QTLs for cell membrane stability and flag leaf area under drought stress in a wheat RIL population

A population of 206 recombinant inbred lines (RILs F₉–F₁₀) derived from wheat cross WL711/C306 was phenotyped for morpho-physiological traits such as flag leaf area (FLA), flag leaf length (FLL), flag leaf width (FLW), and cell membrane stability (CMS) under water deficit stress (WDS) environment. High yielding cultivar, WL711 had higher FLA than the medium yielding cultivar C306 across trials under both environments. Parent cultivar C306 maintained membrane integrity while WL711 showed higher membrane damage under WDS. The RIL population showed considerable variation, normal distribution and transgressive segregation for FLA, FLL, FLW and CMS under WDS. The genetic linkage map of WL711/C306 RIL population was constructed comprising of 346 markers. The total map distance was 4526.8 cM with an averaged interval of 12.9 cM between adjacent markers. Major consistent QTL for FLA, FLL, FLW, and CMS were identified on chromosomes 2DS and 3BS respectively in the WL711/C306 RIL population under WDS. The major QTL for FLA, qFLAWD.2D.1 which expressed in multiple environments and for CMS, qCMSWD.3B.3 and qCMSWD.3B.4, accounted for a large proportion of phenotypic variance (PV) with positive allele being contributed by C306, a drought resistant (DR) parent. QTL qFLAWD.2D.1 for FLA co-located with QTL for grain number (GN) and days to flowering (DTF) while QTL qCMSWD.3B.3 and qCMS.3B.4 co-located with QTL for grain yield and its components, days to flowering, canopy temperature and coleoptiles length as reported in our previous publications on the WL711/C306 population (Shukla et al. in Euphytica 203:449–467, 2015; Singh et al. in J Plant Biochem Biotechnol 24:324–330, 2015). Two candidate genes Ghd7 for grain yield and heading date and OsCDK4 for calcium dependent protein kinases were identified in the 2DS and 3BS QTL regions respectively on comparison with gene content of rice chromosomes 7 and 1 respectively. Hence, QTLs qFLAWD.2D.1 and qCMSWD.3B.3 are potential target regions for fine mapping and marker assisted selection for FLA and CMS respectively in wheat under water deficit environments.

     

Characterization and identification of phytase-producing bacteria isolated from the gastrointestinal tract of four freshwater teleosts

Isolation and enumeration of phytase-producing bacteria in the proximal intestine (PI) and distal intestine (DI) of four freshwater teleosts, Nile tilapia (Oreochromis niloticus), murrel (Channa punctatus), climbing perch (Anabas testudineus), and stinging catfish (Heteropneustes fossilis) have been carried out following enrichment culture technique. The bacterial isolates were screened on the basis of their phytase-producing ability. In modified phytase screening medium (MPSM), phytase-producing strains were recorded at higher densities in the PI of Nile tilapia and climbing perch and at a minimum in the DI of catfish. Out of 32 isolates, 20 phytase-producing strains (9 from the DI and 11 from the PI) were primarily selected on the basis of qualitative assay on MPSM plates. Among these isolates, 3 strains (2 from the PI and 1 from the DI) were selected as potent phytase producers according to quantitative enzyme assay. Maximum phytase activity was detected in the bacterial strain ONF2 isolated from the PI of O. niloticus followed by CPF6 and CPH6, isolated from the PI and DI, respectively of C. punctatus. All the three selected phytase-producing strains were Gram-positive rods, capable of forming endospores, and could tolerate a wide range of temperature (25–42 °C) and pH (6–10). The strain CPF6 was able to grow at temperatures up to 55 °C. On the basis of 16S rDNA sequence analysis, isolates ONF2, CPF6 and CPH6 were identified as Bacillus licheniformis. The strain ONF2 showed 100 % similarity to B. licheniformis strain LCR32 (Accession no. FJ976541.1) whereas CPF6 and CPH6 showed 99 % similarity to B. licheniformis strain LCR32 (Accession no. FJ976541.1).     

Identification, mapping and characterization of two genes ofEscherichia coli K-12 regulating growth and resistance to streptomycin in cold

Escherichia coli MD1157, a routine isolate of AB1157 maintained in our laboratory, was noticed to have spontaneously acquired two conditional cold-dependent phenotypes: C^s (cold sensitivity) and Sm^sc (streptomycin sensitivity in cold). C^s involved delayed appearance of visible colonies on solid (LB or minimal) medium in cold (22° C or below) without any loss of viability, and an extended lag period and longer doubling time following a temperature downshift in liquid medium. Sm^sc involved conditional suppression of therpsL31 -mediated streptomycin (Sm) resistance in cold, resulting in reduced colony forming ability in the presence of Sm. This phenotype was seen only on LB plates and weakly on minimal-medium plates containing some LB, but not on minimal medium alone. Genetic mapping traced these two phenotypes to mutations in two genes mapping to the 14-15 min region of the standardE. coli map, which have been namedgicA (growth in cold) andgicB respectively. Comparison of MD1157 with transductants which had lost either one or both of these mutations showed that whilegicBl contributes only to Sm^sc,gicAl is associated with both C^s and Sm^sc. Comparison of these strains with AB1157 suggested the involvement of a third, as yet unidentified gene in causing these phenotypes.     

Computational modeling suggests binding-induced expansion of Epsin disordered regions upon association with AP2

Intrinsically disordered regions (IDRs) are prevalent in the eukaryotic proteome. Common functional roles of IDRs include forming flexible linkers or undergoing allosteric folding-upon-binding. Recent studies have suggested an additional functional role for IDRs: generating steric pressure on the plasma membrane during endocytosis, via molecular crowding. However, in order to accomplish useful functions, such crowding needs to be regulated in space (e.g., endocytic hotspots) and time (e.g., during vesicle formation). In this work, we explore binding-induced regulation of IDR steric volume. We simulate the IDRs of two proteins from Clathrin-mediated endocytosis (CME) to see if their conformational spaces are regulated via binding-induced expansion. Using Monte-Carlo computational modeling of excluded volumes, we generate large conformational ensembles (3 million) for the IDRs of Epsin and Eps15 and dock the conformers to the alpha subunit of Adaptor Protein 2 (AP2α), their CME binding partner. Our results show that as more molecules of AP2α are bound, the Epsin-derived ensemble shows a significant increase in global dimensions, measured as the radius of Gyration (R_G) and the end-to-end distance (EED). Unlike Epsin, Eps15-derived conformers that permit AP2α binding at one motif were found to be more likely to accommodate binding of AP2α at other motifs, suggesting a tendency toward co-accessibility of binding motifs. Co-accessibility was not observed for any pair of binding motifs in Epsin. Thus, we speculate that the disordered regions of Epsin and Eps15 perform different roles during CME, with accessibility in Eps15 allowing it to act as a recruiter of AP2α molecules, while binding-induced expansion of the Epsin disordered region could impose steric pressure and remodel the plasma membrane during vesicle formation.     

PP1, PKA and DARPP-32 in breast cancer: A retrospective assessment of protein and mRNA expression

Cyclic AMP–dependent protein kinase A (PKA) and protein phosphatase 1 (PP1) are proteins involved in numerous essential signalling pathways that modulate physiological and pathological functions. Both PP1 and PKA can be inhibited by dopamine- and cAMP-regulated phosphoprotein 32 kD (DARPP-32). Using immunohistochemistry, PKA and PP1 expression was determined in a large primary breast tumour cohort to evaluate associations between clinical outcome and clinicopathological criteria (n > 1100). In addition, mRNA expression of PKA and PP1 subunits was assessed in the METABRIC data set (n = 1980). Low protein expression of PKA was significantly associated with adverse survival of breast cancer patients; interestingly, this relationship was stronger in ER-positive breast cancer patients. PP1 protein expression was not associated with patient survival. PKA and PP1 subunit mRNA was also assessed; PPP1CA, PRKACG and PRKAR1B were associated with breast cancer–specific survival. In patients with high expression of DARPP-32, low expression of PP1 was associated with adverse survival when compared to high expression in the same group. PKA expression and PP1 expression are of significant interest in cancer as they are involved in a wide array of cellular processes, and these data indicate PKA and PP1 may play an important role in patient outcome.     

CpG Dinucleotide Frequencies Reveal the Role of Host Methylation Capabilities in Parvovirus Evolution

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to “fractional” methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.     

Discovery of a potent and selective small molecule hGPR91 antagonist

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC₅₀: 0.8 μM)—originally synthesized in Merck for Bradykinin B₁ Receptor (BK₁R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5g (IC₅₀: 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC₅₀: 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.