Non-conservative Substitutions Research Articles

Recent Molecular Epidemiology of Echovirus 11 Throughout North and West Africa Resulted in the First Identification of a Recombinant Strain from an Acute Flaccid Paralysis Case in West Africa

Echovirus 11 has emerged as a major public health concern, causing sepsis in neonates in many European countries in recent years. In Africa, especially West Africa, where resources and diagnostic capacities are limited, only sporadic cases have been reported. To better understand the recent molecular epidemiology of E11 in West Africa, we characterized twenty-three echovirus 11 strains isolated through the acute flaccid paralysis and environmental surveillance systems for polio from 2013 to 2023, using high-throughput sequencing. Our data are noteworthy due to identifying for the first time a recombinant strain from an acute flaccid paralysis case and represent the first focus to date on molecular characterization of echovirus 11 in West Africa. Moreover, our data show that echovirus 11 diverged from 1970 (95% HPD range, 1961–1979) and evolved into four distinct clades, with the virus spread from West Africa to Europe, exhibiting two introductions in France around 2017, from Senegal and Guinea. Furthermore, the in silico analysis reveals four non-conservative amino acid substitutions in the VP1 sequences of the European strains associated with neonatal sepsis in newborns and a conserved amino acid motif in the VP1 protein toward enterovirus genotypes. Our data provide new insights into the epidemiology of echovirus 11 and point to the crucial need to implement specific surveillance programs targeting non-polio enteroviruses for the rapid identification of emerging or re-emerging enterovirus species, particularly in Africa.

Genetic variability of human papillomavirus type 18 based on E6, E7 and L1 genes in central China

BackgroundHigh-risk human papillomavirus (HR-HPV) infection is an important factor for the development of cervical cancer. HPV18 is the second most common HR-HPV after HPV16.MethodsIn this study, MEGA11 software was used to analyze the variation and phylogenetic tree of HPV18 E6-E7 and L1 genes. The selective pressure to E6, E7 and L1 genes was estimated using pamlX. In addition, the B cell epitopes of L1 amino acid sequences and T cell epitopes of E6-E7 amino acid sequences in HPV18 were predicted by ABCpred server and IEDB website, respectively.ResultsA total of 9 single nucleotide variants were found in E6-E7 sequences, of which 2 were nonsynonymous variants and 7 were synonymous variants. Twenty single nucleotide variants were identified in L1 sequence, including 11 nonsynonymous variants and 9 synonymous variants. Phylogenetic analysis showed that E6-E7 and L1 sequences were all distributed in A lineage. In HPV18 E6, E7 and L1 sequences, no positively selected site was found. The nonconservative substitution R545C in L1 affected hypothetical B cell epitope. Two nonconservative substitutions, S82A in E6, and R53Q in E7, impacted multiple hypothetical T cell epitopes.ConclusionThe sequence variation data of HPV18 may lay a foundation for the virus diagnosis, further study of cervical cancer and vaccine design in central China.

Unveiling polymorphism and protein structure prediction insights in diacylglycerol O-acyltransferase 1 and telethonin genes of Egyptian buffalo

BackgroundThe Egyptian buffalo has a sizable impact on Egypt's agricultural sector and food supply. It is regarded as the main dairy animal and an important source of red meat. This study aimed to detect the polymorphisms of the DGAT1 and TCAP genes and assess the potential impact of the discovered nsSNPs on the stability of the tertiary structure polypeptides of selected genes in Egyptian buffalo.MethodsAllele identification was made by the restriction fragment length polymorphism (RFLP), and the single nucleotide polymorphisms (SNPs) were recognized by sequencing the purified PCR products. Protein translation indicated the synonymous and non-synonymous SNPs, and the peptides' 3D tertiary structure of selected genes, as well as the effect of amino acid substitution on the protein structure, was performed using bioinformatics tools.ResultsAnalysis of the data revealed that an nsSNP was detected in a tested region of the DGAT1 gene and caused an amino acid substitution in a polypeptide that was predicted to be neutral and located in the coiled part of the protein. The analysis of the TCAP gene showed four nsSNPs that caused four substitutions located in the α-helix region. Protein prediction analysis showed that the amino acid substitutions in DGAT1 and TCAP were non-conserved with low sensitivity to variation. The non-conservative amino acid substitutions result in amino acids with new properties different from the original amino acid that change the protein's structure and function.ConclusionWe can infer that the DGAT1 and TCAP genes' SNPs may affect meat-related traits and may improve meat quality.

Evolutionary degeneration of septins into pseudoGTPases: impacts on a hetero-oligomeric assembly interface.

The septin family of eukaryotic proteins comprises distinct classes of sequence-related monomers that associate in a defined order into linear hetero-oligomers, which are capable of polymerizing into cytoskeletal filaments. Like actin and ⍺ and β tubulin, most septin monomers require binding of a nucleotide at a monomer-monomer interface (the septin "G" interface) for assembly into higher-order structures. Like ⍺ and β tubulin, where GTP is bound by both subunits but only the GTP at the ⍺-β interface is subject to hydrolysis, the capacity of certain septin monomers to hydrolyze their bound GTP has been lost during evolution. Thus, within septin hetero-oligomers and filaments, certain monomers remain permanently GTP-bound. Unlike tubulins, loss of septin GTPase activity-creating septin "pseudoGTPases"-occurred multiple times in independent evolutionary trajectories, accompanied in some cases by non-conservative substitutions in highly conserved residues in the nucleotide-binding pocket. Here, we used recent septin crystal structures, AlphaFold-generated models, phylogenetics and in silico nucleotide docking to investigate how in some organisms the septin G interface evolved to accommodate changes in nucleotide occupancy. Our analysis suggests that yeast septin monomers expressed only during meiosis and sporulation, when GTP is scarce, are evolving rapidly and might not bind GTP or GDP. Moreover, the G dimerization partners of these sporulation-specific septins appear to carry compensatory changes in residues that form contacts at the G interface to help retain stability despite the absence of bound GDP or GTP in the facing subunit. During septin evolution in nematodes, apparent loss of GTPase activity was also accompanied by changes in predicted G interface contacts. Overall, our observations support the conclusion that the primary function of nucleotide binding and hydrolysis by septins is to ensure formation of G interfaces that impose the proper subunit-subunit order within the hetero-oligomer.

Genetic variation of E6 and E7 genes of human papillomavirus type 16 from central China

BackgroundPersistent high-risk human papillomavirus (HR-HPV) infection is an important factor in the development of cervical cancer, and human papillomavirus type 16 (HPV-16) is the most common HR-HPV type worldwide. The oncogenic potential of HPV-16 is closely related to viral sequence variation.MethodsIn order to clarify the variant characteristics of HPV-16 E6 and E7 genes in central China, E6 and E7 sequences of 205 HPV‐16 positive samples were amplified by polymerase chain reaction. PCR products of E6 and E7 genes were further sequenced and subjected to variation analysis, phylogenetic analysis, selective pressure analysis and B-cell epitope prediction.ResultsTwenty-six single nucleotide variants were observed in E6 sequence, including 21 non-synonymous and 5 synonymous variants. Twelve single nucleotide variants were identified in E7 sequence, including 6 non-synonymous and 6 synonymous variants. Four new variants were found. Furthermore, nucleotide variation A647G (N29S) in E7 was significantly related to the higher risk of HSIL and cervical cancer. Phylogenetic analysis showed that the E6 and E7 sequences were all distributed in A lineage. No positively selected site was found in HPV-16 E6 and E7 sequences. Non-conservative substitutions in E6, H31Y, D32N, D32E, I34M, L35V, E36Q, L45P, N65S and K75T, affected multiple B-cell epitopes. However, the variation of E7 gene had little impact on the corresponding B-cell epitopes (score < 0.85).ConclusionHPV-16 E6 and E7 sequences variation data may contribute to HR-HPV prevention and vaccine development in Jingzhou, central China.

The Bactericidal Activity of a Novel Aneurinibacillus aneurinilyticus Isolate Effectively Controls Foodborne Pathogens Campylobacter jejuni and Listeria monocytogenes

Human zoonotic infection with Campylobacter is a major cause of gastroenteritis in the United States and worldwide. Listeria monocytogenes causes a potentially fatal infection in humans and is often attributed to contaminated food. Genetic typing has demonstrated that Campylobacter infection is often associated with the consumption of contaminated poultry products, with Campylobacter often colonizing the poultry gastrointestinal tract, while listeriosis is commonly associated with the consumption of contaminated ready-to-eat (RTE) foods. In this study, a strain of endospore-forming bacterium (strain NH) that is bactericidal towards the human food pathogens Campylobacter jejuni and Listeria monocytogenes was identified and characterized. Transwell experiments demonstrated that the bactericidal effect on both C. jejuni and L. monocytogenes is due to secretions from the spore former. These foodborne pathogens consistently exhibited 7 log reductions in growth when exposed to the NH cell-free culture filtrate. Sequencing of the 16s rRNA gene V4 variable region and analysis of the full-length 16s rRNA gene sequence from the WGS indicated that strain NH belongs to the species Aneurinibacillus aneurinilyticus. A microplate bioassay demonstrated that a bactericidal substance that is sensitive to protease could be collected from cell-free filtrates by salting out with ammonium sulfate. Gel filtration chromatography indicated a native molecular weight for the bactericidal protein of ca. 50 kDa, consistent with a class III bacteriocin. The active protein bound strongly to a cation-exchange resin and with an isoelectric point of ten, suggesting a positively charged protein. Both cation-exchange chromatography and isoelectric focusing indicated the enrichment of an 11 kDa protein on SDS-PAGE. This protein was identified through mass spectroscopy as the flgM protein, an anti-sigma factor. Analysis of whole genome sequencing (WGS) of the strain NH genome indicated the presence of a number of non-conservative amino acid substitutions in the flgM-gene-derived amino acid sequence of strain NH and A. aneurinilyticus compared to other members of the Aneurinibacillus genus. Further investigation is needed to determine whether these substitutions are correlated with the bactericidal activity. The identified strain may be useful as a feed additive for the pre-harvest control of Campylobacter jejuni in poultry.

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence

ABSTRACT Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.

External amino acid residues of insect GABA receptor channels dictate the action of the isoxazoline ectoparasiticide fluralaner.

Fluralaner is the first isoxazoline ectoparasiticide developed to protect companion animals from fleas and ticks. Fluralaner primarily inhibits arthropod γ-aminobutyric acid receptors (GABARs), which are ligand-gated ion channels comprising five subunits arranged around the channel pore. We previously reported that the action site of fluralaner resides at the M1-M3 transmembrane interface between adjacent GABAR subunits. To investigate whether fluralaner interacts with the second transmembrane segment (M2) located deep in the interface, we generated four housefly RDL GABAR mutants with non-conservative amino acid substitutions in the M2 region. Electrophysiological analysis of GABARs expressed in Xenopus oocytes indicated that S313A and S314A mutants exhibited fluralaner sensitivities similar to that of the wild type. M312S mutant exhibited approximately seven-fold lower sensitivity than that of the wild type. Notably, the N316L mutant was almost insensitive to fluralaner. The findings of this study indicate that the conserved external amino acid residues of insect GABAR channels play a critical role in the antagonistic effect of fluralaner. © 2023 Society of Chemical Industry.

Effect of the DGAT1 K232A mutation and breed on milk traits in cattle populations of Ethiopia

Non-conservative K232A substitution in the diacylglycerol acyl-CoA acyltransferase 1 (DGAT1) gene has been reported to explain variation in milk traits in cattle. The objective of this study was to estimate allele and genotype frequencies and to assess associations between K232A variants and milk yield and composition in cattle populations of Ethiopia. Blood samples for genomic DNA extraction and milk samples for analysis of milk components were collected from 92 randomly selected cattle of five Ethiopian breeds. Gene-specific primers were used to amplify 278 bp of the exon 8 region of DGAT1. Allele and genotype frequencies were calculated using Power Marker, and the GLM function of SAS software was used to assess the associations of detected genetic variation with milk traits. Boran * Holstein Friesian (HF) crosses produced a higher daily milk yield than the other breeds (p&amp;lt; 0.05), whereas the Boran and Begait breeds produced milk with higher fat and protein contents (p&amp;lt; 0.05), and Horro produced milk with a higher content of lactose, than the other breeds studied (p&amp;lt; 0.05). Alleles K and A and genotypes AA, KA, and KK were detected. The frequency of K232 ranged from 0.50 in Boran * HF crosses to 0.97 in the Horro population. The frequency of the KK and KA genotypes in the zebu population ranged from 0.50 to 0.94 and from 0.03 to 0.50, respectively. The AA genotype was associated with higher milk yield in Boran * HF crosses, whereas the KA genotype was associated with higher milk yield in the zebu populations (p&amp;lt; 0.05). The fat and lactose contents of milk produced with the KA genotype were lower than those of milk produced with the KK genotype in all genetic groups (p&amp;lt; 0.05). Substitution of one copy of the K allele led to a significant (p&amp;lt; 0.05) increase in fat content, of up to 0.81%, a decrease in daily milk yield of up to 3 L, and a decrease in lactose content of 0.58% in the sampled populations. The association study confirmed that the DGAT1 K232A marker had significant effects on daily milk yield, milk fat and lactose contents in the investigated cattle. These results suggested that the DGAT1 K232A marker may be utilized to accelerate future molecular breeding of dairy cattle, tropical zebu, their crossbreeds, after validation in a larger population.

Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter.

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.

First detection of a colistin-resistant Klebsiella aerogenes isolate from a critically ill patient with septic shock in Bulgaria.

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only β-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices.

SARS-CoV-2 Delta variant isolates from vaccinated individuals

BackgroundThe SARS-CoV-2 Delta variant was first identified in the U.S. in March 2021 and has rapidly become the predominant lineage across the U.S. due to increased transmissibility, immune evasion and vaccine breakthrough. The aim of this study was to better understand the genetic diversity and the potential impact of mutations observed in SARS-CoV-2 viruses circulating in the U.S. in vaccinated individuals.ResultsWhole genome sequencing was performed on thirty-four SARS-CoV-2 positive samples using the Oxford Nanopore MinION. Evolutionary genomic analysis revealed two novel mutations, ORF1b:V2354F and a premature stop codon, ORF7a:Q94*, identified in a cluster of SARS-CoV-2 Delta isolates collected from vaccinated individuals in Colorado. The ORF1b:V2354F mutation, corresponding to NSP15:V303F, may induce a conformational change and result in a disruption to a flanking beta-sheet structure. The premature stop codon, ORF7a:Q94*, truncates the transmembrane protein and cytosolic tail used to mediate protein transport. This may affect protein localization to the ER-Golgi. In addition to these novel mutations, the cluster of vaccinated isolates contain an additional mutation in the spike protein, at position 112, compared to the Delta variant defining mutations. This mutation, S112L, exists in isolates previously obtained in the U.S. The S112L mutation substitutes a bulky hydrophobic side chain for a polar side chain, which results in a non-conservative substitution within the protein that may affect antibody-binding affinity. Additionally, the vaccinated cluster of isolates contains non-synonymous mutations within ORF8 and NSPs which further distinguish this cluster from the respective ancestral Delta variant.ConclusionsThese results show there is an emerging sub-lineage of the ancestral Delta variant circulating in the U.S. As mutations emerge in constellations, those with a potentially beneficial advantage to the virus may continue to circulate while others will cease.

Predicted impact of the viral mutational landscape on the cytotoxic response against SARS-CoV-2.

The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations.

Genetic variation of E6, E7, and L1 genes of human papillomavirus 51 from central China.

The incidence of cervical cancer is closely related to high-risk human papillomavirus (HR-HPV). Women in Jingzhou had relatively high susceptibility to HPV-51, whose ratio was 9.61% (456/4743) among HR-HPV-positive samples and ranked fifth in all analyzed HR-HPV types. In this study, variations and phylogenetic trees of HPV-51 E6-E7 and L1 sequences were analyzed by MEGA-X. The selective pressure was estimated using PAML. The B-cell epitope of L1 amino acid sequences and T-cell epitope of E6 and E7 amino acid sequences were further predicted by ABCpred server and IEDB website, respectively. In the E6-E7 sequences 14 single nucleotide variants occurred, among which 4 were nonsynonymous variants and 10 were synonymous variants. A total of 41 single nucleotide variants were identified in the L1 sequences, including 10 nonsynonymous variants and 31 synonymous variants. All the isolates of both E6-E7 and L1 were classified into the A variant lineage. In HPV-51E6-E7 and L1 sequences, no positively selected site was found. Two nonconservative substitutions, H119Y and N176S in L1, affected multiple hypothetical B-cell epitopes. Three nonconservative substitutions, T86P, S100L in E6, and F29L in E7, affected multiple hypothetical T-cell epitopes. Elucidation of the HR-HPV prevalence characteristics and genetic variations of HPV-51 in central China may contribute to future investigations of diagnostic probes, therapeutic or preventative vaccines with wider coverage.

Gene Variant of Barrier to Autointegration Factor 2 (Banf2w) Is Concordant with Female Determination in Cichlids.

Oreochromis fishes exhibit variability of sex-determination (SD) genes whose characterization contributes to understanding of the sex differentiation network, and to effective tilapia farming, which requires all-male culture. However, O. niloticus (On) amh is the only master-key regulator (MKR) of SD that has been mapped (XY/XX SD-system on LG23). In O. aureus (Oa), LG3 controls a WZ/ZZ SD-system that has recently been delimited to 9.2 Mbp, with an embedded interval rich with female-specific variation, harboring two paics genes and banf2. Developing genetic markers within this interval and using a hybrid Oa stock that demonstrates no recombination repression in LG3, we mapped the critical SD region to 235 Kbp on the orthologous On physical map (p < 1.5 × 10−26). DNA-seq assembly and peak-proportion analysis of variation based on Sanger chromatograms allowed the characterization of copy-number variation (CNV) of banf2. Oa males had three exons capable of encoding 90-amino-acid polypeptides, yet in Oa females, we found an extra copy with an 89-amino-acid polypeptide and three non-conservative amino acid substitutions, designated as banf2w. CNV analysis suggested the existence of two to five copies of banf2 in diploidic Cichlidae. Disrupting the Hardy–Weinberg equilibrium (p < 4.2 × 10−3), banf2w was concordant with female determination in Oa and in three cichlids with LG3 WZ/ZZ SD-systems (O. tanganicae, O. hornorum and Pelmatolapia mariae). Furthermore, exclusive RNA-seq expression in Oa females strengthened the candidacy of banf2w as the long-sought LG3 SD MKR. As banf genes mediate nuclear assembly, chromatin organization, gene expression and gonad development, banf2w may play a fundamental role inducing female nucleus formation that is essential for WZ/ZZ SD.

Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic mold responsible for severe life-threatening fungal infections in immunocompromised patients. The cell wall, an essential structure composed of glucan, chitin, and galactomannan, is considered to be a target for the development of antifungal drugs. The nucleotide sugar donor GDP-mannose (GDP-Man) is required for the biosynthesis of galactomannan, glycosylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting from fructose-6-phosphate, GDP-Man is produced by the sequential action of the enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP-mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown approaches we demonstrate that the phosphomannomutase encoding gene in A. fumigatus (pmmA) is essential for survival. Reduced expression of pmmA is associated with significant morphological defects including retarded germination, growth, reduced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target.

The origin of the expressed retrotransposed gene ACTBL2 and its influence on human melanoma cells\u2019 motility and focal adhesion formation

We have recently found that β-actin-like protein 2 (actbl2) forms complexes with gelsolin in human melanoma cells and can polymerize. Phylogenetic and bioinformatic analyses showed that actbl2 has a common origin with two non-muscle actins, which share a separate history from the muscle actins. The actin groups’ divergence started at the beginning of vertebrate evolution, and actbl2 actins are characterized by the largest number of non-conserved amino acid substitutions of all actins. We also discovered that ACTBL2 is expressed at a very low level in several melanoma cell lines, but a small subset of cells exhibited a high ACTBL2 expression. We found that clones with knocked-out ACTBL2 (CR-ACTBL2) or overexpressing actbl2 (OE-ACTBL2) differ from control cells in the invasion, focal adhesion formation, and actin polymerization ratio, as well as in the formation of lamellipodia and stress fibers. Thus, we postulate that actbl2 is the seventh actin isoform and is essential for cell motility.

Mass-Based Protein Phylogenetic Approach to Identify Epistasis.

A mass-based protein phylogeny method, known as phylonumerics, is described to build phylogenetic-like trees using a purpose-built MassTree algorithm. These trees are constructed from sets of numerical mass map data for each protein without the need for gene or protein sequences. Such trees have been shown to be highly congruent with conventional sequence-based trees and provide a reliable means to study the evolutionary history of organisms. Mutations determined from the differences in the mass of peptide pairs across different mass sets are computed by the algorithm and displayed at branch nodes across the tree. By definition, since the trees display a phylogeny representing expressed proteins, all mutations are non-synonymous. The frequency of these mutations and a mutation score based on a sum of these frequencies weighted based upon their position to the root of the tree are output. The algorithm also outputs lists of pairs of mutations separated along interconnected branches of the tree. Those which co-occur or which occur consecutively, or near consecutively, and that are separated by a distance less than the average distance for all mutation pairs, are putatively assigned to be epistatic pairs. These pairs are examined further with a focus on non-conservative substitutions given their importance in driving structural and functional change and protein and organismal evolution. The application of the method is demonstrated for the H3 hemagglutinin protein of type A human H3N2 strains of the influenza virus. The most frequent ancestral mutations within epistatic pairs occur within antigenic site domains while the descendant mutations occur either at other antigenic sites or elsewhere in the protein. Both predominate at reported glycosylation sites. The results for this protein further support a "small steps" evolutionary model for the influenza virus where non-conservative mutations that involve the least structural change are favored over those involving substantive change, which may risk the virus's own extinction.

TLR4 and TLR8 variability in Amazonian and West Indian manatee species from Brazil.

Amazonian (Trichechus inunguis) and West Indian (Trichechus manatus) manatees are aquatic mammals vulnerable to extinction found in the Amazon basin and the coastal western Atlantic. Toll-like receptors (TLR) play a key role in recognizing pathogen-associated molecular patterns using leucine-rich repeats (LRRs). We described the diversity of TLR4 and TLR8 genes in these two species of manatee. Amazonian manatee showed seven SNPs in TLR4 and the eight in TLR8, while West Indian manatee shared four and six of those SNPs, respectively. In our analysis, TLR4 showed one non-conservative amino acid replacement substitution in LRR7 and LRR8, on the other hand, TLR8 was less variable and showed only conserved amino acid substitutions. Selection analysis showed that only one TLR4 site was subjected to positive selection and none in TLR8. TLR4 in manatees did not show any evidence of convergent evolution compared to species of the cetacean lineage. Differences in TLR4 and TLR8 polymorphism may be related to distinct selection by pathogens, population reduction of West Indian manatees, or an expected consequence of population expansion in Amazonian manatees. Future studies combining pathogen association and TLR polymorphism may clarify possible roles of these genes and be used for conservation purposes of manatee species.

A novel c.1759T>G variant in follicle-stimulating hormone-receptor gene is concordant with male determination in the flathead grey mullet (Mugil cephalus).

Various master key regulators (MKRs) that control a binary switch of sex determination (SD) have been found in fish; these provide an excellent model for the study of vertebrate genetic SD. The SD region in flathead grey mullet has been previously mapped to a 1 Mbp region harboring 27 genes, of which one is follicle-stimulating hormone receptor (fshr). Although this gene is involved in gonad differentiation and function, it has not been considered as an MKR of SD. We systematically investigated polymorphism in mullet fshr using DNA shotgun sequences, and compared them between males and females. Capable of encoding nonconservative amino acid substitutions, c.1732G>A and c.1759T>G exhibited association with sex on a population level (N = 83; P ≤ 6.7 × 10−19). Hence, 1732 A and 1759 G represent a male-specific haplotype of the gene, designated as “fshry.” Additional flanking SNPs showed a weaker degree of association with sex, delimiting the SD critical region to 143 nucleotides on exon 14. Lack of homozygotes for fshry, and the resulting divergence from Hardy–Weinberg equilibrium (N = 170; P ≤ 3.9 × 10−5), were compatible with a male heterogametic model (XY/XX). Capable of replacing a phenylalanine with valine, c.1759T>G alters a conserved position across the sixth transmembrane domain of vertebrate FSHRs. Amino acid substitutions in this position in vertebrates are frequently associated with constant receptor activation and consequently with FSH/FSHR signaling alteration; thus, indicating a potential role of fshr as an MKR of SD.