In silico structural homology modeling of nif A protein of rhizobial strains in selective legume plants

Symbiosis is a complex genetic regulatory biological evolution which is highly specific pertaining to plant species and microbial strains. Biological nitrogen fixation in legumes is a functional combination of nodulation by nod genes and regulation by nif, fix genes. Three rhizobial strains (Rhizobium leguminosarum, Bradyrhizobium japonicum, and Mesorhizobium ciceri) that we considered for in silico analysis of nif A are proved to be the best isolates with respect to N₂ fixing for ground nut, chick pea and soya bean (in vitro) out of 47 forest soil samples. An attempt has been made to understand the structural characteristics and variations of nif genes that may reveal the factors influencing the nitrogen fixation. The primary, secondary and tertiary structure of nif A protein was analyzed by using multiple bioinformatics tools such as chou-Fasman, GOR, ExPasy ProtParam tools, Prosa -web. Literature shows that the homology modeling of nif A protein have not been explored yet which insisted the immediate development for better understanding of nif A structure and its influence on biological nitrogen fixation. In the present predicted 3D structure, the nif A protein was analyzed by three different software tools (Phyre2, Swiss model, Modeller) and validated accordingly which can be considered as an acceptable model. However further in silico studies are suggested to determine the specific factors responsible for nitrogen fixing in the present three rhizobial strains.     

Relative growth rate,biomass partitioning and nutrient allocation in seedlings of two threatened trees grown under different light conditions

The present study investigates the variation in the relative growth, biomass and nutrient allocation in two threatened tree species viz. Magnolia punduana Hook.f. & Th. and Elaeocarpus prunifolius Wall. ex Müll. Berol. grown under three different levels of irradiance. The irradiance ranged between 1 and 12 mol m^?2 d^?1. Results showed that the highest relative growth rate (RGR) was achieved under the intermediate light treatment for both the species (mean: 0.005 mg mg^?1 d^?1). The growth response coefficient (GRC) model revealed that net assimilation rate (NAR) was the factor driving the RGR in both species. A significant positive correlation was found between NAR and RGR (R² = 0.33, p = 0.000) whereas specific leaf area (SLA) and leaf mass fraction (LMF) was negatively correlated to RGR. Overall, multiple regression of the studied species based on the independent variables viz. NAR, SLA, and LMF showed a significant relation with RGR (F(3,50,53 = 13.001, p = 0.000, R² = 0.43). The biomass distribution in the studied species is in agreement with the “balanced-growth hypothesis” where high irradiance increased allocation to below ground biomass fraction and decreased irradiance increased allocation to the above ground fraction. The highest nitrogen concentration in leaves was observed under the intermediate light treatment. Overall seedlings growth under intermediate light had a higher mean RGR indicating the species' preference for partial light conditions. Long-term experiments under varied light conditions as in the present study would provide useful insight into plant growth strategies in varied environmental conditions.     

Receptor binding,immune escape,and protein stability direct the natural selection of SARS-CoV-2 variants

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.     

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for?the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

in silico analyses of metabolic pathway and protein interaction network for identification of next gen therapeutic targets in Chlamydophila pneumoniae

Chlamydophila pneumoniae, the causative agent of chronic obstructive pulmonary disease (COPD), is presently the fifth mortalitycausing chronic disease in the world. The understanding of disease and treatment options are limited represents a severe concernand a need for better therapeutics. With the advancements in the field of complete genome sequencing and computationalapproaches development have lead to metabolic pathway analysis and protein-protein interaction network which provides vitalevidence to the protein function and has been appropriate to the fields such as systems biology and drug discovery. Proteininteraction network analysis allows us to predict the most potential drug targets among large number of the non-homologousproteins involved in the unique metabolic pathway. A computational comparative metabolic pathway analysis of the host H.sapiens and the pathogen C pneumoniae AR39 has been carried out at three level analyses. Firstly, metabolic pathway analysis wasperformed to identify unique metabolic pathways and non-homologous proteins were identified. Secondly, essentiality of theproteins was checked, where these proteins contribute to the growth and survival of the organism. Finally these proteins werefurther subjected to predict protein interaction networks. Among the total 65 pathways in the C pneumoniae AR39 genome 10 wereidentified as the unique metabolic pathways which were not found in the human host, 32 enzymes were predicted as essential andthese proteins were considered for protein interaction analysis, later using various criteria''s we have narrowed down to prioritizeribonucleotide-diphosphate reductase subunit beta as a potential drug target which facilitate for the successful entry into drugdesigning.     

Isolation,purification and characterization of Cardiolipin synthase from Mycobacterium phlei {PRIVATE}

It has been observed that mycobacterial species has high content of cardiolipin (CL) in their cell membranes more so pathogenicmycobacteria and in bacteria CL activates polymerases, gyrases by removing the bound ADP. Therefore, in the present studycardiolipin synthase (cls) which catalyses the formation of CL was isolated purified and characterized from the cell membrane ofMycobacterium phlei. The purified cls obtained from C-18 RP-HPLC column had a molecular weight of 58 kDa with an isoelectricpoint of 4.5. The enzyme activity (11.5+0.15 µM of CL phosphorous. ml-1 minute-1 for PG as substrate and 14+0.35µM of CLphosphorous. ml-1 minute-1 for CDP-DG as substrate) was optimal at pH 4.8 and showed K_M values of 55+0.05µM and 2.56+0.04µMfor phosphatidyl glycerol and CDP-diacylglycerol, respectively, with an absolute requirement of Mg²⁺ and Mn²⁺ ions for its activityhowever, Ca²⁺ ions inhibited the activity of the cls. The partial amino acid sequence of cls showed significant homology with pgsA3gene of M. tuberculosis and in this organism the CL biosynthesis is very high having three genes coding for PLs biosynthesistherefore, enzymes involved in CL biosynthesis may be an attractive drug target in the development of new antimycobacterialdrugs.     

Stiffness Dependent Separation of Cells in a Microfluidic Device

Abnormal cell mechanical stiffness can point to the development of various diseases including cancers and infections. We report a new microfluidic technique for continuous cell separation utilizing variation in cell stiffness. We use a microfluidic channel decorated by periodic diagonal ridges that compress the flowing cells in rapid succession. The compression in combination with secondary flows in the ridged microfluidic channel translates each cell perpendicular to the channel axis in proportion to its stiffness. We demonstrate the physical principle of the cell sorting mechanism and show that our microfluidic approach can be effectively used to separate a variety of cell types which are similar in size but of different stiffnesses, spanning a range from 210 Pa to 23 kPa. Atomic force microscopy is used to directly measure the stiffness of the separated cells and we found that the trajectories in the microchannel correlated to stiffness. We have demonstrated that the current processing throughput is 250 cells per second. This microfluidic separation technique opens new ways for conducting rapid and low-cost cell analysis and disease diagnostics through biophysical markers.     

Assessment of genetic diversity among soybean genotypes differing in response to aerial blight (Rhizoctonia solani Kuhn) using SSR markers

Forty‐seven genotypes and one wild relative of soybean, Glycine soja, were screened for resistance against aerial blight under epiphytotic conditions in the field during the Kharif season of two consecutive years viz., 2016 and 2017. Out of the 48 genotypes screened, only 18 genotypes exhibited a moderately resistant response to aerial blight during both the years of study. In order to perform molecular screening of the genotypes for aerial blight resistance, the genomic DNA obtained from the seedlings of the forty‐eight soybean genotypes was subjected to PCR amplification with 12 SSR markers. The SSR markers Satt 119, Sat_076, Satt 433, Satt 281, Satt 277, Satt 245 and Satt 520 were able to clearly amplify different banding pattern for resistant and susceptible genotypes, out of which Satt 433 and Satt 520 were found to exhibit a pattern, highly similar to the results of field screening of the genotypes with respect to resistant and susceptible reaction to the disease. The eighteen soybean genotypes that exhibited moderately resistant reaction to RAB under field conditions during both the years showed a banding pattern similar to resistant check PS‐1583 in the amplification profile produced by the SSR markers. The polymorphism information content (PIC) from the analysis of amplification profile of the SSR markers used in the study, ranged from 0.58 to 0.95. The dendrogram constructed using UPGMA cluster analysis clearly differentiated the resistant and susceptible genotypes of soybean into two separate groups.     

The Midline Protein Regulates Axon Guidance by Blocking the Reiteration of Neuroblast Rows within the Drosophila Ventral Nerve Cord

Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.