Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) showing anti-S. aureus and anti-biofilm properties by elevating activities of serine protease (SspA) and cysteine protease staphopain B (SspB).

Staphylococcus aureus biofilms are the pathogenic factor in the spread of infection and are more pronounced in multidrug-resistant strains of S. aureus, where high expression of proteases is observed. Among various proteases, Serine protease (SspA) and cysteine protease Staphopain B (SspB) are known to play a key role in the biofilm formation and removal of biofilms. In earlier studies, we have reported Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) exhibits anti-S. aureus and anti-biofilm properties by elevating the expression of the protease. In this study, the effect of DBTMP on the activities of SspA, and SspB of S. aureus was evaluated. The SspA and SspB genes of S. aureus ATCC12600 were sequenced (Genbank accession numbers: MZ456982 and MW574006). In S. aureus active SspA is formed by proteolytic cleavage of immature SspA, to get this mature SspA (mSspA), we have PCR amplified the mSspA sequence from the SspA gene. The mSspA and SspB genes were cloned, expressed, and characterized. The pure recombinant proteins rSspB and rmSspA exhibited a single band in SDS-PAGE with a molecular weight of 40 and 30 KD, respectively. The activities of rmSspA and rSspB are 32.33 and 35.45 Units/mL correspondingly. DBTMP elevated the activities of rmSspA and rSspB by docking with respective enzymes. This compound disrupted the biofilms formed by the multidrug-resistant strains of S. aureus and further prevented biofilm formation. These findings explain that DBTMP possesses anti-S. aureus and anti-biofilm features.

Mutaciones y variaciones estructurales en el gen catecol-O-metiltransferasa de los pacientes que exhiben dolor quirúrgico persistente crónico / Mutations and structural variations in Catechol-O-methyltransferase gene of patients exhibiting chronic persistent surgical pain

Objetivos: Las mutaciones en el exón 4 del gen COMT están asociadas a dolor quirúrgico persistente crónico (CPSP). En especial G472A (Val158Met), el alelo mutado de COMT, asociado a los pacientes de CPSP, se reporta en diferentes poblaciones étnicas. El objetivo de este estudio es evaluar la prevalencia de las mutaciones genéticas y las variaciones estructurales en el exón 4 de COMT, que puede guardar relación con la aparición de CPSP en pacientes sometidos a esternotomía.Materiales y métodos: Se seleccionaron 100 pacientes con estatus físico i, ii y iii de ASA (American Society of Anesthesiologists) sometidos a esternotomía, para evaluar el desarrollo y magnitud de CPSP mediante cuestionarios de dolor, transcurridos tres meses de la cirugía. Esto guardó relación con la presencia alélica de COMT. Se estudió el exón 4 del gen COMT (que contiene el alelo G472A). Se secuenciaron los productos de la reacción en cadena de la polimerasa (PCR), depositándose las secuencias mutadas en GenBank®. Se realizó el análisis estructural de COMT utilizando ProCheck®, evaluándose las distorsiones de la orientación estructural terciaria tridimensional con la escala RMSD (raíz de la desviación cuadrática media). Resultados: El análisis genético realizado con PCR reflejó amplicones de 220 bp. El 25% de los pacientes con CPSP reflejó una puntuación de dolor < 4 en la escala NRS. El 20% de estos pacientes tenía mutación Val158Met conocida, el 5% de los pacientes reflejó mutaciones nuevas c.382C>G, c.383G>C, p.(Arg128Ala). Las mutaciones del gen COMT contribuyeron a variaciones estructurales mayores de COMT, conducentes a la formación de COMT inactiva que se correlaciona con CPSP. Conclusión: Los resultados del presente estudio mostraron que tanto las nuevas mutaciones, como las previamente reportadas del gen COMT, tienen una fuerte asociación con CPSP.(AU)

Objectives: Mutations in the exon 4 of the COMT gene are associated with chronic persistent surgical pain (CPSP). Especially COMT mutated allele G472A (Val158Met) associated with CPSP patients is reported in different ethnic population. The purpose of this study is to evaluate the prevalence of genetic mutations and structural variations in exon 4 of COMT that can be related to the appearance of CPSP in patients under sternotomy. Materials and methods: One hundred patients with American Society of Anesthesiologists (ASA) physical status grades i, ii and iii, who underwent sternotomy procedures, were selected to assess the development and magnitude of the CPSP evaluated with pain questionaries’ at the end of three months after surgery. This was correlated with COMT allele presence. The exon 4 of COMT gene (that contains the G472A allele) was studied. The polymerase chain reaction (PCR) products were sequenced and mutated sequences were deposited in GenBank®. The structural analysis of COMT was performed using ProCheck® and distortions of three-dimensional tertiary structural orientation was evaluated with root-mean-square deviation (RMSD) score. Results: Genetic analysis carried out through PCR showed 220 bp amplicons. The 25% of patients with CPSP showed a Numeric Rating Scale (NRS) > 4 pain score. The 20% of these patients have known Val158Met mutation, 5% of patients showed novel mutations c.382C>G, c.383G>C, p.(Arg128Ala). The mutations in COMT gene contributed major structural variations in COMT leading to the formation of inactive COMT that correlates with CPSP. Conclusion: The results of the present study showed that both novel and previously reported mutations in COMT gene has strong association with CPSP.(AU)

Mutations and structural variations in Catechol-O-methyltransferase gene of patients exhibiting chronic persistent surgical pain. / Mutaciones y variaciones estructurales en el gen catecol-O-metiltransferasa de los pacientes que exhiben dolor quirúrgico persistente crónico.

OBJECTIVES: Mutations in the exon 4 of the COMT gene are associated with chronic persistent surgical pain (CPSP). Especially COMT mutated allele G472A (Val158Met) associated with CPSP patients is reported in different ethnic population. The purpose of this study is to evaluate the prevalence of genetic mutations and structural variations in exon 4 of COMT that can be related to the appearance of CPSP in patients under sternotomy. MATERIALS AND METHODS: One hundred patients with American Society of Anesthesiologists (ASA) physical status grades i, ii and iii, who underwent sternotomy procedures, were selected to assess the development and magnitude of the CPSP evaluated with pain questionaries' at the end of three months after surgery. This was correlated with COMT allele presence. The exon 4 of COMT gene (that contains the G472A allele) was studied. The polymerase chain reaction (PCR) products were sequenced and mutated sequences were deposited in GenBank®. The structural analysis of COMT was performed using ProCheck® and distortions of three-dimensional tertiary structural orientation was evaluated with root-mean-square deviation (RMSD) score. RESULTS: Genetic analysis carried out through PCR showed 220 bp amplicons. The 25% of patients with CPSP showed a Numeric Rating Scale (NRS) > 4 pain score. The 20% of these patients have known Val158Met mutation, 5% of patients showed novel mutations c.382C>G, c.383G>C, p.(Arg128Ala). The mutations in COMT gene contributed major structural variations in COMT leading to the formation of inactive COMT that correlates with CPSP. CONCLUSION: The results of the present study showed that both novel and previously reported mutations in COMT gene has strong association with CPSP.

Cloning, Expression and Characterization of NAD Kinase from Staphylococcus aureus Involved in the Formation of NADP (H): A Key Molecule in the Maintaining of Redox Status and Biofilm Formation.

BACKGROUND: Staphylococcus aureus has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK) and this gene of S. aureus ATCC 12600 was cloned, sequenced, expressed and characterized. MATERIALS AND METHODS: The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of S. aureus ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in Escherichia coli DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the S. aureus was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure. RESULTS: The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814) revealed presence of only one kind of NADK in all S. aureus strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Å observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity. CONCLUSION: The results of this study suggest increased NADPH concentration in S. aureus plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions.

Biofabrication and spectral characterization of silver nanoparticles and their cytotoxic studies on human CD34 +ve stem cells.

Biosynthesis of plant-mediated silver nanoparticles is gaining significant importance due to environmentally safe 'green method' and it is an efficient alternative method. In the present study, silver nanoparticles were synthesized by using root extract of Glycyrrhiza glabra an important medicinal plant. The AgNPs are characterized by spectral analysis; the surface plasmon resonance (SPR) peak of AgNPs showed maximum absorption at 445 nm. Fourier-transform infrared spectroscopy (FT-IR) data show that the O-H hydroxyl groups, carboxylic acids, ester and ether groups and C-O stretching of alcohols have been utilized in the formation of AgNPs. The X-ray powder diffraction (XRD) data reveal that the AgNPs are face-centered cubic (fcc) in structure. The size was determined by particle size analyzer and atomic force microscope (AFM); the results reveal that AgNPs were spherical in shape and the average grain size is determined as 41.5-46.5 nm. Transmission electron microscopy (TEM) micrographs obtained show that AgNPs were roughly spherical and well dispersed with the sizes ranging from 10 to 45 nm ± 5 nm. The biofabricated AgNPs are extremely stable due to its high negative zeta potential -34.1 mV which indicates that the nanoparticles are polydispered in nature. The cytotoxic studies of AgNPs on human CD34 +ve stem cells in microcarrier culture reveal excellent growth at different concentrations of biosynthesized AgNPs. This is the first report of microcarrier culture of CD34 +ve stem cells on biosynthesized AgNPs.

Phosphorylation controls the functioning of Staphylococcus aureus isocitrate dehydrogenase--favours biofilm formation.

Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring biofilm formation.

In Staphylococcus aureus the regulation of pyruvate kinase activity by serine/threonine protein kinase favors biofilm formation.

Staphylococcus aureus, a natural inhabitant of nasopharyngeal tract, survives mainly as biofilms. Previously we have observed that S. aureus ATCC 12600 grown under anaerobic conditions exhibited high rate of biofilm formation and L-lactate dehydrogenase activity. Thus, the concentration of pyruvate plays a critical role in S. aureus, which is primarily catalyzed by pyruvate kinase (PK). Analyses of the PK gene sequence (JN645815) revealed presence of PknB site in PK gene indicating that phosphorylation may be influencing the functioning of PK. To establish this hypothesis the pure enzymes of S. aureus ATCC 12600 were obtained by expressing these genes in PK 1 and PV 1 (JN695616) clones and passing the cytosolic fractions through nickel metal chelate column. The molecular weights of pure recombinant PK and PknB are 63 and 73 kDa, respectively. The enzyme kinetics of pure PK showed K M of 0.69 ± 0.02 µM, while the K M of PknB for stpks (stpks = NLCNIPCSALLSSDITASVNCAK) substrate was 0.720 ± 0.08 mM and 0.380 ± 0.07 mM for autophosphorylation. The phosphorylated PK exhibited 40 % reduced activity (PK = 0.2 ± 0.015 µM NADH/min/ml to P-PK = 0.12 ± 0.01 µM NADH/min/ml). Elevated synthesis of pyruvate kinase was observed in S. aureus ATCC 12600 grown in anaerobic conditions suggesting that the formed pyruvate is more utilized in the synthesis phase, supporting increased rate of biofilm formation.

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.

Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding - explains hyperglycemia in type 2 diabetic patients.

Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of ß-α-ß units, hairpins, ß-bulges, strands, helices, helix-helix interactions, ß-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.

Comparative Structural and Functional Analysis of Staphylococcus aureus Glucokinase with other Bacterial Glucokinases.

Glucokinase is classified in bacteria based upon having ATP binding site and 'repressor/open reading frames of unknown function/sugar kinases' motif, the sequence of glucokinase gene (JN645812) of Staphylococcus aureus ATCC12600 showed presence of ATP binding site and 'repressor/open reading frames of unknown function/sugar kinases' motif. We have earlier observed glucokinase of S. aureus has higher affinity towards the substrate compared to other bacterial glucokinase and under anaerobic condition with increased glucose concentration S. aureus exhibited higher rate of biofilm formation. To establish this, 3D structure of glucokinase was built using homology modeling method, the PROCHECK and ProSA-Web analysis indicated this built glucokinase structure was close to the crystal structure. This structure was superimposed with different bacterial glucokinase structures and from the root-mean-square deviation values, it is concluded that S. aureus glucokinase exhibited very close homology with Enterococcus faecalis and Clostridium difficle while with other bacteria it showed high degree of variations both in domain and nondomain regions. Glucose docking results indicated -12.3697 kcal/mol for S. aureus glucokinase compared with other bacterial glucokinase suggesting higher affinity of glucose which correlates with enzyme kinetics and higher rate of biofilm formation.

Structural Variations of Human Glucokinase Glu256Lys in MODY2 Condition Using Molecular Dynamics Study.

Glucokinase (GK) is the predominant hexokinase that acts as glucose sensor and catalyses the formation of Glucose-6-phosphate. The mutations in GK gene influence the affinity for glucose and lead to altered glucose levels in blood causing maturity onset diabetes of the young type 2 (MODY2) condition, which is one of the prominent reasons of type 2 diabetic condition. In view of the importance of mutated GK resulting in hyperglycemic condition, in the present study, molecular dynamics simulations were carried out in intact and 256 E-K mutated GK structures and their energy values and conformational variations were correlated. Energy variations were observed in mutated GK (3500 Kcal/mol) structure with respect to intact GK (5000 Kcal/mol), and it showed increased γ -turns, decreased ß -turns, and more helix-helix interactions that affected substrate binding region where its volume increased from 1089.152 Å(2) to 1246.353 Å(2). Molecular docking study revealed variation in docking scores (intact = -12.199 and mutated = -8.383) and binding mode of glucose in the active site of mutated GK where the involvement of A53, S54, K56, K256, D262 and Q286 has resulted in poor glucose binding which probably explains the loss of catalytic activity and the consequent prevailing of high glucose levels in MODY2 condition.

Cloning, expression and characterization of NADP-dependent isocitrate dehydrogenase from Staphylococcus aureus.

The Krebs cycle dictates oxidative and reductive conditions in Staphylococcus aureus and is mainly regulated by isocitrate dehydrogenase (IDH) which plays pivotal role in the growth and pathogenesis of the bacteria. In the present study, IDH gene from S. aureus ATCC12600 was cloned in the Sma I site of pQE 30 vector; the resultant clone was named as UVIDH1. The insert in the clone was sequenced (accession number HM067707), and the sequence showed complete homology with IDH sequence of other S. aureus strains reported in the database indicating presence of single enzyme in S. aureus, and considerable sequence homology with other bacteria was observed; however, only 24% homology was found with NADP-dependent human IDH. Phylogenetically, the S. aureus IDH showed close identity with Bacillus subtilis and high degree of variability with other bacteria and human IDH. The expression of IDH in the clone UVIDH1 was induced with 1 mM IPTG, and the recombinant IDH was purified by passing through nickel metal chelate column; the purified recombinant IDH showed a single band in SDS-PAGE with a molecular weight of 40 kDa; K(m) and V(max) for isocitrate are 8.2 ± 0.28 and 525 ± 25 µM NADPH/mg/min, respectively, and for cofactor NADP 67.5 ± 2.82 µM and V(max) 50.5 ± 2.12 µM NADPH/mg/min.

Factor V Leiden mutation is not a predisposing factor for acute coronary syndromes.

BACKGROUND: The prevalence of Coronary artery disease (CAD) in India has increased considerably over the past few years and could become the number one killer disease if interventions are not done. Factor V Leiden (FVL) mutation and FII G20210A polymorphism are two recently described genetic factors with a propensity towards venous thrombosis. This warrants the investigations for thrombophilia in myocardial infarction patients in India. METHODS: The study cohort consisted of 51 patients aged below 50 years presenting with acute coronary syndromes. In both patient group and normal individuals the major risk factors Protein C deficiency, Protein S deficiency, anticardiolipin antibodies, Fibrinogen and Lipoprotein [a] were studied. Factor V Leiden (FVL) G1691A mutation in both control and patient group was looked by using Polymerase chain reaction (PCR) followed by sequencing of the PCR products. RESULTS: Our results indicated significantly higher levels of anticardiolipin antibodies and fibrinogen in the patients and absence of FVL (G1691A) mutation in our study cohort. One of the patients (H5) showed insertion of an extra A nucleotide in exon 10 of the Factor V gene resulting in frame shift mutation in this patient. CONCLUSION: The results of present study showed absence of FVL mutation in our population. However, there is a need to confirm the above findings on patients from different populations from different parts of the country. The insertion of an extra A in exon 10 in the patient needs to be ascertained to confirm that it is one of its kinds or is prevalent in the population.

Cloning, expression, purification and characterization of UMP kinase from Staphylococcus aureus.

Uridine monophosphate kinase (UMPK) an enzyme of de novo biosynthesis catalyses the formation of UDP and it is involved in cell wall and RNA biosynthesis. In the present study UMPK of Staphylococcus aureus ATCC12600 was characterized. Analysis of purified UMPK by gel filtration chromatography on Sephadex G-200 indicated a molecular weight of 150 kDa and exhibited monomeric form with molecular weight of 25 kDa in SDS-PAGE confirming homohexamer nature of UMPK in solution. The enzyme kinetics of UMPK showed K(m) of 2.80 ± 0.1 µM and Vmax 51.38 ± 1.39 µM of NADH/min/mg. The enzyme exhibited cooperative kinetics with ATP as substrate, as GTP decreased this cooperativity and increased affinity for ATP. The UMPK gene was amplified, sequenced (Accession number: FJ415072), cloned in pQE30 vector and overexpressed in Escherichia coli DH5α. The purified recombinant UMPK showed similar properties of native UMPK. The UMPK gene sequence showed complete homology with pyrH gene sequence of all S. aureus strains reported in the database, the 3D structure of S. aureus UMPK built from the deduced amino acid sequence was super imposed with human UMPK (PDB ID: 1TEV) to find out the structural identity using the MATRAS programme gave an RMSD value 4.24 Å indicating very low homology and extensive structural variations with human UMPK structure. Thus, UMPK may be a potential drug target in the development of antimicrobials.

In vitro cardiogenesis can be initiated in human CD34+ cells.

BACKGROUND: The extensive damage that occurs in the cardiac tissue after myocardial infarct is the major concern in post infarct management. It is very well known that adult stem cells mobilized by administration of G-CSF result in homing of stem cells into the damaged myocardium. This is because of the fact that stem cells have the ability to proliferate and capacity to generate into multiple cell lineages. METHOD: A healthy donor was selected as per the guidelines given by the institutional ethical committee and Helsinki declaration. The donor was given G-CSF 5 microg/kg/day and stem cells were harvested from the peripheral blood using Fresenius ASTec204 cell separator. The PBSC were then evaluated by immunohistochemical staining using anti-human CD34 monoclonal antibodies. The cells were then cultured in DMEM with 10% FCS for 17 weeks and in vitro cardiogenesis was initiated by adding 4 microM/l 5'Azacytidine. RESULTS: In vitro cardiogenesis was initiated in pure CD34+ cells with 5' Azacytidine. The cells showed spontaneous beating after 24 hours of treatment and after 5 weeks, the cells connected with the adjoining cells by a myotube. In these cells, expression of myosin light chain (MLC2v) gene and GATA-4 transcription factor validated the development of cardiomyocytes. CONCLUSION: It is observed that the transplantation of autologous stem cells/fetal cardiomyocytes in the heart scar tissue developed due to infarct, limited the scar expansion, and prevented post infarct heart failures. Homing process due to the transplantation of autologous stem cells is time consuming; therefore, transplantation of cardiomyocytes developed from autologous stem cells could be the future method of correcting the infracted myocardium.