In vitro generation of epidermal keratinocytes from human CD34-positive hematopoietic stem cells.

The epidermis is largely composed of keratinocytes (KCs), and the proliferation and differentiation of KCs from the stratum basale to the stratum corneum is the cellular hierarchy present in the epidermis. In this study, we explore the differentiation abilities of human hematopoietic stem cells (HSCs) into KCs. Cultured HSCs positive for CD34, CD45, and CD133 with prominent telomerase activity were induced with keratinocyte differentiation medium (KDM), which is composed of bovine pituitary extract (BPE), epidermal growth factor (EGF), insulin, hydrocortisone, epinephrine, transferrin, calcium chloride (CaCl2), bone morphogenetic protein 4 (BMP4), and retinoic acid (RA). Differentiation was monitored through the expression of cytokeratin markers K5 (keratin 5), K14 (keratin 14), K10 (keratin 10), K1 (keratin 1), transglutaminase 1 (TGM1), involucrin (IVL), and filaggrin (FLG) on day 0 (D0), day 6 (D6), day 11 (D11), day 18 (D18), day 24 (D24), and day 30 (D30) using immunocytochemistry, fluorescence microscopy, flow cytometry, qPCR, and Western blotting. The results revealed the expression of K5 and K14 genes in D6 cells (early keratinocytes), K10 and K1 genes in D11-D18 cells (mature keratinocytes) with active telomerase enzyme, and FLG, IVL, and TGM1 in D18-D24 cells (terminal keratinocytes), and by D30, the KCs were completely enucleated similar to cornified matrix. This method of differentiation of HSCs to KCs explains the cellular order exists in the normal epidermis and opens the possibility of exploring the use of human HSCs in the epidermal differentiation.

The Therapeutic Potential of 4-Methoxy-1-methyl-2-oxopyridine-3-carbamide (MMOXC) Derived from Ricinine on Macrophage Cell Lines Infected with Methicillin-Resistant Strains of Staphylococcus aureus.

The incidences of methicillin-resistant strains of Staphylococcus aureus (MRSA) and their survival inside the macrophages are the major attributes of the relapsed infections after antimicrobial therapy, and it is a global problem. In this context, we have previously demonstrated 4-methoxy-1-methyl-2-oxopyridine-3-carbamide (MMOXC), a Ricinine derivative exhibiting anti-S. aureus and anti-biofilm characteristics by competitively inhibiting uridine monophosphate kinase (UMPK), UDP-N-acetyl muramyl pentapeptide ligase (Mur-F), and peptidyl deformylase, (PDF). In the present study, the stability of this competitive inhibitor MMOXC was evaluated by showing its ability to remain bound to the active sites of UMPK, Mur-F, and PDF even after increasing the incubation time, temperature, pH, and substrate concentration. On growing MRSA in fewer concentrations of MMOXC, these strains could not attain resistance to MMOXC and at the same time distinct reductions in the expression of UMPK, Mur-F, and PDF genes were noted. In vitro, infective models were generated by infecting MRSA to RAW 264.7 and human monocyte-derived macrophage (hMDM) cell lines. In these infected cell lines, in spite of increased nitric oxide synthase (NOS), NADPH-P450 reductase, superoxide dismutase, catalase, and peroxidase activities, the MRSA survived. At 640 µM/ml, the concentration of MMOXC penetrated into these infected cells and obliterated MRSA. While treating uninfected macrophage cell lines with MMOXC, no appreciable effect was observed indicating that MMOXC is the most suitable drug for the treatment of infections caused by MRSA.

Novel mutations in EPO-R and oxygen-dependent degradation (ODD) domain of EPAS1 genes-a causative reason for Congenital Erythrocytosis.

Congenital Erythrocytosis (CE) can be primary or secondary due to the mutations in genes involved in the erythropoietin receptor and oxygen sensing pathway. In this study, 42 patients with 38 unrelated patients and one family (4 patients) who were JAK-2 mutation (both exon 12 and exon 14) negative with high haematocrit values were investigated. The Endogenous Erythroid colony (EEC) assay was performed in all patients, interestingly EEC colonies were high in EPAS1 and EPOR mutated patients compared to non-mutated patients. The sequence analysis of EPAS1 (exon 12), EPO-R (exon-8), VHL (exon-3), and EGLN1 (exon-1) genes in all these patients showed 19% of patients (8/42) had mutations, in exon12 of EPAS1 and exon 8 of EPO-R genes. Two novel missense mutations MW_600850:c.1183G>C, MW_600851:c.1028A>C in EPO-R gene were observed in the study group. One new MW_600849:c.1969C>T nonsense mutation and five MW_619914:c.1715A>G, MW_619915:c.1694G>T, MW_619916:c.1634T>C, MW_600852:c.1771C>G, MW_600848:c.1859G>A novel missense mutations were observed in the EPAS1 gene. Among them, 4 mutations p. (Gln572Arg), p. (Ser565Ile), p. (Ile545Thr), p. (Gln591Glu) in the ODD (Oxygen-dependent degradation) domain of HIF2α, all these variations contributed to the formation of non-functional HIF2α. No mutations were observed in VHL and EGLN1 genes. Using in silico analysis we observed that these mutations contributed to major conformational changes in the HIF2α protein making it non-functional. The mutations in the EPAS1 gene were heterozygous and show autosomal dominant inheritance patterns and we observed in one family. These novel mutations in the EPAS1 (75% (6/8)) and 25% (2/8) EPO-R genes correlating with EEC positivity were observed for the first time in India in CE patients.

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.

Staphylococcus aureus grown in anaerobic conditions exhibits elevated glutamine biosynthesis and biofilm units.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus, sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited Km of 11.06 ± 0.05 mmol·L-1 for glutamate and 2.4 ± 0.03 mmol·L-1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus.

Novel mutations in the EPO-R, VHL and EPAS1 genes in the Congenital Erythrocytosis patients.

Congenital erythrocytosis (CE) can be classified as primary and secondary and 82 consecutive patients of erythrocytosis who were JAK-2 mutation negative, were further investigated. The genomic DNA was extracted from all the patients and the EPO-R, VHL, EGLN1 and EPAS1 genes were PCR amplified and sequenced. The sequence analysis showed (28/82) 34.14% patients had mutations. Among them, (19/28) 67.86% patients had mutations in exon 8 of EPO-R gene, of which six were novel missense mutations, p.(Gly418Ala), p.(Gly390Ala), p.(Ala411Thr), p.(Gly475Val), p.(Glu490Asp), p.(Glu362Gln) and three were novel frameshift mutations, p.(Glu336*), p.(Pro327Hisfs*68), p.(Gly479Alafs*37). All these EPO-R patients were heterozygotes and were forming endogenous erythrocyte colonies (EEC). Some patients (8/28) 28.57% had mutations in VHL gene, out of which 3 novel homozygous missense mutations in exon 1 of VHL gene, p.Gly80Asp, p.Gln107Glu and p.Gln113Glu, were identified. In addition, (1/28) 3.5% patients had one reported heterozygous missense mutation in exon 12 of EPAS1 gene p.Gly537Arg and one novel frameshift mutation p.(Ala553Glyfs*58). Further, in silico analysis indicated most of the mutations, probably, were damaging the protein structures, causing the CE in these patients. In this study the mutations in EPO-R and EPAS1 genes were identified for the first time in India.

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.

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.

Novel mutations in the kinase domain of BCR-ABL gene causing imatinib resistance in chronic myeloid leukemia patients.

Mutations in the drug binding region of BCR-ABL lead to imatinib resistance during the management of chronic myeloid leukemia (CML). In our study, 62 Philadelphia positive (Ph+) CML patients showing conspicuous expression of BCR-ABL gene were treated with imatinib. At the end of 3 months, 21/62 (33.87%) patients did not obtain complete hematological response (CHR) and also showed no significant decrease in BCR-ABL gene expression. In all the imatinib-resistant patients BCR-ABL gene was PCR amplified and sequenced. The sequence analysis showed four novel missense mutations p.(Leu301Ile), p.(Tyr320His), p.(Glu373Asp), p.(Asp381Asn) and six already reported mutations p.(Val256Gly), p.(Thr315Ile), p.(Gly250Glu), p.(Tyr253His), p.(Phe317Leu), p.(Met351Thr) which contributed in the formation of inactive enzyme and also two novel frameshift mutations p.(Glu281*) and p.(Tyr393*), which resulted in truncated protein formation. Further, the structural analysis revealed all these mutations affected P-loop, gatekeeper, catalytic and activation loop domain regions of the enzyme causing poor imatinib binding in the ATP region. The primary intention of the study was to find out the mutations in the BCR-ABL gene causing imatinib resistance. This study highlights the need for BCR-ABL gene sequence analysis to detect the mutations in CML patients in order to properly guide the therapy.

Association of G472A allele of membrane bound catechol-O-methyltransferase gene with chronic post-sternotomy pain.

Chronic persistent surgical pain (CPSP) is a complex disease with strong genetic component. The studies on revealed association of mutations in membrane bound catechol-O-methyltransferase gene with CPSP were reported indifferent ethnic populations across the globe. We identify that one out of four patients who underwent sternotomy procedure showed CPSP even after 3 months of surgery. The Mb.COMT gene sequence analysis revealed of the four patients, three patients had no mutation in Mb.COMT gene, while in one patient exhibited G472A mutation. Interestingly, this patient showed CPSP even after 90 days of surgery. The magnitude of the CPSP was evaluated with pain questionnaires' at the end of 3 months after discharge from the hospital. In this study 25% (1/4) showed presence G472A allele correlating with CPSP. Further the study suggested that evaluation of G472A allele of Mb.COMT gene in the patients undergoing sternotomy for monitoring pain in pre and post-surgical events.

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.

Clinical characteristics and NF1 gene mutation analysis of three successive generations in three different Indian families with neurofibromatosis type 1 and peripheral nerve sheath tumours.

Neurofibromatosis type 1 (NF1) is a rare autosomal-dominant disorder caused by inactivation of NF1 tumour suppressor gene, which associates in the development of peripheral nerve tumours. NF1 is an important regulator of GAP and RAS proteins, mutations in NF1 results in the impairment in this function causing specific osseous lesions in any organ of the human body. In the present study, we investigated the clinical characteristics and NF1 gene mutation analysis of 3 unrelated Indian families with neurofibromatosis type 1. All the exons of NF1 gene was PCR amplified and sequenced. The structural and functional analysis was performed using molecular modelling tools. The sequence analysis of NF1 gene revealed; in family I five novel mutations p.R103K, p.D105N, p.M108I, p.L114M, p.E116X and p.A131S was observed in exon 4. In family II one missense p.A131S mutation and one silent p.L234L mutation was detected in exon 4. While, in family III one novel frame shift p.E225Rfs∗6 mutation was identified in exon 7 resulting in the truncated protein formation. Further, the structural analysis revealed all these mutations fall in the protein kinase C domain of NF1 gene causing loss of functional GRD and CSRD domains. In conclusion, novel mutations in the exon 4 and exon 7 of NF1 gene in these families correlating with genotype-phenotype characters explaining the neurofibromatosis type 1 and peripheral nerve sheath tumours condition in these patients.

Iron enhances the peptidyl deformylase activity and biofilm formation in Staphylococcus aureus.

Staphylococcus aureus plays a major role in persistent infections and many of these species form structured biofilms on different surfaces which is accompanied by changes in gene expression profiles. Further, iron supplementation plays a critical role in the regulation of several protein(s)/enzyme function, which all aid in the development of active bacterial biofilms. It is well known that for each protein, deformylation is the most crucial step in biosynthesis and is catalyzed by peptidyl deformylase (PDF). Thus, the aim of the current study is to understand the role of iron in biofilm formation and deformylase activity of PDF. Hence, the PDF gene of S. aureus ATCC12600 was PCR amplified using specific primers and sequenced, followed by cloning and expression in Escherichia coli DH5α. The deformylase activity of the purified recombinant PDF was measured in culture supplemented with/without iron where the purified rPDF showed Km of 1.3 mM and Vmax of 0.035 mM/mg/min, which was close to the native PDF (Km = 1.4 mM, Vmax = 0.030 mM/mg/min). Interestingly, the Km decreased and PDF activity increased when the culture was supplemented with iron, corroborating with qPCR results showing 100- to 150-fold more expression compared to control in S. aureus and its drug-resistant strains. Further biofilm-forming units (BU) showed an incredible increase (0.42 ± 0.005 to 6.3 ± 0.05 BU), i.e., almost 15-fold elevation in anaerobic conditions, indicating the significance of iron in S. aureus biofilms.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

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.

Novel mutations in the exon 5, intron 2 and 3' UTR regions of IL-12B gene were observed in clinically proven tuberculosis patients of south India.

Interleukin-12 (IL-12) is formed by the interaction of IL-12p35 and IL-12p40 expressed independently from IL-12A and IL-12B genes. This interleukin plays prominent role in the T-helper type-1 (Th1) response against intracellular pathogens. Variations in IL-12B gene causes disruption of various activities one of them is suppression of Th1 response and is one of the characteristic features observed in patients with active tuberculosis. Hence, in the present study IL-12B gene status was evaluated in 50 new sputum smear-positive pulmonary tuberculosis patients (NSP-PTB) as identified by Ziehl-Nielsen (ZN) staining and 50 apparently healthy control subjects (HCS) who were sputum smear-negative. The sequence analysis showed novel missense mutations p.Ser205Ile, p.Leu206Glu, p.Pro207Ser, p.Glu209Lys, p.Val210Ser, p.(Ser205_Cys327delinsIleGlu) and p.(Lys217_Leu218delinsIle) were found in exon 5 of the IL-12B gene in nine patients resulting formation of inactive IL-12 and three patients showed novel frame shift mutations p.(Asn222Leufs∗23) in exon 5 of causing the formation of truncated protein. Several mutations were noted in intron 2 of the IL-12B gene in 5 patients and in 13 patients mutations were observed in 3' UTR region. All together 30/50 patients (60%) showed mutations in IL-12B gene. Decreased levels of interferon-gamma (IFN-γ) and IL-12 as determined by ELISA and flow cytometry were observed in the peripheral blood mononuclear cell culture supernatants in TB patients having mutations compared with control subjects. Further, in silico analysis revealed due to frame shift mutations in exon 5 at Asn222 resulted in deletion of functional fibronectin type-III (FN3) domain which leads to formation of inactive IL-12 in these patients.

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.