Interaction of human alpha fetoprotein with bilirubin.

Human alpha fetoprotein (AFP) binds bilirubin with an affinity somewhat lower than albumin. Free bilirubin was found to have an extinction maximum at 440 nm with an extinction coefficient of 4.97 x 10(4) M-1cm-1. AFP binding with the bile pigment elicits a blue shift while albumin interaction produced red spectral shift.     

Trypanosoma evansi induces detrimental immuno-catabolic alterations and condition like type-2 diabetes in buffaloes

The present study aimed to investigate the perturbations in immuno-metabolic and redox status of buffaloes with trypanosomosis. Thirteen buffaloes suffering from clinical trypanosomosis and eight apparently healthy buffaloes were included in the present study. Buffaloes with trypanosomosis found to have markedly elevated levels of interleukin-10 (IL-10), nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) in comparison with healthy controls. Whereas, total antioxidant capacity (TAC) and haemoglobin levels of buffaloes with trypanosomosis were significantly lower than the healthy controls. Remarkable elevation in malondialdehyde (MDA) and protein carbonyls (PC) levels were also observed in the diseased buffaloes. Moreover, buffaloes with trypanosomosis were found to have markedly elevated levels of serum glucose, total proteins, globulins, urea and aspartate aminotransferase (AST) and markedly lowered levels of serum calcium, total cholesterol levels and albumin/globulin (A/G) ratio as compared to the controls. Findings of our study evidently suggest that Trypanosoma evansi induces remarkable immunosuppressive and pro-oxidative status with an increased catabolic activity and hyperglycemic condition like type-2 diabetes in naturally infected buffaloes. Therefore, immuno-metabolic and pro-oxidative predicaments should be addressed by the veterinary clinician while managing the clinical cases of trypanosomosis in buffaloes.     

Warming Induces Significant Reprogramming of Beige,but Not Brown,Adipocyte Cellular Identity

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Synthesis of silver nanoparticles by Bacillus clausii and computational profiling of nitrate reductase enzyme involved in production

Biogenic synthesis of silver nanoparticles using microorganisms has found interest recently since last decade because of their prospect to synthesize nanoparticles of various size, shape and morphology which are eco-friendly. Here, an eco-friendly method for production of silver nanoparticles from Bacillus clausii cultured from Enterogermina is explored. Along with the biosynthesis and conformity test, in silico studies was done on NADPH dependent nitrate reductase enzymes from the view point of designing a rational enzymatic strategy for the synthesis. The detailed characterization of the nanoparticles was carried out using UV-Vis spectroscopy, Dynamic Light Scattering (DLS) particle size analysis, Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD) analysis. Computational profiling and in silico characterization of NADH dependent enzymes was carried out based on literature and work done so far. Nitrate reductase sequence was retrieved from NCBI for characterization. Secondary structure was evaluated and verified by JPred as well as SOPMA Tool. Tertiary structure was also modeled by MODELLER and ITASSER parallel and the best structure was selected based on energy values. Structure validation was done by GROMACS and RMSD, RMSF, temperature variation plot were also plotted. Interactions graphs between nitrate reductase and ligand silver nitrate was done through molecular docking using Hex.     

Paradigm for disease deconvolution in rare neurodegenerative disorders in Indian population: insights from studies in cerebellar ataxias

Cerebellar ataxias are a group of rare progressive neurodegenerative disorders with an average prevalence ranges from 4.8 to 13.8 in 100,000 individuals. The inherited disorders affect multiple members of the families, or a community that is endogamous or consanguineous. Presence of more than 3000 mutations in different genes with overlapping clinical symptoms, genetic anticipation and pleiotropy, as well as incomplete penetrance and variable expressivity due to modifiers pose challenges in genotype–phenotype correlation. Development of a diagnostic algorithm could reduce the time as well as cost in clinicogenetic diagnostics and also help in reducing the economic and social burden of the disease. In a unique research collaboration spanning over 20 years, we have been able to develop a paradigm for studying cerebellar ataxias in the Indian population which would also be relevant in other rare diseases. This has involved clinical and genetic analysis of thousands of families from diverse Indian populations. The extensive resource on ataxia has led to the development of a clinicogenetic algorithm for cost-effective screening of ataxia and a unique ataxia clinic in the tertiary referral centre in All India Institute of Medical Sciences. Utilizing a population polymorphism scanning approach, we have been able to dissect the mechanisms of repeat instability and expansion in many ataxias, and also identify founders, and trace the mutational histories in the Indian population. This provides information for genetic testing of at—risk as well as protected individuals and populations. To dissect uncharacterized cases which comprises more than 50% of the cases, we have explored the potential of next-generation sequencing technologies coupled with the extensive resource of baseline data generated in-house and other public domains. We have also developed a repository of patient-derived peripheral blood mononuclear cells, lymphoblastoid cell lines and neuronal lineages (derived from iPSCs) for ascribing functionality to novel genes/mutations. Through integrating these technologies, novel genes have been identified that has broadened the diagnostic panel, increased the diagnostic yield to over 75%, helped in ascribing pathogenicity to novel mutations and enabled understanding of disease mechanisms. It has also provided a platform for testing novel molecules for amelioration of pathophysiological phenotypes. This review through a perspective on CAs suggests a generic paradigm from diagnostics to therapeutic interventions for rare disorders in the context of heterogeneous Indian populations.     

The ‘Checkmate’ for Iron Between Human Host and Invading Bacteria: Chess Game Analogy

Iron is an essential nutrient for all living organisms with critical roles in many biological processes. The mammalian host maintains the iron requirements by dietary intake, while the invading pathogenic bacteria compete with the host to obtain those absorbed irons. In order to limit the iron uptake by the bacteria, the human host employs numerous iron binding proteins and withholding defense mechanisms that capture iron from the microbial invaders. To counteract, the bacteria cope with the iron limitation imposed by the host by expressing various iron acquisition systems, allowing them to achieve effective iron homeostasis. The armamentarium used by the human host and invading bacteria, leads to the dilemma of who wins the ultimate war for iron.     

<Superscript>15</Superscript>N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on <Superscript>15</Superscript>N resulting from solvent exchange

¹⁵N R₂ relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide—water exchange can severely skew Hahn-echo based ¹⁵N R₂ relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the ¹⁵N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent ¹⁵N R₂ rate constants—which should not be mistaken with protein inherent chemical exchange contributions, R_ex, to ¹⁵N R₂. For measurements of ¹⁵N R₂ rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D₂O molar fraction in the sample buffer, as low as 1%, or the use of an external D₂O reference along with a modified ¹⁵N R₂ Hahn-echo based experiment. This combination allows for the measurement of R_ex contributions to ¹⁵N R₂ originating from conformational exchange in a time window from µs to ms.     

Complex interplay of lesion-specific DNA repair enzyme on bistranded clustered DNA damage harboring Tg:G mismatch in nucleosome core particles

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) are major DNA damage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. The processing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNA oligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesions was evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, the yield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when the DNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clustered bistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay between the repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBs under cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the cluster environment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents the processing of the oxidized nucleobases.     

Characterization of a mildly alkalophilic and thermostable recombinant <Emphasis Type="Italic">Thermus thermophilus</Emphasis> laccase with applications in decolourization of dyes

Objective

To examine the potential for applications of TthLAC, a monomeric (~ 53 kDa) laccase encoded by the genome of Thermus thermophilus (strain HB 27) which can be produced at low cost in Escherichia coli.

Result

Functional, thermostable and mildly alkalophilic TthLAC of high purity (> 90%) was produced through simple heating of suspended (TthLAC overexpressing) E.coli cells at 65 °C. For reactions of short duration (< 1 h) the temperature for optimal activity is ~ 90 °C. However, TthLAC undergoes slow partial unfolding and thermal inactivation above 65 °C, making it unsuitable for long incubations above this temperature. With different substrates, optimal function was observed from pH 6 to 8. With the substrate, ABTS, catalytic efficiency (K _m) and maximum velocity (V_max) at 60 °C and pH 6.0 were determined to be 2.4 × 10³ µM and 0.04 × 10³ µM/min respectively. Ultra-pure, affinity-purified TthLAC was used to confirm and characterize the enzyme’s ability to oxidize known (laccase) substrates such as ABTS, syringaldazine and 4-fluoro-2-methylphenol. TthLAC decoloured up to six different industrial dyes, with or without the use of redox mediators such as ABTS.

Conclusions

Unlike versatile laccases from most other sources, which tend to be thermolabile as well as acidophilic, TthLAC is a versatile, thermostable, mildly alkalophilic laccase which can be produced at low cost in E.coli for various redox applications.