Establishment of <Emphasis Type="Italic">lacZ</Emphasis> marked strain of phosphate solubilizing bacterium in the rhizosphere and its effect on plant growth in mungbean

The establishment of lacZ marked strain of P-solubilizing bacterium Pseudomonas in the rhizosphere of mungbean (Vigna radiata) under pothouse conditions was studied. The lacZ marker was transferred to Pseudomonas P-36 on LB medium using donor strain of E. coli. The lacZ marked strain formed blue colonies on selective media and could be identified from soil on the basis of this character. The lacZ marked strain was able to survive in rhizosphere of mungbean under pothouse conditions and maintained a population of about 10⁴ g^?1 of rhizosphere soils up to 60 days study period. Positive effect of inoculation with P-solubilizing bacterium on dry matter yield, P and N-uptake was observed using rock phosphate and single super phosphate as P sources with and without farmyard amendment.     

Functional and crystal structure analysis of active site adaptations of a potent anti-angiogenic human tRNA synthetase

Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.     

Attenuation of cadmium toxicity in mycorrhizal celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.)

A pot-culture experiment was carried out to investigate the effect of arbuscular mycorrhizal (AM) fungus (Glomus macrocarpum Tul. and Tul.) on plant growth and Cd²⁺uptake by Apium graveolens L. in soil with different levels of Cd²⁺. Mycorrhizal (M) and non-mycorrhizal (NM) plants were grown in soil with 0, 5, 10, 40 and 80 Cd²⁺ mg kg⁻¹soil. The infectivity of the fungus was not affected by the presence of Cd²⁺ in the soil. M plants showed better growth and less Cd²⁺ toxicity symptoms. Cd²⁺ root : shoot ratio was higher in M plants than in NM plants. These differences were more evident at highest Cd²⁺ level (80 mg kg⁻¹ soil). Chlorophyll a and chlorophyll b concentrations were significantly higher in AM-inoculated celery leaves. The dilution effect due to increased biomass, immobilization of Cd²⁺ in root and enhanced P-uptake in M plants may be related to attenuation of Cd²⁺toxicity in celery.     

Extraction and purification of an unusual phycoerythrin in a terrestrial desiccation tolerant cyanobacterium <Emphasis Type="BoldItalic">Lyngbya arboricola</Emphasis>

Presence and stability of an unusual phycoerythrin (PE) characteristically similar to R-PE are described in a terrestrial, desiccation-tolerant cyanobacterium, Lyngbya arboricola. Extraction and purification of the PE by using acetone precipitation, gel filtration and ion-exchange chromatography resulted in achieving a purity index (A₅₆₀/A₂₈₀) of up to 5.2. SDS-PAGE of the PE showed presence of 18 kDa, 20 kDa and 32 kDa bands corresponding to α, β and γ subunits of R-PE without any other contaminating phycobiliproteins (PBPs). The absorption spectrum of the PE was distinguished by two major peaks at 499 and 559 nm. The maximum fluorescence emission at room temperature was 578 nm. Spectroscopic and electrophoresis characteristics of PE in the dry mats on storage at 25 ± 1°C over silica gel for 2 years remained almost unaffected. Quantitatively, storage stability of the PE was in the order of dry mats > lyophilized > liquid state and the impact of temperature on loss of PE was in the order of 25°C > −20°C > 4°C. The relevance of L. arboricola for production of stable unusual PE is discussed.     

Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions

A novel cross-linked enzyme aggregates (CLEA) concept called combi-CLEA has been described. It is based upon the fact that CLEA can be made from heterogeneous populations of proteins/enzymes. Porcine pancreatic acetone powder crude extract was used for preparing CLEA in such a way that lipase, -amylase, phospholipase A₂ activities were retained upto 100%. The lipase present in the CLEA showed greater thermal stability at 50 °C as compared to free enzyme. For lipase and phospholipase A₂, V_max/K_m showed no significant change upon combi-CLEA formation but decreased significantly for -amylase activity from 190 to 114 min⁻¹. The lipase activity and -amylase activity in CLEA were completely retained upto three cycles of use. The scanning electron microscopic (SEM) studies showed that morphology of CLEA changed upon inactivation by reuses.     

Differential dynamics of RAS isoforms in GDP‐ and GTP‐bound states

RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP‐bound) and inactive (GDP‐bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP‐ (total of 3.06 μs) and GTP‐bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25–40) and SwitchII (Residues 59–75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP‐bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild‐type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF‐assisted nucleotide exchange. Proteins 2015; 83:1091–1106. © 2015 Wiley Periodicals, Inc.     

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Enhancement in current density and energy conversion efficiency of 3-dimensional MFC anodes using pre-enriched consortium and continuous supply of electron donors

Using a pre-enriched microbial consortium as the inoculum and continuous supply of carbon source, improvement in performance of a three-dimensional, flow-through MFC anode utilizing ferricyanide cathode was investigated. The power density increased from 170 W/m³ (1800 mW/m²) to 580 W/m³ (6130 mW/m²), when the carbon loading increased from 2.5 g/l-day to 50 g/l-day. The coulombic efficiency (CE) decreased from 90% to 23% with increasing carbon loading. The CEs are among the highest reported for glucose and lactate as the substrate with the maximum current density reaching 15.1 A/m². This suggests establishment of a very high performance exoelectrogenic microbial consortium at the anode. A maximum energy conversion efficiency of 54% was observed at a loading of 2.5 g/l-day. Biological characterization of the consortium showed presence of Burkholderiales and Rhodocyclales as the dominant members. Imaging of the biofilms revealed thinner biofilms compared to the inoculum MFC, but a 1.9-fold higher power density.     

Validation of the post sleep questionnaire for assessing subjects with restless legs syndrome: results from two double-blind,multicenter, placebo-controlled clinical trials

Background  

Because of the subjective nature of Restless Legs Syndrome (RLS) symptoms and the impact of these symptoms on sleep, patient-reported outcomes (PROs) play a prominent role as study endpoints in clinical trials investigating RLS treatments. The objective of this study was to validate a new measure, the Post Sleep Questionnaire (PSQ), to assess sleep dysfunction in subjects with moderate-to-severe RLS symptoms.