Amino Acid Sequence Research Articles

The structure of the IL-11 signalling complex provides insight into receptor variants associated with craniosynostosis.

Interleukin 11 (IL-11), a member of the IL-6 family of cytokines, has roles in haematopoiesis, inflammation, bone metabolism, and craniofacial development. IL-11 also has pathological roles in chronic inflammatory diseases, fibrosis, and cancer. In this structural snapshot, we explore our recently published cryo-EM structure of the human IL-11 signalling complex to understand the molecular mechanisms of complex formation and disease-associated mutations. IL-11 signals by binding to its cell surface receptors, the IL-11 receptor α subunit (IL-11Rα) and glycoprotein 130 (gp130), to form a hexameric signalling complex. We examine the locations within the complex of receptor sequence variants that are associated with craniosynostosis and craniosynostosis-like phenotypes and speculate on potential molecular mechanisms leading to defects in signalling function. While these causative amino acid sequence changes in IL-11Rα are generally distal to interfaces between components of the complex, important structural residues are highly represented, including proline residues, cysteine residues involved in disulfide bonds, and residues within or surrounding the tryptophan-arginine ladder. We also note the locations and potential effects of amino acid substitutions within the extracellular domains of gp130 that are associated with craniosynostosis. As focus on the physiological and pathological functions of IL-11 grows, the importance of high-resolution structural knowledge of IL-11 signalling to understand disease-associated mutations and to inform therapeutic strategies will only increase.

ZNF512B binds RBBP4 via a variant NuRD interaction motif and aggregates chromatin in a NuRD complex-independent manner.

The evolutionarily conserved histone variant H2A.Z plays a crucial role in various DNA-based processes, but the mechanisms underlying its activity are not completely understood. Recently, we identified the zinc finger (ZF) protein ZNF512B as a protein associated with H2A.Z, HMG20A and PWWP2A. Here, we report that high levels of ZNF512B expression lead to nuclear protein and chromatin aggregation foci that form in a manner that is dependent on the ZF domains of ZNF512B. Notably, we demonstrate ZNF512B binding to the nucleosome remodeling and deacetylase (NuRD) complex. We discover a conserved amino acid sequence within ZNF512B that resembles the NuRD-interaction motif (NIM) previously identified in FOG-1 and other transcriptional regulators. By solving the crystal structure of this motif bound to the NuRD component RBBP4 and by applying several biochemical and biophysical assays, we demonstrate that this internal NIM is both necessary and sufficient for robust and high-affinity NuRD binding. Transcriptome analyses and reporter assays identify ZNF512B as a repressor of gene expression that can act in both NuRD-dependent and -independent ways. Our study might have implications for diseases in which ZNF512B expression is deregulated, such as cancer and neurodegenerative diseases, and hints at the existence of more proteins as potential NuRD interactors.

Deciphering the 4',7-Dihydroxy-3'-Methylflavone from Boerhavia, Agonist/Antagonist Interactions Against 5-HT2 Receptors using Homology Modeling, Molecular Docking, and Dynamic Simulation Studies.

Seizures, depression, and anxiety are neurological disorders that affected innumerable people worldwide. Recent research has revealed that targeting 5-hydroxytryptamine 2 (5-HT2) receptors can help suppress these conditions. An in-depth literature study has identified that phytocompounds from the Boerhavia genus could reduce seizures. Therefore, homology models of 5-HT2 receptors were generated and validated using techniques such as the alignment of amino acid sequences and the Ramachandran plot. Later, a comparison of modeled structures was made with a non-redundant set of PDB structures. The pharmacokinetics, drug-likeness, blood-brain barrier (BBB) permeability, and Lipinski's rule of five shed light on 22 phytocompounds, which are the potential candidates for molecular docking among 127 Boerhavia's bioactive. Notably, molecular docking analysis revealed 4',7-dihydroxy-3'-methylflavone as the most potent lead compound, which has a strong binding affinity to all modeled receptors. Additionally, with a remarkably high docking score of -9.1 kcal/mol, 4',7-dihydroxy-3'-methylflavone showed promising interactions, particularly with 5-HT2A receptor, as seen from the RMSD, SASA, Rg, and number of hydrogen bonds during 100 ns molecular dynamic (MD) simulation. Principal component analysis (PCA) and Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) further confirmed that 4',7-dihydroxy-3'-methylflavone is the best novel phytocompound in Boerhavia genus for 5-HT2 receptor as agonist/antagonist activity against seizures.

Differential Adjuvant Activity by Flagellins from Escherichia coli, Salmonella enterica Serotype Typhimurium, and Pseudomonas aeruginosa

(1) Background: The adjuvant properties of flagellin from various bacterial species have been extensively studied; however, a systematic comparison of the immunoadjuvant effects of flagellins from different bacterial species is lacking. This study aims to analyze the amino acid sequences and structural features of flagellins from Escherichia coli (FliCE.C), Salmonella enterica serotype Typhimurium (FliCS.T), and Pseudomonas aeruginosa (FliCP.A), and to evaluate their adjuvant activities in terms of Toll-like receptor 5 (TLR5) activation, antibody production, and cytokine responses in a murine model. (2) Methods: Bioinformatics analysis was conducted to compare the amino acid sequences and structural domains (D0, D1, D2, and D3) of flagellins from the three bacterial species. PyMol atomic models were used to confirm structural differences. Toll-like receptor 5 (TLR5) activation assays were performed to measure IL-8 and TNF-α production in vitro. The IgG antibody titers against the model antigen FaeG and cytokine responses, including IL-4 and TNF-α secretion were evaluated in a murine model. (3) Results: Bioinformatics analysis revealed that the D0 and D1 domains are highly conserved, whereas the D2 and D3 domains exhibit significant variability across the three species. Structural analysis via PyMol confirmed these differences, particularly in the D2 and D3 domains. TLR5 activation assays showed that FliCS.T and FliCP.A induced higher levels of IL-8 and TNF-α production compared to FliCE.C, indicating species-specific variations in TLR5 activation. In the murine model, FliCS.T as an adjuvant produced higher antibody titers against FaeG and increased IL-4 secretion in splenocytes compared to FliCE.C and FliCP.A. FliCP.A induced higher TNF-α expression than FliCS.T and FliCE.C, suggesting FliCS.T and FliCP.A are more effective at inducing T-cell responses. (4) Conclusions: This study highlights the potential of FliCS.T and FliCP.A as potent vaccine adjuvants. The results provide insights into the structure–function relationships of these flagellins and support their application in enhancing immune responses against diverse pathogens.

KiNext: a portable and scalable workflow for the identification and classification of protein kinases

BackgroundProtein kinases are a diverse superfamily of proteins common to organisms across the tree of life that are typically involved in signal transduction, allowing organisms to sense and respond to biotic or abiotic environmental factors. They have important roles in organismal physiology, including development, reproduction, acclimation to environmental stress, while their dysregulation can lead to disease, including several forms of cancer. Identifying the complement of protein kinases (the kinome) of any organism is useful for understanding its physiological capabilities, limitations and adaptations to environmental stress. The increasing availability of genomes makes it now possible to examine and compare the kinomes across a broad diversity of organisms. Here we present a pipeline respecting the FAIR principles (findable, accessible, interoperable and reusable) that facilitates the search and identification of protein kinases from a predicted proteome, and classifies them according to group of serine/threonine/tyrosine protein kinases present in eukaryotes.ResultsKiNext is a Nextflow pipeline that regroups a number of existing bioinformatic tools to search for and classify the protein kinases of an organism in a reproducible manner, starting from a set of amino acid sequences. Conventional eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs) are identified by using Hidden Markov Models (HMMs) generated from the catalytic domains of kinases. Furthermore, KiNext categorizes ePKs into the eight kinase groups by employing dedicated Hidden Markov Models (HMMs) tailored for each group. The performance of the KiNext pipeline was validated against previously identified kinomes obtained with other tools that were already published for two marine species, the Pacific oyster Crassostrea gigas and the unicellular green alga Ostreoccocus tauri. KiNext outperformed previous results by finding previously unidentified kinases and by attributing a large proportion of previously unclassified kinases to a group in both species. These results demonstrate improvements in kinase identification and classification, all while providing traceability and reproducibility of results in a FAIR pipeline. The default HMM models provided with KiNext are most suitable for eukaryotes, but the pipeline can be easily modified to include HMM models for other taxa of interest.ConclusionThe KiNext pipeline enables efficient and reproducible identification of kinomes based on predicted amino acid sequences (i.e. proteomes). KiNext was designed to be easy to use, automated, portable and scalable.

Cytochromes b5 Occurrence in Viruses Belonging to the Order Megavirales.

Cytochrome b5 is a small electron transport protein that is found in animals, plants, fungi and photosynthetic proteobacteria where it plays key metabolic roles in energy production, lipid and sterol biosynthesis and cytochrome P450 biochemistry. Previously it was shown that a gene encoding a soluble and functional cytochrome b5 protein was encoded in the large double stranded DNA virus OtV2 that infects the unicellular marine green alga Ostreococcus tauri, the smallest free-living eukaryote described to-date. This single gene represented a unique finding in the virosphere. We now report that genes for soluble and membrane-bound cytochromes b5 also occur in giant viruses in the proposed order Megavirales, particularly the AT-rich Mimiviridae and Tupanviruses. Conversely, other members of the Megaviralestaxa such as the GC-rich Pandoraviridae have not been found to encode cytochrome b5 as yet. Megaviruses encoding cytochrome b5 have been isolated from the deep ocean, from freshwater and terrestrial sources, as well as from human patients. Giant virus cytochrome b5 proteins share high sequence identity with one another (45-95% depending on group) but no more than 25% identity with the cytochrome b5 gene product we identified in Acanthamoeba castellanii, an amoeba host for many giant viruses. Thus, the origin of the unique cytochrome b5 genes in giant viruses remainsunknown. Examination of viral cytochrome b5 primary amino acid sequences revealed that some have either a N- or C-terminal transmembrane anchor, whilst others lack a membrane anchor and are thus predicted to be soluble proteins. This cytochrome b5 topography suggests adapted biochemical functions in those viruses. Our findings raise questions regarding the evolution and diversity of cytochrome b5 proteins in nature, adding to questions about the origin of viral haemoproteins in general.

First Description of Loreto Virus in Three Culicidae Species from the Atlantic Forest, Bahia, Brazil

Loreto virus (LORV) is an insect-specific virus classified into the proposed taxon Negevirus. It was originally described in Iquitos, Peru, in 1977. Here, we describe three novel LORV genomes obtained from the isolates of three pooled samples of Trichoprosopon digitatum, Aedes (Ochlerotatus) fulvus, and Limatus durhamii collected in Ilhéus—Bahia, 2014. Samples were submitted to RNA sequencing on the Illumina platform to recover the LORV genome. The genomes presented, on average, 81.5% nucleotide identity and 92.6% global amino acid identity with the LORV reference genome (NC_034158). Subsequently, phylogenetic analysis was performed based on a multiple sequence alignment of the concatenated amino acid sequences predicted for the three ORFs of the Negevirus genomes, and the target sequences were clustered within the LORV clade. The taxon Negevirus is in constant expansion of its species content and host range. New data about insect specific negeviruses are important for virus evolution studies, along with those approaching interactions with the hosts and their influence in the transmission of arboviruses. Also, the assessment of these data may allow the development of biologic control strategies for arboviral vectors. This is the original report of the identification of LORV in Brazil, infecting three Culicidae species hosts native to the Atlantic Forest biome.

Steered molecular dynamics simulation as a post-process to optimize the iBRAB-designed Fab model.

Therapeutic monoclonal antibodies are an effective method of treating acute infectious diseases. However, knowing which of the produced antibodies in the vast number of human antibodies can cure the disease requires a long time and advanced technology. The previously introduced iBRAB method relies on studied antibodies to design a broad-spectrum antibody capable of neutralizing antigens of many different Influenza A viral strains. To evaluate the antigen-binding fragment as an applicable drug, the therapeutic antibody profiles providing guidelines collected from clinically staged therapeutic antibodies were used to access different measurements. Although the evaluated values were within an accepted range, the modification in the amino acid sequence is required for better properties. Thus, using the steered molecular dynamics(SMD) simulation to determine the binding capacity of amino acids in the functional region, the profile of interacted amino acids of Fab with the antigen was established for modified reference. As a result, the model was modified with amino acids elimination at positions 96-97 in the heavy chain and 26-27, 91, 96-97, and 102-103 in the light chain, which has better Therapeutic Antibody Profiler evaluations than the original designation. Thus again, SMD simulation is a promising computational approach for post-modification in rational drug design.

Characterization of Luciferase from Photorhabdus kayaii and its Application for In vivo Imaging Studies in Mice.

Bioluminescence, or the production of light by luciferases, is the basis of a well-known reporter technology. A quick way to study the efficacy of antimicrobial drugs and vaccines is in vivo bioluminescence imaging (BLI). Photorhabdus spp. represent the only terrestrial group of bioluminescent bacteria. The luciferase obtained from Photorhabdus luminescence has been widely used in BLI studies. However, little information is available about the functions of luciferases obtained from other members of this genera. This study aimed to evaluate the applicability of the luciferase obtained from Photorhabdus kayaii for BLI studies. P. kayaii starE, an Iranian isolate of P. kayaii, was cultivated on NBTA agar plates. The resulting colonies were cultured on McConkey agar to determine the bacterial phase. Bioluminescence emission was measured using a multimode reader. The luciferase genes of this bacterium were sequenced following the PCR amplification, and the corresponding amino acid sequences were determined. The luciferase tertiary structure was then obtained from the TACOS web server and compared to that of P. luminescence in CE software. The lux operon encoding the luciferase (luxA and luxB genes) and substrate synthesis complex was cloned and expressed in Escherichia coli BL21 (DE3) using the pBBR1MCS2_START vector. The luminescence emission during the growth was examined. Moreover, the effects of pH and sodium deoxycholate (bile salt) on bioluminescence emission were investigated. Appropriate conditions for the use of bioluminescent E. coli for BLI studies in mice were demonstrated in terms of cell numbers and injection routes. The bacterium was luminescent and in phase I. Its luciferase monomers (α and β) shared 100% amino acid homology with P. kayaii M-HU2 and more than 92% with P. luminescence. Tertiary structures of the luciferase monomers were 93%- 95% identical to those of P. luminescence. The lux operon was expressed in E. coli, and the maximum bioluminescence signal was observed during the decelerating phase of growth. The bioluminescence at different pH values correlated with the cell survival. The luminescence was emitted by cells exposed to the bile salt. A strong bioluminescent signal was emitted from mice after subcutaneous injection of bioluminescent E. coli at 107 CFU. However, no signals were emitted from mice that were administered the same cell number via intraperitoneal injection. A 2.5-fold increase in the cell number resulted in bioluminescence detection in the abdomen of mice after intraperitoneal injection and a 3.22-fold increase in signal intensity after subcutaneous injection. These results demonstrated the usefulness of P. kayaii luciferase for BLI studies.

Whole-genome sequencing analyses and antibiotic resistance situation of 48 Helicobacter pylori strains isolated in Zhejiang, China

PurposeIn the Zhejiang region, research on Helicobacter pylori is lacking. The purpose of this study was to assess the extent of antibiotic resistance in H. pylori in this region, explore alternative methods for predicting the resistance patterns of H. pylori, and investigate the colonization of native gastric mucosa by other clades of H. pylori in the structure population of this bacterium.MethodsStrains were cultured under microaerobic conditions, and antimicrobial susceptibility testing (AST) was performed via agar dilution. Whole-genome sequencing (WGS) was performed via next-generation sequencing (NGS) technology. Epidemiological data including data from this study and reported articles from Zhejiang, China, were included. Further analyses based on AST, WGS, and epidemiological date include virulence genes, antibiotic resistance-related mutations, and phylogenetic trees based on 7 housekeeping genes and core-genome single nucleotide polymorphisms (SNPs).ResultsThe bacterial isolates in this study presented higher antibiotic resistance rates than previously reported, especially against levofloxacin and clarithromycin. The point mutation A2147G in 23 S rRNA is specific to clarithromycin resistance. Mutations at position/s 87 and/or 91 of the gyrA gene amino acid sequence are highly consistent with levofloxacin resistance highly. The point mutations C1707T in 23 S rRNA and E463K in the gyrB gene have not been previously documented in China. All the bacterial isolates belong to Asian branches in the structure population. The resistance rate to clarithromycin of isolates from hosts born after January 1, 1977 is statistically higher than that of hosts born before 1977.ConclusionEradication therapy based on AST results is urgently needed in Zhejiang. The point mutation A2147G in 23 S rRNA and point mutations in the gyrA gene at amino acid/s 87 and/or 91 are sufficient for predicting resistance to clarithromycin and levofloxacin, respectively. The isolate with the mutation E463K in the gyrB gene represents a significant contribution to the field. Mutations in 23 S rRNA may offer valuable insights into the dynamics of H. pylori transmission among hosts.

Efficacy and safety of the ustekinumab biosimilar ABP 654 in patients with moderate-to-severe plaque psoriasis: a randomized, double-blinded, active-controlled, comparative clinical study over 52 weeks.

ABP 654 is a biosimilar to ustekinumab reference product (RP). ABP 654 has been shown to have an amino acid sequence identical to ustekinumab RP and they are similar in structure, purity, and potency as well as clinical pharmacokinetics and safety in healthy volunteers. This randomized, double-blinded, active-controlled, single-transition, comparative clinical study (ClinicalTrials.gov ID NCT04607980) was conducted to compare the efficacy, safety, and immunogenicity of ABP 654 and ustekinumab RP in patients with moderate-to-severe plaque psoriasis. Patients were randomized in a 1:1 ratio to receive ABP 654 or ustekinumab RP at a weight-based dose of 45 mg or 90 mg administered subcutaneously on day 1 (week 0), week 4, and week 16. At week 28, patients with a ≥75% improvement in Psoriasis Area and Severity Index (PASI) were re-randomized such that those initially randomized to ABP 654 continued to receive ABP 654 and those initially randomized to ustekinumab RP were re-randomized to either continue on ustekinumab RP or transition to ABP 654. The primary efficacy endpoint was PASI percent improvement from baseline to week 12. Secondary endpoints included additional efficacy measurements as well as an assessment of adverse events and antidrug antibodies. At week 12, the observed mean (SD) PASI percent improvement from baseline was 81.9 (19.9) and 81.9 (19.6) for the ABP 654 and ustekinumab RP treatment groups, respectively. The point estimate of the mean difference in PASI percent improvement from baseline to week 12 between the treatment groups was 0.14 with a 2-sided 90% confidence interval (CI) of (-2.6, 2.9), well within the prespecified similarity margin of (-10, +10). In addition, throughout the study, secondary efficacy analyses and safety and immunogenicity profiles were similar across the treatment groups. These results indicate that ABP 654 and ustekinumab RP are clinically similar in efficacy, safety, and immunogenicity in patients with moderate-to-severe plaque psoriasis. Further, a single transition from ustekinumab RP to ABP 654 at week 28 had no impact on the efficacy, safety, or immunogenicity results for the remainder of the 52-week study, supporting a conclusion of no clinically meaningful differences between ABP 654 and ustekinumab RP.

Pre-training with a rational approach for antibody sequence representation.

Antibodies represent a specific class of proteins produced by the adaptive immune system in response to pathogens. Mining the information embedded in antibody amino acid sequences can benefit both antibody property prediction and novel therapeutic development. However, antibodies possess unique features that should be incorporated using specifically designed training methods, leaving room for improvement in pre-training models for antibody sequences. In this study, we present a Pre-trained model of Antibody sequences trained with a Rational Approach for antibodies (PARA). PARA employs a strategy conforming to antibody sequence patterns and an advanced natural language processing self-encoding model structure. This approach addresses the limitations of existing protein pre-training models, which primarily utilize language models without fully considering the differences between protein sequences and language sequences. We demonstrate PARA's performance on several tasks by comparing it to various published pre-training models of antibodies. The results show that PARA significantly outperforms existing models on these tasks, suggesting that PARA has an advantage in capturing antibody sequence information. The antibody latent representation provided by PARA can substantially facilitate studies in relevant areas. We believe that PARA's superior performance in capturing antibody sequence information offers significant potential for both antibody property prediction and the development of novel therapeutics. PARA is available at https://github.com/xtalpi-xic.

Immune History Modifies Disease Severity to HPAI H5N1 Clade 2.3.4.4b Viral Challenge.

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity-either from LAIV vaccination and/or from Cal09 infection-showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

Characterization of the Coriolopsis gallica DyP for Its Potential to Biotransform Various Fluoroquinolones.

Coriolopsis gallica (Cga) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologously expressed in Escherichia coli. We fully characterized the biochemical properties of CgaDyP1 and evaluated its dye-decolorizing potential to confirm that it belongs to the DyP class of enzymes. We also tested its fluoroquinolone antibiotic biotransformation potential for possible biotechnological applications. Alignment of the primary amino acid sequence with DyP homolog sequences showed that CgaDyP1 has high similarity with other fungal DyPs. The recombinant CgaDyP1 exhibited activity on substrates such as ABTS and 2,6-dimethoxyphenol (DMP) with optimal performance at a pH of 3, although activity at pH 2.5, pH 4, and pH5 diminished over time. Thermostability tests indicated that the enzyme remains stable at temperatures between 30 °C and 50 °C and retains 70% of its initial activity after 180 min at 50 °C. Tests on the effect of hydrogen peroxide on CgaDyP1 activity found peak activity at 0.25 mM H2O2. CgaDyP1 decolorized five industrial dyes, and kinetics data confirmed that it belongs to the DyP class of enzymes. CgaDyP1 was shown to biotransform some of the 7 recalcitrant fluoroquinolone antibiotics tested here, including levofloxacin, moxifloxacin, and norfloxacin, and thus holds potential for biotechnological applications.

ECM1 attenuates hepatic fibrosis by interfering with mediators of latent TGF-β1 activation

ObjectiveExtracellular matrix protein 1 (ECM1) serves as a gatekeeper of hepatic fibrosis by maintaining transforming growth factor-β1 (TGF-β1) in its latent form. ECM1 knockout (KO) causes latent (L) TGF-β1 activation,...

Phylogenetic analysis of selected species of Asteraceae on the basis of RPS 11 Gene

The Asteraceae family is a prominent group of flowering plants found across the globe, with the exception of Antarctica. The Asteraceae family is a largest flowering family pivotal group in plant evolution and diversification. Despite its importance, the genetic diversity within this family remains understudied. We focused on the rps-11 gene, a chloroplast marker previously utilized in phylogenetic studies, to investigate its potential in resolving Asteraceae relationships. The focus was on examining genetic diversity within sixteen specifically chosen species from the Asteraceae family. This assessment was based on an analysis of a chloroplast gene responsible for encoding the ribosomal protein of the smaller subunit 11 (rps 11). Nearly 417 bp of rps 11 gene was amplified, sequenced, computationally translated into amino acid sequence and the data was used for phylogenetic analysis as well as for rps 11 protein structure predictions. Based on nucleotide and amino acid sequences phylograms were drawn with the help of Molecular Evolutionary Genetic Analysis (MEGA 6), which exhibited clear genetic relationship among species under investigation. The observed genetic distance was 0.02 for Maximum likelihood tree based on nucleotide sequences whereas it was 0.05 for phylogram based on amino acid sequences. These values revealed that amino acid-based tree has demonstrated greater diversity among selected species in comparison to nucleotides-based tree. On the basis of pair wise distance calculations, genetic divergence values were found within the range of 0.015–0.309. Moreover, 3D protein modeling for rps 11 protein of sixteen selected species was also carried out by iterative threading assembly refinement (I-Tasser) software. The models exhibiting the highest C-score were picked with satisfactory plot statistics (> 90%) and structurally validated by PROCHECK. Furthermore, Ramachandran plots displayed that the rps 11 protein structures of Tagetes minuta, Xanthium strumarium, Lactuca sativa and Chrysanthemum indicum have best feature models with > 90% of residues in the allowed region and ≤ 2% in the disallowed region. The research is not enough to stand alone to validate the viability of the rps11 gene as a prospective contender for phylogenetic analysis. İn future we will focus on the maximum genetic diversity theory for phylogenetic analysis of this family.

Macromolecular Function Emerging from Intramolecular Peptide Stapling of Synthetic Polymers.

Protein function results from the precise folding of polypeptides into bespoke architectures. Taking inspiration from nature, the field of single-chain nanoparticles (SCNPs), intramolecularly crosslinked synthetic polymers, emerged. In contrast to nature, the function of SCNPs is generally defined by the parent polymer or the applied crosslinker, rather than by the crosslinking process itself. This work explores the cyanopyridine-aminothiol click reaction to crosslink peptide-decorated polymers intra-macromolecularly to endow the resulting SCNPs with emerging functionality, resulting from the conversion of N-terminal cysteine units into pyridine-thiazolines. Dimethylacrylamide based polymers with different cysteine-terminated amino acid sequences tethered to their sidechains are investigated (P1 (C),P2 (GDHC),P3 (GDSC)) and intramolecularly crosslinked into SCNPs. Since the deprotection of the parent polymers yields disulfide-based SCNPs, a direct comparison between disulfide and pyridine-thiazolines crosslinked SCNPs is possible. This comparison revealed two emerging properties of the pyridine-thiazoline crosslinked SCNPs: 1) The formation of pyridine-thiazolines gave rise to metal binding sites within the SCNP, which complexed iron. 2) Depending on the peptide sequence in the precursor polymer, the hydrolytic activity of the peptide sequences is either increased (GDHC) or decreased (GDSC) uponpyridine-thiazoline formation compared to identical SCNPs based on disulfide crosslinks.

The host tropism of current zoonotic H7N9 viruses depends mainly on an acid-labile hemagglutinin with a single amino acid mutation in the stalk region.

The incidence of human infection by zoonotic avian influenza viruses, especially H5N1 and H7N9 viruses, has increased. Current zoonotic H7N9 avian influenza viruses (identified since 2013) emerged during reassortment of viruses belonging to different subtypes. Despite analyses of their genetic background, we do not know why current H7N9 viruses are zoonotic. Therefore, there is a need to identify the factor(s) responsible for the extended host tropism that enables these viruses to infect humans as well as birds. To identify H7N9-specific amino acids that confer zoonotic properties on H7N9 viruses, we performed multiple alignment of the hemagglutinin (HA) amino acid sequences of A/Shanghai/1/2013 (H7N9) and A/duck/Zhejiang/12/2011(H7N3) (a putative, non- or less zoonotic HA donor to the zoonotic H7N9 virus). We also analyze the function of an H7N9 HA-specific amino acid with respect to HA acid stability, and evaluated the effect of acid stability on viral infectivity and virulence in a mouse model. HA2-116D, preserved in current zoonotic H7N9 viruses, was crucial for loss of HA acid stability. The acid-labile HA protein in H7 viruses played an important role in infection of human airway epithelial cells; HA2-116D contributed to infection and replication of H7 viruses. Finally, HA2-116D served as a H7 virulence factor in mice. These results suggest that acid-labile HA harboring HA2-116D confers zoonotic characteristics on H7N9 virus and that future novel zoonotic avian viruses could emerge from non-zoonotic H7 viruses via acquisition of mutations that remove HA acid stability.

Identification of receptor-binding protein and host receptor of non-lytic dsRNA phage phiNY.

To date, complete genome sequences of 14 double-stranded RNA (dsRNA) phages are available, and studies have shown that the host range of dsRNA phages is limited. The hosts of most dsRNA phages belong to the genus Pseudomonas. However, the dsRNA phage phiNY, which has a non-lytic life cycle, was isolated from Microvirgula aerodenitrificans. Currently, the interaction between dsRNA phage phiNY and its host bacteria is unclear, which is not beneficial to a comprehensive understanding of dsRNA phage biology and the exploitation of dsRNA phage with non-lytic life cycle for biomedical applications and others. Phage adsorption is a crucial step through the interactions between receptor-binding protein (RBP) of the phage and its receptors to initiate the infection process, which dictates host range specificity. Thus, we identified the RBP and host receptor of phiNY. Through homology alignment, amino acid sequence similarity analysis, and the phylogenetic tree analysis, orf11, located in the M-segment of dsRNA phage phiNY, encodes a putative RBP. We further performed the whole-cell enzyme-linked immunosorbent assay (ELISA), western blotting assay, and indirect immunofluorescence assay and demonstrated that this orf11 is an RBP. Finally, using affinity chromatography, ELISA, and dynamic light scattering, we identified lipopolysaccharides (LPSs) on the surface of the host M. aerodenitrificans strain LH9 as host receptors involved in the adsorption of the dsRNA bacteriophage phiNY and observed the state of phiNY RBP after combining with LPS by atomic force microscopy. These results will guide future studies on phage-host interaction in a dsRNA phage with a non-lytic life cycle.IMPORTANCEThe interactions between the lytic dsRNA phages and their host receptors have been clarified in previous studies. However, the interaction between the dsRNA phage phiNY (which has a non-lytic life cycle) and its host receptors during the dsRNA phage adsorption process was unknown. Here, we found that phiNY uses the orf11 protein as a receptor-binding protein (RBP). In addition, we found that this orf11 recognizes lipopolysaccharide from the host bacterium Microvirgula aerodenitrificans strain LH9 as a specific receptor. These results suggest that phiNY, like lytic dsRNA phages, uses an RBP to bind to a similar host receptor (i.e., lipopolysaccharide). Determining the interaction between the dsRNA phage phiNY and its host receptors will help to elucidate the mechanisms underlying the phiNY non-lytic life cycle and enhance our understanding of its infection mechanism.

Protein folding: Funnel model revised

The spatial structure of proteins, largely determined by their amino acid sequences, is also dependent on the environmental conditions under which the folding process takes place. In aqueous environments, exposure of polar amino acids is the driving factor, whereas protein stabilization in amphipathic membranes requires exposure to hydrophobic residues. This observation can be extended to all other environmental conditions under which proteins exhibit biological activity and, most importantly, to the folding process. The fuzzy oil drop (FOD) model assumes a centric location of hydrophobic residues (hydrophobic core) with exposure of polar residues towards the aqueous environment, as the influence of the aqueous environment is extended to include the contribution of other non-aqueous factors, enabling the assessment of their influence on protein structuring. The application of the modified FOD model (FOD-M) we have developed allows the environment to be represented as an external force field in the form of a continuum. The role of environmental conditions allows modification of the funnel model expressing the localization of the energy minimum as dependent on external conditions expressed by the K scale, where K measures the degree of other than polar water factors participating in folding process.