Long-Lived States Provide Insights from NMR into the ß-Cyclodextrin Drug Assemblies.

In the last two decades, extending spin memory in NMR has been used for several purposes. Long-lived states (LLS) or singlet states are one of the first spin memory enhancement techniques used. LLS have the potential to extract structural information and intra- and intermolecular interactions of complex systems other than studying slow phenomenon. The motional regime of ß-cyclodextrin (ß-CD) drug inclusion complexes generally lies in the intermediate region, where ωτc ≈ 1, and the standard methods of studying these interactions, i.e., NOE and chemical shift monitoring, suffer from insufficient output information. The sensitivity of LLS toward the environmental changes is utilized here to gain insights into the drug assemblies formed by ß-CD. One can use change in relaxation of LLS to study the structural changes during complexation. The examples of ß-CD with the drugs indomethacin, paracetamol, gliclazide, and CI-933 (a precursor 4-methoxybenzamide) were studied. Indomethacin, paracetamol, and 4-methoxybenzamide show strong interaction through the para-substituted benzene ring, unlike gliclazide. Relaxation of LLS in ß-CD-drug complexes is modeled using standard Redfield Relaxation Theory. Computational studies performed support the experimental observations. Docking and molecular dynamics simulation provided the explanation of the relaxation properties of these drug molecules.

Radon and thoron exhalation rate in the soil of Western Haryana, India.

This study reports the exhalation rates of radon and thoron from surface soil collected from 60 rural sites of district Hisar, Haryana, India. The exhalation rates of Rn222 (radon) and Rn220 (thoron) were measured by portable SMART RnDuo (AQTEK SYSTEMS) using a mass accumulation chamber which was equipped with a scintillation material-coated cell. Dose rates due to natural gamma radiations ranged from 0.526 to 1.139 mSv y-1. The Rn222 mass exhalation rate in soil samples varied from 0.14 to 94.65 mBq kg-1 h-1. Thoron surface exhalation rates ranged from 46.42 to 619.88 Bq m-2 h-1. This study gives an idea about the differences in Rn222 and Rn220 exhalation at different locations which may be due to variations in geological features of the locations and characteristics of the topsoil. The findings show that usage of study area soil as building material is safe.

Deciphering the Structural Enigma of HLA Class-II Binding Peptides for Enhanced Immunoinformatics-based Prediction of Vaccine Epitopes.

Vaccines remain the most efficacious means to avoid and eliminate morbid diseases associated with high morbidity and mortality. Clinical trials indicate the gaining impetus of peptide vaccines against diseases for which an effective treatment still remains obscure. CD4 T-cell-based peptide vaccines involve immunization with antigenic determinants from pathogens or neoplastic cells that possess the ability to elicit a robust T helper cell response, which subsequently activates other arms of the immune system. The available in silico predictors of human leukocyte antigen II (HLA-II) binding peptides are sequence-based techniques, which ostensibly have balanced sensitivity and specificity. Structural analysis and understanding of the cognate peptide and HLA-II interactions are essential to empirically derive a successful peptide vaccine. However, the availability of structure-based epitope prediction algorithms is inadequate compared with sequence-based prediction methods. The present study is an attempt to understand the structural aspects of HLA-II binders by analyzing the Protein Data Bank (PDB) complexes of pHLA-II. Furthermore, we mimic the peptide exchange mechanism and demonstrate the structural implication of an acidic environment on HLA-II binders. Finally, we discuss a structure-guided approach to decipher potential HLA-II binders within an antigenic protein. This strategy may accurately predict the peptide epitopes and thus aid in designing successful peptide vaccines.

Clinical and epidemiologic profile of the initial COVID-19 patients at a tertiary care centre in India.

COVID-19 has now become a pandemic. It has spread from Wuhan, China, in December 2019 to a large number of countries within three months. The objective of this work is to report the initial experience with epidemiologic and clinical features, as well as with the management of COVID-19 patients in India. This is a descriptive case series of the first 21 COVID-19 infected patients confirmed with polymerase chain reaction (PCR) and admitted to a tertiary care centre in India from 01.02.2020 to 19.03.2020. Clinical, laboratory, and radiologic data were collected, including age, sex, nationality, travel history, symptoms, duration of stay, and comorbidities. The mean age of the population was 40.3 years with a male preponderance. Thirteen (62%) patients had recent travel history outside India in the previous 30 days, two thirds of whom had travelled to Italy. The most common symptoms were fever and cough (42.9%) followed by sore throat, headache and breathlessness. Vital and laboratory parameters were preserved in all patients and none of them required ventilatory support. Among the first 21 patients diagnosed with COVID-19 infection in India, the typical clinical presentation consisted in a mild upper respiratory tract infection predominantly affecting the young male population. One patient required supplemental oxygen. All patients recovered with no residual symptoms.   *The Safdarjung Hospital COVID 2019 working group: Nitesh Gupta, Sumita Agrawal, Pranav Ish, Suruchi Mishra, Rajni Gaind, Ganapathy Usha, Balvinder Singh, Manas Kamal Sen, Shibdas Chakrabarti (Consultant and Head, Pulmonary Medicine); NK Gupta (Professor, Pulmonary medicine); Dipak Bhattacharya (Consultant, Pulmonary medicine); Rohit Kumar (Assistant Professor, Pulmonary Medicine); Siddharth R. Yadav (Assistant Professor, Pulmonary Medicine); Rushika Saksena (Specialist, Microbiology); Rojaleen Das (Assistant Professor, Microbiology); Vikramjeet Dutta (Assistant Professor, Microbiology); Anupam Kr Anveshi (Senior Resident, Microbiology); Santvana Kohli (Assistant Professor, Anaesthesiology); Naveen KV (Assistant Professor,  Anaesthesiology); Amandeep Jaswal (Assistant Professor, Anaesthesiology).

Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant.

Although unglycosylated HuEpo is fully functional, it has very short serum half-life. However, the mechanism of in vivo clearance of human Epo (HuEpo) remains largely unknown. In this study, the relative importance of protease-sensitive sites of recombinant HuEpo (rHuEpo) has been investigated by analysis of structural data coupled with in vivo half-life measurements. Our results identify α3-α4 inter-helical loop region as a target site of lysosomal protease Cathepsin L. Consistent with previously-reported lysosomal degradation of HuEpo, these results for the first time identify cleavage sites of rHuEpo by specific lysosomal proteases. Furthermore, in agreement with the lowered exposure of the peptide backbone around the cleavage site, remarkably substitutions of residues with bulkier amino acids result in significantly improved in vivo stability. Together, these results have implications for the mechanism of in vivo clearance of the protein in humans.

Cosmetic arm lengthening with monorail fixator.

Upper limb length discrepancy is a rare occurrence. Humerus shortening may need specialized treatment to restore the functional and cosmetic status of upper limb. We report a case of humerus lengthening of 9 cm with a monorail external fixator and the result was observed during a 2-year follow-up. Humerus lengthening needs specialized focus as it is not only a cosmetic issue but also a functional demand. The monorail unilateral fixator is more functional and cosmetically acceptable, and thus becomes an effective treatment option.

Quantification of minerals and trace elements in raw caprine milk using flame atomic absorption spectrophotometry and flame photometry.

This study reports minerals and trace elements quantification in raw caprine milk of Beetal breed, reared in Northern India and their feed, fodder & water using flame atomic absorption spectrophotometry and flame photometry. The mineral and trace elements' concentration in the milk was in the order: K > Ca > Na > Fe > Zn > Cu. The results showed that minerals concentration in caprine milk was lesser than reference values. But trace elements concentration (Fe and Zn) was higher than reference values. Multivariate statistical techniques, viz., Pearsons' correlation, Cluster analysis (CA) and Principal component analysis (PCA) were applied to analyze the interdependences within studied variables in caprine milk. Significantly positive correlations were observed between Fe - Zn, Zn - K, Ca - Na and Ca - pH. The results of correlation matrix were further supported by Cluster analysis and Principal component analysis as primary cluster pairs were found for Ca - pH, Ca - Na and Fe - Zn in the raw milk. No correlation was found between mineral & trace elements content of the milk and feed.

The length of glycine-rich linker in DNA-binding domain is critical for optimal functioning of quorum-sensing master regulatory protein HapR.

HapR is a quorum-sensing master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of this wild-type protein. While unraveling the underlying cause of functional inertness of a natural variant (HapRV2), the significance of a conserved glycine residue at position 39 in a glycine-rich linker in DNA-binding domain comes into light. This work aims at investigating how the length of glycine-rich linker (R(33)GIGRGG(39)) bridging helices α1 and α2 modulates the functionality of HapR. In pursuit of our interest, glycine residues were inserted after terminal glycine (G39) of the linker in a sequential manner. To evaluate functionality, all the glycine linker variants were subjected to a battery of performance tests under various conditions. Combined in vitro and in vivo results clearly demonstrated a gradual functional impairment of HapR linker variants coupled with increasing length of glycine-rich linker and finally, linker variant harboring four glycine residues resulted in a functionally compromised protein with significant loss of communication with cognate DNAs. Molecular dynamics studies of modeled HapR linker variants in complex with cognate promoter region show that residues namely Ser50, Thr53 and Asn56 are involved in varying degree of interactions with different nucleotides of HapR-DNA complex. The diminished functionality between variants and DNA appears to result from reduced or no interactions between Phe55 and nucleotides of cognate DNA as observed during simulations.

An unusual cause of radial nerve palsy.

Neurapraxia frequently occurs following traction injury to the nerve intraoperatively, leading to radial nerve palsy which usually recovers in 5-30 weeks. In our case, we had operated a distal one-third of humeral shaft fracture and fixed it with 4.5 mm limited contact dynamic compression plate. The distal neurovascular status of the limb was assessed postoperatively in the recovery room and was found to be intact and all the sensory-motor functions of the radial nerve were normal. On the second postoperative day, following the suction drain removal and dressing, patient developed immediate radial nerve palsy along with wrist drop. We reviewed the literature and found no obvious cause for the nerve palsy and concluded that it was due to traction injury to the radial nerve while removing the suction drain in negative pressure.

Comprehending conformational changes in EmrE, multidrug transporter at different pH: insights from molecular dynamics simulations.

EmrE is a small multidrug resistance (SMR) pump of antiparallel topology that confers resistance to a broad range of polyaromatic cations in Escherichia coli. Atomic-level understanding of conformational changes for the selectivity of substrate and transport of a diverse array of drugs through the smallest known efflux pumps is crucial to multi-drug resistance. Therefore, the present study aims to provide insights into conformational changes during the transport through EmrE transporter at different pH. Molecular dynamics simulations have been carried out on the complete structure of EmrE in the absence of substrate. Computational analyses such as secondary structure, principal component, dynamic cross-correlation matrix, and hydrogen bond calculations have been performed. Analysis of MD trajectories in this study revealed pH-dependent interactions that influenced the structural dynamics of EmrE. Notably, at high pH, Glu14 and Tyr60 in both monomers formed electrostatic interactions, while these interactions decreased significantly at a low pH. Interestingly, a kink at helix 3 (H3) and dual open conformation of EmrE at low pH were also observed in contrast to a closed state discerned towards the periplasmic side at high pH. Significant interactions between C-terminal residues and residues at the edge of H1 & Loop1 and H3 & Loop3 were identified, suggesting their role in stabilizing the closed conformation of EmrE at the periplasmic end under high pH conditions. The present study enhances our understanding of EmrE's conformational changes, shedding light on the pH-dependent mechanisms that are likely to impact its function in multi-drug resistance.Communicated by Ramaswamy H. Sarma.

Molecular characterization and expression analysis of a novel cystatin-like gene in a hypoxia-tolerant Indian catfish, Clarias batrachus [Linnaeus, 1758].

A novel member of Cystatin superfamily was identified from Indian catfish, Clarias batrachus, in response to oxidation stress induced by environmental hypoxia. Integrated genomic approaches, expression profiling and computational techniques showed that CbCystatin had putative cystatin/monelin like domain and might be a transmembrane and/or intermediate protein in signaling pathways. CbCystatin was found to be clustered into family 2 Cystatins. At transcriptional level, its expression was significantly up-regulated in response to short as well as long periods (more than 20 fold) of hypoxia, suggesting its positive association with oxygen concentrations lower than physiological concentrations.

Role of Cys-298 in specific recognition of glutathione by aldose reductase.

Aldose reductase (AR) is an NADPH-dependent oxidoreductase that is well-studied for its role in Diabetes Mellitus. Glutathione conjugated aldehydes are efficiently catalysed by AR. We have employed molecular dynamics simulations to investigate the dynamics of a glutathione analog, γ-glutamyl-S-(1,2-di-carboxyethyl)-cysteinyl-glycine (DCEG), into the binding pocket of AR. Study revealed that backbone nitrogens of Ala-299 and Leu-300 form a tiny pocket gated by thiol group of Cys-298. The glycine moiety of DCEG was able to displace the thiol group of Cys-298 to make hydrogen bond interactions with backbone of Ala-299, Leu-300, and Leu-301. This study provides the details of the dynamic interactions of DCEG in the binding pocket of AR, and shall aid in the design/discovery of differential inhibitors against AR.Communicated by Ramaswamy H. Sarma.

Structurally disordered C-terminal residues of GTP cyclohydrolase II are essential for its enzymatic activity.

GTP cyclohydrolase II (GCHII) is one of the rate limiting enzymes in riboflavin biosynthesis pathway and is shown to be a potential drug target for most of the pathogens. Previous biochemical and structural studies have identified the active site residues and elucidated the steps involved in the catalytic mechanism of GCHII. However, the last ∼20-25 C-terminal residues of GCHII remains unstructured in all the crystal structures determined to date and their role in the catalytic activity, if any, remains elusive. Therefore, to understand the role of these unstructured C-terminal residues, a series of C-terminal deletion mutants of GCHII from Helicobacter pylori (hGCHII) were generated and their catalytic activity was compared with its wild-type. Surprisingly, none of the C-terminal deletion mutants shows any enzymatic activity indicating that these are essential for GCHII function. To get additional insights for such loss of activity, homology models of full-length and deletion mutants of hGCHII in complex with GTP, Mg2+, and Zn2+ were generated and subjected to molecular dynamics simulation studies. The simulation studies show that a conserved histidine at 190th position from the unstructured C-terminal region of hGCHII interacts with α-phosphate of GTP. We propose that His-190 may play a role in the hydrolysis of pyrophosphate from GTP and in releasing the product, DARP. In summary, we demonstrate that the unstructured C-terminal residues of GCHII are important for its enzymatic activity and must be considered during rational drug designing. Communicated by Ramaswamy H. Sarma.

Convergence of two global regulators to coordinate expression of essential virulence determinants of Mycobacterium tuberculosis.

Cyclic AMP (cAMP) is known to function as a global regulator of Mycobacterium tuberculosis gene expression. Sequence-based transcriptomic profiling identified the mycobacterial regulon controlled by the cAMP receptor protein, CRP. In this study, we identified a new subset of CRP-associated genes including virulence determinants which are also under the control of a major regulator, PhoP. Our results suggest that PhoP as a DNA binding transcription factor, impacts expression of these genes, and phosphorylated PhoP promotes CRP recruitment at the target promoters. Further, we uncover a distinct regulatory mechanism showing that activation of these genes requires direct recruitment of both PhoP and CRP at their target promoters. The most fundamental biological insight is derived from the inhibition of CRP binding at the regulatory regions in a PhoP-deleted strain owing to CRP-PhoP protein-protein interactions. Based on these results, a model is proposed suggesting how CRP and PhoP function as co-activators of the essential pathogenic determinants. Taken together, these results uncover a novel mode of regulation where a complex of two interacting virulence factors impact expression of virulence determinants. These results have significant implications on TB pathogenesis.

Assessment of association between smoking and all-cause mortality among Malaysian adult population: Findings from a retrospective cohort study.

INTRODUCTION: Smoking is a known risk factor for many chronic diseases. Illness and death due to smoking are a significant public health burden in many countries. This study aims to address the information gap in smoking-related mortality in Malaysia by estimating the risk of cardiovascular disease and all-cause mortalities due to smoking among Malaysian adults. METHODS: We analyzed data on 2525 respondents, aged 24-64 years, of the Malaysian Non-Communicable Disease Surveillance survey conducted September 2005 to February 2006. Mortality records from the Malaysian National Registration Department were linked to the MYNCDS-1 data to determine respondents' mortality status over 12 years from 2006 to 2018. Associations between smoking and all-cause mortalities were assessed using Cox proportional hazards regression with adjustments for non-communicable disease and sociodemographic and lifestyle factors. RESULTS: The prevalence of daily smoking was 21.2% (95% CI: 19.0-23.7). During the 31668 person-years follow-up, 213 deaths from all causes occurred, where 68 deaths were among smokers (13.2%), and 452 were among non-smokers (6.3%). Smoking was associated with a significantly increased risk of all-cause mortality (adjusted hazard ration, AHR=1.79; 95% CI: 1.12- 2.97). These associations remained significant after excluding mortalities in the first two years of follow-up. CONCLUSIONS: Daily smoking is associated with a significantly higher risk of all-cause death. Behavioral and pharmacological smoking cessation interventions should be intensified among smokers to reduce the risk of mortality.

HSPMdb: a computational repository of heat shock protein modulators.

Heat shock proteins (Hsp) are among highly conserved proteins across all domains of life. Though originally discovered as a cellular response to stress, these proteins are also involved in a wide range of cellular functions such as protein refolding, protein trafficking and cellular signalling. A large number of potential Hsp modulators are under clinical trials against various human diseases. As the number of modulators targeting Hsps is growing, there is a need to develop a comprehensive knowledge repository of these findings which is largely scattered. We have thus developed a web-accessible database, HSPMdb, which is a first of its kind manually curated repository of experimentally validated Hsp modulators (activators and inhibitors). The data was collected from 176 research articles and current version of HSPMdb holds 10 223 entries of compounds that are known to modulate activities of five major Hsps (Hsp100, Hsp90, Hsp70, Hsp60 and Hsp40) originated from 15 different organisms (i.e. human, yeast, bacteria, virus, mouse, rat, bovine, porcine, canine, chicken, Trypanosoma brucei and Plasmodium falciparum). HSPMdb provides comprehensive information on biological activities as well as the chemical properties of Hsp modulators. The biological activities of modulators are presented as enzymatic activity and cellular activity. Under the enzymatic activity field, parameters such as IC50, EC50, DC50, Ki and KD have been provided. In the cellular activity field, complete information on cellular activities (percentage cell growth inhibition, EC50 and GI50), type of cell viability assays and cell line used has been provided. One of the important features of HSPMdb is that it allows users to screen whether or not their compound of interest has any similarity with the previously known Hsp modulators. We anticipate that HSPMdb would become a valuable resource for the broader scientific community working in the area of chaperone biology and protein misfolding diseases. HSPMdb is freely accessible at http://bioinfo.imtech.res.in/bvs/hspmdb/index.php.

Tobacco use and other aspects related to smoking among school-going adolescents aged 13-15 years in Malaysia: Analysis of three cross-sectional nationally representative surveys in 2003, 2009 and 2016.

INTRODUCTION: Periodic surveys on tobacco use patterns and other aspects of tobacco use among school-going adolescents in Malaysia provide information on the effectiveness of anti-smoking measures implemented. However, such information is limited in Malaysia. We investigated the prevalence of smoking and other related aspects among middle-secondary school students in Malaysia from the years 2003-2016 to fill this gap. METHODS: We analyzed data from the Global Youth Tobacco Survey (GYTS) 2003, GYTS 2009, and the Tobacco and Electronic Cigarette Survey among Malaysia Adolescents (TECMA) 2016. The surveys employed multistage sampling to select representative samples of adolescents attending secondary school in Malaysia. Data were collected using a pre-validated self-administered anonymous questionnaire adopted from the GYTS. RESULTS: Between 2003 and 2016, major changes occurred in which there were reductions in the prevalence of ever smoking, current smoking, and susceptibility to smoking. Reductions were also observed in exposure to SHS in public places and in the home. The proportion of school-going adolescents who support a ban on smoking in public places increased between 2013 to 2016, and there was a significant reduction in the proportion of respondents that were offered 'free' cigarettes by tobacco company representatives. However, there was no difference in the proportion of adolescents who initiated smoking before the age of 10 years and current smokers seeking advice to quit smoking across the time period. CONCLUSIONS: Our study indicates that the smoking policies and measures have been effective in reducing smoking prevalence, secondhand smoke exposure, and access to cigarettes, among school-going adolescents in Malaysia. However, measures to reduce smoking initiation and increase smoking cessation need to be strengthened to reduce the burden of smoking-related diseases in Malaysia in the long-term.

Role of the 88-97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis.

The role of a prominent surface-exposed loop (residues 88-97) in the alpha domain of streptokinase (SK), in human plasminogen (HPG) activation was explored through its selective mutagenesis and deletion studies. We first made a conformationally constrained derivative of the loop by the substitution of sequences known to possess a strong propensity for beta-turn formation. The mutant so formed (termed SK88-97-Beta Turn), when tested for co-factor activity against substrate HPG, after first forming a 1:1 molar complex with human plasmin (HPN), showed a nearly 6-fold decreased co-factor activity compared to the wild-type, native SK. The major catalytic change was observed to be at the k(cat) level, with relatively minor changes in Km values against HPG. Real-time binary interaction (i.e. the 1:1 complexation between SK, or its mutant/s, with HPG), and ternary complexation studies (i.e. the docking of a substrate HPG molecule into the preformed SK-HPG complex) using Surface Plasmon Resonance were done. These studies revealed minor alterations in binary complex formation but the ternary interactions of the substitution and/or deletion mutants were found to be decreased for full-length HPG compared to that for native SK.HPG. In contrast, their ternary interactions with the isolated five-kringle domain unit of plasminogen (K1-5) showed Kd values comparable to that seen with the native SK.HPG complex. Taking into consideration the overall alterations observed in catalytic levels after site-specific mutagenesis and complete loop deletion of the 88-97 loop, on the one hand, and its known position at the SK-HPG interface in the binary complex, suggests the importance of this loop. The present results suggest that the 88-97 loop of the alpha domain of SK contributes towards catalytic turn-over, even though its individual contribution towards enzyme-substrate affinity per se is minimal.

Conformational behavior of polypeptides derived through simultaneous global conservative site-directed mutagenesis of chymotrypsin inhibitor 2.

The natural occurrence of conservative residue substitutions in proteins suggests that side-chain packing schemes in protein interiors can accommodate mutational replacements of residues by others of similar nature. To explore the extent to which such substitutions are tolerated, especially when introduced simultaneously and globally over the entire length of a polypeptide chain, we examined the conformational behavior of a model 65 residues-long protein, wild-type chymotrypsin inhibitor 2 (WTCI2), and two globally-mutated (GM) variants named GMCI2-1 and GMCI2-2, each incorporating 55 conservative residue substitutions. GMCI2-1, was soluble over a wide range of pH, and folded into a compact, spherical, monomer marked by (i) complete absence of surface hydrophobicity, (ii) a WTCI2-like betaII-type CD spectrum, (iii) high WTCI2-like thermal stability, and (d) 1D and 2D NMR spectra characteristic of folded protein structure. GMCI2-2 was insoluble over a wide range of pH, and could be solubilized only at pH 4.0, showing non-WTCI2-like far-UV CD spectra characterized by high helical content. These results tentatively indicate that polypeptides incorporating residues of identical nature at equivalent chain locations can show the potential to fold with similar characteristics. However, further detailed investigations would be required to determine whether indeed the structural fold of GMCI2-1 resembles that of WTCI2, and to evaluate the extent to which it does so.

Replacement of the active surface of a thermophile protein by that of a homologous mesophile protein through structure-guided 'protein surface grafting'.

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.