Exploring the Role of Serum Hydrogen Sulphide (H2S) Levels in Manic Depressive Psychosis in Terms of Its Association, Diagnostic Ability, and Severity Prediction: Findings From a Tertiary Care Center in North Bengal.

INTRODUCTION: Manic depressive psychosis (MDP) or bipolar disorder, a prevalent psychiatric condition globally and in the Indian population, has been attributed to various pathological mechanisms. Hydrogen sulphide (H2S), a member of the gasotransmitter family, may be linked to the development of bipolar disorder because it plays a crucial role in maintaining proper neuronal function in terms of excitability, plasticity, and homeostatic functions. There is very little data regarding the role of the gasotransmitter H2S in MDP in terms of its association, diagnostic ability, and severity prediction, which led us to conduct this study among MDP patients in the Sub-Himalayan region of West Bengal. METHODS: This was an observational case-control study performed in the Department of Biochemistry, North Bengal Medical College and Hospital, Siliguri, West Bengal, India, from January 2022 to December 2022. Fifty diagnosed MDP patients and 50 healthy age- and sex-matched control subjects satisfying the inclusion and exclusion criteria were studied. The H2S level in the blood was assayed using the standardised spectrophotometric methylene blue method. The severity of depression was assessed by Hamilton Depression Rating Scale (HAM-D) scoring. RESULTS: Of the 50 MDP patients, 45 (90%) were in the depressive phase, and five (10%) were in the manic phase. Of the 45 depressive patients, eight (17.8%) had mild depression, 12 (26.7%) had moderate depression, 19 (42.2%) had severe depression, and six (13.3%) had very severe depression. The mean H2S level in MDP patients (41.98±18.88 µmol/l) was significantly (P<0.05) lower than that in control subjects (99.20± 15.20 µmol/l). It was also observed that the mean H2S level in MDP patients decreased with the duration of the disease but was not statistically significant. The mean H2S levels in the different depression severity groups were found to be significantly different (P<0.001). Receiver operating characteristic (ROC) curve analysis revealed that a cut-off value of H2S <78.5 µmol/l was associated with MDP, with a sensitivity of 96% and a specificity of 88%, and a cut-off value of H2S < 53 µmol/l predicted the severity of depression with a sensitivity of 89.3% and a specificity of 76.5%. CONCLUSION: The significant association of the gasotransmitter H2S in MDP patients and its role as a diagnostic and severity predictive marker can help us to employ proper measures for better management of MDP and improving quality of life.

Tuning gold-based surface functionalization for streptavidin detection: A combined simulative and experimental study.

A rationally designed gold-functionalized surface capable of capturing a target protein is presented using the biotin-streptavidin pair as a proof-of-concept. We carried out multiscale simulations to shed light on the binding mechanism of streptavidin on four differently biotinylated surfaces. Brownian Dynamics simulations were used to reveal the preferred initial orientation of streptavidin over the surfaces, whereas classical molecular dynamics was used to refine the binding poses and to investigate the fundamental forces involved in binding, and the binding kinetics. We assessed the binding events and the stability of the streptavidin attachment through a quartz crystal microbalance with dissipation monitoring (QCM-D). The sensing element comprises of biotinylated polyethylene glycol chains grafted on the sensor's gold surface via thiol-Au chemistry. Finally, we compared the results from experiments and simulations. We found that the confined biotin moieties can specifically capture streptavidin from the liquid phase and provide guidelines on how to exploit the microscopic parameters obtained from simulations to guide the design of further biosensors with enhanced sensitivity.

Atomistic Simulations of Functionalized Nano-Materials for Biosensors Applications.

Nanoscale biosensors, a highly promising technique in clinical analysis, can provide sensitive yet label-free detection of biomolecules. The spatial and chemical specificity of the surface coverage, the proper immobilization of the bioreceptor as well as the underlying interfacial phenomena are crucial elements for optimizing the performance of a biosensor. Due to experimental limitations at the microscopic level, integrated cross-disciplinary approaches that combine in silico design with experimental measurements have the potential to present a powerful new paradigm that tackles the issue of developing novel biosensors. In some cases, computational studies can be seen as alternative approaches to assess the microscopic working mechanisms of biosensors. Nonetheless, the complex architecture of a biosensor, associated with the collective contribution from "substrate-receptor-analyte" conjugate in a solvent, often requires extensive atomistic simulations and systems of prohibitive size which need to be addressed. In silico studies of functionalized surfaces also require ad hoc force field parameterization, as existing force fields for biomolecules are usually unable to correctly describe the biomolecule/surface interface. Thus, the computational studies in this field are limited to date. In this review, we aim to introduce fundamental principles that govern the absorption of biomolecules onto functionalized nanomaterials and to report state-of-the-art computational strategies to rationally design nanoscale biosensors. A detailed account of available in silico strategies used to drive and/or optimize the synthesis of functionalized nanomaterials for biosensing will be presented. The insights will not only stimulate the field to rationally design functionalized nanomaterials with improved biosensing performance but also foster research on the required functionalization to improve biomolecule-surface complex formation as a whole.

Molecular Dynamics Simulations of a Catalytic Multivalent Peptide-Nanoparticle Complex.

Molecular modeling of a supramolecular catalytic system is conducted resulting from the assembling between a small peptide and the surface of cationic self-assembled monolayers on gold nanoparticles, through a multiscale iterative approach including atomistic force field development, flexible docking with Brownian Dynamics and µs-long Molecular Dynamics simulations. Self-assembly is a prerequisite for the catalysis, since the catalytic peptides do not display any activity in the absence of the gold nanocluster. Atomistic simulations reveal details of the association dynamics as regulated by defined conformational changes of the peptide due to peptide length and sequence. Our results show the importance of a rational design of the peptide to enhance the catalytic activity of peptide-nanoparticle conjugates and present a viable computational approach toward the design of enzyme mimics having a complex structure-function relationship, for technological and nanomedical applications.

Atomistic simulations of gold surface functionalization for nanoscale biosensors applications.

A wide class of biosensors can be built via functionalization of gold surface with proper bio conjugation element capable of interacting with the analyte in solution, and the detection can be performed either optically, mechanically or electrically. Any change in physico-chemical environment or any slight variation in mass localization near the surface of the sensor can cause differences in nature of the transduction mechanism. The optimization of such sensors may require multiple experiments to determine suitable experimental conditions for the immobilization and detection of the analyte. Here, we employ molecular modeling techniques to assist the optimization of a gold-surface biosensor. The gold surface of a quartz-crystal-microbalance sensor is functionalized using polymeric chains of poly(ethylene glycol) (PEG) of 2 KDa molecular weight, which is an inert long chain amphiphilic molecule, supporting biotin molecules (bPEG) as the ligand molecules for streptavidin analyte. The PEG linkers are immobilized onto the gold surface through sulphur chemistry. Four gold surfaces with different PEG linker density and different biotinylation ratio between bPEG and PEG, are investigated by means of state-of-the art atomistic simulations and compared with available experimental data. Results suggest that the amount of biotin molecules accessible for the binding with the protein increases upon increasing the linkers density. At the high density a 1:1 ratio of bPEG/PEG can further improve the accessibility of the biotin ligand due to a strong repulsion between linker chains and different degree of hydrophobicity between bPEG and PEG linkers. The study provides a computaional protocol to model sensors at the level of single molecular interactions, and for optimizing the physical properties of surface conjugated ligand which is crucial to enhance output of the sensor.

In-silico studies on conformational stability of flagellin-receptor complexes.

Flagellin is a protein, responsible for virulent activities of bacteria. The host cell surface receptor protein TLR5 is known to interact with flagellin in order to activate immune response. However, the underlying microscopic details of this immune response are still elusive. In this study, we report on conformational stability of flagellin of two different organisms known as fliC and flaD in bilayer with reference to water. We find that both the flagellin is conformationally more stable in bilayer than in water. We also observe that fliC-TLR5 and flaD-TLR5 complexes are conformationally stable when the extracellular domain of the protein binds to conserved D1 domain of both fliC and flaD, although the binding interface between fliC-TLR5 and flaD-TLR5 is not identical. Our studies suggest that this might lead to differences in coreceptor bindings involved in immune response and thus have potential application in pharmaceutical developments. AbbreviationsA2Aadenosine receptorDPPCdipalmitoyl phosphatidylcholineecdextracellular domainecl2extracellular loop 2eLRRextracellular Leucine rich repeat domainflaDflagellin of Vibrio choleraefliCflagellin of Salmonella typhimuriumHPVhyper-variableMDmolecular dynamicsRMSDroot means squared deviationTIRtoll-interleukin receptorTLR5toll like receptor 5VPAC1vasoactive intestinal peptide receptorCommunicated by Ramaswamy H. Sarma.

Self-assembly in amphiphilic macromolecules with solvent exposed hydrophobic moieties.

Self-assembly by amphiphilic molecules with solvent exposed hydrophobic groups are relevant in biomolecular systems as well as in technological applications. Here we study such self-assembly in these systems using a model system of spherical particles having charge at core but solvent repelling surface, using Monte-Carlo simulations and mean field treatment. We find that solvophobicity mediated attraction leads aggregation, while electrostatic repulsions control stability of finite clusters. The aggregation threshold relates the parameters of two interactions through an algebraic dependence. The study also qualitatively explains experimental observations on aggregation of misfolded proteins and can be useful guide to tune stability of nm sized self-assembly in systems with exposed hydrophobic groups.

Allostery in Orai1 binding to calmodulin revealed from conformational thermodynamics.

Here, we study microscopic mechanism of complex formation between Ca2+-bound calmodulin (holoCaM) and Orai1 that regulates Ca2+-dependent inactivation process in eukaryotic cells. We compute conformational thermodynamic changes in holoCaM with respect to complex of Orai1 bound to C-terminal domain of holoCaM using histograms of dihedral angles of the proteins over trajectories from molecular dynamics simulations. Our analysis shows that the N-terminal domain residues L4, T5, Q41, N42, T44 and E67 of holoCaM get destabilized and disordered due to Orai1 binding to C-terminal domain of calmodulin affect the N-terminal domain residues. Among these residues, polar T44, having maximum destabilization and disorder via backbone fluctuations, shows the largest change in solvent exposure. This suggests that N-terminal domain is allosterically regulated via T44 by the binding of Orai1 to the C-terminal domain.

Dynamic signature of ligand binding over a protein surface.

We study the motion of Zn^{2+} in the presence of ubiquitin by all-atom molecular-dynamics simulations. We observe that unlike normal diffusive liquid, metal ions show an exponential tail in the self-van Hove function (self-vHf). Moreover, the metal ions are trapped strongly by acidic residues which form a binding pocket over the protein surface. The exponential tail disappears by mutation of trapping residues, suggesting that the tail appears due to trapped motion of the ions. The mean-squared displacements, however, in all the cases show linear dependence on time. Our model establishes that ligand binding generically results in an exponential tail of self-vHf. The self-vHf may give an approach to find binding pockets over a protein surface.

A high-density intraspecific SNP linkage map of pigeonpea (Cajanas cajan L. Millsp.).

Pigeonpea (Cajanus cajan (L.) Millsp.) is a major food legume cultivated in semi-arid tropical regions including the Indian subcontinent, Africa, and Southeast Asia. It is an important source of protein, minerals, and vitamins for nearly 20% of the world population. Due to high carbon sequestration and drought tolerance, pigeonpea is an important crop for the development of climate resilient agriculture and nutritional security. However, pigeonpea productivity has remained low for decades because of limited genetic and genomic resources, and sparse utilization of landraces and wild pigeonpea germplasm. Here, we present a dense intraspecific linkage map of pigeonpea comprising 932 markers that span a total adjusted map length of 1,411.83 cM. The consensus map is based on three different linkage maps that incorporate a large number of single nucleotide polymorphism (SNP) markers derived from next generation sequencing data, using Illumina GoldenGate bead arrays, and genotyping with restriction site associated DNA (RAD) sequencing. The genotyping-by-sequencing enhanced the marker density but was met with limited success due to lack of common markers across the genotypes of mapping population. The integrated map has 547 bead-array SNP, 319 RAD-SNP, and 65 simple sequence repeat (SSR) marker loci. We also show here correspondence between our linkage map and published genome pseudomolecules of pigeonpea. The availability of a high-density linkage map will help improve the anchoring of the pigeonpea genome to its chromosomes and the mapping of genes and quantitative trait loci associated with useful agronomic traits.

Spatio-temporal coordination among functional residues in protein.

The microscopic basis of communication among the functional sites in bio-macromolecules is a fundamental challenge in uncovering their functions. We study the communication through temporal cross-correlation among the binding sites. We illustrate via Molecular Dynamics simulations the properties of the temporal cross-correlation between the dihedrals of a small protein, ubiquitin which participates in protein degradation in eukaryotes. We show that the dihedral angles of the residues possess non-trivial temporal cross-correlations with asymmetry with respect to exchange of the dihedrals, having peaks at low frequencies with time scales in nano-seconds and an algebraic tail with a universal exponent for large frequencies. We show the existence of path for temporally correlated degrees of freedom among the functional residues. We explain the qualitative features of the cross-correlations through a general mathematical model. The generality of our analysis suggests that temporal cross-correlation functions may provide convenient theoretical framework to understand bio-molecular functions on microscopic basis.

Genomic Selection in the Era of Next Generation Sequencing for Complex Traits in Plant Breeding.

Genomic selection (GS) is a promising approach exploiting molecular genetic markers to design novel breeding programs and to develop new markers-based models for genetic evaluation. In plant breeding, it provides opportunities to increase genetic gain of complex traits per unit time and cost. The cost-benefit balance was an important consideration for GS to work in crop plants. Availability of genome-wide high-throughput, cost-effective and flexible markers, having low ascertainment bias, suitable for large population size as well for both model and non-model crop species with or without the reference genome sequence was the most important factor for its successful and effective implementation in crop species. These factors were the major limitations to earlier marker systems viz., SSR and array-based, and was unimaginable before the availability of next-generation sequencing (NGS) technologies which have provided novel SNP genotyping platforms especially the genotyping by sequencing. These marker technologies have changed the entire scenario of marker applications and made the use of GS a routine work for crop improvement in both model and non-model crop species. The NGS-based genotyping have increased genomic-estimated breeding value prediction accuracies over other established marker platform in cereals and other crop species, and made the dream of GS true in crop breeding. But to harness the true benefits from GS, these marker technologies will be combined with high-throughput phenotyping for achieving the valuable genetic gain from complex traits. Moreover, the continuous decline in sequencing cost will make the WGS feasible and cost effective for GS in near future. Till that time matures the targeted sequencing seems to be more cost-effective option for large scale marker discovery and GS, particularly in case of large and un-decoded genomes.

A comprehensive transcriptome assembly of Pigeonpea (Cajanus cajan L.) using sanger and second-generation sequencing platforms.

A comprehensive transcriptome assembly for pigeonpea has been developed by analyzing 128.9 million short Illumina GA IIx single end reads, 2.19 million single end FLX/454 reads, and 18 353 Sanger expressed sequenced tags from more than 16 genotypes. The resultant transcriptome assembly, referred to as CcTA v2, comprised 21 434 transcript assembly contigs (TACs) with an N50 of 1510 bp, the largest one being ~8 kb. Of the 21 434 TACs, 16 622 (77.5%) could be mapped on to the soybean genome build 1.0.9 under fairly stringent alignment parameters. Based on knowledge of intron junctions, 10 009 primer pairs were designed from 5033 TACs for amplifying intron spanning regions (ISRs). By using in silico mapping of BAC-end-derived SSR loci of pigeonpea on the soybean genome as a reference, putative mapping positions at the chromosome level were predicted for 6284 ISR markers, covering all 11 pigeonpea chromosomes. A subset of 128 ISR markers were analyzed on a set of eight genotypes. While 116 markers were validated, 70 markers showed one to three alleles, with an average of 0.16 polymorphism information content (PIC) value. In summary, the CcTA v2 transcript assembly and ISR markers will serve as a useful resource to accelerate genetic research and breeding applications in pigeonpea.

The first draft of the pigeonpea genome sequence.

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety 'Asha'. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable 'Arhar' simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.

Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh].

BACKGROUND: Pigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping. RESULTS: In this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥ 18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population. CONCLUSION: We developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.