Development of a Spectral Library for the Discovery of Altered Genomic Events in Mycobacterium avium Associated With Virulence Using Mass Spectrometry-Based Proteogenomic Analysis.

Mycobacterium avium is one of the prominent disease-causing bacteria in humans. It causes lymphadenitis, chronic and extrapulmonary, and disseminated infections in adults, children, and immunocompromised patients. M. avium has ∼4500 predicted protein-coding regions on average, which can help discover several variants at the proteome level. Many of them are potentially associated with virulence; thus, identifying such proteins can be a helpful feature in developing panel-based theranostics. In line with such a long-term goal, we carried out an in-depth proteomic analysis of M. avium with both data-dependent and data-independent acquisition methods. Further, a set of proteogenomic investigations were carried out using (i) a protein database for Mycobacterium tuberculosis, (ii) an M. avium genome six-frame-translated database, and (iii) a variant protein database of M. avium. A search of mass spectrometry data against M. avium protein database resulted in identifying 2954 proteins. Further, proteogenomic analyses aided in identifying 1301 novel peptide sequences and correcting translation start sites for 15 proteins. Ultimately, we created a spectral library of M. avium proteins, including novel genome search-specific peptides and variant peptides detected in this study. We validated the spectral library by a data-independent acquisition of the M. avium proteome. Thus, we present an M. avium spectral library of 29,033 peptide precursors supported by 0.4 million fragment ions for further use by the biomedical community.

Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble Mycobacterium tuberculosis. Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with M. tuberculosis in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, morbidity, and mortality. Although a high-resolution LC-MS/MS-based multiprotein panel assay can be developed due to its specificity and sensitivity, it needs a library of species-specific peptides as a platform. Toward this, we performed an analysis of proteomes of 9 NTM species with more than 20 million peptide spectrum matches gathered from 26 proteome data sets. Our metaproteomic analyses determined 48,172 species-specific proteotypic peptides across 9 NTMs. Notably, M. smegmatis (26,008), M. abscessus (12,442), M. vaccae (6487), M. fortuitum (1623), M. avium subsp. paratuberculosis (844), M. avium subsp. hominissuis (580), and M. marinum (112) displayed >100 species-specific proteotypic peptides. Finally, these peptides and corresponding spectra have been compiled into a spectral library, FASTA, and JSON formats for future reference and validation in clinical cohorts by the biomedical community for further translation.

Susceptibility of Rice Crop to Salt Threat: Proteomic, Metabolomic, and Physiological Inspections.

Rice is a staple food crop worldwide; however, salinity stress is estimated to reduce its global production by 50%. Knowledge about initial molecular signaling and proteins associated with sensing salinity among crop plants is limited. We characterized early salt effects on the proteome and metabolome of rice tissues. Omics results were validated by western blotting and multiple reaction monitoring assays and integrated with physiological changes. We identified 8160 proteins and 2045 metabolites in rice tissues. Numerous signaling pathways were induced rapidly or partially by salinity. Combined data showed the most susceptible proteins or metabolites in each pathway that likely affected the sensitivity of rice to salinity, such as PLA1, BON3 (involved in sensing stress), SnRK2, pro-resilin, GDT1, G-proteins, calmodulin activators (Ca2+ and abscisic acid signaling), MAPK3/5, MAPKK1/3 (MAPK pathway), SOS1, ABC F/D, PIP2-7, and K+ transporter-23 (transporters), OPR1, JAR1, COL1, ABA2, and MAPKK3 (phytohormones). Additionally, our results expanded the stress-sensing function of receptor-like kinases, phosphatidylinositols, and Na+ sensing proteins (IPUT1). Combined analyses revealed the most sensitive components of signaling pathways causing salt-susceptibility in rice and suggested potential targets for crop improvement.

Physiological Temperature and Osmotic Changes Drive Dynamic Proteome Alterations in the Leptospiral Outer Membrane and Enhance Protein Export Systems.

Leptospirosis, a remerging zoonosis, has no effective vaccine or an unambiguous early diagnostic reagent. Proteins differentially expressed (DE) under pathogenic conditions will be useful candidates for antileptospiral measures. We employed a multipronged approach comprising high-resolution TMT-labeled LC-MS/MS-based proteome analysis coupled with bioinformatics on leptospiral proteins following Triton X-114 subcellular fractionation of leptospires treated under physiological temperature and osmolarity that mimic infection. Although there were significant changes in the DE proteins at the level of the entire cell, there were notable changes in proteins at the subcellular level, particularly on the outer membrane (OM), that show the significance of subcellular proteome analysis. The detergent-enriched proteins, representing outer membrane proteins (OMPs), exhibited a dynamic nature and upregulation under various physiological conditions. It was found that pathogenic proteins showed a higher proportion of upregulation compared to the nonpathogenic proteins in the OM. Further analysis identified 17 virulent proteins exclusively upregulated in the outer membrane during infection that could be useful for vaccine and diagnostic targets. The DE proteins may aid in metabolic adaptation and are enriched in pathways related to signal transduction and antibiotic biosynthesis. Many upregulated proteins belong to protein export systems such as SEC translocase, T2SSs, and T1SSs, indicating their sequential participation in protein transport to the outer leaflet of the OM. Further studies on OM-localized proteins may shed light on the pathogenesis of leptospirosis and serve as the basis for effective countermeasures.

Helicobacter pylori regulated microRNA map of human gastric cells.

BACKGROUND: Helicobacter pylori is an infection of concern for its chronic colonization leading to peptic ulcers and gastric cancer. In recent times, microRNAs have been extensively studied to understand their role in the pathogenesis of this bacteria in diverse contexts of gastric diseases. The current analysis reports the microRNA-mRNA interactions that are associated with effective survival and virulence of this pathogen. MATERIALS AND METHODS: We convened differentially regulated human microRNAs responsive to H. pylori infection (HP-hDEmiRs) at different multiplicity of infection and time points in human gastric cell lines through retrospective data mining of experimental studies. In view of the molecular disparity of clinical samples and animal models, data from tissue, serum/plasma, urine, and ascites were excluded. Further, we utilized diverse bioinformatics approaches to retrieve experimentally validated, high-confidence targets of the HP-hDEmiRs to analyze the microRNA-mRNA interactions that are relevant to H. pylori pathogenesis. RESULTS: A total of 39 HP-hDEmiRs that showed unidirectional expression of either overexpression or downregulation were identified to modulate 23 targets explicitly studied under this infection. We also identified 476 experimentally validated targets regulated by at least 4 of the HP-hDEmiRs. In addition to the pathways prior-associated with H. pylori infection, the microRNA-mRNA interactome analysis identified several cellular processes and pathways highly associated with cell cycle, cell division, migration, and carcinogenesis. CONCLUSION: This study generated a platform to study the mechanisms utilized by this pathogen using microRNAs as surrogate.

Proteomic Analysis of Adult Human Hippocampal Subfields Demonstrates Regional Heterogeneity in the Protein Expression.

Background: Distinct hippocampal subfields are known to get affected during aging, psychiatric disorders, and various neurological and neurodegenerative conditions. To understand the biological processes associated with each subfield, it is important to understand its heterogeneity at the molecular level. To address this lacuna, we investigated the proteomic analysis of hippocampal subfields─the cornu ammonis sectors (CA1, CA2, CA3, CA4) and dentate gyrus (DG) from healthy adult human cohorts. Findings: Microdissection of hippocampal subfields from archived formalin-fixed paraffin-embedded tissue sections followed by TMT-based multiplexed proteomic analysis resulted in the identification of 5,593 proteins. Out of these, 890 proteins were found to be differentially abundant among the subfields. Further bioinformatics analysis suggested proteins related to gene splicing, transportation, myelination, structural activity, and learning processes to be differentially abundant in DG, CA4, CA3, CA2, and CA1, respectively. A subset of proteins was selected for immunohistochemistry-based validation in an independent set of hippocampal samples. Conclusions: We believe that our findings will effectively pave the way for further analysis of the hippocampal subdivisions and provide awareness of its subfield-specific association to various neurofunctional anomalies in the future. The current mass spectrometry data is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029697.

Quantitative phosphoproteomics reveals diverse stimuli activate distinct signaling pathways during neutrophil activation.

Neutrophils display functional heterogeneity upon responding diversely to physiological and pathological stimulations. During type 2 diabetes (T2D), hyperglycemia constitutively activates neutrophils, leading to reduced response to infections and on the other hand, elevated metabolic intermediates such as homocysteine induce bidirectional activation of platelets and neutrophils leading to thrombosis. Hence, in the context of T2D-associated complications, we examined the influence of high glucose, homocysteine, and LPS representing effector molecules of hyperglycemia, thrombosis, and infection, respectively, on human neutrophil activation to identify distinct signaling pathways by quantitative phosphoproteomics approach. High glucose activated C-Jun-N-Terminal Kinase, NTRK1, SYK, and PRKACA kinases associated with Rho GTPase signaling and phagocytosis, whereas LPS induced AKT1, SRPK2, CSNK2A1, and TTN kinases involved in cytokine signaling and inflammatory response. Homocysteine treatment led to activatation of  LRRK2, FGR, MAPK3, and PRKCD kinases which are associated with neutrophil degranulation and cytoskeletal remodeling. Diverse inducers differentially modulated phosphorylation of proteins associated with neutrophil functions such as oxidative burst, degranulation, extracellular traps, and phagocytosis. Further validation of phosphoproteomics data on selected kinases revealed neutrophils pre-cultured under high glucose showed impeded response to LPS to phosphorylate p-ERK1/2Thr202/Tyr204, p-AKTSer473, and C-Jun-N-Terminal KinaseSer63 kinases. Our study provides novel phosphoproteome signatures that may be explored to understand neutrophil biology in T2D-associated complications.

Metabolite profiling reveals overexpression of the global regulator, MoLAEA leads to increased synthesis of metabolites in Magnaporthe oryzae.

AIMS: To study the altered metabolic pathways and metabolites produced in overexpression and knockdown mutants of a global regulator named MoLAEA, which was recently found to regulate the expression of the genes involved in secondary metabolism in one of the most destructive plant pathogens, Magnaporthe oryzae. METHODS AND RESULTS: Mass spectrometry-based global untargeted metabolomic profiling was used to identify altered metabolites. Metabolites were extracted from the mutant strains of MoLAEA using two extraction methods viz., aqueous and organic extraction and data acquired using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative polarities. Levels of metabolites involved in various biological pathways such as amino acid as well as polyamine biosynthesis, fatty acid and pyrimidine metabolism showed a remarkable change in the mutant strains. Interestingly, metabolites involved in stress responses were produced in higher quantities in the overexpression strain, whereas certain overproduced metabolites were associated with distinctive phenotypic changes in the overexpression strain compared with the wild type. Further, the expression of several genes involved in the stress responses was found to have higher expression in the overexpression strain. CONCLUSIONS: The global regulator MoLAEA is involved in secondary metabolism in the plant pathogen M. oryzae such that the mutant strains showed an altered level of several metabolites involved in the biosynthesis pathways compared with the wild type. Also, metabolites involved in stress responses were overproduced in the overexpression strain and this can be seen in the higher growth in media amended with stress-inducing agents or a higher expression of genes involved in stress response in the overexpression strain compared with the wild type. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of metabolite profiling relative to the global regulation of secondary metabolism in M. oryzae, where secondary metabolism is poorly understood. It opens up avenues for more relevant investigations on the genetic regulation of several of the metabolites found in the analysis, which have not been previously characterized in M. oryzae.

Metabolomics analysis highlights Yashtimadhu (Glycyrrhiza glabra L.)-mediated neuroprotection in a rotenone-induced cellular model of Parkinson's disease by restoring the mTORC1-AMPK1 axis in autophagic regulation.

Parkinson's disease (PD) is an age-associated progressive neurodegenerative movement disorder, and its management strategies are known to cause complications with prolonged usage. We aimed to explore the neuroprotective mechanism of the Indian traditional medicine Yashtimadhu, prepared from the dried roots of Glycyrrhiza glabra L. (licorice) in the rotenone-induced cellular model of PD. Retinoic acid-differentiated IMR-32 cells were treated with rotenone (PD model) and Yashtimadhu extract. Mass spectrometry-based untargeted and targeted metabolomic profiling was carried out to discover altered metabolites. The untargeted metabolomics analysis highlighted the rotenone-induced dysregulation and Yashtimadhu-mediated restoration of metabolites involved in the metabolism of nucleic acids, amino acids, lipids, and citric acid cycle. Targeted validation of citric acid cycle metabolites showed decreased α-ketoglutarate and succinate with rotenone treatment and rescued by Yashtimadhu co-treatment. The dysregulation of the citric acid cycle by rotenone-induced energetic stress via dysregulation of the mTORC1-AMPK1 axis was prevented by Yashtimadhu. Yashtimadhu co-treatment restored rotenone-induced ATG7-dependent autophagy and eventually caspases-mediated cell death. Our analysis links the metabolic alterations modulating energy stress and autophagy, which underlies the Yashtimadhu-mediated neuroprotection in the rotenone-induced cellular model of PD.

Deep Proteome Profiling of Semen of Indian Indigenous Malnad Gidda (Bos indicus) Cattle.

Malnad Gidda is a dwarf indigenous cattle breed of India, which is known for its uniqueness of calving every year under a low input grazing system of rearing. Bulls of Malnad Gidda are known to be highly fertile even in stress conditions. However, the proteomic profiling of semen of this breed has not been investigated so far, which might provide a platform for a better understanding of its semen quality and male fertility. Therefore, we made an effort to characterize and quantify the proteome of seminal plasma and spermatozoa components of Malnad Gidda semen using a high-resolution mass spectrometry platform. We identified 2814 proteins from spermatozoa and 1974 proteins from the seminal plasma of this breed. Furthermore, >90% of proteins from each fraction were quantified using the intensity-based absolute quantification. We observed signal peptides in 33% of seminal plasma proteins, indicating their secretory nature. Gene Ontology analysis revealed their involvement in cytoskeletal assembly associated with sperm head, sperm motility, acrosome reaction, seminal plasma binding, and spermatogenesis-associated protein. An in-depth proteome profiling of semen of a unique indigenous cattle breed of India was carried out. Our findings could provide a reference for further studies on sperm functions, semen quality, and reproductive health of Bos indicus cattle. Mass spectrometry data generated in this study is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014172.

Metabolomic fingerprinting of bull spermatozoa for identification of fertility signature metabolites.

The objective of the study was to identify the fertility-associated metabolites in bovine spermatozoa using liquid chromatography-mass spectrometry (LC-MS). Six Holstein Friesian crossbred bulls (three high-fertile and three low-fertile bulls) were the experimental animals. Sperm proteins were isolated and protein-normalized samples were processed for metabolite extraction and subjected to LC-MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high- and low-fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high-fertile compared to low-fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d-cysteine, selenocystine. In addition, metabolites such as spermine and l-cysteine were identified exclusively in high-fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high- and low-fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l-malic acid, d-cysteine, and chondroitin 4-sulfate hold the potential to be recognized as fertility-associated metabolites.

Multi-Omics Driven Assembly and Annotation of the Sandalwood (Santalum album) Genome.

Indian sandalwood (Santalum album) is an important tropical evergreen tree known for its fragrant heartwood-derived essential oil and its valuable carving wood. Here, we applied an integrated genomic, transcriptomic, and proteomic approach to assemble and annotate the Indian sandalwood genome. Our genome sequencing resulted in the establishment of a draft map of the smallest genome for any woody tree species to date (221 Mb). The genome annotation predicted 38,119 protein-coding genes and 27.42% repetitive DNA elements. In-depth proteome analysis revealed the identities of 72,325 unique peptides, which confirmed 10,076 of the predicted genes. The addition of transcriptomic and proteogenomic approaches resulted in the identification of 53 novel proteins and 34 gene-correction events that were missed by genomic approaches. Proteogenomic analysis also helped in reassigning 1,348 potential noncoding RNAs as bona fide protein-coding messenger RNAs. Gene expression patterns at the RNA and protein levels indicated that peptide sequencing was useful in capturing proteins encoded by nuclear and organellar genomes alike. Mass spectrometry-based proteomic evidence provided an unbiased approach toward the identification of proteins encoded by organellar genomes. Such proteins are often missed in transcriptome data sets due to the enrichment of only messenger RNAs that contain poly(A) tails. Overall, the use of integrated omic approaches enhanced the quality of the assembly and annotation of this nonmodel plant genome. The availability of genomic, transcriptomic, and proteomic data will enhance genomics-assisted breeding, germplasm characterization, and conservation of sandalwood trees.

Phosphoproteomics of Retinoblastoma: A Pilot Study Identifies Aberrant Kinases.

Retinoblastoma is a malignant tumour of the retina which most often occurs in children. Earlier studies on retinoblastoma have concentrated on the identification of key players in the disease and have not provided information on activated/inhibited signalling pathways. The dysregulation of protein phosphorylation in cancer provides clues about the affected signalling cascades in cancer. Phosphoproteomics is an ideal tool for the study of phosphorylation changes in proteins. Hence, global phosphoproteomics of retinoblastoma (RB) was carried out to identify signalling events associated with this cancer. Over 350 proteins showed differential phosphorylation in RB compared to control retina. Our study identified stress response proteins to be hyperphosphorylated in RB which included H2A histone family member X (H2AFX) and sirtuin 1. In particular, Ser140 of H2AFX also known as gamma-H2AX was found to be hyperphosphorylated in retinoblastoma, which indicated the activation of DNA damage response pathways. We also observed the activation of anti-apoptosis in retinoblastoma compared to control. These observations showed the activation of survival pathways in retinoblastoma. The identification of hyperphosphorylated protein kinases including Bromodomain containing 4 (BRD4), Lysine deficient protein kinase 1 (WNK1), and Cyclin-dependent kinase 1 (CDK1) in RB opens new avenues for the treatment of RB. These kinases can be considered as probable therapeutic targets for RB, as small-molecule inhibitors for some of these kinases are already in clinical trials for the treatment other cancers.

Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

Host response profile of human brain proteome in toxoplasma encephalitis co-infected with HIV.

BACKGROUND: Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection. RESULTS: We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism. CONCLUSIONS: Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.

Proteogenomic analysis of pathogenic yeast Cryptococcus neoformans using high resolution mass spectrometry.

BACKGROUND: Cryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis. Owing to an increase in the number of immunosuppressed individuals along with emergence of drug-resistant strains, C. neoformans is gaining importance as a pathogen. Although, whole genome sequencing of three varieties of C. neoformans has been completed recently, no global proteomic studies have yet been reported. RESULTS: We performed a comprehensive proteomic analysis of C. neoformans var. grubii (Serotype A), which is the most virulent variety, in order to provide protein-level evidence for computationally predicted gene models and to refine the existing annotations. We confirmed the protein-coding potential of 3,674 genes from a total of 6,980 predicted protein-coding genes. We also identified 4 novel genes and corrected 104 predicted gene models. In addition, our studies led to the correction of translational start site, splice junctions and reading frame used for translation in a number of proteins. Finally, we validated a subset of our novel findings by RT-PCR and sequencing. CONCLUSIONS: Proteogenomic investigation described here facilitated the validation and refinement of computationally derived gene models in the intron-rich genome of C. neoformans, an important fungal pathogen in humans.

Guardians Turned Culprits: NETosis and Its Influence on Pulmonary Fibrosis Development.

Idiopathic pulmonary fibrosis (IPF) is a debilitating, life-threatening irreversible lung disease characterized by the excessive accumulation of fibrotic tissue in the lungs, impairing their function. The exact mechanisms underlying Pulmonary fibrosis (PF) are multifaceted and not yet fully understood. Reports show that during COVID-19 pandemic, PF was dramatically increased due to the hyperactivation of the immune system. Neutrophils and macrophages are the patrolling immune cells that keep the microenvironment balanced. Neutrophil extracellular traps (NETs) are a normal protective mechanism of neutrophils. The chief components of the NETs include DNA, citrullinated histones, and anti-microbial peptides which are released by the activated neutrophils. However, it is becoming increasingly evident that hyperactivation of immune cells can also turn into criminals when it comes to pathological state. Dysregulated NETosis may contribute to sustained inflammation, overactivation of fibroblasts, and ultimately promoting collagen deposition which is the characteristic feature of PF. The role of NETs along with inflammation is attaining greater attention. However, seldom researches are related to the relationship between NETs causing PF. This review highlights the cellular mechanism of NETs-induced pulmonary fibrosis, which could give a better understanding of molecular targets which may be helpful for treating NETs-induced PF.

Unpacking Immune Modulation as a Site of Therapeutics Innovation for Nematode Parasite Wuchereria bancrofti: A Temporal Quantitative Phosphoproteomics Profiling of Macrophage Migration Inhibitory Factor 2.

Nematode infections are common in both humans and livestock, with major adverse planetary health and economic impacts. Wuchereria bancrofti is a parasitic nematode that causes lymphatic filariasis, a neglected tropical disease that can lead to severe disability and deformity worldwide. For the long-term survival of the bancroftian parasites in the host, a complex immune invasion strategy is involved through immunomodulation. Therefore, immunomodulation can serve as a site of research and innovation for molecular targets. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine crucial to the host antimicrobial alarm system and stress response. Interestingly, the nematode parasite W. bancrofti also produces two homologs of MIF (Wba-MIF1 and 2). Using a mass spectrometry-based phosphoproteomics approach, we report new findings on the immunomodulatory effect and signaling mechanism of Wba-MIF2 in macrophage cells. Accordingly, we observed 1201 phosphorylated sites on 467 proteins. Out of the 1201 phosphorylated sites, 1075, 117, and 9 were found on serine (S), threonine (T), and tyrosine (Y) residues, respectively. Our bioinformatics analysis led to identification of major pathways, including spliceosomes, T cell receptor signaling pathway, Th17 differentiation pathway, interleukin-17 signaling pathway, and insulin signaling pathway upon Wba-MIF2 treatment. Wba-MIF2 treatment also enriched CDK4, CDK1, and DNAPK kinases. The comparison of the signaling pathway of Wba-MIF2 with that of human-MIF suggests both share similar signaling pathways. These findings collectively offer new insights into the role and mechanism of Wba-MIF2 as an immunomodulator and inform future diagnostics and drug discovery research for W. bancrofti.

Cisplatin and Procaterol Combination in Gastric Cancer? Targeting Checkpoint Kinase 1 for Cancer Drug Discovery and Repurposing by an Integrated Computational and Experimental Approach.

Checkpoint kinase 1 (CHK1), a serine/threonine kinase, plays a crucial role in cell cycle arrest and is a promising therapeutic target for drug development against cancers. CHK1 coordinates cell cycle checkpoints in response to DNA damage, facilitating repair of single-strand breaks, and maintains the genome integrity in response to replication stress. In this study, we employed an integrated computational and experimental approach to drug discovery and repurposing, aiming to identify a potent CHK1 inhibitor among existing drugs. An e-pharmacophore model was developed based on the three-dimensional crystal structure of the CHK1 protein in complex with CCT245737. This model, characterized by seven key molecular features, guided the screening of a library of drugs through molecular docking. The top 10% of scored ligands were further examined, with procaterol emerging as the leading candidate. Procaterol demonstrated interaction patterns with the CHK1 active site similar to CHK1 inhibitor (CCT245737), as shown by molecular dynamics analysis. Subsequent in vitro assays, including cell proliferation, colony formation, and cell cycle analysis, were conducted on gastric adenocarcinoma cells treated with procaterol, both as a monotherapy and in combination with cisplatin. Procaterol, in synergy with cisplatin, significantly inhibited cell growth, suggesting a potentiated therapeutic effect. Thus, we propose the combined application of cisplatin and procaterol as a novel potential therapeutic strategy against human gastric cancer. The findings also highlight the relevance of CHK1 kinase as a drug target for enhancing the sensitivity of cytotoxic agents in cancer.

Comparative Metabolomics and Network Pharmacology Analysis Reveal Shared Neuroprotective Mechanisms of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb.

Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., two nootropics, are recognized in Indian Ayurvedic texts. Studies have attempted to understand their action as memory enhancers and neuroprotectants, but many molecular aspects remain unknown. We propose that Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb. share common neuroprotective mechanisms. Mass spectrometry-based untargeted metabolomics and network pharmacology approach were used to identify potential protein targets for the metabolites from each extract. Phytochemical analyses and cell culture validation studies were also used to assess apoptosis and ROS activity using aqueous extracts prepared from both herbal powders. Further, docking studies were also performed using the LibDock protocol. Untargeted metabolomics and network pharmacology approach unveiled 2751 shared metabolites and 3439 and 2928 non-redundant metabolites from Bacopa monnieri and Centella asiatica extracts, respectively, suggesting a potential common neuroprotective mechanism among these extracts. Protein-target prediction highlighted 92.4% similarity among the proteins interacting with metabolites for these extracts. Among them, kinases mapped to MAPK, mTOR, and PI3K-AKT signaling pathways represented a predominant population. Our results highlight a significant similarity in the metabolome of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., and their potential protein targets may be attributed to their common neuroprotective functions.