Role of Hemigraphis alternata in wound healing: metabolomic profiling and molecular insights into mechanisms.

Hemigraphis alternata (H. alternata), commonly known as Red Flame Ivy, is widely recognized for its wound healing capabilities. However, the pharmacologically active plant components and their mechanisms of action in wound healing are yet to be determined. This study presents the mass spectrometry-based global metabolite profiling of aqueous and ethanolic extract of H. alternata leaves. The analysis identified 2285 metabolites from 24,203 spectra obtained in both positive and negative polarities. The identified metabolites were classified under ketones, carboxylic acids, primary aliphatic amines, steroids and steroid derivatives. We performed network pharmacology analysis to explore metabolite-protein interactions and identified 124 human proteins as targets for H. alternata metabolites. Among these, several of them were implicated in wound healing including prothrombin (F2), alpha-2A adrenergic receptor (ADRA2A) and fibroblast growth factor receptor 1 (FGFR1). Gene ontology analysis of target proteins enriched cellular functions related to glucose metabolic process, platelet activation, membrane organization and response to wounding. Additionally, pathway enrichment analysis revealed potential molecular network involved in wound healing. Moreover, in-silico docking analysis showed strong binding energy between H. alternata metabolites with identified protein targets (F2 and PTPN11). Furthermore, the key metabolites involved in wound healing were further validated by multiple reaction monitoring-based targeted analysis.

A comparative proteomic analysis for non-invasive early prediction of hypoxic-ischemic injury in asphyxiated neonates.

AIM: Hypoxic Ischemic Encephalopathy (HIE) is one of the principal causes of neonatal mortality and long-term morbidity worldwide. The neonatal signs of mild cerebral injury are subtle, making an early precise diagnosis difficult. Delayed detection, poor prognosis, and lack of specific biomarkers for the disease are increasing mortality rates. In this study, we intended to identify specific biomarkers using comparative proteomic analysis to predict the severity of perinatal asphyxia so that its outcome can also be prevented. EXPERIMENTAL DESIGN: A case-control study was conducted on 38 neonates, and urine samples were collected within 24 and 72 h of life. A tandem mass spectrometry-based quantitative proteomics approach, followed by validation via sandwich ELISA, was performed. RESULTS: The LC-MS/MS-based proteomics analysis resulted in the identification of 1201 proteins in urine, with 229, 244, and 426 being differentially expressed in HIE-1, HIE-2, and HIE-3, respectively. Axon guidance, Diseases of programmed cell death, and Detoxification of reactive oxygen species pathways were significantly enriched in mild HIE versus severe HIE. Among the differentially expressed proteins in various stages of HIE, we chose to validate four proteins - APP, AGT, FABP1, and FN1 - via sandwich ELISA. Individual and cumulative ROC curves were plotted. AGT and FABP1 together showed high sensitivity, specificity, and accuracy as potential biomarkers for early diagnosis of HIE. CONCLUSION: Establishing putative urinary biomarkers will facilitate clinicians to more accurately screen neonates for brain injury and monitor the disease progression. Prompt treatment of neonates may reduce mortality and neurodevelopmental impairment.

Rifampicin-Mediated Metabolic Changes in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is considered to be a devastating pathogen worldwide, affecting millions of people globally. Several drugs targeting distinct pathways are utilized for the treatment of tuberculosis. Despite the monumental efforts being directed at the discovery of drugs for Mtb, the pathogen has also developed mechanisms to evade the drug action and host processes. Rifampicin was an early anti-tuberculosis drug, and is still being used as the first line of treatment. This study was carried out in order to characterize the in-depth rifampicin-mediated metabolic changes in Mtb, facilitating a better understanding of the physiological processes based on the metabolic pathways and predicted protein interactors associated with the dysregulated metabolome. Although there are various metabolomic studies that have been carried out on rifampicin mutants, this is the first study that reports a large number of significantly altered metabolites in wild type Mtb upon rifampicin treatment. In this study, a total of 173 metabolites, associated with pyrimidine, purine, arginine, phenylalanine, tyrosine, and tryptophan metabolic pathways, were significantly altered by rifampicin. The predicted host protein interactors of the rifampicin-dysregulated Mtb metabolome were implicated in transcription, inflammation, apoptosis, proteolysis, and DNA replication. Further, tricarboxylic acidcycle metabolites, arginine, and phosphoenolpyruvate were validated by multiple-reaction monitoring. This study provides a comprehensive list of altered metabolites that serves as a basis for understanding the rifampicin-mediated metabolic changes, and associated functional processes, in Mtb, which holds therapeutic potential for the treatment of Mtb.

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.

Deciphering metabolomic alterations in seminal plasma of crossbred (Bos taurus X Bos indicus) bulls through comparative deep metabolomic analysis.

The incidence of sub-fertility is higher in crossbred bulls compared to zebu bulls. In the present study, we analysed the metabolomic profile of seminal plasma from crossbred and zebu bulls and uncovered differentially expressed metabolites between these two breeds. Using a high-throughput LC-MS/MS-based approach, we identified 990 and 1,002 metabolites in crossbred and zebu bull seminal plasma respectively. After excluding the exogenous metabolites, we found that 50 and 68 putative metabolites were unique to crossbred and zebu bull seminal plasma, respectively, whilst 87 metabolites were common to both. After data normalisation, 63 metabolites were found to be dysregulated between crossbred and zebu bull seminal plasma. Observed pathways included Linoleic acid metabolism (observed metabolite was phosphatidylcholine) in crossbred bull seminal plasma whereas inositol phosphate metabolism (observed metabolites were phosphatidylinositol-3,4,5-trisphosphate/inositol 1,3,4,5,6-pentakisphosphate/myo-inositol hexakisphosphate) was observed in zebu bull seminal plasma. Abundance of Tetradecanoyl-CoA was significantly higher, whilst abundance of Taurine was significantly lower in crossbred bull seminal plasma. In conclusion, the present study established the seminal plasma metabolomic profile in crossbred and zebu bulls and suggest that increased lipid peroxidation coupled with low concentrations of antioxidants in seminal plasma might be associated with high incidence of sub-fertility in crossbred bulls.

Data on dose-dependent cytotoxicity of rotenone and neuroprotection conferred by Yashtimadhu (Glycyrrhiza glabra L.) in an in vitro Parkinson's disease model.

The data described in this article presents the toxicity of rotenone and the neuroprotective effect of Yashtimadhu choorna (powder) in an in vitro Parkinson's disease model [1]. Yashtimadhu choorna is prepared from the roots of Glycyrrhiza glabra L., commonly known as licorice/ liquorice. The effects of rotenone and Yashtimadhu was assessed using cellular and molecular assays such as cell cytotoxicity assay, live-dead cell staining assay, cell cycle analysis, and western blotting. Protein-protein interaction was studied using ANAT plug-in in Cytoscape. Rotenone displayed time and dose-dependent toxicity, as evidenced by cell cytotoxicity assay and live-dead cell staining assay. Yashtimadhu showed no toxicity and prevented rotenone-induced toxicity. Rotenone and Yashtimadhu displayed differential control on the cell cycle. The Protein-interaction network showed the proteins interacting with ERK-1/2 and the pathways regulated by these interactions. The pathways regulated were primarily involved in cellular oxidative stress and apoptosis response. The data described here will enable the extent of cellular toxicity as a result of rotenone treatment and the neuroprotection conferred by Yashtimadhu choorna. This will enable understanding and exploring the effect of traditional and complementary medicine and aiding the identification of molecular targets to confer neuroprotection in Parkinson's disease.

MS2Compound: A User-Friendly Compound Identification Tool for LC-MS/MS-Based Metabolomics Data.

Metabolomics is a leading frontier of systems science and biomedical innovation. However, metabolite identification in mass spectrometry (MS)-based global metabolomics investigations remains a formidable challenge. Moreover, lack of comprehensive spectral databases hinders accurate identification of compounds in global MS-based metabolomics. Creating experiment-derived metabolite spectral libraries tailored to each experiment is labor-intensive. Therefore, predicted spectral libraries could serve as a better alternative. User-friendly tools are much needed, as the currently available metabolomic analysis tools do not offer adequate provision for users to create or choose context-specific databases. Here, we introduce the MS2Compound, a metabolite identification tool, which can be used to generate a custom database of predicted spectra using the Competitive Fragmentation Modeling-ID (CFM-ID) algorithm, and identify metabolites or compounds from the generated database. The database generator can create databases of the model/context/species used in the metabolomics study. The MS2Compound is also powered with mS-score, a scoring function for matching raw fragment spectra to a predicted spectra database. We demonstrated that mS-score is robust in par with dot product and hypergeometric score in identifying metabolites using benchmarking datasets. We evaluated and highlight here the unique features of the MS2Compound by a re-analysis of a publicly available metabolomic dataset (MassIVE id: MSV000086784) for a complex traditional drug formulation called Triphala. In conclusion, we believe that the omics systems science and biomedical research and innovation community in the field of metabolomics will find the MS2Compound as a user-friendly analysis tool of choice to accelerate future metabolomic analyses.

Preliminary comparative deep metabolomic analysis of spermatozoa from zebu and crossbred cattle suggests associations between metabolites, sperm quality and fertility.

Poor semen quality and infertility/subfertility are more frequent in crossbred than zebu bulls. Using a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach, we established the preliminary metabolomic profile of crossbred and zebu bull spermatozoa (n=3 bulls each) and identified changes in sperm metabolomics between the two groups. In all, 1732 and 1240 metabolites were detected in zebu and crossbred bull spermatozoa respectively. After excluding exogenous metabolites, 115 and 87 metabolites were found to be unique to zebu and crossbred bull spermatozoa respectively whereas 71 metabolites were common to both. In the normalised data, 49 metabolites were found to be differentially expressed between zebu and crossbred bull spermatozoa. The significantly enriched (P<0.05) pathways in spermatozoa were taurine and hypotaurine metabolism (observed metabolites taurine and hypotaurine) in zebu and glycerophospholipid metabolism (observed metabolites phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine) in crossbred bulls. The abundance of nitroprusside (variable importance in projection (VIP) score >1.5) was downregulated, whereas that of l-cysteine, acetyl coenzyme A and 2'-deoxyribonucleoside 5'-diphosphate (VIP scores >1.0) was upregulated in crossbred bull spermatozoa. In conclusion, this study established the metabolomic profile of zebu and crossbred bull spermatozoa and suggests that aberrations in taurine, hypotaurine and glycerophospholipid metabolism may be associated with the higher incidence of infertility/subfertility in crossbred bulls.

Prevention of MEK-ERK-1/2 hyper-activation underlines the neuroprotective effect of Glycyrrhiza glabra L. (Yashtimadhu) against rotenone-induced cellular and molecular aberrations.

ETHNOPHARMACOLOGICAL RELEVANCE: Yashtimadhu choorna (powder) is prepared from the dried root of Glycyrrhiza glabra L., commonly known as licorice. The Indian Ayurvedic system classifies Yashtimadhu as a Medhya Rasayana that can enhance brain function, improves memory, and possess neuroprotective functions, which can be used against neurodegenerative diseases like Parkinson's disease (PD). AIM OF THE STUDY: We aimed to decipher the neuroprotective effects of G. glabra L., i.e., Yashtimadhu, in a rotenone-induced PD model. MATERIALS AND METHODS: Retinoic acid-differentiated IMR-32 cells were treated with rotenone (PD model) and Yashtimadhu, and were assessed for cellular toxicity, live-dead staining, cell cycle, oxidative stress, protein abundance, and kinase phosphorylation. RESULTS: Yashtimadhu conferred protection against rotenone-induced cytotoxicity, countered cell death, reduced expression of pro-apoptotic proteins (cleaved-caspases-9, and 3, cleaved-PARP, BAX, and BAK) and increased anti-apoptotic protein, BCL-2. Rotenone-induced cell cycle re-entry (G2/M transition), was negated by Yashtimadhu and was confirmed with PCNA levels. Yashtimadhu countered rotenone-mediated activation of mitochondrial proteins involved in oxidative stress, cytochrome-C, PDHA1, and HSP60. Inhibition of rotenone-induced ERK-1/2 hyperphosphorylation prevented activation of apoptosis, which was confirmed with MEK-inhibitor, highlighted the action of Yashtimadhu via ERK-1/2 modulation. CONCLUSIONS: We provide the evidence for neuroprotection conferred by G. glabra L. (Yashtimadhu) and its mechanism via inhibiting MEK-ERK-1/2 hyper-phosphorylation, prevention of mitochondrial stress, and subsequent prevention of apoptosis. The study highlights Yashtimadhu as a promising candidate with neuroprotective effects, the potential of which can be harnessed for identifying novel therapeutic targets.

Differences in milk metabolites in Malnad Gidda (Bos indicus) cows reared under pasture-based feeding system.

The milk and milk products from cows reared under grazing system are believed to be healthier and hence have high demand compared to milk from cows reared in the non-grazing system. However, the effect of grazing on milk metabolites, specifically lipids has not been fully understood. In this study, we used acetonitrile precipitation and methanol:chloroform methods for extracting the milk metabolites followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) run to identify the different metabolites between the milk of grazing and non-grazing early lactating Malnad Gidda cows. Various carbohydrates, amino acids, nucleosides and vitamin derivatives were found to be differentially abundant in grazing cows. A total of 35 metabolites were differentially regulated (fold change above 1.5) between the two groups. Tyrosyl-threonine, histidinyl-cysteine, 1-methyladenine, L-cysteine and selenocysteine showed fold change above 3 in grazing cows. The lipid profile of milk showed a lesser difference between grazing and non-grazing cows as compared to polar metabolites. To the best of our knowledge, this is the largest inventory of milk metabolomics data of an Indian cattle (Bos indicus) breed. We believe that our study would help to emerge a field of Nutri-metabolomics and veterinary omics research.

A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway.

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca2+/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca2+/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein-protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database ( https://www.wikipathways.org/index.php/Pathway:WP4874 ). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.

From LC-MS/MS metabolomics profiling of Kanchanara Guggulu to molecular docking and dynamics simulation of quercetin pentaacetate with aldose reductase.

Kanchanara Guggulu (KG) is an important traditional medicine that is prescribed by the Ayurveda physicians for the treatment of swellings in various organs such as the thyroid, and lymph nodes. High-resolution mass-spectrometry-based metabolomics found metabolites in KG. LC-MS/MS-based metabolomics analysis of KG identified 2,579 compounds including quercetin and kaempferol derivatives. The molecular docking and dynamics analysis of quercetin pentaacetate with aldose reductase is documented for further consideration in drug discovery.

Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls.

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic bulls.

Plant Omics: Metabolomics and Network Pharmacology of Liquorice, Indian Ayurvedic Medicine Yashtimadhu.

Plant omics is an emerging field of systems science and offers the prospects of evidence-based evaluation of traditional herbal medicines in human diseases. To this end, the powdered root of Yashtimadhu (Glycyrrhiza glabra L.), commonly known as liquorice, is frequently used in Indian Ayurvedic medicine with an eye to neuroprotection but its target proteins, mechanisms of action, and metabolites remain to be determined. Using a metabolomics and network pharmacology approach, we identified 98,097 spectra from positive and negative polarities that matched to ∼1600 known metabolites. These metabolites belong to terpenoids, alkaloids, and flavonoids, including both novel and previously reported active metabolites such as glycyrrhizin, glabridin, liquiritin, and other terpenoid saponins. Novel metabolites were also identified such as quercetin glucosides, coumarin derivatives, beta-carotene, and asiatic acid, which were previously not reported in relation to liquorice. Metabolite-protein interaction-based network pharmacology analyses enriched 107 human proteins, which included dopamine, serotonin, and acetylcholine neurotransmitter receptors among other regulatory proteins. Pathway analysis highlighted the regulation of signaling kinases, growth factor receptors, cell cycle, and inflammatory pathways. In vitro validation confirmed the regulation of cell cycle, MAPK1/3, PI3K/AKT pathways by liquorice. The present data-driven, metabolomics and network pharmacology study paves the way for further translational clinical research on neuropharmacology of liquorice and other traditional medicines.

Identification of Molecular Network Associated with Neuroprotective Effects of Yashtimadhu (Glycyrrhiza glabra L.) by Quantitative Proteomics of Rotenone-Induced Parkinson's Disease Model.

Parkinson's disease (PD) is a progressive neurodegenerative disorder, whose treatment with modern therapeutics leads to a plethora of side effects with prolonged usage. Therefore, the management of PD with complementary and alternative medicine is often pursued. In the Ayurveda system of alternative medicine, Yashtimadhu choorna, a Medhya Rasayana (nootropic), prepared from the dried roots of Glycyrrhiza glabra L. (licorice), is prescribed for the management of PD with a favorable outcome. We pursued to understand the neuroprotective effects of Yashtimadhu choorna against a rotenone-induced cellular model of PD using differentiated IMR-32 cells. Cotreatment with Yashtimadhu choorna extract rescued rotenone-induced apoptosis and hyperphosphorylation of ERK-1/2. Quantitative proteomic analysis of six peptide fractions from independent biological replicates acquired 1,561,169 mass spectra, which when searched resulted in 565,008 peptide-spectrum matches mapping to 30,554 unique peptides that belonged to 4864 human proteins. Proteins commonly identified in biological replicates and >4 PSMs were considered for further analysis, leading to a refined set of 3720 proteins. Rotenone treatment differentially altered 144 proteins (fold ≥1.25 or ≤0.8), involved in mitochondrial, endoplasmic reticulum, and autophagy functions. Cotreatment with Yashtimadhu choorna extract rescued 84 proteins from the effect of rotenone and an additional regulation of 4 proteins. Network analysis highlighted the interaction of proteins and pathways regulated by them, which can be targeted for neuroprotection. Validation of proteomics data highlighted that Yashtimadhu confers neuroprotection by preventing mitochondrial oxidative stress and apoptosis. This discovery will pave the way for understanding the molecular action of Ayurveda drugs and developing novel therapeutics for PD.

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.

Lowered Decorin With Aberrant Extracellular Matrix Remodeling in Aqueous Humor and Tenon's Tissue From Primary Glaucoma Patients.

Purpose: To evaluate the inflammatory cytokine, growth factors, extracellular matrix (ECM) remodeling genes, profibrotic and antifibrotic molecules in patients undergoing glaucoma filtration surgery (GFS). Additionally, the effect of preoperative antiglaucoma medications (AGMs) and postoperative bleb status were related to these parameters. Methods: Tenon's tissue and aqueous humour (AH) were collected from 207 patients undergoing GFS with primary open-angle glaucoma (POAG) (n = 77), primary angle-closure glaucoma (PACG) (n = 62), and cataract controls (n = 68). Monocyte chemoattractant protein-1 (MCP-1), connective tissue growth factor (CTGF), transforming growth factor ß1/2 (TGF-ß1/2), lysyl oxidase (LOX), lysyl oxidase L2 (LOXL2), elastin (ELN), collagen type 1 α 1 (COL1A1), secreted protein acidic and rich in cysteine (SPARC), α-smooth muscle actin (α-SMA), and decorin (DCN) were determined in tenon's tissue by real-time PCR and in AH using ELISA. Results: A significant increase was observed in the transcripts of MCP-1, TGF-ß2, and SPARC in POAG and PACG (P < 0.05); CTGF, TGF-ß1, LOX, LOXL2, ELN, COL1A1, and α-SMA in PACG (P < 0.05) compared with control. DCN transcript was significantly decreased in POAG and PACG (P < 0.05) compared with control. The protein levels of CTGF, TGF-ß1/ß2, ELN, SPARC, and LOXL2 was significantly elevated in POAG and PACG (P < 0.05); DCN was decreased (P < 0.05) compared with control. These parameters showed significant association with duration of preoperative AGMs and postoperative bleb status. Conclusions: This study demonstrates increased expression of growth factors and ECM molecules, both at protein and transcript levels in GFS patients. A decreased DCN in AH seems striking, and if restored might have a therapeutic role in minimizing postoperative scarring to improve GFS outcome.

Effect of brilliant Blue-G on cellular stress response in retinal pigment epithelial cells: In vitro.

To assess the cellular stress evoked by exposure of Brilliant Blue-G (BBG), adult retinal pigment epithelial (ARPE-19) cells were treated with various dilutions of BBG in balanced salt solution plus (BSS-PLUS) with and without endoillumination (Alcon Constellation Vision System). The treatments lasted for acute periods of 2 and 5 min. MTT and presto blue assays were performed to assess the changes in cell viability; reactive oxygen species (ROS) production was quantified by DCFDA (dichlorofluorescin diacetate) assay, and the expression of inflammatory stress and endoplasmic reticulum (ER) genes were quantified by qPCR. We observed no reduction in cell viability at 2 min of dye treatment with and without endoillumination while at 5 min exposure, a reduction in cell viability at all concentrations of the dye was observed compared to control. Though there was an increase in ROS with endoillumination, it was insignificant. There was no change in the mRNA expression of TNF-α while that of GRP78, and inflammatory genes viz. IL-8, IL-1ß showed a significant increase at 0.5 mg/ml dye with endoillumination. BBG reduced cell viability with increasing concentration and time. The undiluted concentration of the dye results in inflammatory stress compared to the diluted formulations. Interestingly, increased GRP78 at undiluted concentration indicates a protective response in cells exposed to light. However, further studies are needed to evaluate the effect of cellular stress on the visual outcome. We infer that the commercially available formulation of BBG is safe for the RPE, at the recommended dose for a short duration however its toxicity to other cell types need to be addressed.