Optimization and Identification of Single Mutation in Hemoglobin Variants with 2,2,2 Trifluoroethanol Modified Digestion Method and Nano-LC Coupled MALDI MS/MS.

Background: Hemoglobin (Hb) variants arise due to point mutations in globin chains and their pathological treatments rely heavily on the identification of the nature and location of the mutation in the globin chains. Traditional methods for diagnosis such as HPLC and electrophoresis have their own limitations. Therefore, the present study aims to develop and optimize a specific method of sample processing that could lead to improved sequence coverage and analysis of Hb variants by nano LC-MALDI MS/MS. Methods: In our study, we primarily standardized various sample processing methods such as conventional digestion with trypsin followed by 10% acetonitrile treatment, digestion with multiple proteases like trypsin, Glu-C, Lys-C, and trypsin digestion subsequent to 2,2,2 trifluoroethanol (TFE) treatment. Finally, the peptides were identified by LC-MALDI MS/MS. All of these sample processing steps were primarily tested with recombinant Hb samples. After initial optimization, we found that the TFE method was the most suitable one and the efficiency of this method was applied in Hb variant identification based on high sequence coverage. Results: We developed and optimized a method using an organic solvent TFE and heat denaturation prior to digestion, resulting in 100% sequence coverage in the ß-chains and 95% sequence coverage in the α-chains, which further helped in the identification of Hb mutations. A Hb variant protein sequence database was created to specify the search and reduce the search time. Conclusion: All of the mutations were identified using a bottom-up non-target approach. Therefore, a sensitive, robust and reproducible method was developed to identify single substitution mutations in the Hb variants from the sequence of the entire globin chains. Biological Significance: Over 330,000 infants are born annually with hemoglobinopathies and it is the major cause of morbidity and mortality in early childhood. Hb variants generally arise due to point mutation in the globin chains. There is high sequence homology between normal Hb and Hb variant chains. Due to this high homology between the two forms, identification of variants by mass spectrometry is very difficult and requires the full sequence coverage of α- and ß-chains. As such, there is a need for a suitable method that provides 100% sequence coverage of globin chains for variant analysis by mass spectrometry. Our study provides a simple, robust, and reproducible method that is suitable for LC-MALDI and provides nearly complete sequence coverage in the globin chains. This method may be used in the near future in routine diagnosis for Hb variant analysis.

Efficacy of add-on Ayurveda and Yoga intervention in health care workers of tertiary care hospital during COVID-19: Randomized controlled trial.

BACKGROUND: The present study aimed to evaluate the safety and prophylactic efficacy of add-on Comprehensive Ayurveda and mindfulness-based Yoga (CAY) regimen to standard care among HealthCare Workers (HCWs) against COVID-19. MATERIALS AND METHODS: This prospective single-blind (outcome assessor-blinded) RCT was conducted in tertiary care hospital in Delhi during July 2020-April 2021. HCWs of both sexes were randomized to add-on CAY intervention or control group. The primary outcomes were the incidence of confirmed COVID-19 positive cases and influenza-like illness events (ILI). Secondary outcomes were anxiety (GAD-7), depression (PHQ-9), and quality of life (SF-36) at the end of 12 weeks. RESULTS: Three hundred fifty-six participants (181 in intervention and 175 in the control group) were randomized. With the modified intention to treat approach, we analyzed 309 participants. The mean age for the intervention and control group was 39.3 ± 10.1 and 36.6 ± 10 years, respectively. Incidence of COVID-19 event was higher in control group compared to CAY group (16 of 164 [9.8%] vs. 11 of 145 [7.6%]; P = 0.50). The incidence of ILI events was also higher in the control group as compared to the CAY group (14 of 164 [8.5%] vs 9 of 145 [6.2%]). The health change domain of the SF-36 questionnaire showed statistically significant improvement in the CAY group as compared to the control group (P < 0.01). CONCLUSION: Incidence of COVID-19 and ILI events was lower in the CAY group compared with the contr ol group, though the difference is not statistically significant.

A sustainable approach towards utilization of plastic waste for an efficient electrode in microbial fuel cell applications.

Microbial fuel cells (MFC) are a novel technique for power generation from wastewater. A number of approaches for the modification of physical as well as chemical properties of the electrodes can be employed to attain the maximum output power density and high power electricity. The use of an active organic linker, extracted from waste residue (plastic), for the synthesis of porous nanostructured materials would be beneficial in the fabrication of electrodes for MFC. Herein, terephthalic acid monomer (t) derived from plastic waste was successfully applied as an electrochemically active linking unit to form an iron-based metal-organic framework (Fe-t-MOF: MIL-53(Fe)). The synthesized Fe-t-MOF was further modified with conducting polymer (polyaniline (PANI)). The produced nanocomposite (Fe-t-MOF/PANI) was coated on stainless steel (SS) disk (as a current collector) for use as an electrode component of the MFC system. The power density, open circuit potential (OCP), and a limiting current density of the MFC are 680 mW/m2, 0.67 V, and 3500mA/m2, respectively. The technique opted here should help search a novel, efficient, sustainable, and cost-effective route for the modification of the plastic waste into an MFC electrode to achieve bioenergy production through wastewater treatment.

Advances in mass spectrometric methods for detection of hemoglobin disorders.

Hemoglobin disorders are caused due to alterations in the hemoglobin molecules. These disorders are categorized in two broad classes - hemoglobin variants and thalassemias. The hemoglobin variants arise due to point mutations in the alpha (α), beta (ß), gamma (γ), delta (δ), or epsilon (ε) globin chains of these proteins, while thalassemias are caused due to the under-production of α or ß globin chain. Hemoglobin disorders account for 7 % of the major health issues globally. Mass Spectrometry is an extensively used analytical tool in the field of protein identification, protein-protein interaction, biomarker discovery and diagnosis of several impairments including hemoglobin related disorders. The remarkable advancements in the technology and method development have enormously augmented the clinical significance of mass spectrometry in these fields. The present review describes hemoglobin disorders and the recent advancements in mass spectrometry in the detection of such disorders, including its advantages, lacunae, and future directions. The literature evidence concludes that mass spectrometry can be potentially used as a 'First Line Screening Assay' for the detection of hemoglobin disorders in the near future.

PANI/PbS QD nanocomposite structure for visible light driven photocatalytic degradation of rhodamine 6G.

Among conducting polymers, polyaniline (PANI) is one of the most widely used materials due to its unique properties (e.g., high electrical conductivity, outstanding electrochemical properties, easy polymerization, high stability, and low-cost synthesis). In this study, we report the synthesis of a composite of polyaniline with lead sulfide quantum dots (PbS QDs), which was subsequently employed for photocatalysis of a dye, rhodamine 6G (Rh-6G). This PANI/PbS composite was prepared by employing the chemical oxidative polymerization of aniline monomer in the presence of PbS QDs. The composite has been characterized by X-ray powder diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and ultraviolet-visible spectroscopy. The composite formation turned out to be beneficial not only for the dispersion of PbS QDs but also for increasing the conductivity of the whole catalyst. They exhibited ~87% degradation of the dye content for 50 min. The kinetic rate for its destruction is 5.03 mmol g-1 h-1 with the quantum efficiency (QE) of 7.98E-06 molec/photon. Due to enhanced charge transfer characteristics, the PANI/PbS photocatalyst was capable of efficiently degrading the dye molecules across varying concentrations. The electron-hole pair generated after the visible light irradiation on the PANI/PbS composite led to an efficient oxidative degradation of Rh 6G.

Hypobaric hypoxia induced renal damage is mediated by altering redox pathway.

Systemic hypobaric hypoxia is reported to cause renal damage; nevertheless the exact pathophysiological mechanisms are not completely understood. Therefore, the present study aims to explore renal pathophysiology by using proteomics approach under hypobaric hypoxia. Six to eight week old male Sprague Dawley rats were exposed to hypobaric hypoxia equivalent to altitude of 7628 metres (pO2-282mmhg) at 28°C and 55% humidity in decompression chamber for different time intervals; 1, 3, and7 days. Various physiological, proteomic and bioinformatic studies were carried out to examine the effect of chronic hypobaric hypoxia on kidney. Our data demonstrated mild to moderate degenerative tubular changes, altered renal function, injury biomarkers and systolic blood pressure with increase in duration of hypobaric hypoxia exposure. Renal proteomic analysis showed 38 differential expressed spots, out of which 25 spots were down regulated and 13 were up regulated in 7 dayhypobarichypoxic exposure group of rats as compared to normoxia control. Identified proteins were involved in specific molecular changes pertinent to endogenous redox pathways, cellular integrity and energy metabolism. The study provides an empirical evidence of renal homeostasis under hypobaric hypoxia by investigating both physiological and proteomics changes. The identification of explicit key proteins provides a valuable clue about redox signalling mediated renal damage under hypobaric hypoxia.

Synthesis and spectroscopic studies of functionalized graphene quantum dots with diverse fluorescence characteristics.

In this research, we report a facile method for synthesizing a series of carboxyl functionalized graphene quantum dots (GQDs) using graphite flakes (300 meshes) as raw material. These highly luminescent GQDs emitted blue, light blue, green, yellow, and red light (400-700 nm intensity peaks) under ultraviolet irradiation conditions, while exhibiting quantum yields in the range of 50-70%. The products were comprehensively characterized using ultraviolet-visible, photoluminescence, infrared, Raman, and dynamic light scattering spectroscopies. The GQDs were found to remain highly stable against photobleaching when stored over a prolonged period of more than three months. The proposed method for the synthesis of high quality, multicolor GQDs can be utilized to extend the application of these nanoparticles to molecular biotechnology and bioengineering; for example, the immobilization of cancer markers on their surface. As such, carboxylic acid groups present on the surface of these GQDs help create complexes for in vivo sensing applications.

Graphene modified screen printed immunosensor for highly sensitive detection of parathion.

Due to indiscriminate use of pesticides, there is a growing need to develop sensors that can sensitively detect the trace amount of pesticides in food and water samples. Parathion, identified as an acetylcholinesterase inhibitor, had been one of the most widely used pesticides throughout the world. Symptoms of its poisoning are found to be diverse enough to include nausea, vomiting, diarrhea, muscle cramping/twitching, and shortness of breath. In this work, a graphene based impedimetric immunosensor has been fabricated and employed for highly sensitive and specific detection of parathion. The fabrication proceeded through the modification of the screen-printed carbon electrodes (SPE) with graphene sheets, followed by their functionalization with 2-aminobenzyl amine (2-ABA) via an electrochemical reaction. These amine functionalized graphene electrodes were then bio-interfaced with the anti-parathion antibodies. In the impedimetric mode, this biosensor detected parathion in a broad linear range, i.e. 0.1-1000ng/L with a very low limit of detection (52pg/L). It also showed high selectivity towards parathion in the presence of malathion, paraoxon, and fenitrothion. The viability of this biosensor was demonstrated by detecting parathion in real samples (e.g., tomato and carrot) and through cross-calibration against HPLC.