Towards a comprehensive understanding of the muscle proteome in Schizothorax labiatus: Insights from seasonal variations, metabolic responses, and reproductive signatures in the River Jhelum.

Proteomics is a very advanced technique used for defining correlations, compositions and activities of hundreds of proteins from organisms as well as effectively used in identifying particular proteins with varying peptide lengths and amino acid counts. In the present study, an endeavour has been put forth to create muscle proteome expression of snow trout, Schizothorax labiatus. Liquid chromatography-mass spectrometry (LC-MS) using label free quantification (LFQ) technique has extensively been carried out to explore changes in protein metabolism and its composition to discriminate across species, clarify functions and pinpoint protein biomarkers from organisms. In LFQ technique, the abundances of proteins are determined based on the signal intensities of their corresponding peptides in mass spectrometry. The main benefit of using this method is that it doesn't require pre-labelling proteins with isotopic tags, which streamlines the experimental procedure and gets rid of any bias that might have been caused by the labelling process. LFQ techniques frequently offer a wider dynamic range, making it possible to detect and quantify proteins over a broad range of abundances obtained from the complex biological materials including fish muscle. The results of proteomic analysis could provide an insight in understanding about how various proteins are expressed in response to environmental challenges. For proteomic study, two different weight groups of S. labiatus were taken from River Jhelum based on biological, physiological and logistical factors. These groups corresponded to different life stages, such as younger size and adults/brooders in order to capture potential variations in the muscle proteome related to growth and development. The proteomic analysis of S. labiatus depicted that an overall of 220 proteins in male and 228 in female fish of group 1 were noted. However, when male and female S. labiatus were examined based on spectral count and peptide abundance using ProteinLynx Global Software, a total of 10 downregulated and 32 upregulated proteins were found. In group 2 of S. labiatus, a total of 249 proteins in male and 301 in female fish were documented. When the two genders of S. labiatus were likened to one another by LFQ technique, a total of 41 downregulated and 06 upregulated proteins were identified. The variability in the protein numbers between two fish weight groups reflected biological differences, influenced by factors such as age, developmental stages, physiological condition and reproductive activities. During the study, it was observed that S. labiatus exhibited downregulated levels of proteins that were involved in feeding and growth. The contributing factors to this manifestation could be explained by lower feeding and metabolic activity of fish and decreased food availability during winter in River Jhelum. Contrarily, the fish immune response proteins were found to be significantly over-expressed in S. labiatus, indicating that the environment was more likely to undergo increased microbial infection, pollution load and anthropogenic activities. In addition, it was also discovered that there was an upregulated expression of the reproductive proteins in S. labiatus, which could be linked to the fish's pre-spawning time as the fish used in this study was collected in the winter season which is the pre-spawning period of the fish. Therefore, the present study would be useful in obtaining new insights regarding the molecular makeup of species, methods of adaptation and reactions to environmental stresses. This information contributes to our understanding of basic science and may have applications in environmental monitoring, conservation and preservation of fish species.

LC-MS/MS based characterisation and differential expression of proteins in Himalayan snow trout, Schizothorax labiatus using LFQ technique.

Molecular characterization of fish muscle proteins are nowadays considered as a key component to understand the role of specific proteins involved in various physiological and metabolic processes including their up and down regulation in the organisms. Coldwater fish specimens including snow trouts hold different types of proteins which help them to survive in highly diversified temperatures fluctuating from 0 to 20 °C. So, in current study, the liquid chromatography mass spectrometry using label free quantification technique has been used to investigate the muscle proteome profile of Schizothorax labiatus. For proteomic study, two weight groups of S. labiatus were taken from river Sindh. The proteomic analysis of group 1 revealed that a total of 235 proteins in male and 238 in female fish were recorded. However, when male and female S. labiatus were compared with each other on the basis of spectral count and abundance of peptides by ProteinLynx Global Server software, a total of 14 down-regulated and 22 up-regulated proteins were noted in this group. The highly down-regulated ones included homeodomain protein HoxA2b, retinol-binding protein 4, MHC class II beta chain and proopiomelanocortin while as the highly expressed up-regulated proteins comprised of gonadotropin I beta subunit, NADH dehydrogenase subunit 4, manganese superoxide dismutase, recombinase-activating protein 2, glycosyltransferase, chymotrypsin and cytochrome b. On the other hand, the proteomic characterisation of group 2 of S. labiatus revealed that a total of 227 proteins in male and 194 in female fish were recorded. When male and female S. labiatus were compared with each other by label free quantification, a total of 20 down-regulated and 18 up-regulated proteins were recorded. The down-regulated protein expression of group 2 comprised hepatic lipase, allograft inflammatory factor-1, NADH dehydrogenase subunit 4 and myostatin 1 while the highly expressed up-regulated proteins included glycogen synthase kinase-3 beta variant 2, glycogen synthase kinase-3 beta variant 5, cholecystokinin, glycogen synthase kinase-3 beta variant 3 and cytochrome b. Significant (P < 0.05) difference in the expression of down-regulated and up-regulated proteins was also noted between the two sexes of S. labiatus in each group. According to MS analysis, the proteins primarily concerned with the growth, skeletal muscle development and metabolism were down-regulated in river Sindh, which indicates that growth of fish during the season of collection i.e., winter was slow owing to less food availability, gonad development and low metabolic activity. While, the proteins related to immune response of fish were also noted to be down-regulated thereby signifying that the ecosystem has less pollution loads, microbial, pathogenic and anthropogenic activities. It was also found that the proteins involved in glycogen metabolism, reproductive and metabolic processes, particularly lipid metabolism were up-regulated in S. labiatus. The significant expression of these proteins may be connected to pre-spawning, gonad development and use of stored food as source of energy. The information generated in this study can be applied to future research aimed at enhancing food traceability, food safety, risk management and authenticity analysis.

Comparative structural study of selective and non-selective NSAIDs against the enzyme cyclooxygenase-2 through real-time molecular dynamics linked to post-dynamics MM-GBSA and e-pharmacophores mapping.

BACKGROUND: Inflammation-provoked disorders including cancer are arbitrated by cyclooxygenase-2 (COX-2). Celecoxib and niflumic acid are among the potent and selective inhibitors of this enzyme while aspirin (acetylsalicylic acid) and sodium salicylate are its non-selective and lesser potent inhibitors. Despite these proven studies, the comparative structural study of these selective and non-selective molecules at atomistic scale in complex state with COX-2 that may answer this differential inhibitory behavior has not been accomplished spotlighting the imperative need of additional research in this area. Thus, this study was framed to provide a strong explanation for the enigma of higher inhibitory activity of celecoxib-niflumic acid duo in comparison to aspirin and sodium salicylate towards COX-2. METHODS: A contemporary approach including advanced molecular docking against COX2, molecular dynamics of receptor-ligand complexes, simulation-trajectory-backed MMGBSA for different time points, radius of gyration (Rg) calculations, and e-pharmacophores approach was employed to attain a rational conclusion. RESULTS: Our findings demonstrated the higher binding affinity of celecoxib and niflumic acid over aspirin and sodium salicylate against COX-2. Although both selective and non-selective COX-2 inhibitors manifested nearly the same stability in the active site of this enzyme but the e-pharmocophoric features found in the case of selective inhibitors scored over non-selective ones. Thus, our findings excluded the differential stability to be the cause of stronger potency of selective inhibitors but attributed their potency to greater number of complementary features present in these inhibitors against the active site of inflammation engendering COX-2.

Histone deacetylase inhibitors as sanguine epitherapeutics against the deadliest lung cancer.

The back-breaking resistance mechanisms generated by lung cancer cells against epidermal growth factor receptor (EGFR), KRAS and Janus kinase 2 (JAK2) directed therapies strongly prioritizes the requirement of novel therapies which are perfectly tolerated, potentially cytotoxic and can reinstate the drug-sensitivity in lung cancer cells. Enzymatic proteins modifying the post-translational modifications of nucleosome-integrated histone substrates are appearing as current targets for defeating various malignancies. Histone deacetylases (HDACs) are hyperexpressed in diverse lung cancer types. Blocking the active pocket of these acetylation erasers through HDAC inhibitors (HDACi) has come out as an optimistic therapeutic recourse for annihilating lung cancer. This article in the beginning gives an overview about lung cancer statistics and predominant lung cancer types. Succeeding this, compendium about conventional therapies and their serious drawbacks has been provided. Then, connection of uncommon expression of classical HDACs in lung cancer onset and expansion has been detailed. Moreover, keeping the main theme in view this article deeply discusses HDACi in the context of aggressive lung cancer as single agents and spotlights various molecular targets suppressed or induced by these inhibitors for engendering cytotoxic effect. Most particularly, the raised pharmacological effects achieved on using these inhibitors in concerted form with other therapeutic molecules and the cancer-linked pathways altered by this procedure are described. The positive direction towards further heightening of efficacy and the pressing requirement of exhaustive clinical assessment has been proposed as a new focus point.

Evaluation of free radical quenching, anti-inflammatory activity together with anticancer potential of Lychnis coronaria and characterization of novel molecules from its extract through high resolution-liquid chromatography mass spectrometry coupled to structural biochemistry approach.

Lychnis coronaria, a perennial (herbaceous) belonging to Caryophyllaceae has been traditionally used for treating different complications. However, the free radical scavenging effect, anti-inflammatory activity and anticancer property of methanolic extract of this plant has not been addressed. Most importantly, the chemical constituents present in the extract of Lychnis coronaria responsible for its diverse activities have not been scrutinized till date. Here, we used a complex approach for exploring the above mentioned effects of Lychnis coronaria. We performed rigorous phytochemical screening followed by quantification of tannins, phenols, alkaloids, quinones and sterols from the extract. Moreover we employed in vitro DPPH, ABTS , FRAP assay, albumin denaturation inhibition experiment, MTT assay, high resolution liquid chromatography mass spectrometry for measurng the reactive oxygen species quenching, anti-inflammatory and anticancer strength of Lychnis coronaria and for identifying the possible bioactive molecules. We identified two novel molecules panaxynol (polyacetylenic alcohol) and norharman (9H-Pyrido [3, 4-B] indole) following rigorous analysis of the extract. Following this, the binding affinity of these molecules was estimated using human cyclooxygenase (COX)-2 enzyme as target. Among the constituents of Lychnis coronaria norharman manifested stronger binding towards COX-2 compared to panaxynol. Most importantly, norharman showed high stability in the groove of COX2 as confirmed by molecular dynamics simulation. Collectively, Lychnis coronaria manifested free radical neutralizing, inflammation soothing and anticancer effect in concentration dependent manner and thus may serve as a promising phytotherapeutic in future.Communicated by Ramaswamy H. Sarma.

Isolation of high-quality RNA for high throughput applications from secondary metabolite-rich Crocus sativus L.

OBJECTIVE: Isolating high-quality RNA is a basic requirement while performing high throughput sequencing, microarray, and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides, and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction including CTAB, TriZol, and SDS-based methods, which invariably yield less and poor quality RNA and hence it necessitated the isolation of high-quality RNA suitable for high throughput applications. RESULTS: In the present study we made certain adjustments to the available protocols including modifications in the extraction buffer itself and the procedure employed. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) value greater than 7.5. The quality of the RNA was further assessed by qPCR-based amplification of mRNA and mature miRNAs such as Cs-MIR166c and Cs-MIR396a.

Exploring novel and potent molecules for disrupting DEPTOR-mTOR interaction through structure-steered screening, extra-exactitude molecular docking, prime binding free energy estimation and voguish molecular dynamics.

Dep domain containing mTOR interacting protein (DEPTOR) has critical implications in the development and progression of human malignancies. Increased expression of DEPTOR promotes the growth of tumor cells by inhibiting the mTORC1, which alleviates the negative feedback inhibition by mTORC1 downstream target S6Ks on PI3K/AKT pathway thereby promotes cell survival and prevents apoptosis. This clearly suggests that targetting DEPTOR-mTOR interactions through small molecules may prove as an effective strategy for circumventing distinct cancers. In this study, we employed a top-down approach for finding three novel molecules which may prove effective in disrupting Deptor-mTOR interaction. Following DEPTOR modelling and validation we performed grid-directed structure-based screening by specifying the residues of DEPTOR known to interact with mTOR. A library of 10,000 protein-protein disrupting molecules was screened against the defined region of DEPTOR. From the screened molecules, 30 molecules with highest binding affinity were chosen for molecular docking. Thirty (30) extra-precision molecular docking experiments and 30 molecular mechanics generalized born surface area (MMGBSA) assays were performed. Following this top 10 molecules in terms of binding affinity were selected and the interaction profile of their corresponding docked files was generated. The top three molecules were finally selected after taking all the three parameters including docking score, binding energy value and interaction profile into consideration. For atomistic insights regarding DEPTOR-topmost hit interactions, molecular dynamics was performed for 100 ns. This molecule after further evaluation may prove as promising candidate for anticancer therapy.Communicated by Ramaswamy H. Sarma.

Delineating binding potential, stability of Sulforaphane-N-acetyl-cysteine in the active site of histone deacetylase 2 and testing its cytotoxicity against distinct cancer lines through stringent molecular dynamics, DFT and cell-based assays.

Histone deacetylase 2 (HDAC2), an isozyme of Class I HDACs has potent imputations in actuating neurodegenerative signaling. Currently, there are sizeable therapeutic disquiets with the use of synthetic histone deacetylase inhibitors in disease management. This strongly suggests the unfulfilled medical necessity of plant substitutes for therapeutic intervention. Sulforaphane-N-acetyl-cysteine (SFN-N-acetylcysteine or SFN-NAC), a sulforaphane metabolite has shown significantly worthier activity against HDACs under in vitro conditions. However, the atomistic studies of SFN-NAC against HDAC2 are currently lacking. Thus, the present study employed a hybrid strategy including extra-precision (XP) grid-based flexible molecular docking, molecular mechanics generalized born surface area (MM-GBSA), e-Pharmacophores method, and molecular dynamics simulation for exploring the binding strengh, mode of interaction, e-Pharmacophoric features, and stability of SFN-NAC towards HDAC2. Further, the globally acknowledged density functional theory (DFT) study was performed on SFN-NAC and entinostat individually in complex state with HDAC2. Apart from this, these inhibitors were tested against three distinct cancer cell models and one transformed cell line for cytotoxic activity. Moreover, double mutant of HDAC2 was generated and the binding orientation and interaction of SFN-NAC was scrutinized in this state. On the whole, this study unbosomed and explained the comparatively higher binding affinity of entinostat for HDAC2 and its wide spectrum cytotoxicity than SFN-NAC.

Irigenin, a novel lead from Western Himalayan chemiome inhibits Fibronectin-Extra Domain A induced metastasis in Lung cancer cells.

Several lines of evidence indicate that Fibronectin Extra Domain A (EDA) promotes metastatic capacity of tumor cells by engaging cell surface α9ß1 integrins. This interaction mediated by the C-C loop of EDA activates pro-oncogenic signaling pathways leading to epithelial to mesenchymal transition (EMT) of tumor cells, thus signifying its importance in control of metastatic progression. In this context the present study was designed to explore the active compounds from selected ethno-medicinal plants of western Himalayan region for targeting EDA of Fibronectin in lung carcinoma cells. Structure based informatics for drug designing and screening was employed to generate a lead compound(s) feed that were conformationally and energetically viable. Out of 120 compounds selected, Irigenin showed best binding-affinity with C-C loop of EDA. Irigenin specifically targeted α9ß1 and α4ß1 integrin binding sites on EDA comprising LEU46, PHE47, PRO48, GLU58, LEU59 and GLN60 in its C-C loop as evaluated by energy decomposition per residue of Irigenin-EDA complex. In-vitro cell motility assays complemented with EDA knock-in and knockdown assays distinctively demonstrated that Irigenin prevents metastatic capacity of lung cancer cells by selectively blocking EDA. The results presented thus project Irigenin as a lead compound to overcome Fibronectin EDA induced metastatic progression in lung carcinoma cells.