Assessment of Genetic Diversity Among Rice Genotypes with Differential Adaptations to Salinity Using Physio-morphological and Molecular Markers

Breeding for salt tolerance using traditional screening and selection methods have been limited by the complex and polygenic nature of salt tolerance trait. This study was designed to evaluate some of the premium Basmati rice varieties for salt tolerance and to characterize genetic diversity among the rice varieties with different adaptations to saline soils using microsatellite (SSR) and ISSR markers. Plants of nine rice varieties including salt tolerant, salt sensitive and traditional Basmati, were grown in hydroponics using Yoshida solution containing 0 (control, pH 5.0) and 30 mM NaCl (Electrical conductivity 4.8 d/S, pH 5.0) and assessed for salinity tolerance on 1–9 scale as per IRRI standard evaluation system using seedling growth parameters, visual salt injuries and Na-K ratio. Physio-morphological studies showed that traditional Basmati rice varieties (Basmati 370 and HBC19) were more sensitive than the salt sensitive control variety, MI-48. SSR as well as ISSR marker systems generated higher levels of polymorphism and could distinguish between all the 9 rice cultivars. A total of 299 (225 polymorphic) and 437 (430 polymorphic) bands were detected using 28 UBC ISSR primers and 100 welldistributed mapped SSR markers, respectively. ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.43). The ISSR and SSR marker data analyzed using clustering algorithms showed two distinct clusters separating the Basmati (Basmati 370, HBC19 and CSR-30) from other non-aromatic indica (IR36, Pokkali, CSR10 and MI-48) rice varieties indicating greater divergence between Basmati and non-aromatic indica rice genotypes. Marker analysis showed a close relationship among the two traditional (Basmati 370 and HBC19) and cross-bred (CSR30) Basmati rice varieties and greater diversity between the two salt-tolerant genotypes, Pokkali and BR4-10.     

Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis

MicroRNA398 targets two Cu/Zn superoxide dismutases (CSD1 and CSD2) in higher plants. Previous investigations revealed both decreased miR398 expression during high Cu²⁺ or paraquat stress and increased expression under low Cu²⁺ or high sucrose in the growth medium. Here, we show that additional abiotic stresses such as ozone and salinity also affect miR398 levels. Ozone fumigation decreased miR398 levels that were gradually restored to normal levels after relieved from the stress. Furthermore, miR398 levels decreased in Arabidopsis leaves infiltrated with avirulent strains of Pseudomonas syringae pv. tomato, Pst DC3000 (avrRpm1 or avrRpt2) but not the virulent strain Pst DC3000. To our knowledge, miR398 is the first miRNA shown to be down-regulated in response to biotic stress (P. syringae). CSD1, but not CSD2, mRNA levels were negatively correlated with miR398 levels during ozone, salinity and biotic stress, suggesting that CSD2 regulation is not strictly under miR398 control during diverse stresses. Overall, this study further establishes a link between oxidative stress and miR398 in Arabidopsis.     

EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains

Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen causing significant economic losses to the cattle industry. Glycoprotein E-deleted marker vaccines form the basis for BoHV-1 control programs widely, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In the present study, we report an EvaGreen-based multiplex real-time polymerase chain reaction (EGRT-PCR) assay for rapid differentiation of wild-type and glycoprotein E-deleted strains of BoHV-1. The EGRT-PCR assay could simultaneously detect two viral genes (glycoprotein B and E) and an internal positive control gene (bovine growth hormone- bGH), in a single-tube reaction. The analytical sensitivity of the EGRT-PCR assay was as little as 10 copies of the BoHV-1 DNA per reaction. The modified real-time PCR assay could successfully differentiate wild-type and gE-deleted BoHV-1 strains based on gene specific melting temperatures (T_m) peaks. Our results have shown that the EGRT-PCR developed in this study might prove to be a promising tool in disease management by enabling rapid differentiation of wild-type and gE-deleted strains of BoHV-1.     

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.     

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.     

Elicitors,SA and MJ enhance carotenoids and tocopherol biosynthesis and expression of antioxidant related genes in Moringa oleifera Lam. leaves

Moringa oleifera Lam. leaves are rich source of carotenoids (provitamin A) and α-tocopherol (vitamin E), and there is a scope for their further enhancement, through elicitor mediation, thereby a great potential for addressing these vitamins deficiency. In the present study, we report the efficacy of foliar administration of biotic elicitors, carboxy-methyl chitosan and chitosan, and signaling molecules, methyl jasmonate (MJ) and salicylic acid (SA) for enhancement of major carotenoids and α-tocopherol. Highest α-tocopherol content of 49.7 mg/100 g FW was recorded upon foliar application of 0.1 mM SA after 24 h of treatment, which represented a 187.5 % increase in comparison to the untreated control. Similarly, a maximum of 52.6 mg/100 g FW lutein, and 21.8 mg/100 g FW β-carotene content were observed in leaves after 24 h of treatment with MJ, which represented a 54.0 and 20.3 % increase in comparison to the untreated control, respectively. Among the major genes of carotenoid biosynthetic pathway, the expression of lycopene β-cyclase (LCY-β) was maximum influenced after treatment with elicitors and signaling molecules, compared to phytoene synthase and phytoene desaturase, suggesting the LCY-β-mediated enhancement in the production of β-carotene in elicitor treated M. oleifera leaves. Enhanced production of α-tocopherol under respective elicitor treatment was further supported by 2.0–2.7 fold up-regulation of γ-tocopherol methyl transferase, compared to untreated control. This is the first report on elicitor-mediated enhanced production of tocopherol and carotenoids in foliage of economically important food plant.     

Consumers using the Internet for insomnia information: The who,what, and why

Information obtained from the Internet often influences the treatment choices of patients with insomnia. This study explored patterns of online information seeking and utilization among patients with insomnia. A total of 1013 participants took part in an online survey about sleep health information between July 2012 and March 2013. Participants also completed the Insomnia Severity Index and the Dysfunctional Beliefs and Attitudes about Sleep Scale. The results showed that those seeking insomnia-related information resources frequently searched online, and the information found appeared to influence important health behaviors such as treatment decisions, taking medication and whether to seek professional care. Information of interest revolved around insomnia treatment options and symptomology. While no predictors for Internet use were identified, the Internet does represent an important health-care portal for insomnia patients and warrants further investigation as targeted e-health interventions become more prominent in the routine management of insomnia.

     

Purification and Characterization of an Extracellular,Thermo-Alkali-Stable,Metal Tolerant Laccase from Bacillus tequilensis SN4

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k _cat/K _m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu²⁺, Co²⁺, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.     

Matrix-Assisted Refolding,Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.     

A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus‐oocyte‐complex (COC)

Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non‐invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell‐safe with no inherent cell toxicity or light‐induced phototoxicity indicated by negative DNA damage staining. Pre‐measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01‐0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.