In-silico and structure-based assessment to evaluate pathogenicity of missense mutations associated with non-small cell lung cancer identified in the Eph-ephrin class of proteins.

Ephs belong to the largest family of receptor tyrosine kinase and are highly conserved both sequentially and structurally. The structural organization of Eph is similar to other receptor tyrosine kinases; constituting the extracellular ligand binding domain, a fibronectin domain followed by intracellular juxtamembrane kinase, and SAM domain. Eph binds to respective ephrin ligand, through the ligand binding domain and forms a tetrameric complex to activate the kinase domain. Eph-ephrin regulates many downstream pathways that lead to physiological events such as cell migration, proliferation, and growth. Therefore, considering the importance of Eph-ephrin class of protein in tumorigenesis, 7,620 clinically reported missense mutations belonging to the class of variables of unknown significance were retrieved from cBioPortal and evaluated for pathogenicity. Thirty-two mutations predicted to be pathogenic using SIFT, Polyphen-2, PROVEAN, SNPs&GO, PMut, iSTABLE, and PremPS in-silico tools were found located either in critical functional regions or encompassing interactions at the binding interface of Eph-ephrin. However, seven were reported in nonsmall cell lung cancer (NSCLC). Considering the relevance of receptor tyrosine kinases and Eph in NSCLC, these seven mutations were assessed for change in the folding pattern using molecular dynamic simulation. Structural alterations, stability, flexibility, compactness, and solvent-exposed area was observed in EphA3 Trp790Cys, EphA7 Leu749Phe, EphB1 Gly685Cys, EphB4 Val748Ala, and Ephrin A2 Trp112Cys. Hence, it can be concluded that the evaluated mutations have potential to alter the folding pattern and thus can be further validated by in-vitro, structural and in-vivo studies for clinical management.

In Silico and Structure-Based Assessment of Similar Variants Discovered in Tandem Repeats of BRCT Domains of BRCA1 and BARD1 To Characterize the Folding Pattern.

BRCA1 and BARD1 are important proteins in the homologous DNA damage repair pathways. Different genetic variants identified in these proteins have been clinically correlated with the occurrence of hereditary breast and ovarian cancer (HBOC). Variants of unknown significance (VUS) reported in the BRCT domains of BRCA1 and BARD1 substantiate the importance of BRCT domain-containing proteins for genomic integrity. To classify the pathogenicity of variants, in silico, structural and molecular dynamics (MD)-based approaches were explored. Different variants reported in the BRCT region were retrieved from cBioPortal, LOVD3, BRCA Exchange, and COSMIC databases to evaluate the pathogenicity. Multiple sequence alignment and superimposition of the structures of BRCA1 BRCT and BARD1 BRCT domains were performed to compare alterations in folding patterns. From 11 in silico predictions servers, variants reported to be pathogenic by 70% of the servers were considered for structural analysis. To our observations, four residue pairs of both the proteins were reported, harboring 11 variants, H1686Y, W1718L, P1749L, P1749S, and W1837L variants for BRCA1 BRCT and H606D, H606N, W635L, P657L, P657S, and W762F for BARD1 BRCT. MD simulations of the BRCT repeat regions of these variants and wild-type proteins were performed to evaluate the differences of folding patterns. Root mean square deviation (RMSD), R g, solvent-accessible surface area (SASA), and root mean square fluctuation (RMSF) of variants showed slight differences in the folding patterns from the wild-type proteins. Furthermore, principal components analysis of H1686Y, P1749S, and W1718L variants of BRCA1 showed less flexibility than the wild type, whereas that of H606D, W635L, and W762F of BARD1 showed more flexibility than the wild type. Normal mode analysis of the energy minima from the simulation trajectories revealed that most of the variants do not show much differences in the flexibility compared to the wild-type proteins, except for the discrete regions in the BRCT repeats, most prominently in the 1798-1801 amino acid region of BRCA1 and at the residue 744 in BARD1.

Comprehensive Assessment of Indian Variations in the Druggable Kinome Landscape Highlights Distinct Insights at the Sequence, Structure and Pharmacogenomic Stratum.

India confines more than 17% of the world's population and has a diverse genetic makeup with several clinically relevant rare mutations belonging to many sub-group which are undervalued in global sequencing datasets like the 1000 Genome data (1KG) containing limited samples for Indian ethnicity. Such databases are critical for the pharmaceutical and drug development industry where diversity plays a crucial role in identifying genetic disposition towards adverse drug reactions. A qualitative and comparative sequence and structural study utilizing variant information present in the recently published, largest curated Indian genome database (IndiGen) and the 1000 Genome data was performed for variants belonging to the kinase coding genes, the second most targeted group of drug targets. The sequence-level analysis identified similarities and differences among different populations based on the nsSNVs and amino acid exchange frequencies whereas a comparative structural analysis of IndiGen variants was performed with pathogenic variants reported in UniProtKB Humsavar data. The influence of these variations on structural features of the protein, such as structural stability, solvent accessibility, hydrophobicity, and the hydrogen-bond network was investigated. In-silico screening of the known drugs to these Indian variation-containing proteins reveals critical differences imparted in the strength of binding due to the variations present in the Indian population. In conclusion, this study constitutes a comprehensive investigation into the understanding of common variations present in the second largest population in the world and investigating its implications in the sequence, structural and pharmacogenomic landscape. The preliminary investigation reported in this paper, supporting the screening and detection of ADRs specific to the Indian population could aid in the development of techniques for pre-clinical and post-market screening of drug-related adverse events in the Indian population.

Cost-effective microabsorbance detection based nanoparticle immobilized microfluidic system for potential investigation of diverse chemical contaminants present in drinking water.

The measurement of the concentration of different heavy-metal ions present in the water environments is becoming increasingly essential as water-pollution concerns worsen. The optical sensor has become a good platform for detecting heavy-metal-ion concentration due to its compact size; chemical inertness; and anti-electromagnetic interference. Here, we propose to fabricate a simple and cost-effective microfluidic device for the detection of aqueous-heavy-metal ions such as lead(II), chromium(III) and mercury(II) using an optical-micro-absorbance-spectroscopy/LSPR based principle. Firstly, a disposable-PDMS-micro-device with a rectangular "Z-shaped microfluidic channel" integrated with micro-lens-structure and optical-fibre-coupler-structure was fabricated via cost-effective soft-lithography-technique using a microfabricated SU8 master. Further, the synthesized-Silver-Nanoparticles were also immobilized inside the microchannel structure in some of the micro-devices for nanoparticle-based-sensing studies. The real-time presence of heavy metal ions in the minuscule sample volume was analyzed by passing different-sample concentrations intermittently through the abovementioned microfluidic structure and measuring the bulk-micro-absorbance across its enhanced optical path length coupler-structure. The results specify that the fabricated micro-device can be easily utilized for label-free detection of a minimum of 0.5 ppb for all the aforesaid sample-heavy metal ions. The absorbance-change observed per unit concentration-change of Lead ion, mercury ion and chromium ion (from 0.001 to ∼50 µg/ml) is found on average-1.8 × 10-2 ΔA/µg/ml, 1.1 × 10-2 ΔA/µg/ml, 4.2 × 10-3 ΔA/µg/ml, respectively. For silver nanoparticle-based studies, the absorbance-change observed per unit concentration change of aforesaid heavy-metal-ions (i.e. the sensitivity) was found on average ∼2 times higher in comparison to simple micro-absorbance-based studies. Additionally, the micro-device has a capability for simplistic incessant(real-time)investigation, a preset-analyte-quantity-interface, and management over the injected analyte-evaporation.

Cost-effective smart microfluidic device with immobilized silver nanoparticles and embedded UV-light sources for synergistic water disinfection effects.

A novel microfluidic-device for water disinfection via diverse physiochemical effects has been demonstrated. Firstly, a microfluidic device with embedded, multiple germicidal UV-LEDs was fabricated through the innovatively modified cost-effective soft-lithography process. Further, synthesised silver nanoparticles were immobilized within its inner microchannel surface. Disinfection results proved the synergistic bactericidal effect of coated AgNPs and coupled UV-light, while a suspension of bacterial strains, were passed through the micro-device.

Detection of silent myocardial ischaemia in asymptomatic diabetic patients during treadmill exercise testing.

BACKGROUND: Diabetic patients have an increased prevalence of atherosclerosis and coronary artery disease. They may also experience higher morbidity and mortality after acute coronary syndrome compared with non-diabetic subjects. AIM: The objective of this study was to determine the presence of silent myocardial ischaemia by treadmill test in asymptomatic diabetic patients and to compare it with age- and sex-matched subjects without diabetes mellitus. METHODS: The study design was cross-sectional and the setting was a tertiary care centre. Fifty (42 males, 8 females) asymptomatic patients with diabetes in the age group of 30-70 years were included in the study group and 30 (24 males, 6 females) non-diabetic subjects of comparable age, sex and physical activity were the control group. They were assessed for the presence of silent ischaemia by a standard treadmill test using the Bruce protocol. RESULTS: Twenty-five of 50 diabetic patients showed a positive response to the exercise stress test, while 7 of 30 controls showed stress test positivity (p < 0.05). The stress test positivity showed a female predilection among diabetic patients (50% in diabetic patients and 16.67% in controls; p < 0.05). Diabetic patients with a positive stress test showed higher prevalence of hypertension (36%) and dyslipidaemia (84%) compared with diabetic patients with a negative stress test (12% and 28%, respectively) [p < 0.001]. The controls showed a better exercise capacity compared with diabetic patients; diabetic patients with a negative stress test had better exercise capacity than those with a positive stress test. CONCLUSIONS: The treadmill test is a useful, specific, cost-effective, non-invasive tool for detection of silent myocardial ischaemia in asymptomatic diabetic patients.