Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response.

BACKGROUND: Bovine milk contains extracellular vesicles (EVs) that play a role in cellular communication, acting in either an autocrine, paracrine, or an exocrine manner. The unique properties of the EVs protect the cargo against degradation. We profiled the ncRNAs (non-coding RNA) present in the EVs from seven dairy products - raw whole milk, heat-treated skim milk, homogenized heat-treated skim milk, pasteurized homogenized skim milk, pasteurized heavy whipping cream, sweet cream buttermilk and cultured buttermilk with four replicates each, obtained at different processing steps from a commercial dairy plant. EVs and their cargo were extracted by using a validated commercial kit that has been shown to be efficient and specific for EVs. Further, to find the annotation of ncRNAs, we probed bovine and other organism repositories(such as miRBase, miRTarBase, Ensemble) to find homolog ncRNA annotation in case the annotations of ncRNA are not available in Bos Taurus database. RESULTS: Specifically, 30 microRNAs (miRNAs), were isolated throughout all the seven milk samples, which later when annotated with their corresponding 1546 putative gene targets have functions associated with immune response and growth and development. This indicates the potential for these ncRNAs to beneficially support mammary health and growth for the cow as well as neonatal gut maturation. The most abundant miRNAs were bta-miR-125a and human homolog miR-718 based on the abundance values of read count obtained from the milk samples.bta-miR-125a is involved in host bacterial and viral immune response, and human homolog miR-718 is involved in the regulation of p53, VEGF, and IGF signaling pathways, respectively. Sixty-two miRNAs were up-regulated and 121 miRNAs were down-regulated throughout all the milk samples when compared to raw whole milk. In addition, our study explored the putative roles of other ncRNAs which included 88 piRNAs (piwi-interacting RNA), 64 antisense RNAs, and 105 lincRNAs (long-intergenic ncRNAs) contained in the bovine exosomes. CONCLUSION: Together, the results indicate that bovine milk contains significant numbers of ncRNAs with putative regulatory targets associated with immune- and developmental-functions important for neonatal bovine health, and that processing significantly affects the ncRNA expression values; but statistical testing of overall abundance(read counts) of all miRNA samples suggests abundance values aren't much affected. This can be attributed to the breakage of exosomal vesicles during the processing stages. It is worth noting, however, that these gene regulatory targets are putative, and further evidence could be generated through experimental validation.

Pharmacokinetics, dose proportionality and permeability of S002-333 and its enantiomers, a potent antithrombotic agent, in rabbits.

1. S002-333 [(2-(4'-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3-carboxylic acid amide)] is a novel and potent antithrombotic active agent. The present work investigates the pharmacokinetics, bioavailability, dose proportionality and permeability of the racemate, S002-333 in male New Zealand White (NZW) rabbits. 2. Rabbits were administered single intravenous (i.v.) (2 mg/kg) and three oral doses of 10, 20 and 40 mg/kg of S002-333, respectively, at different occasions to evaluate dose proportionality. Serial blood samples were collected and analyzed by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Since S002-333 is a racemate consisting of S004-1032 (R) and S007-1558 (S), same samples were analyzed using a chiralcel column so as to evaluate the respective enantiomers. 3. The peak plasma concentration, after oral administration, occurred at ∼10 h post-dose. The clearance (CL) and volume of distribution (Vd) after i.v. dose were found to be 3.05 ± 0.09 l/h/kg and 6.73 ± 1.16 l/kg, respectively. The absolute oral bioavailability of S002-333 was 16.32%, whereas it was 6.62 and 5.90% for R- and S-enantiomers, respectively. The absolute bioavailability of 10, 20 and 40 mg/kg doses were found to be 27.91, 14.39 and 16.91%, respectively. The PAMPA (parallel artificial membrane permeability assay) assay shows that S002-333 has a low-passive permeability at gastric and intestinal environment. 4. In conclusion, S002-333 has low-passive permeability, low CL and large Vd. The R-enantiomer has a "slightly" greater bioavailability than the S-enantiomer.

Profiling the structural determinants for the selectivity of representative factor-Xa and thrombin inhibitors using combined ligand-based and structure-based approaches.

The current study deciphers the combined ligand- and structure-based computational insights to profile structural determinants for the selectivity of representative diverse classes of FXa-selective and thrombin-selective as well as dual FXa-thrombin high affinity inhibitors. The thrombin-exclusive insertion 60-loop (D-pocket) was observed to be one of the most notable recognition sites for the known thrombin-selective inhibitors. Based on the topological comparison of four common active-site pockets (S1-S4) of FXa and thrombin, the greater structural disparity was observed in the S4-pocket, which was more symmetrical (U-shaped) in FXa as compared to thrombin mainly due to the presence of L99 and I174 residues in latter in place of Y99 and F174 respectively in former protease. The S2 pocket forming partial roof at the entry of 12 Å deep S1-pocket, with two extended ß-sheets running antiparallel to each other by undergoing U-turn (∼180̊), has two conserved glycine residues forming H-bonds with the bound ligand for governing ligand binding affinity. The docking, scoring, and binding pose comparison of the representative high-affinity and selective inhibitors into the active sites of FXa and thrombin revealed critical residues (S214, Y99, W60D) mediating selectivity through direct- and long-range electrostatic interactions. Interestingly, most of the thrombin-selective inhibitors attained S-shaped conformation in thrombin, while FXa-selective inhibitors attained L-shaped conformations in FXa. The role of residue at 99th position of FXa and thrombin toward governing protease selectivity was further substantiated using molecular dynamics simulations on the wild-type and mutated Y99L FXa bound to thrombin-selective inhibitor 2. Furthermore, predictive CoMFA (FXa q² = 0.814; thrombin q² = 0.667) and CoMSIA (FXa q² = 0.807; thrombin q² = 0.624) models were developed and validated (FXa r²(test) = 0.823; thrombin r(2)(test) = 0.816) to feature molecular determinants of ligand binding affinity using the docking-based conformational alignments (DBCA) of 141 (88(train)+53(test)) and 39 (27(train)+11(test)) nonamidine class of potent FXa (0.004 ≤ K(i) (nM) ≤ 4700) and thrombin (0.001 ≤ K(i) (nM) ≤ 940) inhibitors, respectively. Interestingly, the ligand-based insights well corroborated with the structure-based insights in terms of the role of steric, electrostatic, and hydrophobic parameters for governing the selectivity for the two proteases. The new computational insights presented in this study are expected to be valuable for understanding and designing potent and selective antithrombotic agents.

Efficiency of Homology Modeling Assisted Molecular Docking in G-protein Coupled Receptors.

BACKGROUND: Molecular docking is in regular practice to assess ligand affinity on a target protein crystal structure. In the absence of protein crystal structure, the homology modeling or comparative modeling is the best alternative to elucidate the relationship details between a ligand and protein at the molecular level. The development of accurate homology modeling (HM) and its integration with molecular docking (MD) is essential for successful, rational drug discovery. OBJECTIVE: The G-protein coupled receptors (GPCRs) are attractive therapeutic targets due to their immense role in human pharmacology. The GPCRs are membrane-bound proteins with the complex constitution, and the understanding of their activation and inactivation mechanisms is quite challenging. Over the past decade, there has been a rapid expansion in the number of solved G-protein-coupled receptor (GPCR) crystal structures; however, the majority of the GPCR structures remain unsolved. In this context, HM guided MD has been widely used for structure-based drug design (SBDD) of GPCRs. METHODS: The focus of this review is on the recent (i) developments on HM supported GPCR drug discovery in the absence of GPCR crystal structures and (ii) application of HM in understanding the ligand interactions at the binding site, virtual screening, determining receptor subtype selectivity and receptor behaviour in comparison with GPCR crystal structures. RESULTS: The HM in GPCRs has been extremely challenging due to the scarcity in template structures. In such a scenario, it is difficult to get accurate HM that can facilitate understanding of the ligand-receptor interactions. This problem has been alleviated to some extent by developing refined HM based on incorporating active /inactive ligand information and inducing protein flexibility. In some cases, HM proteins were found to outscore crystal structures. CONCLUSION: The developments in HM have been highly operative to gain insights about the ligand interaction at the binding site and receptor functioning at the molecular level. Thus, HM guided molecular docking may be useful for rational drug discovery for the GPCRs mediated diseases.

Influence of socio-economic status and television watching on childhood obesity in Kolkata.

Obesity is fast becoming an epidemic among the urban children and it has its adverse effect on the status of health even during adulthood. In this paper an attempt is made to assess the percentage of obesity among 6-10 year children and assess the effect of different socio-economic variables and TV watching on childhood obesity. We restricted our study to primary school-going children who attended classes I-IV. The sample consisted of 5216 children from 20 different Bengali medium and English medium schools in Kolkata. Categorical logistic regression of obesity on the socio-economic factors namely type of medium school, religion, parent's education, duration of television watching etc., has been carried out. The categorical logistic regression shows the significant effect of some of the socio-economic or demographic variables including the duration of television watching on obesity. We have seen a positive association between obesity and TV watching and also between obesity and consumption of fast food. This calls for making the parents aware and taking action as early as possible.

Novel Glycoprotein VI Antagonists as Antithrombotics: Synthesis, Biological Evaluation, and Molecular Modeling Studies on 2,3-Disubstituted Tetrahydropyrido(3,4-b)indoles.

The development of small molecule inhibitors targeting GPVI has promising therapeutic role, as they inhibit arterial thrombosis with limited risk of bleeding. Among the compounds showing in vivo antithrombotic activity, the most active compound 6b (ED50 = 28.36 µmol/kg po in mice) showed improved inhibition for collagen (IC50 = 6.7 µM), CRP-XL (IC50 = 53.5 µM), and convulxin (CVX) (IC50 = 5.7 µM) mediated platelet aggregation as compared to losartan (LOS) (collagen, IC50 = 10.4 µM; CRP-XL, IC50 = 158 µM; CVX, IC50 = 11 µM) than any of its enantiomers S (6c) (collagen, IC50 = 25.3 µM; CRP-XL, IC50 = 181.4 µM; CVX, IC50 = 9 µM) and R (6d) (collagen, IC50 = 126.3 µM; CRP-XL, IC50 > 500 µM; CVX, IC50 = 86.8 µM). Compound 6b also inhibited platelet P-selectin expression and thus may diminish atherosclerosis. The molecular interactions of both enantiomers 6c and 6d at the GPVI receptor have been explained through docking studies.

Modelling passive cardiac conductivity during ischaemia.

The results of a geometric model of cardiac tissue, used to compute the bidomain conductivity tensors during three phases of ischaemia, are described. Ischaemic conditions were simulated by model parameters being changed to match the morphological and electrical changes of three phases of ischaemia reported in literature. The simulated changes included collapse of the interstitial space, cell swelling and the closure of gap junctions. The model contained 64 myocytes described by 2 million tetrahedral elements, to which an external electric field was applied, and then the finite element method was used to compute the associated current density. In the first case, a reduction in the amount of interstitial space led to a reduction in extracellular longitudinal conductivity by about 20%, which is in the range of reported literature values. Moderate cell swelling in the order of 10-20% did not affect extracellular conductivity considerably. To match the reported drop in total tissue conductance reported in experimental studies during the third phase of ischaemia, a ten fold increase in the gap junction resistance was simulated. This ten-fold increase correlates well with the reported changes in gap junction densities in the literature.

Pharmacophore Modeling, Docking and Molecular Dynamics Studies on Caspase-3 Activators Binding at ß-Tubulin Site.

Induction of apoptosis by the activation of caspase 3 makes it a promising target for designing anticancer drugs hence an investigation for the essential structural features mandatory for caspase 3 activation has been carried out using a dataset comprising of caspase 3 activator candidate drug Azixa in phase II clinical trial and its analogs using DS2.0. A training set of 40 compounds was selected for the purpose of model generation from 76 molecules with an activity range spanning from 0.002µM to 6.9µM. Among the generated pharmacophore models, the best model Hypo1 constituted by two hydrophobic aliphatic (Hal), two hydrophobic aromatic (Har), and one hydrogen bond acceptor (HBA) features with a correlation coefficient of 0.85, and a cost difference (null cost - total cost) of 46 bits well predicted the test set of 36 compounds (Rpred = 0.8). The key mechanism conferring caspase 3 activation is due to binding of Azixa at ß-tubulin site that is located close to or at same site as colchicine. In the absence of co-crystal structure we have proposed a binding mode of Azixa at the tubulin site by performing docking studies and performed molecular dynamics simulation to ascertain the temporal changes of the protein-ligand complex.

Novel Glycoconjugate of 8-Fluoro Norfloxacin Derivatives as Gentamicin-resistant Staphylococcus aureus Inhibitors: Synthesis and Molecular Modelling Studies.

Antibiotic resistance has been the subject of interest in clinical practice due to high prevalence of antibiotic-resistant pathogenic organisms. In view of the prevalence of lesser resistance in antibiotics belonging to aminoglycoside class of compounds viz. Food and Drug Administration-approved gentamicin for the treatment of Staphylococcus infections, which also has instances of resistance in the clinical isolates of Staphylococcus aureus, a series of novel glycoconjugates of 8-fluoro norfloxacin analogues with high regio-selectivity by employing copper (I)-catalyzed 1, 3-dipolar cycloaddition of 1-O-propargyl monosaccharides has been synthesized and evaluated for the antibacterial activity against gentamicin resistance Staphylococcus aureus. Among these compounds, the compound 10g showed better antibacterial activity (MIC = 3.12 µg/ml) than gentamicin (Escherichia coli (12.5 µg/ml), Staphylococcus aureus (6.25 µg/ml) and Klebsiella pneumonia (6.25 µg/ml), including gentamicin resistant (>50 µg/ml) strain in vitro). The docking studies suggest DNA gyrase of Staphylococcus aureus as a probable target for the antibacterial action of compound 10g.

N-Phenyl-N'-acetylthiourea-a specific reagent for photometric determination of ruthenium.

N-Phenyl-N'-acetylthiourea is recommended as a specific chelating agent for the spectrophotometric determination of ruthenium. The blue complex formed in 5-8M hydrochloroic acid is not extractable into common organic solvents but is soluble in 30% ethanol solution. The colour is stable for up to 48 hr and Beer's law is obeyed at 650 nm over the metal concentration range 2-18 mug/ml. The molar absorptivity is 4.7 x 10(3) l.mole(-1). cm(-1).

Use of 3-hydroxy-1,3-diphenyltriazine in the spectrophotometric determination of copper, palladium, iron, cobalt, nickel, and molybdenum.

3-Hydroxy-1,3-diphenyltriazine (HDPTA) forms brightly coloured complexes with copper, palladium, iron, cobalt and nickel ions, which are readily extractable in benzene. The deep yellow molybdenum complex is completely soluble in 60% ethanol. All the chelates have a definite absorption maximum between 398 and 422 nm, the reagent absorption being negligible above 415 nm. The molar composition of the chelates is ML(2), except for iron and cobalt which form ML(3) complexes. Microquantities of these metals can be determined spectrophotometrically with HDPTA.

Extraction and spectrophotometric determination of ruthenium and osmium with thiobenzhydrazide.

Ruthenium forms a pink complex with thiobenzhydrazide in hot 1.0-4.5M hydrochloric acid medium, which can be extracted with chloroform, and the extract shows maximal absorbance at 520 nm. The chloroform-extractable osmium-thiobenzhydrazide complex formed at pH 2.3-4.8 shows maximal absorption at 385 nm as well as at 480-490 nm. The colour of the extracts of both the complexes is stable for more than 24 hr and can be employed for the spectrophotometry of ruthenium and osmium in the presence of a considerable excess of diverse ions commonly associated with them. Ruthenium and osmium can be quantitatively separated from one another with thiobenzhydrazide.

CoMFA, CoMSIA, and docking studies on thiolactone-class of potent anti-malarials: identification of essential structural features modulating anti-malarial activity.

The integrated ligand- and structure-based drug design techniques have been applied on a homogeneous dataset of thiolactone-class of potent anti-malarials, to explore the essential structural features for the inhibition of Plasmodium falciparum. Developed CoMFA (q(2) = 0.716) and CoMSIA (q(2) = 0.632) models well explained structure-activity variation in both the training (CoMFA R(2) = 0.948 & CoMSIA R(2) = 0.849) and test set (CoMFA R(2) (pred) = 0.789 & CoMSIA R(2) (pred) = 0.733) compounds. The docking and scoring of the most active compound 10 into the active site of high-resolution (2.35 Å) structure of FabB-TLM binary complex (PDB-ID: 1FJ4) indicated that thiolactone core of this compound forms bifurcated H-bonding with two catalytic residues His298 and His333, and its saturated decyl side group is stabilized by hydrophobic interactions with the residues of a small hydrophobic groove, illustrating that the active site architecture, including two catalytic histidines and a small hydrophobic groove, is vital for protein-ligand interaction. In particular, the length and flexibility of the side group attached to the position 5 of thiolactone have been observed to play a significant role in the interaction with FabB enzyme. These results present scope for rational design of thiolactone-class of compounds that could furnish improved anti-malarial activity.