Cloning and comparative modeling identifies a highly stress tolerant Cu/Zn cytosolic super oxide dismutase 2 from a drought tolerant maize inbred line.

Plants have a complex system of stress response that deals with different types of stresses. Maize (Zea mays L.), one of the most important crops grown throughout the world, across a range of agro-ecological environments, employs complex mechanisms of gene regulation in response to drought stress. HKI 335 is a tropical maize inbred line showing remarkable adaptation to drought stress. Abiotic stresses, like drought, trigger the production of reactive oxygen species (ROS) due to the incomplete reduction or excitation of molecular oxygen, eventually leading to cell damage. Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme that acts as the first line of defense against ROS. We cloned the Sod2 gene from HKI 335 inbred line and analyzed its protein through detailed in silico characterization. Our comparative modeling revealed that at the level of tertiary structure, the HKI 335 SOD2 protein is highly similar to Potentilla atrosanguinea SOD2, which had been previously identified as highly thermostable SOD that can tolerate autoclaving as well as sub-zero temperatures. We performed phylogenetic analysis, estimated physicochemical properties, post-translational modifications, protein-protein interactions, and domain composition of this SOD2. The phylogenetic analysis showed that orthologous sequences of SOD from different species were clustered into two clusters. Secondary structure prediction indicates that SOD2 is a soluble protein and no transmembrane domains have been found. Most of the beta sheets have RSA value greater than 2. The Ramachandran plot from PDBsum revealed that most of the residues fall in the highly favored region. It was estimated that the value of the instability index was less than 40, the value of the aliphatic index was extremely high and the GRAVY value lies between -2 and +2. We could identify only one phosphorylation site, located at position 20 with a score of 0.692. Overall, the unique stress-tolerant properties of the HKI 335 SOD2, may be one of the reasons contributing to the high drought tolerance trait exhibited by HKI 335 maize inbred line. Further research may reveal more insights into the drought adaptation mechanism in maize and the eventual deployment of the trait in maize hybrids.

Synthesis of Alginate Nanogels with Polyvalent 3D Transition Metal Cations: Applications in Urease Immobilization.

Biocompatible nanogels are highly in demand and have the potential to be used in various applications, e.g., for the encapsulation of sensitive biomacromolecules. In the present study, we have developed water-in-oil microemulsions of sodium alginate sol/hexane/Span 20 as a template for controlled synthesis of alginate nanogels, cross-linked with 3d transition metal cations (Mn2+, Fe3+, and Co2+). The results suggest that the stable template of 110 nm dimensions can be obtained by microemulsion technique using Span 20 at concentrations of 10mM and above, showing a zeta potential of -57.3 mV. A comparison of the effects of the cross-links on the morphology, surface charge, protein (urease enzyme) encapsulation properties, and stability of the resulting nanogels were studied. Alginate nanogels, cross-linked with Mn2+, Fe3+, or Co2+ did not show any gradation in the hydrodynamic diameter. The shape of alginate nanogels, cross-linked with Mn2+ or Co2+, were spherical; whereas, nanogels cross-linked with Fe3+ (Fe-alginate) were non-spherical and rice-shaped. The zeta potential, enzyme loading efficiency, and enzyme activity of Fe-alginate was the highest among all the nanogels studied. It was found that the morphology of particles influenced the percent immobilization, loading capacity, and loading efficiency of encapsulated enzymes. These particles are promising candidates for biosensing and efficient drug delivery due to their relatively high loading capacity, biocompatibility, easy fabrication, and easy handling.

Elucidating potential molecular signatures through host-microbe interactions for reactive arthritis and inflammatory bowel disease using combinatorial approach.

Reactive Arthritis (ReA), a rare seronegative inflammatory arthritis, lacks exquisite classification under rheumatic autoimmunity. ReA is solely established using differential clinical diagnosis of the patient cohorts, where pathogenic triggers linked to enteric and urogenital microorganisms e.g. Salmonella, Shigella, Yersinia, Campylobacter, Chlamydia have been reported. Inflammatory Bowel Disease (IBD), an idiopathic enteric disorder co-evolved and attuned to present gut microbiome dysbiosis, can be correlated to the genesis of enteropathic arthropathies like ReA. Gut microbes symbolically modulate immune system homeostasis and are elementary for varied disease patterns in autoimmune disorders. The gut-microbiota axis structured on the core host-microbe interactions execute an imperative role in discerning the etiopathogenesis of ReA and IBD. This study predicts the molecular signatures for ReA with co-evolved IBD through the enveloped host-microbe interactions and microbe-microbe 'interspecies communication', using synonymous gene expression data for selective microbes. We have utilized a combinatorial approach that have concomitant in-silico work-pipeline and experimental validation to corroborate the findings. In-silico analysis involving text mining, metabolic network reconstruction, simulation, filtering, host-microbe interaction, docking and molecular mimicry studies results in robust drug target/s and biomarker/s for co-evolved IBD and ReA. Cross validation of the target/s or biomarker/s was done by targeted gene expression analysis following a non-probabilistic convenience sampling. Studies were performed to substantiate the host-microbe disease network consisting of protein-marker-symptom/disease-pathway-drug associations resulting in possible identification of vital drug targets, biomarkers, pathways and inhibitors for IBD and ReA.Our study identified Na(+)/H(+) anti-porter (NHAA) and Kynureninase (KYNU) to be robust early and essential host-microbe interacting targets for IBD co-evolved ReA. Other vital host-microbe interacting genes, proteins, pathways and drugs include Adenosine Deaminase (ADA), Superoxide Dismutase 2 (SOD2), Catalase (CAT), Angiotensin I Converting Enzyme (ACE), carbon metabolism (folate biosynthesis) and methotrexate. These can serve as potential prognostic/theranostic biomarkers and signatures that can be extrapolated to stratify ReA and related autoimmunity patient cohorts for further pilot studies.

In-silico investigations of selective miRNA-gene targets and their validation studies in obstructive sleep apnea (OSA) patient cohorts.

BACKGROUND: Obstructive sleep apnoea (OSA) is a prevalent form of sleep disordered breathing which results in sleep fragmentation and deprivation. Obesity and cardiovascular disorders are the major risk factors associated with OSA. Molecular analysis of the factors associated with OSA could demarcate the clinical analysis pattern in a population. OBJECTIVE: This study pertains to in-silico analyses of miRNA and their gene targets with validation for their potential role in OSA as putative biomarker candidates. METHODS: miRDB, TargetScan and miRanda databases were used to identify targets of miR-27 and let-7 that have documented role in OSA and co-related obesity and cardiovascular disorders. Quantitative PCR was used to analyze expression pattern of miR-27 and let-7 in obese and non-obese OSA patient cohorts with respective controls. In-silico analysis was done using PatchDoc to obtain atomic contact energy (ACE) scores that indicated the docked gene targets to the predicted miRNA structures. The docked structures were analysed using Maestro Suite 11 for the hydrogen and aromatic interactions. RESULTS: Downregulation of miR-27 and let-7 in OSA compared to controls was observed. In-silico data analysis was performed for gene targets (TGFBR1, TGFBR2, SMAD2, SMAD4, CRY2 and CNR1) of the selected miRNAs (miR-27 and let-7). Among all, CNR1 and CRY2 were found to be better targets for miR-27 and let-7 respectively as per ACE scores, ROC scores and expression fold change in OSA. CONCLUSION: Our study gives insights to the expression profiling of miR-27 and let-7 and explore a set of potential target genes (CNR1 and CRY2) of these two miRNAs for a promising clinical relevance in OSA.

Association of Inflammatory Bowel Disease with Arthritis: Evidence from In Silico Gene Expression Patterns and Network Topological Analysis.

Inflammatory bowel disease (IBD) is an idiopathic prolonged ailment accountable for inflammatory conditions of the intestine. Moreover, arthritis is responsible for joints' stiffness and painful inflammation. IBD shows certain articular extra-intestinal manifestations associating IBD with arthritis. IBD associated arthritis is found to be linked with ankylosing spondylitis (AS). The present study insights for the potential and putative drug targets and biomarkers of IBD associated with arthritis using in silico approaches. Microarray data analysis of datasets involving IBD affected and AS affected vs controls were done to explore the differentially expressed genes (DEGs). In majority of the datasets, the common DEGs found were sterile alpha motif domain containing 9 like (SAMD9L), inhibin beta A subunit (INHBA), transmembrane protein 45A (TMEM45A) and transmembrane and tetratricopeptide repeat containing 1 (TMTC1). The common functions and pathways found between the DEGs were control of macromolecule metabolism process, control of metabolic process, control of primary metabolic process, and control of protein metabolic process, cell differentiation, organ development, single-organism development process, multicellular organism development process, development of system, single-multicellular organism development process, developmental process, development of anatomical structure, multicellular organismal development process, control of biological process, cell proliferation, hematopoietic progenitor cell differentiation and immune system process. TMTC1 and INBHA were found to be more biologically significant genes according to the topological properties of the network. This study also suggests that TMTC1, INBHA, TMEM45A and SAMD9L DEGs and their accompanying pathways might have the potential to be exploited as drug targets and biomarkers in the diagnosis and/or treatment of IBD linked arthritis and warrants for further experimental validation.

Canvassing the aetiology, prognosis and molecular signatures of obstructive sleep apnoea.

CONTEXT: Obstructive sleep apnoea (OSA) is a sleep respiration disorder with multiple pathophysiological risks. The study of OSA is important from a public health perspective due to increased risk of cardiovascular morbidity and metabolic disorders. OBJECTIVE: The review content contributes to amalgamate the clinico-molecular analysis of microRNAs, single-nucleotide polymorphisms (SNPs), transcriptome profiles and epigenetics in the prognosis of OSA. The conceptual focus here is to demarcate the involvement of regulatory players like miRNAs that have most probable contributions towards other changes in patients leading to clinical outcomes. METHODS: Literature survey was done by retrieving data from databases such as Google Scholar, PubMed and Science Direct. RESULTS: Abundant reports have suggested the involvement and role of biomarkers such as CRP, TNF-α, IL-6, IL-8 and CAMs but there are interspersed speculations about the involvement of epigenetics in OSA. CONCLUSIONS: miRNA and transcriptome profiling, DNA methylation and SNPs are some of the less researched aspects that aim to bridge the gap in the molecular mechanisms underlying the pathophysiology of OSA. The quest for biomarkers in OSA is now distinctly achieving new heights. In the context of diagnosis, the above mentioned epigenetic regulators are likely to emerge as viable contenders.

A non-hydrolyzable ATP derivative generates a stable complex in a light-inducible two-component system.

Isothermal calorimetry (ITC) measurements yielded the binding constants during complex formation of light-inducible histidine kinases (HK) and their cognate CheY-type response regulators (RR). HK-RR interactions represent the core function of the bacterial two-component system, which is also present in many bacterial phytochromes. Here, we have studied the recombinant forms of phytochromes CphA and CphB from the cyanobacterium Tolypothrix PCC7601 and their cognate RRs RcpA and RcpB. The interaction between the two reaction partners (HK and RR) was studied in the presence and absence of ATP. A complex formation was observable in the presence of ATP, but specific interactions were only found when a non-hydrolyzable ATP derivative was added to the mixture. Also, the incubation of the HK domain alone (expressed as a recombinant protein) with the RR did not yield specific interactions, indicating that the HK domain is only active as a component of the full-length phytochrome. Considering also previous studies on the same proteins (Hübschmann, T., Jorissen, H. J. M. M., Börner, T., Gärtner, W., and de Marsac, N. (2001) Eur. J. Biochem. 268, 3383-3389) we now conclude that the HK domains of these phytochromes are active only when the chromophore domain is in its Pr form. The formerly documented phosphate transfer between the HK domain and the RR takes place via a transiently formed protein-protein complex, which becomes detectable by ITC in the presence of a non-hydrolyzable ATP derivative. This finding is of interest also in relation to the function of some (blue light-sensitive) photoreceptors that carry the HK domain and the RR fused together in one single protein.

The photoreactions of recombinant phytochrome CphA from the cyanobacterium Calothrix PCC7601: a low-temperature UV-Vis and FTIR study.

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV-Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV-Vis and FTIR experiments. Three intermediates have been trapped at -25, -45 and -120 degrees C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at -70 and -140 degrees C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore-protein interactions.

The chromophore structures of the Pr States in plant and bacterial phytochromes.

The resonance Raman spectra of the Pr state of the N-terminal 65-kDa fragment of plant phytochrome phyA have been measured and analyzed in terms of the configuration and conformation of the tetrapyrroles methine bridges. Spectra were obtained from phyA adducts reconstituted with the natural chromophore phytochromobilin as well as phycocyanobilin and its isotopomers labeled at the terminal methine bridges through (13)C/(12)C and D/H substitution. Upon comparing the resonance Raman spectra of the various phyA adducts, it was possible to identify the bands that originate from normal modes dominated by the stretching coordinates of the terminal methine bridges A-B and C-D. Quantum chemical calculations of the isolated tetrapyrroles reveal that these modes are sensitive indicators for the methine bridge configuration and conformation. For all phyA adducts, the experimental spectra of Pr including this marker band region are well reproduced by the calculated spectra obtained for the ZZZasa configuration. In contrast, there are substantial discrepancies between the experimental spectra and the spectra calculated for the ZZZssa configuration, which has been previously shown to be the chromophore geometry in the Pr state of the bacterial, biliverdin-binding phytochrome from Deinococcus radiodurans (Wagner, J. R., J. S. Brunzelle, K. T. Forest, R. D. Vierstra. 2005. Nature. 438:325-331). The results of this work, therefore, suggest that plant and bacterial (biliverdin-binding) phytochromes exhibit different structures in the parent state although the mechanism of the photoinduced reaction cycle may be quite similar.

Homologous expression of a bacterial phytochrome. The cyanobacterium Fremyella diplosiphon incorporates biliverdin as a genuine, functional chromophore.

Bacteriophytochromes constitute a light-sensing subgroup of sensory kinases with a chromophore-binding motif in the N-terminal half and a C-terminally located histidine kinase activity. The cyanobacterium Fremyella diplosiphon (also designated Calothrix sp.) expresses two sequentially very similar bacteriophytochromes, cyanobacterial phytochrome A (CphA) and cyanobacterial phytochrome B (CphB). Cyanobacterial phytochrome A has the canonical cysteine residue, by which covalent chromophore attachment is accomplished in the same manner as in plant phytochromes; however, its paralog cyanobacterial phytochrome B carries a leucine residue at that position. On the basis of in vitro experiments that showed, for both cyanobacterial phytochrome A and cyanobacterial phytochrome B, light-induced autophosphorylation and phosphate transfer to their cognate response regulator proteins RcpA and RcpB [Hübschmann T, Jorissen HJMM, Börner T, Gärtner W & deMarsac NT (2001) Eur J Biochem268, 3383-3389], we aimed at the identification of a chromophore that is incorporated in vivo into cyanobacterial phytochrome B within the cyanobacterial cell. The approach was based on the introduction of a copy of cphB into the cyanobacterium via triparental conjugation. The His-tagged purified, recombinant protein (CphBcy) showed photoreversible absorption bands similar to those of plant and bacterial phytochromes, but with remarkably red-shifted maxima [lambda(max) 700 and 748 nm, red-absorbing (P(r)) and far red-absorbing (P(fr)) forms of phytochrome, respectively]. A comparison of the absorption maxima with those of the heterologously generated apoprotein, assembled with phycocyanobilin (lambda(max) 686 and 734 nm) or with biliverdin IXalpha (lambda(max) 700 and 750 +/- 2 nm), shows biliverdin IXalpha to be a genuine chromophore. The kinase activity of CphBcy and phosphotransfer to its cognate response regulator was found to be strictly P(r)-dependent. As an N-terminally located cysteine was found as an alternative covalent binding site for several bacteriophytochrome photoreceptors that bind biliverdin and lack the canonical cysteine residue (e.g. Agrobacterium tumefaciens and Deinococcus radiodurans), this corresponding residue in heterologously expressed cyanobacterial phytochrome B was mutated into a serine (C24S); however, there was no change in its spectral properties. On the other hand, the mutation of His267, which is located directly after the canonical cysteine, into alanine (H267A), caused complete loss of the capability of cyanobacterial phytochrome B to form a chromoprotein.