Untargeted metabolomics of buffalo urine reveals hydracyrlic acid, 3-bromo-1-propanol and benzyl serine as potential estrus biomarkers.

Buffalo is a silent heat animal and doesn't show prominent signs of estrous like cattle so it becomes difficult for farmers to determine the receptivity of the animal based purely on the animal behaviour. India, having a huge population size, needs to produce more milk for the population. Successful artificial insemination greatly depends on the receptivity of the animal. Hence the present study aimed to identify the changes in the metabolome of the buffalo. GC-MS based mass spectrometric analysis was deployed for the determination of estrous by differential expression of metabolites. It was found that hydracrylic acid, 3-bromo-1-propanol and benzyl serine were significantly upregulated in the estrous phase of buffalo (p.value ≤0.05, FC ≥ 2). The pathway enrichment analysis also supported the same as pathways related to amino acid metabolism and fatty acid metabolism were up regulated along with the Warburg effect which is linked to the rapid cell proliferation which might help prepare animals to meet the energy requirement during the estrous. Further analysis of the metabolic biomarkers using ROC analysis also supported these three metabolites as probable biomarkers as they were identified with AUC values of 0.7 or greater. SIGNIFICANCE: The present study focuses on the untargeted metabolomics studies of buffalo urine with special reference to the estrous phase of reproductive cycle. The estrous signals are more prominent in cattle, where animals show clear estrous signals such as mounting and discharge along with vocal signals. Buffalo is a silent heat animal and it becomes difficult for farmers to detect the estrous based on the physical and behavioral signals. Hence the present study focuses on GC-MS based untargeted metabolomics to identify differentially expressed urine metabolites. In this study, hydracrylic acid, 3-bromo-1-propanol and benzyl serine were found to be significantly upregulated in the estrous phase of buffalo (p-value ≤0.05, FC ≥ 2). Further confirmation of the metabolic biomarkers was done using Receiver operating characteristics (ROC) analysis which also supported these three metabolites as probable biomarkers as they had AUC values of 0.7 or greater. Hence, this study will be of prime importance for the people working in the area of animal metabolomics.

Fluorescent Carbon Dots: Aggregation-Induced Emission Enhancement, Application as Probe for CN- and Cr2O7-2, Sensing Strips and Bio-imaging Agent.

Fluorescent carbon dots (Trp-CDs) were prepared using tryptophan as precursor and were characterized on the basis of elemental analysis, powder-XRD, IR, Raman spectroscopy, 13C-NMR, UV-Vis, fluorescence and TEM. Trp-CDs exhibit poor fluorescence in 100% water but showed strong Aggregation Induced Emission (AIE) in ethanol and higher alcohols. The anion sensing study of Trp-CD revealed that it selectively detects CN- and Cr2O7-2 and from fluorescence quenching titration study, quenching constant, LOD and range of detection were evaluated. The emission life-time of Trp-CD before and after addition of CN- and Cr2O7-2 were measured, the decay curve before addition of anion was best fitted with a bi-exponential function with life-time of τ1 2.79 ns (10.74%) and τ2 18.93 ns (89.26%). The mechanistic study revealed that for CN-, the fluorescence quenching is due to its interaction with protons attached to surface functional groups and for Cr2O7-2, it is due to inner filter effect (IFE). Sensing strips were prepared by coating Trp-CDs onto various solid surfaces including agarose films and were used for detection of CN- and Cr2O7-. Trp-CD was found to be nontoxic and biocompatible and used as staining agent for Artemia and Bacteria (Bacillus Subtilis, Pseudomonas) and detection of CN- and Cr2O7-.

lncRNA-miRNA-mRNA network in kidney transcriptome of Labeo rohita under hypersaline environment.

The present study describes the kidney transcriptome of Labeo rohita, a freshwater fish, exposed to gradually increased salinity concentrations (2, 4, 6 and 8ppt). A total of 10.25 Gbps data was generated, and a suite of bioinformatics tools, including FEELnc, CPC2 and BLASTn were employed for identification of long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). Our analysis revealed a total of 170, 118, 99, and 269 differentially expressed lncRNA and 120, 118, 99, and 124 differentially expressed miRNAs in 2, 4, 6 and 8 ppt treatment groups respectively. Two competing endogenous RNA (ceRNA) networks were constructed i.e. A* ceRNA network with up-regulated lncRNAs and mRNAs, down-regulated miRNAs; and B* ceRNA network vice versa. 2ppt group had 131 and 83 lncRNA-miRNA-mRNA pairs in A* and B* networks, respectively. 4ppt group featured 163 pairs in A* network and 191 in B* network, while the 6ppt had 103 and 105 pairs. 8ppt group included 192 and 174 pairs. These networks illuminate the intricate RNA interactions in freshwater fish to varying salinity conditions.

Integrative miRNA-mRNA network analysis to identify crucial pathways of salinity adaptation in brain transcriptome of Labeo rohita.

Introduction: Brain being the master regulator of the physiology of animal, the current study focuses on the gene expression pattern of the brain tissue with special emphasis on regulation of growth, developmental process of an organism and cellular adaptation of Labeo rohita against unfavourable environmental conditions. Methods: RNA-seq study was performed on collected brain samples at 8ppt salt concentration and analyzed for differential gene expression, functional annotation and miRNA-mRNA regulatory network. Results: We found that 2450 genes were having significant differential up and down regulation. The study identified 20 hub genes based on maximal clique centrality algorithm. These hub genes were mainly involved in various signaling pathways, energy metabolism and ion transportation. Further, 326 up and 1214 down regulated genes were found to be targeted by 7 differentially expressed miRNAs i.e., oni-miR-10712, oni-miR-10736, ssa-miR-221-3p, ssa-miR-130d-1-5p, ssa-miR-144-5p and oni-miR-10628. Gene ontology analysis of these differentially expressed genes led to the finding that these genes were involved in signal transduction i.e., calcium, FOXO, PI3K-AKT, TGF-ß, Wnt and p53 signalling pathways. Differentially expressed genes were also involved in regulation of immune response, environmental adaptation i.e., neuroactive ligand-receptor interaction, ECM-receptor interaction, cell adhesion molecules and circadian entrainment, osmoregulation and energy metabolism, which are critical for salinity adaptation. Discussion: The findings of whole transcriptomic study on brain deciphered the miRNA-mRNA interaction patterns and pathways associated with salinity adaptation of L. rohita.

Interplay of gene expression and regulators under salinity stress in gill of Labeo rohita.

BACKGROUND: Labeo rohita is the most preferred freshwater carp species in India. The concern of increasing salinity concentration in freshwater bodies due to climate change may greatly impact the aquatic environment. Gills are one of the important osmoregulatory organs and have direct contact with external environment. Hence, the current study is conducted to understand the gill transcriptomic response of L. rohita under hypersalinity environment. RESULTS: Comprehensive analysis of differentially expressed long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs was performed in gills of L. rohita treated with 2, 4, 6 and 8ppt salinity concentrations. Networks of lncRNA-miRNA-mRNA revealed involvement of 20, 33, 52 and 61 differentially expressed lncRNAs, 11, 13, 26 and 21 differentially expressed miRNAs in 2, 4, 6 and 8ppt groups between control and treatment respectively. These lncRNA-miRNA pairs were regulating 87, 214, 499 and 435 differentially expressed mRNAs (DE mRNAs) in 2, 4, 6 and 8ppt treatments respectively. Functional analysis of these genes showed enrichment in pathways related to ion transportation and osmolyte production to cope with induced osmotic pressure due to high salt concentration. Pathways related to signal transduction (MAPK, FOXO and phosphatidylinositol signaling), and environmental information processing were also upregulated under hypersalinity. Energy metabolism and innate immune response pathways also appear to be regulated. Protein turnover was high at 8ppt as evidenced by enrichment of the proteasome and aminoacyl tRNA synthesis pathways, along with other enriched KEGG terms such as apoptosis, cellular senescence and cell cycle. CONCLUSION: Altogether, the RNA-seq analysis provided valuable insights into competitive endogenous (lncRNA-miRNA-mRNA) regulatory network of L. rohita under salinity stress. L. rohita is adapting to the salinity stress by means of upregulating protein turnover, osmolyte production and removing the damaged cells using apoptotic pathway and regulating the cell growth and hence diverting the essential energy for coping with salinity stress.

Heterologous expression of recombinant nattokinase in Escherichia coli BL21(DE3) and media optimization for overproduction of nattokinase using RSM.

Nattokinase, a serine protease, was discovered in Bacillus subtilis during the fermentation of a soybean byproduct. Nattokinase is essential for the lysis of blood clots and the treatment of cardiac diseases including atherosclerosis, thrombosis, high blood pressure, and stroke. The demand for thrombolytic drugs rises as the prevalence of cardiovascular disease rises, and nattokinase is particularly effective for the treatment of cardiovascular diseases due to its long duration of action. In this study, we cloned the nattokinase gene from the Bacillus subtilis strain into the pET32a vector and expressed the protein in the E. coli BL21(DE3) strain. The active recombinant nattokinase was purified using Ni-NTA affinity chromatography and then evaluated for fibrinolytic and blood clot lysis activity. Physiological parameters for optimizing protein production at optimal pH, temperature, IPTG concentration, and incubation time were investigated. A statistical technique was used to optimize media components for nattokinase overproduction, and Central Composite Design-Response Surface Methodology-based optimization was used to select significant components for protein production. The optimized media produced 1805.50 mg/L of expressed nattokinase and 42.80 gm/L of bacterial mass. The fibrinolytic activity obtained from refolded native protein was 58FU/mg, which was five times higher than the available orokinase drug (11FU/mg). The efficiency with which a statistical technique for media optimization was implemented improved recombinant nattokinase production and provides new information for scale - up nattokinase toward industrial applications.

Kidney transcriptome response to salinity adaptation in Labeo rohita.

The increasing salinization of freshwater resources, owing to global warming, has caused concern to freshwater aquaculturists. In this regard, the present study is aimed at economically important freshwater fish, L. rohita (rohu) adapting to varying degrees of salinity concentrations. The RNA-seq analysis of kidney tissue samples of L. rohita maintained at 2, 4, 6, and 8 ppt salinity was performed, and differentially expressed genes involved in various pathways were studied. A total of 755, 834, 738, and 716 transcripts were downregulated and 660, 926, 576, and 908 transcripts were up-regulated in 2, 4, 6, and 8 ppt salinity treatment groups, respectively, with reference to the control. Gene ontology enrichment analysis categorized the differentially expressed genes into 69, 154, 92, and 157 numbers of biological processes with the p value < 0.05 for 2, 4, 6, and 8 ppt salinity groups, respectively, based on gene functions. The present study found 26 differentially expressed solute carrier family genes involved in ion transportation and glucose transportation which play a significant role in osmoregulation. In addition, the upregulation of inositol-3-phosphate synthase 1A (INO1) enzyme indicated the role of osmolytes in salinity acclimatization of L. rohita. Apart from this, the study has also found a significant number of genes involved in the pathways related to salinity adaptation including energy metabolism, calcium ion regulation, immune response, structural reorganization, and apoptosis. The kidney transcriptome analysis elucidates a step forward in understanding the osmoregulatory process in L. rohita and their adaptation to salinity changes.

Recent advances in the potential applications of luminescence-based, SPR-based, and carbon-based biosensors.

The need for biosensors has evolved in the detection of molecules, diseases, and pollution from various sources. This requirement has headed to the development of accurate and powerful equipment for analysis using biological sensing component as a biosensor. Biosensors have the advantage of rapid detection that can beat the conventional methods for the detection of the same molecules. Bio-chemiluminescence-based sensors are very sensitive during use in biological immune assay systems. Optical biosensors are emerging with time as they have the advantage that they act with a change in the refractive index. Carbon nanotube-based sensors are another area that has an important role in the biosensor field. Bioluminescence gives much higher quantum yields than classical chemiluminescence. Electro-generated bioluminescence has the advantage of miniature size and can produce a high signal-to-noise ratio and the controlled emission. Recent advances in biological techniques and instrumentation involving fluorescence tag to nanomaterials have increased the sensitivity limit of biosensors. Integrated approaches provided a better perspective for developing specific and sensitive biosensors with high regenerative potentials. This paper mainly focuses on sensors that are important for the detection of multiple molecules related to clinical and environmental applications. KEY POINTS: • The review focusses on the applications of luminescence-based, surface plasmon resonance-based, carbon nanotube-based, and graphene-based biosensors • Potential clinical, environmental, agricultural, and food industry applications/uses of biosensors have been critically reviewed • The current limitations in this field are discussed, as well as the prospects for future advancement.

Characterization of VopJ by modelling, docking and molecular dynamics simulation with reference to its role in infection of enteropathogen Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a renowned enteropathogen known for infecting humans. The infection usually involves various genes which help bacterium bypass the immune system of the host. Type III secretion system (T3SS) is an essential factor for the infection. The present study introduces the probable structure of VopJ, a T3SS effector of V. parahaemolyticus. The vopJ gene was amplified and sequenced from V. parahaemolyticus. The model generated through homology modelling showed a Z score of around 2.5, which fits quite near in the standard model available in the databases. The model has only a couple of outlier amino acids, which indicate a good fit of the model. Docking studies with small molecules like Acetyl-CoA, Inositol hexakisphosphate, GTP, and AMP have shown negative ΔG - 10.49, -52.80, -8.36 and -9.02, respectively, which indicates spontaneous binding. The molecular simulation studies have also supported the binding with a low RMSD value of less than 0.5 nm. The RMSF values obtained using the modelling were also quite low (>0.35 nm), which indicates the consistency achieved using the docking studies. These small molecules are very crucial in the MAPK pathways, which is essential for the immune response from the host cell. This effector can thus have an ability to highjack the immune system and help the bacterium in the potent infection. Up to our understanding, this is the first report which describes the in-silico model to understand the mode of infection of T3SS in enteropathogen V. parahaemolyticus. Communicated by Ramaswamy H. Sarma.

Estimation of bisulfate in edible plant foods, dog urine, and drugs: picomolar level detection and bio-imaging in living organisms.

In order to explore the properties of any species in solution, the actual, i.e. equilibrium concentration of the free species should be taken into account. Researchers have not paid attention to the deprotonation equilibrium between HSO4- and SO42- while probing bisulfate ion. In this study, we have addressed this concern and developed two zwitterions, CG (coumarin-integrated glycine) and CA (coumarin-integrated alanine), for the selective detection of HSO4- at a picomolar level (50 to 325 pM) with very high binding affinity (∼108 M-1) in pure water at physiological pH. The principle of HSO4- recognition was established via UV-vis and fluorescence techniques. DFT calculations suggested that the H-bonding interactions between the probes and HSO4- are the driving force for this unforeseen selectivity. The membrane penetration ability and nontoxicity of CG/CA enable them to function as staining agents in living brine shrimps and bacteria. The use of these probes for the estimation of HSO4- in various day-to-day edible foods and drugs along with urine samples is unprecedented. The significance and novelty of this study lies in the application and development of assays for estimating bisulfate in several real-world samples that are predominantly aqueous in nature, which are the first of their kind.

An Anthracene Excimer Fluorescence Probe on Mesoporous Silica for Dual Functions of Detection and Adsorption of Mercury (II) and Copper (II) with Biological In Vivo Applications.

Dual functional activity by the same organic-inorganic hybrid material toward selective metal ion detection and its adsorption has drawn more attraction in the field of sensing. However, most of the hybrid materials in the literature are either for sensing studies or adsorption studies. In this manuscript, a fluorescent active hybrid material SiO2 @PBATPA is synthesized by covalent coupling of anthracene-based chelating ligand N,N'-(propane-1,3-diyl) bis(N-(anthracen-9-ylmethyl)-2-((3-(triethoxysilyl)propyl) amino) acetamide) (PBATPA) within the mesopores of newly synthesized cubic mesoporous silica. The synthetic strategy is designed to form an exclusively intramolecular excimer on a solid surface, which is then used as a sensory tool for selective detection of metal ions through fluorescence quenching by the destruction of excimer upon metal ion binding. The dual functions of sensing and adsorption studies show selectivity toward Hg2+ and Cu2+ among various metal ions with detection limits of 37 and 6 ppb, respectively, and adsorption capacities of 482 and 246 mg g-1 , respectively. This material can be used as a sensory cum adsorbent material in real food samples and living organisms such as the brine shrimp Artemia salina without any toxic effects from the material.

Self-Assembly of Partially Alkylated Dextran- graft-poly[(2-dimethylamino)ethyl methacrylate] Copolymer Facilitating Hydrophobic/Hydrophilic Drug Delivery and Improving Conetwork Hydrogel Properties.

Key issues of injectable hydrogels are incapability of loading hydrophobic drugs due to insolubility of drugs in aqueous prepolymer solution as well as in hydrogel matrix, and high water swelling, which leads to poor mechanical and bioadhesive properties. Herein, we report that self-assembly of partially long-chain alkylated dextran- graft-poly[(2-dimethylamino)ethyl methacrylate] copolymer in aqueous solution could encapsulate pyrene, a hydrophobic probe, griseofulvin, a hydrophobic antifungal drug, and ornidazole, a hydrophilic antibiotic. Addition of activated chloride terminated poly(ethylene glycol) (PEG) into the guest molecules loaded copolymer solution produced an injectable dextran- graft-poly[(2-dimethylamino)ethyl methacrylate]-linked-PEG conetwork hydrogel. The alkylated hydrogels exhibited zero order release kinetics and were mechanically tough (50-54 kPa storage modulus) and bioadhesive (8-9 kPa). The roles of alkyl chains and dextran on the drug loading-release behavior, degradation behavior, gelation time, and the mechanical property of the hydrogels have been studied in details. Additionally, DNA hybrid composite hydrogel was formed owing to the cationic nature of the prepolymer solution and the hydrogel. Controlled alkylation of a prepolymer thus highlights the potential to induce and enhance the hydrogel property.

Siderophore coated magnetic iron nanoparticles: Rational designing of water soluble nanobiosensor for visualizing Al3+ in live organism.

This article aims to establish the judicious use of iron-binding chemistry of microbial chelators in order to functionalize the surface of iron nanoparticles to develop non-toxic nanobiosensor. Anchoring a simple siderophore 2,3-dihydroxybenzoylglycine (H3L), which bears catechol and carboxyl functionalities in tandem, on to the surface of Fe3O4 nanoparticles has developed a unique nanobiosensor HL-FeNPs which showed highly selective and sensitive detection of Al3+ in 100% water at physiological pH. The biosensor HL-FeNPs, with 20nM limit of detection, behaves reversibly and instantly. In-vivo bio-imaging in live brine shrimp Artemia confirmed that HL-FeNPs could be used as fluorescent biomarker for Al3+ in live whole organisms. Magnetic nature of the nanosensor enabled HL-FeNPs to remove excess Al3+ by using external magnet. To our knowledge, the possibility of microbial chelator in the practical development of Al3+ selective nanobiosensor is unprecedented.

Collection of mullet fish (Mugil cephalus) from west coast of India: evaluation of its quality with relation to food safety.

The present study focuses on bacterial and selected heavy metal contaminations of economically important mullet fish (Mugil cephalus) collected from West coast of Gujarat, India. Molecular identification using 16S rRNA sequencing revealed the presence of Aeromonas veronii, A. mollusorum, A. cavae, A. bivalvum, and V.alginolyticus in the gill and the intestine along with some other non-pathogenic bacteria. Pathogenicity of different Aeromonas species was confirmed by hemolysin assay. Apart from pathogenicity, multidrug resistance pattern was also reported against some commonly used antibiotics. Heavy metal analysis of different parts such as ventral and dorsal muscles as well as gills of M. cephalus revealed maximum concentration of Pb (24.08 ± 4.40 mg/kg), Cd (8.25 ± 3.04 mg/kg), and Cu (33.67 ± 5.34 mg/kg), which were higher than the permissible limit. To the best of our knowledge, this is the first study analyzing different heavy metals and associated bacteria in M. cephalus fish in India. Further, the distribution of heavy metals in M. cephalus fish from other countries was also compared.

Reporting a new siderophore based Ca(2+) selective chemosensor that works as a staining agent in the live organism Artemia.

A Ca(2+)-specific chemosensor involving acyclic non-ether and non-carboxylato-type metal chelating ligands is rare. The tetradentate OONO artificial receptor, HL, possessing a sulfur-containing intermediate siderophore aeruginic acid, tethered to a rhodamine 6G based signalling unit in a single molecule has been synthesized. The fluoroionophore required excitation in the visible wavelength (510 nm) and showed highly selective and sensitive detection of Ca(2+) ions in 100% water solution in HEPES buffer at physiological pH (7.4). The probe HL, with LOD as low as 70 nM, behaves reversibly and showed nearly 17-fold enhanced selectivity for Ca(2+) over other cell abundant alkali and alkaline metal ions such as Na(+), K(+), Li(+), and Mg(2+) without any intervention. Job's plot, (1)H NMR titration and ESI-MS data provided corroborative evidence in support of 1 : 1 association between HL and Ca(2+). From a wide range of transition and heavy metal ions series, HL also binds Cu(2+). However, the use of l-cysteine removes the interference from Cu(2+) and results in highly selective detection specificity of HL for Ca(2+). As a reversible "off-on-off" fluorescent chemosensor, it is possible to detect Ca(2+) at as low as 5 µM in the midgut region of the gastrointestinal tract of the live animal Artemia, a brine shrimp.

Naked eye instant reversible sensing of Cu(2+) and its in situ imaging in live brine shrimp Artemia.

A Cu(2+)-specific colorimetric reversible fluorescent receptor was designed and synthesized which showed a naked eye observable colour change from colourless to pink on addition of an aqueous buffer (pH 7.4) solution of 30 ppb Cu(2+). Short response time (≤5 s) and low detection limit (nearly 3 ppb) make suitable as a reliable "dip-in" open eye sensor for Cu(2+). Bio-imaging application in live brine shrimp Artemia enabled to detect Cu(2+) at as low as 10 ppb exposure.

Study on gluco-regulatory potential of glimepiride sulfonamide using in silico, in vitro and in vivo approaches.

Glimepiride sulfonamide (GS) is a drug intermediate to synthesize glimepiride (antidiabetic drug). We hypothesized that GS exerts gluco-regulatory effect because GS is comprised of the structural components of dipeptidyl peptidase-IV (DPP-IV) inhibitors sitagliptin and DPP-728and glimepiride (sulfonylurea based antidiabetic drug).We analyzed the drug-likeness and docking efficiency of GS with DPP-IV using in silico tools. Also DPP-IV inhibition assays were conducted in vitro. Gluco-regulatory potential and parameters of drug safety were evaluated on normal euglycemic and streptozotocin (STZ) induced diabetic rats. We observed strong drug-likeness and DPP-IV binding efficiency of GS. Similarly, profound DPP-IV inhibition was also observed in vitro. Studies on euglycemic and STZ induced diabetic rats revealed antidiabetic potential for GS without significant change in the studied toxicological parameters. Glimepiride sulfonamide has antidiabetic potential mainly through DPP-IV inhibition, but also through insulin stimulation and alpha-amylase inhibition.