In silico identification of potential phytochemical inhibitors for mpox virus: molecular docking, MD simulation, and ADMET studies.

The mpox virus (MPXV), a member of the Poxviridae family, which recently appeared outside of the African continent has emerged as a global threat to public health. Given the scarcity of antiviral treatments for mpox disease, there is a pressing need to identify and develop new therapeutics. We investigated 5715 phytochemicals from 266 species available in IMMPAT database as potential inhibitors for six MPXV targets namely thymidylate kinase (A48R), DNA ligase (A50R), rifampicin resistance protein (D13L), palmytilated EEV membrane protein (F13L), viral core cysteine proteinase (I7L), and DNA polymerase (E9L) using molecular docking. The best-performing phytochemicals were also subjected to molecular dynamics (MD) simulations and in silico ADMET analysis. The top phytochemicals were forsythiaside for A48R, ruberythric acid for A50R, theasinensin F for D13L, theasinensin A for F13L, isocinchophyllamine for I7L, and terchebin for E9L. Interestingly, the binding energies of these potential phytochemical inhibitors were far lower than brincidofovir and tecovirimat, the standard drugs used against MPXV, hinting at better binding properties of the former. These findings may pave the way for developing new MPXV inhibitors based on natural product scaffolds. However, they must be further studied to establish their inhibitory efficacy and toxicity in in vitro and in vivo models.

Extent of Virulence and Antibiotic Resistance Genes in Helicobacter pylori and Campylobacteria.

Helicobacter pylori, a member of the clade campylobacteria, is the leading cause of chronic gastritis and gastric cancer. Virulence and antibiotic resistance of H. pylori are of great concern to public health. However, the relationship between virulence and antibiotic resistance genes in H. pylori in relation to other campylobacteria remains unclear. Using the virulence and comprehensive antibiotic resistance databases, we explored all available 354 complete genomes of H. pylori and compared it with 90 species of campylobacteria for virulence and antibiotic resistance genes/proteins. On average, H. pylori had 129 virulence genes, highest among Helicobacter spp. and 71 antibiotic resistance genes, one of the lowest among campylobacteria. Just 2.6% of virulence genes were shared by all campylobacterial members, whereas 9.4% were unique to H. pylori. The cytotoxin-associated genes (cags) seemed to be exclusive to H. pylori. Majority of the isolates from Asia and South America were cag2-negative and many antibiotic resistance genes showed isolate-specific patterns of occurrence. Just 15 (8.8%) antibiotic resistance genes, but 103 (66%) virulence genes including 25 cags were proteomically identified in H. pylori. Arcobacterial members showed large variation in the number of antibiotic resistance genes and there was a positive relation with the genome size. Large repository of antibiotic resistance genes in campylobacteria and a unique set of virulence genes might have important implications in shaping the course of virulence and antibiotic resistance in H. pylori.

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Unveiling the role of hub proteins in controlling quorum sensing regulated virulence through analogues in Pseudomonas aeruginosa PAO1: A functional protein-protein network biology approach.

The protein-protein interaction (PPI) network analysis of specific genes identified for biofilm production and virulence/secretion system mediated by quorum sensing. The PPI depicted 13 hub proteins (namely rhlR, lasR, pscU, vfr, exsA, lasI, gacA, toxA, pilJ, pscC, fleQ, algR, and chpA) out of 160 nodes involving 627 edges. The PPI network analysis based on topographical features depicted pcrD with the highest degree value and vfr gene with the greatest betweenness centrality and closeness centrality (BC and CC) values. Based on in silico results, curcumin used as an Acyl homo-serine lactone (AHL) mimicker in P. aeruginosa, was also found effective in suppressing the quorum sensing regulated virulence factors such as elastase and pyocyanin. Based on in vitro experiment, curcumin suppressed biofilm formation at 62 µg/ml concentration. Host-pathogen interaction experiment showed that curcumin was also proved to be efficient in saving C. elegans from paralysis and killing effects of P. aeruginosa PAO1.

Functional network analysis identifies multiple virulence and antibiotic resistance targets in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia, an emerging multidrug-resistant opportunistic bacterium in humans is of major concern for immunocompromised individuals for causing pneumonia and bloodborne infections. This bacterial pathogen is associated with a considerable fatality/case ratio, with up to 100%, when presented as hemorrhagic fever. It is resistant to commonly used drugs as well as to antibiotic combinations. In-silico based functional network analysis is a key approach to get novel insights into virulence and resistance in pathogenic organisms. This study included the protein-protein interaction (PPI) network analysis of 150 specific genes identified for antibiotic resistance mechanism and virulence pathways. Eight proteins, namely, PilL, FliA, Smlt2260, Smlt2267, CheW, Smlt2318, CheZ, and FliM were identified as hub proteins. Further docking studies of 58 selected phytochemicals were performed against the identified hub proteins. Deoxytubulosine and corosolic acid were found to be potent inhibitors of hub proteins of pathogenic S. maltophilia based on protein-ligand interactive study. Further pharmacophore studies are warranted with these molecules to develop them as novel antibiotics against S. maltophilia.

Prevalence and heterogeneity of antibiotic resistance genes in Orientia tsutsugamushi and other rickettsial genomes.

Despite a million infections every year and an estimated one billion people at risk, scrub typhus is regarded as a neglected tropical disease. The causative bacterium Orientia tsutsugamushi, a member of rickettsiae, seems to be intrinsically resistant to several classes of antibiotics. The emergence of antibiotic-resistant scrub typhus is likely to become a global public health concern. Yet, it is unknown as to how common antibiotic resistance genes are in O. tsutsugamushi, and how variable these loci are among the genomes of rickettsiae. By using the comprehensive antibiotic resistance database, we explored 79 complete genomes from 24 species of rickettsiae for antibiotic resistance loci. There were 244 unique antibiotic resistance genes in rickettsiae. Both the total and unique antibiotic resistance genes in O. tsutsugamushi were significantly less compared to other members of rickettsiae. However, antibiotic resistance genes in O. tsutsugamushi genomes were more unique and highly variable. Many genes such as resistant variants of evgS, and vanS A/G were present in numerous copies. These results will have important implications in the context of antibiotic-resistant scrub typhus.

Overexpression of a membrane transport system MSMEG_1381 and MSMEG_1382 confers multidrug resistance in Mycobacterium smegmatis.

Mycobacterium tuberculosis is a leading cause of human mortality worldwide, and the emergence of drug-resistant strains demands the discovery of new classes of antimycobacterial that can be employed in the therapeutic pipeline. Previously, a secondary metabolite, chrysomycin A, isolated from Streptomyces sp. OA161 displayed potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis and different species of mycobacteria. The antibiotic inhibits mycobacterial topoisomerase I and DNA gyrase, leading to bacterial death, but the mechanisms that could cause resistance to this antibiotic are currently unknown. To further understand the resistance mechanism, using M. smegmatis as a model, spontaneous resistance mutants were isolated and subjected to whole-genome sequencing. Mutation in a TetR family transcriptional regulator MSMEG_1380 was identified in the resistant isolates wherein the gene was adjacent to an operon encoding membrane proteins MSMEG_1381 and MSMEG_1382. Sequence analysis and modeling studies indicated that MSMEG_1381 and MSMEG_1382 are components of the Mmp family of efflux pumps and over-expression of either the operon or individual genes conferred resistance to chrysomycin A, isoniazid, and ethambutol. Our study highlights the role of membrane transporter proteins in conferring multiple drug resistance and the utility of recombinant strains overexpressing membrane transporters in the drug screening pipeline.

Molecular detection of monkeypox and related viruses: challenges and opportunities.

The recent widespread emergence of monkeypox (mpox), a rare and endemic zoonotic disease by monkeypox virus (MPXV), has made global headlines. While transmissibility (R0 ≈ 0.58) and fatality rate (0-3%) are low, as it causes prolonged morbidity, the World Health Organization has declared monkeypox as a public health emergency of international concern. Thus, effective containment and disease management require quick and efficient detection of MPXV. In this bioinformatic overview, we summarize the numerous molecular tests available for MPXV, and discuss the diversity of genes and primers used in the polymerase chain reaction-based detection. Over 90 primer/probe sets are used for the detection of poxviruses. While hemagglutinin and A-type inclusion protein are the most common target genes, tumor necrosis factor receptor and complement binding protein genes are frequently used for distinguishing Clade I and Clade II of MPXV. Problems and possibilities in the detection of MPXV have been discussed.

Inferring Recombination Events in SARS-CoV-2 Variants In Silico.

Over the last 34 months, at least 10 severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) distinct variants have evolved. Among these, some were more infectious while others were not. These variants may serve as candidates for identification of the signature sequences linked to infectivity and viral transgressions. Based on our previous hijacking and transgression hypothesis, we aimed to investigate whether SARS-CoV-2 sequences associated with infectivity and trespassing of long noncoding RNAs (lncRNAs) provide a possible recombination mechanism to drive the formation of new variants. This work involved a sequence and structure-based approach to screen SARS-CoV-2 variants in silico, taking into account effects of glycosylation and links to known lncRNAs. Taken together, the findings suggest that transgressions involving lncRNAs may be linked with changes in SARS-CoV-2-host interactions driven by glycosylation events.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized ß-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

Mass Spectrometry-Based Identification of Phospho-Tyr in Plant Proteomics.

O-Phosphorylation (phosphorylation of the hydroxyl-group of S, T, and Y residues) is among the first described and most thoroughly studied posttranslational modification (PTM). Y-Phosphorylation, catalyzed by Y-kinases, is a key step in both signal transduction and regulation of enzymatic activity in mammalian systems. Canonical Y-kinase sequences are absent from plant genomes/kinomes, often leading to the assumption that plant cells lack O-phospho-l-tyrosine (pY). However, recent improvements in sample preparation, coupled with advances in instrument sensitivity and accessibility, have led to results that unequivocally disproved this assumption. Identification of hundreds of pY-peptides/proteins, followed by validation using genomic, molecular, and biochemical approaches, implies previously unappreciated roles for this "animal PTM" in plants. Herein, we review extant results from studies of pY in plants and propose a strategy for preparation and analysis of pY-peptides that will allow a depth of coverage of the plant pY-proteome comparable to that achieved in mammalian systems.

CarbonylDB: a curated data-resource of protein carbonylation sites.

Motivation: Oxidative stress and protein damage have been associated with over 200 human ailments including cancer, stroke, neuro-degenerative diseases and aging. Protein carbonylation, a chemically diverse oxidative post-translational modification, is widely considered as the biomarker for oxidative stress and protein damage. Despite their importance and extensive studies, no database/resource on carbonylated proteins/sites exists. As such information is very useful to research in biology/medicine, we have manually curated a data-resource (CarbonylDB) of experimentally-confirmed carbonylated proteins/sites. Results: The CarbonylDB currently contains 1495 carbonylated proteins and 3781 sites from 21 species, with human, rat and yeast as the top three species. We have made further analyses of these carbonylated proteins/sites and presented their occurrence and occupancy patterns. Carbonylation site data on serum albumin, in particular, provides a fine model system to understand the dynamics of oxidative protein modifications/damage. Availability and implementation: The CarbonylDB is available as a web-resource and for download at http://digbio.missouri.edu/CarbonylDB/. Supplementary information: Supplementary data are available at Bioinformatics online.

Comparative proteomic analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale and different monocot species.

The rhizome is responsible for the invasiveness and competitiveness of many plants with great economic and agricultural impact worldwide. Besides its value as an invasive organ, the rhizome plays a role in the establishment and massive growth of forage, providing biomass for biofuel production. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development, and function in plants. In this work, we characterized the proteome of rhizome apical tips and elongation zones from different species using a GeLC-MS/MS (one-dimensional electrophoresis in combination with liquid chromatography coupled online with tandem mass spectrometry) spectral-counting proteomics strategy. Five rhizomatous grasses and an ancient species were compared to study the protein regulation in rhizomes. An average of 2200 rhizome proteins per species were confidently identified and quantified. Rhizome-characteristic proteins showed similar functional distributions across all species analyzed. The over-representation of proteins associated with central roles in cellular, metabolic, and developmental processes indicated accelerated metabolism in growing rhizomes. Moreover, 61 rhizome-characteristic proteins appeared to be regulated similarly among analyzed plants. In addition, 36 showed conserved regulation between rhizome apical tips and elongation zones across species. These proteins were preferentially expressed in rhizome tissues regardless of the species analyzed, making them interesting candidates for more detailed investigative studies about their roles in rhizome development.

The potato tuber mitochondrial proteome.

Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum 'Folva') and their proteome investigated. Proteins were resolved by one-dimensional gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different search programs, a total of 1,060 nonredundant proteins were identified in a quantitative manner using normalized spectral counts including as many as 5-fold more "extreme" proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression spanned 1,800-fold and included nearly all components of the electron transport chain, tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of prominent posttranslational modifications revealed that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms.

Reconstructing dynamic gene regulatory networks from sample-based transcriptional data.

The current method for reconstructing gene regulatory networks faces a dilemma concerning the study of bio-medical problems. On the one hand, static approaches assume that genes are expressed in a steady state and thus cannot exploit and describe the dynamic patterns of an evolving process. On the other hand, approaches that can describe the dynamical behaviours require time-course data, which are normally not available in many bio-medical studies. To overcome the limitations of both the static and dynamic approaches, we propose a dynamic cascaded method (DCM) to reconstruct dynamic gene networks from sample-based transcriptional data. Our method is based on the intra-stage steady-rate assumption and the continuity assumption, which can properly characterize the dynamic and continuous nature of gene transcription in a biological process. Our simulation study showed that compared with static approaches, the DCM not only can reconstruct dynamical network but also can significantly improve network inference performance. We further applied our method to reconstruct the dynamic gene networks of hepatocellular carcinoma (HCC) progression. The derived HCC networks were verified by functional analysis and network enrichment analysis. Furthermore, it was shown that the modularity and network rewiring in the HCC networks can clearly characterize the dynamic patterns of HCC progression.

Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry.

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins.

Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~100,281) in a large set of eukaryotic proteins (~22,995). Phosphorylation probability was found to be much higher in both the termini of protein sequences and this is much pronounced in transmembrane proteins. A large proportion (51.3%) of occupied sites had a nearby phosphorylation within a distance of 10 amino acids; however, this proportion is very high compared to the expected one (16.9%). The distribution of phosphorylated sites in proteins showed a strong deviation from the expected maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.

Seasonal dynamics and polyphenism of butterfly communities in the coastal plains of central Western Ghats, India.

Long-term socioeconomic progress requires a healthy environment/ecosystem, but anthropogenic activities cause environmental degradation and biodiversity loss. Constant ecological monitoring is, therefore, necessary to assess the state of biodiversity and ecological health. However, baseline data are lacking even for ecologically sensitive regions such as the Western Ghats. We looked at the seasonality and polyphenism of butterflies of the central Western Ghats to obtain baseline population patterns on these charismatic taxa. We recorded 43118 individuals (175 species) using fortnightly time-constrained counts for two consecutive years and found the peak abundance (49% of the total individuals) in the post-monsoon period (October to January). Seasonal abundance was correlated with the overall increase in species richness. Habitat differences were stronger than seasonality as samples clustered based on sites. Several species also displayed polyphenism with distinct distributions of wet and dry season forms. Seasonal equitability and indicator species analysis showed distinct inter-species differences in seasonality patterns. This work provides key baseline data on the seasonal dynamics of butterflies of the Western Ghats in the context of climate change and conservation. It will help monitor this ecologically sensitive region using butterflies.

Pattern of occurrence and occupancy of carbonylation sites in proteins.

Proteins are targets for modification by reactive oxygen species, and carbonylation is an important irreversible modification that increases during oxidative stress. While information on protein carbonylation is accumulating, its pattern is not yet understood. We have made a meta-analysis of the available literature data (456 carbonylation sites on 208 proteins) to appreciate the nature of carbonylation sites in proteins. Of the carbonylated (Arg, Lys, Pro, and Thr - RKPT) amino acids, Lys is the most abundant, whereas Pro is the most susceptible and Thr is the least susceptible. The incidence of carbonylation is lower in the N-terminal part of the protein primary sequence. Although a significantly higher number of carbonylated sites occur in Arg-, Lys-, Pro- and Thr-rich regions of proteins, the hydropathy environment of carbonylated sites is not significantly different from potential carbonylation sites. Comparison of metal-catalyzed oxidation of two closely related proteins indicates that this type of carbonylation might not be very specific in proteins. Interestingly, carbonylated sites show a very strong tendency to cluster together in the protein primary sequence hinting at some sort of discerning mechanism. While some attributes of protein carbonylation appear to be random, further investigations are warranted to appreciate the deterministic nature of protein carbonylation sites.