Identification and validation of cold responsive microRNAs of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using high throughput sequencing

Cold stress is one of the major abiotic factors that influence the productivity and geographical distribution of many agriculturally important crops like Hevea brasiliensis. Cultivation of H. brasiliensis in India is being extended to northeastern regions, where low temperature during winter adversely affects its survival, growth, and productivity. Developing cold-tolerant genotypes is a primary requisite to maximize the productivity under such challenging environmental conditions. However, lack of methods for early evaluation of cold tolerance in the newly developed clones and the extensive time required for assessing their tolerance in the field are major constraints for clonal selection. The present study was initiated with an objective to identify and characterize cold stress responsive miRNAs from H. brasiliensis that show stronger association with cold tolerance. Next generation sequencing using Illumina HiSeq method revealed the expression of 21 and 29 conserved miRNA (from clone RRIM 600) families in cold-stressed and control samples, respectively. Forty-two novel miRNAs were identified from this study. Upon differential expression analysis, eight conserved miRNAs were found commonly expressed in both the samples. When expression analyses were performed subsequently with six selected miRNAs in two Hevea clones (viz. RRII 105 and RRIM 600), miR169 showed a strong association with cold tolerance. miRNAs such as miR482 and miR159 also exhibited association with cold tolerance. This study suggests the possibility of employing these miRNAs as markers for cold tolerance after validation in more number of genotypes with varying levels of cold tolerance.     

Crystal structure of the long-chain fatty acid transporter FadL

The mechanisms by which hydrophobic molecules, such as long-chain fatty acids, enter cells are poorly understood. In Gram-negative bacteria, the lipopolysaccharide layer in the outer membrane is an efficient barrier for fatty acids and aromatic hydrocarbons destined for biodegradation. We report crystal structures of the long-chain fatty acid transporter FadL from Escherichia coli at 2.6 and 2.8 angstrom resolution. FadL forms a 14-stranded beta barrel that is occluded by a central hatch domain. The structures suggest that hydrophobic compounds bind to multiple sites in FadL and use a transport mechanism that involves spontaneous conformational changes in the hatch.     

Crystal structure of a lipid G protein-coupled receptor

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P(1)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P(1), resulting in the modulation of immune and stromal cell responses.     

Formation of Biogenic Amines and Associated Biochemical and Microbial Attributes of Whole Sutchi Catfish (Pangasianodon hypophthalmus) during Iced Storage

Sutchi catfish (Pangasianodon hypophthalmus) is a highly preferred farmed species that is produced in huge quantities. Meat from ice-stored (4 ± 2°C) whole Sutchi catfish was evaluated for formation of biogenic amines, such as putrescine, cadaverine, histamine (HIM), agmatine, tyramine, spermine, and spermidine, and compared with biochemical, microbial, and sensory attributes during 22 days. Analysis of content of biogenic amines in the meat was carried out by liquid chromatography with tandem mass spectrometry using a without derivatization method. Three amines, namely, tyramine, spermidine, and spermine, were only present on 0^th day of storage. Presence of cadaverine was noticed from 9^th day onwards, where the aerobic plate count (APC) reached 4.85 log CFU/g. Putrescine was detected on the 22^nd day of storage, where the APC crossed 7 log CFU/g. HIM was detected in lower quantities from 3^rd day onwards. A shelf life of 15 days was determined based on sensory and microbiological evaluation. Although the samples were biochemically acceptable throughout the storage period, APC exceeded the limit on day 19, and the gradual increase of H₂S-producing bacteria, Brochothrix thermosphacta, Aeromonas, and Enterobacteriaceae, was observed during storage.     

Expression of kit ligand and insulin‐like growth factor binding protein 3 during in vivo or in vitro development of ovarian follicles in sheep

Expression of Kit ligand (KL) and insulin‐like growth factor binding protein (IGFBP3) genes was studied at different in vivo and corresponding in vitro stages of development of the ovarian follicles in sheep. The expression of both KL and IGFBP3 was significantly higher in the primordial follicles relative to any other stage of development. Compared to the other stages, the KL expression in the cumulus cells from in vivo grown large antral follicles and that of IGFBP3 in COCs’ isolated from large antral follicles matured in vitro for 24 hr were significantly higher. In the oocytes from in vivo grown ovarian follicles, the expression of KL was the same at all the stages of development. Insulin‐like growth factor binding protein 3 expression was also the same in the oocytes at all the stages of the development except for a significantly lower expression in those from antral follicles. The expression of KL in the cumulus cells decreased significantly in the in vitro grown early antral follicles but did not change further as the development progressed. The expression of IGFBP3 in the cumulus cells from in vitro grown ovarian follicles appeared to increase as the development progressed although the increase was not significant between any two consecutive stages of development. In the oocytes in in vitro grown ovarian follicles, the expression levels of KL and IGFBP3 genes did not change with development. It is concluded that (i) KL and IGFBP3 genes follow specific patterns of expression during ovarian folliculogenesis and (ii) in vitro culture of preantral follicles compromises the development potential through alterations in the stage‐specific patterns of expression of these and other developmentally important genes.     

Quantitative studies of erythropoiesis in the clinically normal, phlebotomized, and feline leukemia virus-infected cat

Erythropoiesis was evaluated in 5 cats at base line with normal PCV and then in the same cats with anemia induced by phlebotomy and in 5 other cats with nonregenerative anemia from community-acquired feline leukemia virus (FeLV) infection. The hematologic evaluation included complete blood cell and reticulocyte counts, marrow morphologic features, determination of serum erythropoietin concentrations by radioimmunoassay, ferrokinetic studies, and in vitro marrow culture of early erythroid progenitors (erythroid burst-forming units; BFU-E) and late erythroid progenitors (erythroid colony-forming units; CFU-E). Phlebotomized cats developed marrow erythroid hyperplasia and an increased reticulocyte count. Ferrokinetic studies revealed an increase in plasma iron turnover from 1.4 to 3.8 mg of Fe/dl of blood/day and RBC use from 50.4% to 78.5%. The mean CFU-E number and CFU-E/BFU-E ratio increased after phlebotomy, but the increase was not significant (P greater than 0.05). Serum erythropoietin values did increase significantly. In FeLV-infected cats, a nonregenerative anemia was demonstrated by marrow erythroid hypoplasia and a low total reticulocyte count. An increased percentage of rubriblasts and prorubricytes was observed in 4 of the 5 cats. Although serum erythropoietin values were high (321 +/- 123 mU/ml vs normal 14 +/- 1 mU/ml), ferrokinetic data revealed decreased erythropoiesis. Marrow culture studies in the FeLV-infected cats also revealed low numbers of BFU-E and CFU-E, but normal numbers of granulocyte-macrophage progenitors remained. Seemingly, the FeLV infection impaired the ability of feline marrow to respond physiologically to anemia.     

Detection of bicarbonate administration (milkshake) in Standardbred horses

SUMMARY Total plasma carbon dioxide (TCO₂) concentrations were measured in Standardbred horses to determine criteria to discriminate between normal horses and horses with excessive TCO₂ concentrations on raceday. TCO₂ concentrations from stabled horses were distributed normally with a mean of 30.2 mmol/L and a standard deviation of 1.2 (n = 192) while pre-race TCO₂ concentrations were not normally distributed. The results indicate that about 50 horses per million are likely to have TCO₂ concentrations greater than or equal to 35 mmol/L and that it is extremely unlikely that a normal horse would have a resting TCO₂ concentration above 36 mmol/L. These values were associated with sensitivities of 67% and 59%, respectively, and with a specificity of 100%. TCO₂ concentrations were relatively stable in blood samples stored at 4°C for 4 days, whereas the TCO₂ in specimens stored at room temperature (25°C) and at ambient temperature (16–28°C) declined progressively over 5 days. The accuracy and precision of the Beckman EL-ISE Auto Analyser were acceptable and within the manufacturers specified range. Paired specimens analysed using a Beckman EL-ISE Auto Analyser and a Kodak Dry Chemistry Analyser were not significantly different. However, the measurements made using the Kodak Dry Chemistry Analyser averaged 0.5 mmol/L higher than those analysed on the Beckman EL-ISE. The significance of these sources of variation in TCO₂ concentration in relation to the testing of horses for ‘milkshake’ administration are discussed.     

Studies on itraconazole delivery and pharmacokinetics in mallard ducks (Anas platyrhynchos)

Avian aspergillosis is commonly treated with itraconazole (ITZ). This paper describes two studies using mallard ducks (Anas platyrhynchos). The first study evaluated in vivo release of ITZ from subcutaneously injected controlled-release gel formulations and the second study compared pharmacokinetic parameters for two ITZ oral suspensions. ITZ-A suspension was prepared by mixing contents of commercially available capsules with hydrochloric acid and orange juice. ITZ-B suspension was prepared by dispersing the complex of the drug with hydroxypropyl-beta-cyclodextrin in water. Concentrations of ITZ and its active metabolite, hydroxyitraconazole (OH-ITZ), in plasma and tissue samples were measured using high-performance liquid chromatography. In the second study, drug concentrations in plasma samples were also analyzed using a bioassay. After administration of two ITZ controlled-release formulations, plasma and tissue concentrations of ITZ and OH-ITZ were either very low (< or = 52 ng/mL) or undetectable. Exceptions included skin, subcutaneous fat, and muscle adjacent to the injection site. The drug from ITZ-A and ITZ-B suspensions was absorbed after oral administration. ITZ pharmacokinetic parameters for both suspensions in mallard ducks were similar and the bioassay successfully measured ITZ equivalents in plasma samples from ducks.