant that fails to phase-separate at physiological concentrations can still efficiently mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. This suggests that the ability of TDP43 to phase-separate is not essential for its splicing function.

An evolutionary approach to optimizing glucose-6-phosphatase-α enzymatic activity for gene therapy of glycogen storage disease type Ia.

Glycogen storage disease type-Ia (GSD-Ia), caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), is characterized by impaired glucose homeostasis with a hallmark hypoglycemia, following a short fast. We have shown that G6pc-deficient (G6pc-/-) mice treated with recombinant adeno-associated virus (rAAV) vectors expressing either wild-type (WT) (rAAV-hG6PC-WT) or codon-optimized (co) (rAAV-co-hG6PC) human (h) G6Pase-α maintain glucose homeostasis if they restore ≥3% of normal hepatic G6Pase-α activity. The co vector, which has a higher potency, is currently being used in a phase I/II clinical trial for human GSD-Ia (NCT03517085). While routinely used in clinical therapies, co vectors may not always be optimal. Codon-optimization can impact RNA secondary structure, change RNA/DNA protein-binding sites, affect protein conformation and function, and alter posttranscriptional modifications that may reduce potency or efficacy. We therefore sought to develop alternative approaches to increase the potency of the G6PC gene transfer vectors. Using an evolutionary sequence analysis, we identified a Ser-298 to Cys-298 substitution naturally found in canine, mouse, rat, and several primate G6Pase-α isozymes, that when incorporated into the WT hG6Pase-α sequence, markedly enhanced enzymatic activity. Using G6pc-/- mice, we show that the efficacy of the rAAV-hG6PC-S298C vector was 3-fold higher than that of the rAAV-hG6PC-WT vector. The rAAV-hG6PC-S298C vector with increased efficacy, that minimizes the potential problems associated with codon-optimization, offers a valuable vector for clinical translation in human GSD-Ia.

The Complete Chloroplast Genomes of Echinacanthus Species (Acanthaceae): Phylogenetic Relationships, Adaptive Evolution, and Screening of Molecular Markers.

Among the four species of Echinacanthus (Acanthaceae), one distributed in the West Himalayan region and three restricted to the Sino-Vietnamese karst region. Because of its ecological significance, molecular markers are necessary for proper assessment of its genetic diversity and phylogenetic relationships. Herein, the complete chloroplast genomes of four Echinacanthus species were determined for the first time. The results indicated that all the chloroplast genomes were mapped as a circular structure and each genomes included 113 unique genes, of which 80 were protein-coding, 29 were tRNAs, and 4 were rRNAs. However, the four cp genomes ranged from 151,333 to 152,672 bp in length. Comparison of the four cp genomes showed that the divergence level was greater between geographic groups. We also analyzed IR expansion or contraction in the four cp genomes and the fifth type of the large single copy/inverted repeat region in Lamiales was suggested. Furthermore, based on the analyses of comparison and nucleotide variability, six most divergent sequences (rrn16, ycf1, ndhA, rps16-trnQ-UUG, trnS-GCU-trnG-UCC, and psaA-ycf3) were identified. A total of 37–45 simple sequence repeats were discovered in the four species and 22 SSRs were identified as candidate effective molecular markers for detecting interspecies polymorphisms. These SSRs and hotspot regions could be used as potential molecular markers for future study. Phylogenetic analysis based on Bayesian and parsimony methods did not support the monophyly of Echinacanthus. The phylogenetic relationships among the four species were clearly resolved and the results supported the recognition of the Sino-Vietnamese Echinacanthus species as a new genus. Based on the protein sequence evolution analysis, 12 genes (rpl14, rpl16, rps4, rps15, rps18, rps19, psbK, psbN, ndhC, ndhJ, rpoB, and infA) were detected under positive selection in branch of Sino-Vietnamese Echinacanthus species. These genes will lead to understanding the adaptation of Echinacanthus species to karst environment. The study will help to resolve the phylogenetic relationship and understand the adaptive evolution of Echinacanthus. It will also provide genomic resources and potential markers suitable for future species identification and speciation studies of the genus.

Evolutionary relationship analysis of Middle East respiratory syndrome coronavirus 4a and 4b protein coding sequences.

The 4a and 4b proteins of the Middle East respiratory syndrome coronavirus (MERS-CoV) have been described for their antagonism on host innate immunity. However, unlike clustering patterns of the complete gene sequences of human and camel MERS-CoVs, the 4a and 4b protein coding regions did not constitute species-specific phylogenetic groups. Moreover, given the estimated evolutionary rates of the complete, 4a, and 4b gene sequences, the 4a and 4b proteins might be less affected by species-specific innate immune pressures. These results suggest that the 4a and 4b proteins of MERS-CoV may function against host innate immunity in a manner independent of host species and/or evolutionary clustering patterns.

First Detection of Proteus cibarius sp. of clinical significance

The rapidly changing resistance pattern of multidrug resistant Gram negative bacilli over the last decade is challenging. Their ability to develop resistance to β-lactam antibiotics and produce extended spectrum β-lactamases is most current clinical issue in antibiotic resistance, globally. This study was carried out to identify Gram negative bacterial responsible for β-lactam antibiotics treatment failure in an 88-year-old patient not responding to cephalosporin treatment. Wound sample was obtained from patient with long term infected diabetic wound and cultured on MacConkey agar. Antimicrobial susceptibility and double disk synergistic test were carried out to ascertain antibiotics resistance profile of isolate and production of extended spectrum beta-lactamases, respectively. Isolate was presumptively identified using standard biochemical tests. Genomic DNA extraction, amplification of the 16S rRNA gene with 27F (f5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (r5′-GGTTACCCTTGTTACGACTT-3′) primers and sequencing analysis were carried out to confirm presumptive identification. Nucleotides sequences were aligned with bioedit 7.2.5 and aligned sequences were compared with similar sequences on Gene bank. Construction of phylogenetic tree and evolutionary analysis of related sequences was conducted in MEGA7, using the Neighbor-Joining method. Isolate was identified to be Proteus cibarius strain ADE 1 with ascension number MH037129. Proteus cibarius strain ADE 1 was resistant to all β-lactam antibiotics and positive for ESBL production. This is the first report on Proteus cibarius associated with human pathogenicity. It is also the first detection of antibiotics resistance and ESBL production in Proteus cibarius. Proteus cibarius has a pathogenic potential and is an emerging opportunistic pathogen capable of spreading multiple antibiotics resistance.

PoSE: visualization of patterns of sequence evolution using PAML and MATLAB

BackgroundDetermining patterns of nucleotide and amino acid substitution is the first step during sequence evolution analysis. However, it is not easy to visualize the different phylogenetic signatures imprinted in aligned nucleotide and amino acid sequences.ResultsHere we present PoSE (Pattern of Sequence Evolution), a reliable resource for unveiling the evolutionary history of sequence alignments and for graphically displaying their contents. Substitutions are displayed by category (transitions and transversions), codon position, and phenotypic effect (synonymous and nonsynonymous). Visualization is accomplished using MATLAB scripts wrapped around PAML (Phylogenetic Analysis by Maximum Likelihood), implemented in an easy-to-use graphical user interface. The application displays inferred substitutions estimated by baseml or codeml, two programs included in the PAML software package. PoSE organizes patterns of substitution in eleven plots, including estimated non-synonymous/synonymous ratios (dN/dS) along the sequence alignment. In addition, PoSE provides visualization and annotation of patterns of amino acid substitutions along groups of related sequences that can be graphically inspected in a phylogenetic tree window.ConclusionsPoSE is a useful tool to help determine major patterns during sequence evolution of protein-coding sequences, hypervariable regions, or changes in dN/dS ratios. PoSE is publicly available at https://github.com/CDCgov/PoSE