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The latex of the medicinal plant Artocarpus lakoocha (A. lakoocha) , which has been shown to have potential anti-inflammatory and wound-healing capabilities, contains a novel heme-peroxidase. This protein was subjected to activity assays, fluorescence spectroscopy, and far-UV circular dichroism to investigate its structure, dynamics, and stability. The results demonstrated the presence of three folding states: the native state (N) at neutral pH, intermediate states including molten globule (MG) at pH 2 and acid-unfolded (UA) at pH 1.5 or lower, and acid-refolded (A) at pH 0.5, along with alkaline denatured (UB) at pH 8-12 and the third denatured state (D) at GuHCl concentrations exceeding 5 M. Absorbance studies indicated the presence of loosely associated form of heme in the pH range of 1-2. The protein showed stability and structural integrity across a wide pH range (3-10), temperature (70°C), and high concentrations of GuHCl (5 M) and urea (8 M). This study is the first to report multiple 'partially folded intermediate states' of A. lakoocha peroxidase, with varying amounts of secondary structure, stability, and compactness. These results demonstrate the high stability of A. lakoocha peroxidase and its potential for biotechnological and industrial applications, making it a valuable model system for further studies on its structure-function relationship., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sonkar, Pachauri, Kumar, Choudhary and Jagannadham.)

Membrane vesicles (MVs) serve as an essential virulence factor in several pathogenic bacteria. The release of MVs by Listeria monocytogenes is only recently recognized; still, the enigmatic role of MVs in pathogenesis is yet to be established. We report the transcriptome response of Caco-2 cells upon exposure to MVs and the L. monocytogenes that leads to observe the up-regulation of autophagy-related genes in the early phase of exposure to MVs. Transcription of inflammatory cytokines is to the peak at the fourth hour of exposure. An array of differentially expressed genes was associated with actin cytoskeleton rearrangement, autophagy, cell cycle arrest, and induction of oxidative stress. At a later time point, transcriptional programs are generated upon interaction with MVs to evade innate immune signals, by modulating the expression of anti-inflammatory genes. KEGG pathway analysis is palpably confirming that MVs appear principally responsible for the induction of immune signaling pathways. Besides, MVs induced the expression of cell cycle regulatory genes, likely responsible for the ability to prolong host cell survival, thus protecting the replicative niche for L. monocytogenes . Notably, we identified several non-coding RNAs (ncRNAs), possibly involved in the regulation of early manipulation of the host gene expression, essential for the persistence of L. monocytogenes ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

Outer membrane vesicles (OMVs) of Acinetobacter baumannii DS002 carry proteins which perform selective biological functions. The proteins involved in cell wall/membrane biogenesis and inorganic ion transport and metabolism occupied a significant portion of the 302 proteins associated with OMVs. Interestingly, the TonB-dependent transporters (TonRs), linked to the active transport of nutrients across the energy-deprived outer membrane, are predominant among proteins involved in inorganic ion transport and metabolism. The OMVs of DS002 contain TonRs capable of transporting iron complexed to catecholate, hydroximate, and mixed types of siderophores. Consistent with this observation, the OMVs were firmly bound to ferric-enterobactin ( 55 Fe-Ent) and successfully transported iron into A. baumannii DS002 cells grown under iron-limiting conditions. In addition to the TonRs, OMVs also carry proteins known to promote pathogenesis, immune evasion, and biofilm formation. Our findings provide conclusive evidence for the role of OMVs in the transport of nutrients such as iron and show the presence of proteins with proven roles in pathogenicity and immune response. IMPORTANCE TonB-dependent transporters (TonRs) play a crucial role in transporting nutrients such as iron, nickel, copper, and complex carbohydrates across the energy-deprived outer membrane. Due to their unique structural features, TonRs capture nutrients in an energy-independent manner and transport them across the outer membrane by harvesting energy derived from the inner membrane-localized Ton-complex. In this study, we report the presence of TonRs capable of transporting various nutrients in OMVs and demonstrate their role in capturing and transporting ferric iron complexed with enterobactin into A. baumannii DS002 cells. The OMV-associated TonRs appear to play a critical role in the survival of A. baumannii, listed as a priority pathogen, under nutrient-deprived conditions.

Exploring the molecular mechanisms behind bacterial adaptation to extreme temperatures has potential biotechnological applications. In the present study, Pseudomonas sp. Lz4W, a Gram-negative psychrophilic bacterium adapted to survive in Antarctica, was selected to decipher the molecular mechanism underlying the cold adaptation. Proteome analysis of the isolates grown at 4°C was performed to identify the proteins and pathways that are responsible for the adaptation. However, many proteins from the expressed proteome were found to be hypothetical proteins (HPs), whose function is unknown. Investigating the functional roles of these proteins may provide additional information in the biological understanding of the bacterial cold adaptation. Thus, our study aimed to assign functions to these HPs and understand their role at the molecular level. We used a structured insilico workflow combining different bioinformatics tools and databases for functional annotation. Pseudomonas sp. Lz4W genome (CP017432, version 1) contains 4493 genes and 4412 coding sequences (CDS), of which 743 CDS were annotated as HPs. Of these, from the proteome analysis, 61 HPs were found to be expressed consistently at the protein level. The amino acid sequences of these 61 HPs were submitted to our workflow and we could successfully assign a function to 18 HPs. Most of these proteins were predicted to be involved in biological mechanisms of cold adaptations such as peptidoglycan metabolism, cell wall organization, ATP hydrolysis, outer membrane fluidity, catalysis, and others. This study provided a better understanding of the functional significance of HPs in cold adaptation of Pseudomonas sp. Lz4W. Our approach emphasizes the importance of addressing the "hypothetical protein problem" for a thorough understanding of mechanisms at the cellular level, as well as, provided the assessment of integrating proteomics methods with various annotation and curation approaches to characterize hypothetical or uncharacterized protein data. The MS proteomics data generated from this study has been deposited to the ProteomeXchange through PRIDE with the dataset identifier-PXD029741., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ijaq, Chandra, Ray and Jagannadham.)

Sequence determination of peptides is a crucial step in mass spectrometry-based proteomics. Peptide sequences are determined either by database search or by de novo sequencing using tandem mass spectrometry. Determination of all the theoretical expected peptide fragments and eliminating false discoveries remains a challenge in proteomics. Developing standards for evaluating the performance of mass spectrometers and algorithms used for identification of proteins is important for proteomics studies. The current study is focused on these aspects by using synthetic peptides. A total of 599 peptides were designed from in silico tryptic digest with 1 or 2 missed cleavages from 199 human proteins, and synthetic peptides corresponding to these sequences were obtained. The peptides were mixed together, and analysis was carried out using liquid chromatography-electrospray ionization tandem mass spectrometry on a Q-Exactive HF mass spectrometer. The peptides and proteins were identified with SEQUEST program. The analysis was carried out using the proteomics workflows. A total of 573 peptides representing 196 proteins could be identified, and a spectral library was created for these peptides. Analysis parameters such as "no enzyme selection" gave the maximum number of detected peptides as compared with trypsin in the selection. False discoveries could be identified. This study highlights the limitations of peptide detection and the need for developing powerful algorithms along with tools to evaluate mass spectrometers and algorithms. It also shows the limitations of peptide detection even with high-end mass spectrometers. The mass spectral data are available in ProteomeXchange with accession no. PXD017992., (© Association of Biomolecular Resource Facilities.)

Membrane vesicles (MVs) are naturally secreted by many pathogenic organisms and have various functions that include the release of microbial virulence factors that contributes to pathogenesis. However, very little is known regarding the function of Gram-positive bacteria membrane vesicles. Here, we investigated the functional role of membrane vesicles of Listeria monocytogenes. We found that L. monocytogenes secreted MVs are spherical and diameter size around 192.3 nm. Here, we investigated the role of L. monocytogenes membrane vesicles in interbacterial communication to cope with antibiotic stress. We found that MVs are protecting the bacteria against the antibiotics trimethoprim and streptomycin. These MVs enabled streptomycin-susceptible L. monocytogenes 1143 to survive in the presence of streptomycin. The zeta potential, dynamic light scattering (DLS) and 1-Nphenylnapthylamine (NPN)-uptake assay reveals that MVs protect the bacterium from active antibiotics by different strategies. Exposure to environmental stressors was shown to increase the level of MV production in L. monocytogenes. The biological activity of MV-associated listeriolysin O, internalin B, and phosphatidylinositol-specific phospholipase C (PI-PLC) was investigated using epithelial cell cytotoxicity. The reduced cytotoxicity was observed in Δhly MVs on Caco-2 cells suggesting that MVs are biologically active. It is shown that a potent toxin LLO contributes to the MV mediated pathogenesis of L. monocytogenes., Competing Interests: Declaration of competing interest The authors declare no competing interests of this study., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose : Glycoproteins play an important role in human mucosal defenses and immunity-related cell-to-cell interactions. The aim of the present study is to investigate the presence and patterns of lacrimal sac glycoproteins involved in defense mechanisms with a special reference to prolactin-inducible protein (PIP). Methods : The study was performed on healthy lacrimal sacs obtained from exenteration samples immediately after surgery and frozen at -80 degrees for subsequent analysis. Four lectins namely Concanavalin A (Con A), Dolichos lablab lectin (DLL), Wheat Germ agglutinin (WGA), and Momordica charantia lectin (MCL) were purified by affinity chromatography. Soluble proteins extract of the lacrimal sac was subjected to chromatography on lectin-affigel columns. Eluted samples from each of the lectin coupled-affigels were analyzed by 10% SDS-PAGE under reducing conditions and the protein bands were visualized using Coomassie blue stain. The protein gel bands were further subjected to mass spectrometry for glycoprotein analysis. Results : Mass spectrometry identified several glycoproteins from the lacrimal sac extracts, with known roles in defense mechanisms. The number of such glycoproteins identified were 9 each from Con A and DLL-I affinity eluted gel bands and 8 and 14 from MCL and WGA affinity eluted gel bands, respectively. Interestingly, PIP was detected in significant proportions in all the eluted gel bands with WGA showing the highest expression. Conclusions : This study is the first step towards the lacrimal sac glycoprotein profiling. PIP could be a major lead for further work on the etiopathogenesis of lacrimal drainage obstructions.

Membrane vesicles (MVs) are produced by various Gram positive and Gram negative pathogenic bacteria and play an important role in virulence. In this study, the membrane vesicles (MVs) of L. monocytogenes were isolated from the culture supernatant. High-resolution electron microscopy and dynamic light scattering analysis revealed that L. monocytogenes MVs are spherical with a diameter of 200 to 300 nm in size. Further, comprehensive proteomic analyses of MVs and whole cells of L. monocytogenes were performed using LC/MS/MS. A total of 1355 and 312 proteins were identified in the L. monocytogenes cells and MVs, respectively. We identified that 296 proteins are found in both whole cells, and MV proteome and 16 proteins were identified only in the MVs. Also, we have identified the virulence factors such as listeriolysin O (LLO), internalin B (InlB), autolysin, p60, NLP/P60 family protein, UPF0356 protein, and PLC-A in MVs. Computational prediction of host-MV interactions revealed a total of 1841 possible interactions with the host involving 99 MV proteins and 1513 host proteins. We elucidated the possible pathway that mediates internalization of L. monocytogenes MV to host cells and the subsequent pathogenesis mechanisms. The in vitro infection assays showed that the purified MVs could induce cytotoxicity in Caco-2 cells. Using endocytosis inhibitors, we demonstrated that MVs are internalized via actin-mediated endocytosis. These results suggest that L. monocytogenes MVs can interact with host cell and contribute to the pathogenesis of L. monocytogenes during infection., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Microorganisms thrive in extreme environments and are known for synthesizing valuable metabolites. Salt-loving microorganisms can flourish in saline environments which inhibit the growth of other microbial life, and they possess the potential to produce stable and novel biomolecules for the use in biotechnological applications, including anticancer compounds. Sambhar Lake is the largest inland soda lake in India and is an appropriate habitat for halophilic bacterial and archaeal strains in terms of diversity and potential production of bioactive compounds. In the present study, a moderately halo-alkaliphilic bacterial strain C12A1 was isolated from Sambhar Lake, located in Rajasthan, India. C12A1 was gram-positive, motile, rod-shaped, formed oval endospores, produced carotenoids, and exhibited optimal growth at 37 °C in 10⁻15% NaCl (pH 8). C12A1 was found to be able to hydrolyze skimmed milk, gelatin, and Tween 80 but unable to hydrolyze starch and carboxymethylcellulose. C12A1 showed 98.87% and 98.50% identity in 16S rRNA gene sequence to P. halophilus and P. salipiscarius , respectively. Nevertheless, C12A1 was clustered within the clade consisting of P. salipiscarius strains, but it showed a distinct lineage. Thus, C12A1 was designated as Piscibacillus sp. Cell proliferation assay results showed that C12A1 broth extract (BEP) decreased cell viability in breast cancer MDA-MB-231 cells, which was confirmed by the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Induction of cell toxicity was visualized by microscopy. Reverse Transcriptase PCR (RT-PCR) analysis demonstrated that BEP inhibited the expression of proliferative B-cell lymphoma-extra large (Bcl-xL) and cell cycle marker Cyclin-dependent kinase 2 (CDK2) at transcript levels. Similarly, cell migration and colony formation along with mesenchymal marker vimentin and stem cell marker BMI transcripts were found to be inhibited when cells were treated with the BEP. The anti-breast cancer potential of C12A1 indicates that microorganisms inhabiting saline-alkaline habitats, with Piscibacillus sp. in particular, are a promising source for discovery of novel bioactive substances.

The detection of post-translational modifications of proteins is an important comprehensive research area. Over the years, proteomic studies involving protein acetylation have attracted a great deal of attention. In the present study, we have focussed on the acetylation of histidine and the intrinsic stability of b 1 -ion of oxazolone ring and/or with side chain imidazole bicyclic product. The formation of oxazolone structure may occur when an amino moiety undergoes acetylation reaction and when it is present in the vicinity of the side chain imidazole moiety. Tryptic peptides generated from the proteins of Acenitobacter radioresistens MMC5-containing N-terminal histidine were explored in a standard proteomic workflow. Formation of [Formula: see text] ion with an oxazolone ring in these peptides has been supported by a tandem mass spectrometric study of a synthetic peptide and density functional theory calculations. The results obtained from this study have implications in understanding the fragmentation of the peptides generated in the proteomic workflows.

A wide variety of post-translational modifications such as oxidation, phosphorylation, glycosylation, methylation, and acetylation play critical roles in cellular functions. Detection of post-translational modifications in proteins is important to understand their crucial roles in cellular functions. Identifying each modification requires special attention in mass spectral acquisition and analysis. Here, we report a mass spectral method for the detection of multiple phosphorylations in peptides by analyzing their products after fragmentation. Synthetic peptides were used to identify these modifications by matrix-assisted laser desorption/ionization (MALDI) TOF/TOF. Peptides with serine, threonine, and tyrosine were used with mono- to tetra-phosphorylation sites in different combinations to get insights into their fragmentation and identify the location of these sites. The y-ion series were observed without the loss of phosphate groups and were thus very useful in determining the localization and sequence of the phosphate residues. Acetylation of the peptides was found to be useful in detecting the b1-ion and helped in identifying the N-terminus. When a mixture of the phosphorylated peptides (from mouse protein sequences) were analyzed by LC-MS/MS on a Velos Orbitrap Mass Spectrometer and the data subjected to analysis by Sequest using the mouse database, the peptides were identified along with the parent proteins. A comparison of MALDI TOF/TOF spectra with ESI MS/MS helped in eliminating falsely discovered peptides using the database search.

Euphorbia tirucalli peroxidase (ETP) was immobilized on chitosan beads having magnetic properties for the ease of separation and increasing the reusability of ETP for cost effective assay conditions. The present work reports immobilization of ETP on polymeric support chitosan-cobalt oxide beads subsequently activated with 0.05% cynuric chloride. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM). The immobilized ETP can be reused up to 10 cycles with retention of more than 60% activity. The optimum pH was shifted from 6.0 to 5.5 for soluble ETP to immobilized ETP and optimum temperature from 50°C and 55°C for the immobilized ETP. Based on response surface methodology, the optimal immobilization conditions obtained were: enzyme concentration, 2mg/286mg beads; optimal pH, 4.93; temperature, 28.88; cynuric chloride concentration, 0.17%; reaction time, 14.4h, which resulted 74.51% maximum immobilization. The enzyme magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of ETP onto the magnetic nanoparticles could be useful for biotechnological applications and bioassay due to its reusability and improved stability., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, an eco-friendly and sustainable green route was employed for the synthesis of stable silver nanoparticles (AgNPs) using aqueous leaf extract of Euphorbia hirta (AEE) as both reducing as well as a stabilizing agent. The synthesis of AgNPs was confirmed by UV-visible spectroscopy which produced a prominent SPR band at λmax 425nm after 25min of sunlight exposure. The AgNPs thus synthesized were optimized using one factor at a time approach, and these optimized conditions were 25min of sunlight exposure time, 5.0% (v/v) of AEE inoculum dose and 3.0mM of AgNO3 concentration. The Field Emission Scanning Electron Microscopy (FE-SEM) and High Resolution Transmission Electron Microscopy (HRTEM) analysis confirmed the presence of spherical AgNPs with average size 15.5nm. The crystallinity was determined by X-ray Diffractometer (XRD) and Selected Area Electron Diffraction (SAED) pattern. Chemical and elemental compositions were determined by Fourier Transformed Infrared Spectroscopy (FTIR) and Energy Dispersive X-ray Spectroscopy (EDX) respectively. The Atomic Force Microscopy (AFM) images with average roughness 1.15nm represented the lateral and 3D topological characteristic of AgNPs. The AgNPs thus synthesized showed effective antibacterial activity against gram negative and gram positive bacteria as well as hydrogen peroxide sensing property with a minimum detection limit of 10(-7)M., (Copyright © 2016. Published by Elsevier B.V.)

The explanation for Vedanta offered by Bhakti Niskama Santa (BNS) 1 is valid from both scientific and philosophical grounds. It seems that the published critique of Gustavo Caetano-Anollés (GCA) 2 to Shanta's paper is purely emotional and does not have any valid scientific or philosophical justification. In his rebuttal to Caetano-Anollés's critique, Shanta 3 highlighted how the concept of 'Organic Whole' in Vedanta is completely different than that of Creationist Movement and Intelligent Design. Thus Caetano-Anollé's attempt to equate Vedanta with Creationist Movement and Intelligent Design is merely superfluous. This article highlights the validity of the argument made by Bhakti Niskama Shanta 1 and thus also intends to clarify why the Caetano-Anollés critique is groundless.

The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Outer membrane vesicles (OMVs) of gram-negative bacteria are released during all growth phases and play an important role in bacterial physiology. They consist of lipids, proteins, lipopolysaccharides and other molecules. The OMVs of the Antarctic bacterium Pseudomonas syringae Lz 4W were isolated and identified their proteins. The mass spectral data set deposited with PRIDE, accession number PXD 000221 is presented in this report. The proteins identified from the OMVs of P. syringae Lz4W, data of this study were published in the Journal of proteome research [1].

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging., Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research., Availability and Implementation: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)