Thrombophilic risk factors and ABO blood group profile for arteriovenous access failure in end stage kidney disease patients: a single-center experience.

INTRODUCTION: Thrombosis of fistula occurs most frequently in end-stage kidney disease (ESKD) patients receiving hemodialysis. However, the role of thrombophilia in arteriovenous fistula (AVF) failure has not been well established. Hence, this study was aimed at assessing the roles of hereditary and acquired thrombophilic factors in association with AVF failure among patients with ESKD undergoing hemodialysis. METHODS: A cross-sectional study was conducted on 100 ESKD patients, of whom 50 patients with well-functioning AVFs with no fistula failures earlier were enrolled as Group 1, and 50 patients who have had AVF failure were enrolled as Group 2. The hereditary factors as factor V Leiden, factor XIII, prothrombin, and methylene tetrahydrofolate reductase and the acquired factors as lipoprotein (a), fibrinogen, homocysteine, and anticardiolipin antibodies IgG and IgM were studied. RESULTS: Among the hereditary factors, no statistically significant difference was observed in relation to factor V Leiden and Prothrombin (p > 0.05). However, for factor XIII and methylene tetrahydrofolate reductase, a statistically significant difference was observed between patients with well-functioning AVFs and patients who have had AVF failure (p < 0.05). We found a statistically significant increase in all the acquired factors in patients who have had AVF failure in comparison with patients with well-functioning AVFs (p < 0.001). Association between ABO blood groups and thrombophilic factors showed significant association between factor V Leiden, anticardiolipin antibody IgG and IgM and ABO blood groups (p < 0.05), whereas none of the other thrombophilic factors showed significant association (p > 0.05). CONCLUSION: Thus, our study suggests significant role of acquired factors in causing AVF failure.

Andrographolide binds to spike glycoprotein and RNA-dependent RNA polymerase (NSP12) of SARS-CoV-2 by in silico approach: a probable molecule in the development of anti-coronaviral drug.

The SARS-CoV-2 belongs to Coronaviridae family infects host cells by the interaction of its spike glycoprotein and angiotensin-converting enzyme 2 (ACE 2) of host cells. Upon entry, the virus uses its RNA dependent RNA polymerase (NSP12) for transcribing its genome to survive in the cell and spread its infection. The protein sequences of receptor-binding domain (RBD) of spike glycoprotein, and NSP12 exhibits high homology in the family of Coronoviridae and are ideal candidates for the development of anti-coronaviral drugs. In the quest to identify inhibitory molecules against these proteins, we searched several molecules that are present in naturally occurring medicinal plants database. Andrographolide which is largely present in the leaf extracts of Andrographis paniculata (AP) and is known to exhibit antiviral, antibacterial, and stabilizes Th1/Th2/Th17 responses; taking this clue, we used in silico approaches to see the binding of andrographolide to RBD and NSP12 molecules. Our docking results showed very strong affinity of andrographolide to RBD and NSP12 of the SARS-CoV-2 virus with dock scores of -10.3460 for RBD and -10.7313 for NSP12 indicating andrographolide acts as an inhibitor of RBD and NSP12. These unique properties of andrographolide, AP extract, can be tested as anti-coronaviral drug.

Down-regulation of SOX17, GATA4 and FoxA2 promotes differentiation potential of hepatocytes from human hematopoietic stem cells.

The failure of regeneration of damaged liver in various end-stage liver diseases results in high morbidity and mortality. In this context, we have demonstrated the differentiation ability of human hematopoietic stem cells (HSCs) into hepatocytes. In this study, HSCs were isolated from a donor and cultured which exhibited the presence of CD34 and CD133 and absence of CD90 and CD73 markers. These CD34+ HSCs were induced for 21 days in hepatocyte differentiation medium (HDM). The obtained cells were characterized by immunocytochemical, immunofluorescence, western blot, qRT-PCR and flow cytometry analysis. Further, functional assays were done to accentuate the differentiated cells are hepatocytes. In HDM at day 6 differentiated cells showed the expression of definitive endodermal (DE) markers, SOX17, GATA4 and FoxA2 indicating the beginning of differentiation process. At day 21 the flow cytometry analysis showed 84.2 % positive to ALB-PE, 75.4 % positive to HNF4α-PE, and 77.3% positive to AFP-PE. Further, the qRT-PCR and western blot analysis presented prominent expression of hepatocyte-specific genes AAT, ALB, AFP, CK18, CK19, HNF4α, TFR2, and Hepcidin confirms the generation of hepatocytes in HDM. The ability of albumin secretion, urea production, glycogen storage, uptake of LDL, high ALDH enzyme activity describes the functionality of differentiated hepatocytes. Distinct expression of UGT1A1, CYP2B6, CYP2C9, CYP3A4, and CYP7A1 genes explains the ability to clear toxins and bilirubin as observed in normal hepatocytes. All these results indicate HSCs were differentiated into hepatocytes thus, autologous transplantation of HSCs could be a better option in the regeneration of the damaged liver.

Chronic persistent surgical pain is strongly associated with COMT alleles in patients undergoing cardiac surgery with median sternotomy.

OBJECTIVES: Catechol-O-methyl transferase (COMT), a catechol-dependent enzyme, plays pivotal role in the development of pain. In different ethnic populations, it is associated with chronic persistent surgical pain (CPSP). In this context, the present study is aimed to assess involvement of COMT allele (Val158Met) in the development of CPSP. METHODOLOGY: The patients (n = 216) underwent cardiac surgery with median sternotomy were selected to assess the magnitude of the CPSP evaluated with pain questionnaires' after 3 months from surgery. The exon 4 of COMT gene was PCR amplified and sequenced. The quantitative gene expression of COMT using RT-PCR corroborated the COMT enzyme activity. RESULTS: Among 216 patients who underwent sternotomy procedure, 54 patients showed CPSP even after 3 months from surgery. The sequence analysis revealed that, in 25% (54/216) patients having following one or more alleles: c.472G>A (Val158Met) (reported), and novel c.382C>G;c.383G>C (Arg128Ala), c.373C>G (Arg125Gly), c.370G>A (Val124Met), c.359G>C (Gly120Ala), c.349G>A, c.350G>A(Ala117Ser), c.349G>C, c.351C>A (Ala117Pro), c.349G>A (Ala117Thr), c.350G>C (Ala117Gly), and c.405G>C (Ala135Ser) were observed for the first time in Indian population. Distinct CPSP (≥ 4 NRS pain score) was observed in these patients correlating with COMT enzyme activity (7.80 ± 0.92 units/mg) which is 14 times lowered when compared with non-CPSP patient's (n = 162) 110.15 ± 6.41 units/mg. The findings of COMT gene expression using quantitative RT-PCR corroborated the COMT enzyme activity. CONCLUSION: The dominant effect of mutant COMT alleles connecting with low enzyme activity resulted in CPSP, warrants COMT genetic analysis prior to surgery was useful to predict the occurrence of CPSP.

Novel mutations in the RING-finger domain of BRCA1 gene in clinically diagnosed breast cancer patients.

In the present study, we investigated the frequency of BRCA1 gene mutations in 30 breast cancer (BC) patients of independent family history and 30 healthy control subjects. The immunohistochemistry (IHC) of BC patients showed duct cell carcinoma and distinct expression of the human epidermal growth factor receptor 2 (HER2). The genomic DNA was extracted from the BC patients and control subjects, the BRCA1 gene was PCR amplified and sequenced. The sequence analysis revealed that BRCA1 gene mutations were detected in 5/30 (16.6%) unrelated patients. One novel deleterious c.53delT mutation was detected in 3/30 (10%) unrelated patients leading to p.Met18Serfs*5 frame shift mutation in exon 2. Two patients 2/30 (6%) had novel c.297_301delinsCTCAA mutation in exon 5 leading to p.Leu99_Tyr101delinsPheSerAsn. Interestingly, the qRT-PCR analysis showed high expression of BRCA1 gene in all these patients having mutations compared with control subjects. Further, in silico analysis revealed loss of zinc-binding region of the RING-finger domain in BRCA1 structure due to these mutations, variable number of helices, helix-helix interactions, ß-turns, and γ-turns were identified in the secondary structure, resulted in the formation of non-functional protein which is unable to activate BRCA1-associated genome surveillance complex (BASC) leading to uncontrolled cell proliferation. Moreover, the molecular dynamics (MD) simulations of mutated BRCA1 protein demonstrated extensive variations in the domain and non-domain regions compared with the wild-type structure as indicated by RMSD values. All these results conclusively explain that the c.53delT mutation may be the probable founder of deleterious mutation in this ethnic group.

In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

Down-regulation of PAX2 promotes in vitro differentiation of podocytes from human CD34+ cells.

Podocytes are major kidney cells that help in glomerular filtration and any damage or loss is a major event in the progression of kidney diseases. Understanding podocytes development will help in designing therapeutic strategies against these renal diseases. Therefore, in vitro generation of podocytes from adult hematopoietic CD34+ stem cells is explored in the present study. Apheretically, isolated human HSCs from peripheral blood showed the presence of CD34 surface glycoprotein through immunocytochemistry (ICC) and flowcytometry. Initially, these HSCs were induced with activin-A (10 ng/ml), retinoic acid (RA) (10 ng/ml) and bone morphogenic protein (BMP-7) (2.5 ng/ml) for 5 days. Transdifferentiation of HSCs to podocytes through intermediate mesoderm was studied with positive selection of Osr1+ cells. Subsequently, thus-obtained Osr1+ cells were induced further with activin-A (10 ng/ml), RA (10 ng/ml), BMP-7 (2.5 ng/ml), EGF (30 ng/ml) and bFGF (30 ng/ml) for 9 days. Distinct cobblestone morphological changes were observed on staining with Leishman's stain. Consequently, differentiated cells were immunopositive for anti-podocin, anti-synaptopodin and anti-GLEPP1 monoclonal antibodies. These cells showed expression of early podocyte markers PAX2 and Wt1 at day 3 followed by day 6 and mature podocyte markers NPHS1, SULT1B1, NPHS2 and Synaptopodin at day 9. Interestingly, on day 9, diminished expression of PAX2 was noted. Differentiated cells showed high tyrosine kinase activity signifying that phosphorylation controls slit diaphragm proteins. Synaptopodin regulates the integrity of cytoskeleton and cell motility of podocytes and this phenomenon was confirmed through scratch assay using agarose molds that showed high cell mobility and migration. These findings establish HSCs as ideal candidates for regenerative therapies of damaged podocytes.

Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

BACKGROUND: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. METHODS: Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. RESULTS: Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. CONCLUSION: The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

Gel based in vitro 3D model exploring the osteocytic potentiality of human CD34+ stem cells.

Osteocytic potentiality of human CD34+ stem cells explored in the present study by generating in vitro agarose gel 3D model to understand the bone ossification process. The G-CSF and IL-3 mobilized human CD34+ stem cells isolated apheretically from donor peripheral blood and purity of the cells was assessed by FACS and immunocytochemical (ICC) studies. The CD34+ stem cells were cultured in gel based 3D model with osteogenic stimulating medium for 21 days. The transition stages from undifferentiated to differentiated osteocytes through osteoblasts were studied with expression markers Differentiated cells at Day 7 showed positive reactivity with monoclonal anti-Runx2, an early osteoblastic marker. qPCR expression analysis showed early and mature osteoblastic markers like RUNX2, Osterix, RANKL, along with osteocyte markers SPARC, Sclerostin. While poor expression of OSCAR genes was observed apart from conspicuous expression of alkaline phosphatase. The expression of sclerostin and SPARC suggests that these differentiated cells are behaving like true osteocytes, sclerostin expression causes transformation of osteoblast into osteocytes and negligible expression of OSCAR, RANK, NFATc and cathepsin K genes explains there are no osteoclasts in the differentiated culture. These cells showed positive reaction with Alizarin red stain indicating expression of calcium bound bone morphogenic proteins like osteonectin. All these results clearly confirm the human CD34+ stem cells possess unique osteogenic differentiation potential and can be used in the early regeneration of injured bone.

In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic ß-cells.

Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into ß-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific ß-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained ß-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into ß-cells of islets of langerhans.

In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells.

OBJECTIVES: Human CD34(+) stem cells differentiated into type-II pneumocytes in Dulbecco's modified Eagle medium (DMEM) having hydrocortisone, insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and bovine serum albumin (BSA), expressing surfactant proteins-B (SP-B) and C (SP-C), alkaline phosphatase (ALP) and lysozyme. RESULTS: FACS-enumerated pure CD34(+) cells, isolated from human peripheral blood, were cultured in DMEM and showed positive reaction with anti-human CD34 monoclonal antibodies in immunocytochemistry. These cells were cultured in DMEM having hydrocortisone, insulin, FGF, EGF and BSA (HIFEB-D) medium having an air-liquid interface. They differentiated into type-II pneumocytes with expression of SP-B and SP-C genes and disappearance of CD34 expression as assessed using real-time PCR. In reverse transcription-PCR amplicons showed 208 and 907 bp confirming SP-B and SP-C expressions. These cells expressed ALP with an activity of 1.05 ± 0.09 mM ml(-1) min(-1) and lysozyme that killed E. coli. CONCLUSION: The successful differentiation of human CD34(+) stem cells into type-II pneumocytes, and transplantation of such cells obtained from the patient's stem cell could be the futuristic approach to regenerate diseased lung alveoli.

Role of STPK in Maintaining the Quiescent Nature of Human CD34+ Stem Cells.

Haematopoietic stem cell normally exists in the hypoxic niche of bone marrow and in this high anaerobic condition phosphorylation is vital in understanding the stemness of these stem cells in bone marrow. Analysis of human aldehyde dehydrogenase (ALDH) and isocitrate dehydrogenase (IDH) we have observed the presence of serine threonine protein kinase (STPK) sites in the protein sequence of these enzymes conferring that functioning of ALDH and IDH is regulated largely by STPK through phosphorylation. Human CD34+ stem cells and mononuclear cells as a control isolated from peripheral blood and were propagated in DMEM media at 5% CO2, 95% humidity and at 37°C. Thus obtained cells showed high enzyme activity for STPK, ALDH and low enzyme activity for IDH in CD34+ cells compare to control cells. These results were concurred with qRT-PCR studies with high gene expression levels of hypoxia inducing factor-1-alpha (HIF1α), STPK, ALDH and low IDH expression in CD34+ cells while normalized with ß-actin. In addition the phosphorylating sites on ALDH and IDH proteins were identified and their importance in maintaining the anaerobic conditions in HSCs was demonstrated. In view of the importance of STPK signalling in the present study mechanism in cell division was addressed with phosphorylation of key regulating enzymes in the metabolic pathway of cell cycle was explored.

In vitro transdifferentiation of human cultured CD34+ stem cells into oligodendrocyte precursors using thyroid hormones.

The extent of myelination on the axon promotes transmission of impulses in the neural network, any disturbances in this process results in the neurodegenerative condition. Transplantation of oligodendrocyte precursors that supports in the regeneration of axons through myelination is an important step in the restoration of damaged neurons. Therefore, in the present study, the differentiation of human CD34+ stem cells into oligodendrocytes was carried out. The pure human CD34+ culture developed from the stem cells obtained from a peripheral blood of a donor were subjected to oligodendrocyte differentiation medium (ODM). The ODM at a concentration of 40ng/ml thyroxine, 40ng/ml 3,3',5-tri-iodo-thyronine showed distinct morphological changes from day 6 to 9 with cells exhibiting conspicuous stellate morphology and extensive foot processes. The real-time PCR analysis showed prominent expression of Olig2, CNPase, PDGFRα and PLP1/DM20 in the differentiated cells confirming the formed cells are oligodendrocyte precursors. The expression of these genes increased from days 6 to 9 corresponding to the morphological changes observed with almost no expression of GFAP+ cells. The distinct CNPase activity was observed in these differentiated cells compared to normal CD34+ stem cells correlating with results of real-time PCR conclusively explains the development of oligodendrocytes from human CD34+ stem cells.

Anaerobic Glycolysis and HIF1α Expression in Haematopoietic Stem Cells Explains Its Quiescence Nature.

Metabolic alteration that a stem cell undergoes during proliferation and quiescence are decisive. These cells survive in extreme hypoxic environment that prevails in bone marrow. The present study is aimed to understand this nature in hematopoietic stem cells. These stem cells were mobilized from bone marrow into peripheral blood by giving G-CSF at a concentration of 5 µg/Kg/d and the cells were isolated by apheresis technique. The morphological analysis of these cells using Giemsa stain and SEM showed presence of only single type of cells with conspicuous nuclei, the hematopoietic nature was assessed by the presence of CD34, a glycoprotein using anti-CD34 monoclonal antibodies. The ICC results revealed presence of CD34 marker further; pure population of CD34+ stem cells was described by FACS. These cells were cultured separately in DMEM having 5.5mM, 11.1mM and 25mM glucose respectively. In these cells GK, PK and L-LDH enzyme activities were estimated which showed increased activities at 5.5mM glucose concentration and further elevation of glucose concentration the activities were fallen considerably. Similarly, qPCR analysis of HIF1α and GAPDH genes showed very high expression of HIF1α at 5.5mM glucose concentration which reduced with increased glucose concentration. While GAPDH gene expression enhanced on elevation of glucose concentration. Thus, these results indicate high HIF1α expression in low glucose condition with improved anaerobic glycolysis seems to be one of the key factors in maintaining the quiescent state of CD34+ stem cells.

Mutations in exons 3 and 7 resulting in truncated expression of human ATP6V1B1 gene showing structural variations contributing to poor substrate binding-causative reason for distal renal tubular acidosis with sensorineural deafness.

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H(+) secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding ß1 subunit of H(+)-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A > G, c.308 C > A, c.310 C > G, c.704 T > C, c.705 G > T, c.709 A > G, c.710 A > G, c.714 G > A, c.716 C > A, c.717delC, c.722 C > G, c.728insG, c.741insT, c.753G > C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H(+) ions contributing to dRTA with sensorineural deafness.

In silico designing and molecular docking of a potent analog against Staphylococcus aureus porphobilinogen synthase.

BACKGROUND: The emergence of multidrug-resistant strains of Staphylococcus aureus, there is an urgent need for the development of new antimicrobials which are narrow and pathogen specific. AIM: In this context, the present study is aimed to have a control on the staphylococcal infections by targeting the unique and essential enzyme; porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of δ-aminolevulinic acid, an essential step in the tetrapyrrole biosynthesis. Hence developing therapeutics targeting PBGS will be the promising choice to control and manage the staphylococcal infections. 4,5-dioxovalerate (DV) is known to inhibit PBGS. MATERIALS AND METHODS: In view of this, in this study, novel dioxovalerate derivatives (DVDs) molecules were designed so as to inhibit PBGS, a potential target of S. aureus and their inhibitory activity was predicted using molecular docking studies by molecular operating environment. The 3D model of PBGS was constructed using Chlorobium vibrioform (Protein Data Bank 1W1Z) as a template by homology modeling method. RESULTS: The built structure was close to the crystal structure with Z score - 8.97. Molecular docking of DVDs into the S. aureus PBGS active site revealed that they are showing strong interaction forming H-bonds with the active sites of K248 and R217. The ligand-receptor complex of DVD13 showed a best docking score of - 14.4555 kcal/mol among DV and all its analogs while the substrate showed docking score of - 13.0392 kcal/mol showing interactions with S199, K217 indicating that DVD13 can influence structural variations on the enzyme and thereby inhibiting the enzyme. CONCLUSION: The substrate analog DVD13 is showing significant interactions with active site of PBGS and it may be used as a potent inhibitor to control S. aureus infections.

Novel three missense mutations observed in Von Hippel-Lindau gene in a patient reported with renal cell carcinoma.

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumors, especially cerebellar hemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). We have identified of VHL gene using immunohistochemistry in a patient who was diagnosed for RCC. In order to understand the involvement of mutation in the VHL gene exon 1 was amplified and sequenced (accession number: JX 401534). The sequence analysis revealed the presence of novel missense mutations c.194 C>T, c.239 G>A, c.278 G>A, c.319 C>G, c. 337 C > G leading to the following variations p.Ala 65 Val, p.Gly 80 Asp, p.Gly 93 Glu, p.Gln 107 Glu, p.Gln 113 Glu in the protein.

In vitro differentiation of cultured human CD34+ cells into astrocytes.

BACKGROUND: Astrocytes are abundantly present as glial cells in the brain and play an important role in the regenerative processes. The possible role of stem cell derived astrocytes in the spinal cord injuries is possible related to their influence at the synaptic junctions. AIM: The present study is focused on in vitro differentiation of cultured human CD34+ cells into astrocytes. MATERIALS AND METHODS: Granulocyte-colony stimulating factor mobilized human CD34+ cells were isolated from peripheral blood using apheresis method from a donor. These cells were further purified by fluorescence-activated cell sorting and cultured in Dulbecco's modified eagle's medium. Thus, cultured cells were induced with astrocyte defined medium (ADM) and in the differentiated astrocytes serine/threonine protein kinases (STPK) and glutamine synthetase (GLUL) activities were estimated. The expression of glial fibrillary acidic protein (GFAP) and GLUL were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The cultured human CD34+ cells differentiated into astrocytes after 11 h of incubation in ADM. The RT-PCR experiment showed the expression of GLUL (1.5 kb) and GFAP (2.9 kb) in differentiated astrocytes. The high enzyme activities of GLUL and STPK in differentiated astrocytes compared with cultured human CD34+ cells confirmed astrocyte formation. CONCLUSION: In the present study, in vitro differentiation of stem cells with retinoic acid induction may result in the formation of astrocytes.