Unmasking the morphological alteration of erythrocytes among women suffering from PCOS.

Dyslipidemia is frequently observed in polycystic ovarian syndrome (PCOS). Changes in plasma lipid levels potentially alter erythrocyte membrane lipid composition due to lack of inbuilt lipid synthesis machinery. Therefore, development of morphologically altered erythrocytes in PCOS patients with dyslipidemia is expected. However, this has not been established so far. So, we took this opportunity to explore the morphological alterations among dyslipidemic PCO women. We recruited thirty-five dyslipidemic PCOS women (satisfying Rotterdam criteria, without medication) and twenty-five age-matched healthy controls. Scanning electron microscopy revealed a significant increase in the number of stomatocytes, acanthocytes, and echinocytes in the PCO group. PCO group showed a considerable decrease in plasma antioxidant levels. Elevated lipid peroxidation, protein carbonylation, and decreased free thiol group in erythrocyte membrane in PCOS suggest oxidative degradation of the erythrocyte membrane. Elevated intracellular ROS levels, increased methemoglobin formation, and a decrease in NADPH methemoglobin reductase in PCOS also indicate altered physicochemical property of hemoglobin due to oxidative overload. Additionally, these patients exhibit a rise in erythrocyte membrane cholesterol and triglyceride, which promotes the membrane to become less fluidic and less fragile. Thus, these results corroborate a potential role in altering erythrocyte morphology among dyslipidemic PCO women.

Identification of approved drugs with ALDH1A1 inhibitory potential aimed at enhancing chemotherapy sensitivity in cancer cells: an in-silico drug repurposing approach.

The aldehyde dehydrogenase 1A1 (ALDH1A1) also known as retinal dehydrogenase, is an enzyme normally involved in the cellular metabolism, development and detoxification processes in healthy cells. However, it's also considered a cancer stem cell marker and its high levels of expression in several cancers, including breast, lung, ovarian, and colon cancer have been associated with poor prognosis and resistance to chemotherapy. Given its crucial role in chemotherapy resistance by detoxification of chemotherapeutic drugs, ALDH1A1 has attracted significant research interest as a potential therapeutic target for cancer. Though a few synthetic inhibitors of ALDH1A1 have been synthesized and their efficacy has been proved in-vitro and in-vivo studies, none of them have passed clinical trials so far. In this scenario, we have performed an in-silico study to verify whether any of the already approved drugs used for various purposes has the ability to inhibit catalytic activity of ALDH1A1, so that they can be repurposed for cancer therapy. Keeping in mind the feasibility of repurposing in a larger population we have selected the approved drugs from five widely used drug categories such as antibiotic, antiviral, antifungal, anti diabetic and antihypertensive for screening. Computational techniques like molecular docking, molecular dynamics simulations and MM-PBSA binding energy calculation have been used in this study to screen the approved drugs. Based on the logical analysis of results, we propose that three drugs - telmisartan, irbesartan and maraviroc can inhibit the catalytic activity of ALDH1A1 and thus can be repurposed to increase chemotherapy sensitivity in cancer cells.Communicated by Ramaswamy H. Sarma.

Exploring the possibility of drug repurposing for cancer therapy targeting human lactate dehydrogenase A: a computational approach.

Human lactate dehydrogenase A (LDHA) is an anaerobic glycolytic enzyme involved in the inter-conversion of pyruvate to lactate. The level of LDHA in various types of cancer cells is found to be elevated and the dependence of cancer cells on anaerobic glycolysis is viewed as the reason for this elevation. Moreover, inhibition of LDHA activity has been shown to be effective in impairing the growth of tumors, making the LDHA as a potential target for cancer therapy. In this computational study, we have performed a pharmacophore based screening of approved drugs followed by a molecular docking based screening to find a few potential LDHA inhibitors. Molecular dynamics simulations have also been performed to examine the stability of the LDHA-drug complexes as obtained from the docking study. The result of the study showed that darunavir, moxalactam and eprosartan can bind to the active site of LDHA with high affinity in comparison to two known synthetic inhibitors of LDHA. The results of the molecular dynamics simulation showed that these drugs can bind stably with the enzyme through hydrogen bond and hydrophobic interactions. Hence, it is concluded that darunavir, moxalactam and eprosartan may be considered as potential inhibitors of LDHA and can be used for cancer therapy after proper validation of their effectiveness through in vitro, in vivo and clinical trials.Communicated by Ramaswamy H. Sarma.

Cord and peripheral blood erythrocyte analysis by scanning electron microscopy and flow cytometry.

INTRODUCTION: Human umbilical cord blood is rich in hematopoietic cells. We aimed to focus on the morphological, biochemical, membrane protein profile and surface protein expression differences of erythrocytes, isolated from cord and adult peripheral blood using techniques such as high-resolution scanning electron microscopy (SEM), gel electrophoresis (SDS-PAGE) and flow cytometry. METHODS: Adult peripheral blood was collected from consenting adults, and umbilical cord blood was procured from consenting mothers, post-delivery at Medical College, Kolkata. We emphasized on cord and adult peripheral blood erythrocytes' morphological variations using SEM images and protein expression by flow cytometric analysis. Some conventional biochemical analyses such as osmotic fragility of the cell membrane, haemoglobin co-oxidation study and lipid peroxidation assay were done for supporting evidence along with membrane protein content using gel electrophoresis. RESULTS: Our SEM images indicated clear morphological variations in cord erythrocyte with a higher degree of cellular deformities and difference in membrane texture. Flow cytometric analysis of cord erythrocyte showed a significant difference in CD235a expression than adults. We observed an overexpression of GLUT1 and decreased expression of Band 3 in cord erythrocyte membrane. Our results also showed cord erythrocytes have low osmotic fragility, a slower rate of co-oxidation of cord haemoglobin and a lesser lipid peroxidation level than that of adults. CONCLUSION: Cord blood erythrocytes have deeper indentations leading to higher flexibility, more oxygen-carrying capacity and less osmotic fragility in comparison with adult erythrocytes. The expression of CD235a and Band 4.5 (GLUT 1) was significantly higher in cord erythrocytes than peripheral adult erythrocytes.

Oxidative degradation perturbs physico-chemical properties of hemoglobin in cigarette smokers: a threat to different biomolecules.

CONTEXT: Cigarette smokers develop structural modification in hemoglobin (Hb) and this modification enable Hb to undergo higher rate of auto-oxidation, leading to generation of further intracellular ROS. OBJECTIVE: In this study, we exhibited the possible cause and consequences of Hb modification in cigarette smokers. METHODS: Twenty-two smokers and 16 nonsmokers, aged 25 to 35 years, having a smoking history of 7-10 years were recruited in this study. Carbonyl content, ferryl form, peroxidase-like and esterase-like activities of Hb were assayed. Free iron release by Hb, erythrocyte membrane-bound Hb and plasma Hb were also measured along with assessment of important biomolecular degradations by Hb. RESULTS AND DISCUSSION: Increase in carbonyl content in Hb indicates its oxidative degradation. Increase in ferryl Hb formation, peroxidase-like activity and decrease in esterase like activity of Hb along with increased release of nonheme iron (from Hb) clearly indicates alteration in physico-chemical properties of Hb in smokers. Moreover, increase in erythrocyte membrane-bound Hb and plasma-free Hb provide further evidences for higher rate of Hb oxidation in smokers' erythrocyte. The rates of protein, lipid, sugar and DNA degradation were noticed to be higher by smokers' Hb; and were further attenuated by desferrioxamine as well as mannitol. CONCLUSION: We conclude that in cigarette smokers, there is oxidative degradation of Hb and the degradation causes alteration in its physico-chemical properties, which in turn may degrade different biomolecules in its close vicinity by releasing more iron and production of more superoxide as well as hydroxyl radical.

Cigarette smokers develop altered erythrocyte membrane composition: an investigation unmasking the role of membrane bound integral protein GLUT 1.

Erythrocytes in cigarette smokers are prone to oxidative damage. Here, we sought to elucidate the facts behind modifications and possible defense system developed in erythrocyte of cigarette smokers. We observed significant increase in stomatocytes and spherocytes, and osmotic fragility of erythrocyte, along with reduced level of protein thiol and increased fluorescence anisotropy in isolated membrane. Denaturing gel electrophoresis indicated alterations in band 3, band 4.2 and band 4.5. Among those, Glut 1 (i.e. band 4.5), which transports glucose (insulin independent) and dehydroascorbate (DHA), was selectively chosen for its long history in reducing reactive oxygen species (ROS). The increased Glut 1 level in smokers was confirmed by immunoblotting and immunocytochemistry. Furthermore, smokers showed significantly higher glucose uptake in whole blood. The intracellular (Ic) ROS (as indicated by 2',7'-dichlorofluorescin) was significantly higher in smokers as evidenced by flow cytometric assay. Glucose and DHA alone or together significantly reduced IcROS at higher rate in smokers. However, in presence of Glut 1 specific blocker, phloretin, neither glucose nor DHA could reduce IcROS in both non-smokers and smokers. This confirms that Glut 1 by transporting glucose or DHA attenuates IcROS. Therefore, we conclude that erythrocytes, although altered morphologically, also develop a defense system by upregulating Glut 1 to combat with enhanced Ic oxidative insult in cigarette smokers.

Cigarette smokers develop structurally modified hemoglobin: a possible way of increasing oxidative stress.

CONTEXT: Increased levels of free radicals and various reactive species along with reduced antioxidant defence system are the major threat to erythrocyte in tobacco smokers. Thus, the hemoglobin (Hb) within the erythrocyte is very prone to oxidative damage. Earlier reports suggest that cigarette smoking is related with the glutathionylation and formation of adducts of Hb. OBJECTIVE: We have highlighted the possible changes in secondary and tertiary structures of Hb in cigarette smokers and its physiological consequences. MATERIAL AND METHOD: Twenty smokers and 18 non-smokers, aged 25-35 years were volunteered in this study. We used flow cytometry for measuring intracellular reactive oxygen species (IcROS). The purified Hb was subjected to different spectrophotometric, fluoremetric and circular dichroic (CD) analysis. The hydrophobicity, thermal stability, heme release and oxidation of purified Hb were also studied. RESULT: We observed that the IcROS was also higher in cigarette smokers than non-smokers. The data of intrinsic fluorescence, synchronous fluorescence and tryptophan quenching studies showed that the microenvironments of ß37 tryptophan and tyrosine residues of Hb were moved toward more hydrophobic region in cigarette smokers. Increased hydrophobicity and thermal stability furthermore indicated more compactness of smokers' hemoglobin. From CD spectra, we confirmed an overall modification of the secondary and tertiary structures of hemoglobin in smokers. Both auto- and co-oxidation rates of purified Hb were found to be higher in cigarette smokers. DISCUSSION AND CONCLUSION: We conclude that the modified Hb in cigarette smokers may further enhance the oxidative insult within the cell.

Analysis of the topology of Vibrio cholerae NorM and identification of amino acid residues involved in norfloxacin resistance.

NorM, a putative efflux pump of Vibrio cholerae, is a member of the multidrug and toxic compound extrusion family of transporters. We demonstrate that NorM confers resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Inactivation of norM rendered V. cholerae hypersensitive towards these fluoroquinolones. Multiple sequence alignment of members of its family identified several regions of high sequence conservation. The topology of NorM was determined using beta-lactamase and chloramphenicol acetyltransferase fusions. The amino acid residues G(184), K(185), G(187), P(189), E(190), G(192), and G(195) in the periplasmic loops and L(381), R(382), G(383), Y(384), K(385), and D(386) in the cytoplasmic loops, as well as all the acidic and cysteine residues of NorM, were mutated. Mutants G184V, G184W, K185I, P189S, E190K, and E190A lost the norfloxacin resistance-imparting phenotype characteristic of NorM. Mutants E124V, D155V, G187V, G187R, C196S, Y384H, Y384S, and Y384F exhibited partial resistance to norfloxacin. Mutants with replacements of G(184) or G(187) by A, K(185) by R, and E(190) by D retained the norfloxacin resistance phenotype of NorM. Analysis of the accumulation of norfloxacin in intact cells of Escherichia coli expressing NorM or its mutants in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone supported the results obtained through susceptibility testing and argued in favor of NorM-mediated efflux as the determining factor in norfloxacin susceptibility in the genetically manipulated strains. Taken together, these results suggested that E(124), D(155), G(184), K(185), G(187), P(189), E(190), C(196), and Y(384) are likely involved in NorM-dependent norfloxacin efflux. Except for D(155), C(196), and Y(384), all of these residues are located in periplasmic loops.