Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study

Heat shock protein 70 (HSP70) is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70) has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state) and with HSP70 protein of E. coli 70kDa DnaK (close state) and relaxed them for 100 nanoseconds (ns) using all-atom Molecular Dynamics (MD) Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD) in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA) and Minimum Distance Matrix (MDM). The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD) and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.     

Genome-wide association mapping of salinity tolerance in rice (Oryza sativa)

Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na⁺/K⁺ ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5–18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na⁺/K⁺ ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice.     

shRNAPred (version 1.0): An open source and standalone software for short hairpin RNA (shRNA) prediction

The small hairpin RNAs (shRNA) are useful in many ways like identification of trait specific molecular markers, gene silencing and characterization of a species. In public domain, hardly there exists any standalone software for shRNA prediction. Hence, a software shRNAPred (1.0) is proposed here to offer a user-friendly Command-line User Interface (CUI) to predict 'shRNA-like' regions from a large set of nucleotide sequences. The software is developed using PERL Version 5.12.5 taking into account the parameters such as stem and loop length combinations, specific loop sequence, GC content, melting temperature, position specific nucleotides, low complexity filter, etc. Each of the parameters is assigned with a specific score and based on which the software ranks the predicted shRNAs. The high scored shRNAs obtained from the software are depicted as potential shRNAs and provided to the user in the form of a text file. The proposed software also allows the user to customize certain parameters while predicting specific shRNAs of his interest. The shRNAPred (1.0) is open access software available for academic users. It can be downloaded freely along with user manual, example dataset and output for easy understanding and implementation. AVAILABILITY: The database is available for free at http://bioinformatics.iasri.res.in/EDA/downloads/shRNAPred_v1.0.exe.     

Iodine-sodium cyanoborohydride-mediated reductive ring opening of 4,6-O-benzylidene acetals of hexopyranosides

A quick, efficient and convenient method for the regiospecific reductive ring opening of 4,6-O-benzylidene acetals of O-/S-alkyl/aryl glycosides of mono- and disaccharides, leading to the exclusive formation of the corresponding 6-O-benzyl ethers, using sodium cyanoborohydride in the presence of molecular iodine, is reported. It has been observed that common protecting groups such as ethers and esters are well tolerated under the conditions studied. The reaction was proved unsuccessful when applied to a glucosamine-derived benzylidene acetal.     

VirE2, a type IV secretion substrate,interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens

Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens - a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) - and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2-terminal membrane-spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB-encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector-coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.     

HNH family subclassification leads to identification of commonality in the His-Me endonuclease superfamily

The HNHc (SMART ID: SM00507) domain (SCOP nomenclature: HNH family) can be subclassified into at least eight subsets by iterative refinement of HMM profiles. An initial clustering of 323 proteins containing the HNHc domain helped identify the subsets. The subsets could be differentiated on the basis of the pattern of occurrence of seven defining features. Domain association is also different between the subsets. The subsets show organism as well as domain-based clustering, suggestive of propagation by both duplication and horizontal transfer events. Structure-based sequence analysis of the subsets led to the identification of common structural and sequence motifs in the HNH family with the other three families under the His-Me endonuclease superfamily.     

The outs and ins of bacterial type IV secretion substrates

Bacteria use type IV secretion systems (T4SS) to translocate macromolecular substrates destined for bacterial, plant or human target cells. The T4SS are medically important, contributing to virulence-gene spread, genome plasticity and the alteration of host cellular processes during infection. The T4SS are ancestrally related to bacterial conjugation machines, but present-day functions include (i) conjugal transfer of DNA by cell-to-cell contact, (ii) translocation of effector molecules to eukaryotic target cells, and (iii) DNA uptake from or release to the extracellular milieu. Rapid progress has been made toward identification of type IV secretion substrates and the requirements for substrate recognition.     

Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion

Bacteria use type IV secretion systems (T4SS) to translocate DNA (T-DNA) and protein substrates across the cell envelope. By transfer DNA immunoprecipitation (TrIP), we recently showed that T-DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9. Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis. In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP-dependent mechanism. By co-immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10. We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co-synthesis of previously identified 'core' components of the VirB/D4 T4SS. Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway.     

Effect of diet on serotonergic neurotransmission in depression

Depression is characterized by sadness, purposelessness, irritability, and impaired body functions. Depression causes severe symptoms for several weeks, and dysthymia, which may cause chronic, low-grade symptoms. Treatment of depression involves psychotherapy, medications, or phototherapy. Clinical and experimental evidence indicates that an appropriate diet can reduce symptoms of depression. The neurotransmitter, serotonin (5-HT), synthesized in the brain, plays an important role in mood alleviation, satiety, and sleep regulation. Although certain fruits and vegetables are rich in 5-HT, it is not easily accessible to the CNS due to blood brain barrier. However the serotonin precursor, tryptophan, can readily pass through the blood brain barrier. Tryptophan is converted to 5-HT by tryptophan hydroxylase and 5-HTP decarboxylase, respectively, in the presence of pyridoxal phosphate, derived from vitamin B₆. Hence diets poor in tryptophan may induce depression as this essential amino acid is not naturally abundant even in protein-rich foods. Tryptophan-rich diet is important in patients susceptible to depression such as certain females during pre and postmenstrual phase, post-traumatic stress disorder, chronic pain, cancer, epilepsy, Parkinson’s disease, Alzheimer’s disease, schizophrenia, and drug addiction. Carbohydrate-rich diet triggers insulin response to enhance the bioavailability of tryptophan in the CNS which is responsible for increased craving of carbohydrate diets. Although serotonin reuptake inhibitors (SSRIs) are prescribed to obese patients with depressive symptoms, these agents are incapable of precisely regulating the CNS serotonin and may cause life-threatening adverse effects in the presence of monoamine oxidase inhibitors. However, CNS serotonin synthesis can be controlled by proper intake of tryptophan-rich diet. This report highlights the clinical significance of tryptophan-rich diet and vitamin B₆ to boost serotonergic neurotransmission in depression observed in various neurodegenerative diseases. However pharmacological interventions to modulate serotonergic neurotransmission in depression, remains clinically significant. Depression may involve several other molecular mechanisms as discussed briefly in this report.     

Mulberry biodiversity conservation through cryopreservation

A 238 mulberry germplasm accession collection from diverse regions maintained under tropical conditions was identified from an ex situ field gene bank. The purpose was to prioritize the in vitro conservation and cryopreservation to develop long-term biodiversity conservation for ensuring sustainable utilization of these valuable resources. Reliable cryo techniques using desiccation and slow freezing of winter-dormant buds were used. Storage potential of bud grafts of different Morus species at −1.5°C for 90 d indicated species-specific variation, and most of the wild species were found sensitive. In vitro regeneration and cryopreservation (−196°C) protocols using differentiated bud meristems, like axillary winter-dormant buds, were worked out for a wide range of landraces, wild, and cultivated varieties of Morus. Buds maintained under subtropical location are also amenable for cryopreservation. Successful cryopreservation of winter-dormant buds belonging to Morus indica, Morus alba, Morus latifolia, Morus cathayana, Morus laevigata, Morus nigra, Morus australis, Morus bombycis, Morus sinensis, Morus multicaulis, and Morus rotundiloba was achieved. Among wild species, Morus tiliaefolia and Morus serrata showed moderate recovery after cryopreservation. Survival rates did not alter after 3 yr of cryopreservation. Inter-simple sequence repeat markers were used to ascertain the genetic stability of cryopreserved mulberry germplasm accessions, which showed no difference detected among the plantlets regenerated from frozen apices in comparison to the nonfrozen material.