Slippery liquid infused fluoropolymer coating for central lines to reduce catheter associated clotting and infections.

Thrombosis and infections are two grave, interrelated problems associated with the use of central venous catheters (CVL). Currently used antibiotic coated CVL has limited clinical success in resisting blood stream infection and may increase the risk of emerging antibiotic resistant strains. We report an antibiotic-free, fluoropolymer-immobilized, liquid perfluorocarbon-coated peripherally inserted central catheter (PICC) line and its effectiveness in reducing catheter associated thrombosis and pathogen colonization, as an alternative to antibiotic coated CVL. Commercially available polyurethane PICC catheter was modified by a three-step lamination process, with thin fluoropolymer layers to yield fluoropolymer-polyurethane-fluoropolymer composite structure before applying the liquid perfluorocarbon (LP). This high throughput process of modifying commercial PICC catheters with fluoropolymer is quicker, safer and shows higher thromboresistance than fluorinated, omniphobic catheter surfaces, produced by previously reported self-assembled monolayer deposition techniques. The LP immobilized on the fluoropolymer is highly durable in physiological flow conditions for over 60 days and continue to resist Staphylococcus colonization.

Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity.

Biosynthesis of the peptidyl nucleoside antifungal agent blasticidin S in Streptomyces griseochromogenes requires the hydrolytic function of a nucleotide hydrolase, BlsM, to excise the free cytosine from the 5'-monophosphate cytosine nucleotide. In addition to its hydrolytic activity, interestingly, BlsM has also been shown to possess a novel cytidine deaminase activity, converting cytidine, and deoxycytidine to uridine and deoxyuridine. To gain insight into the substrate specificity of BlsM and the mechanism by which it performs these dual function, the solution structure of BlsM was determined by multi-dimensional nuclear magnetic resonance approaches. BlsM displays a nucleoside deoxyribosyltransferase-like dimeric topology, with each monomer consisting of a five-stranded ß-sheet that is sandwiched by five α-helixes. Compared with the purine nucleotide hydrolase RCL, each monomer of BlsM has a smaller active site pocket, enclosed by a group of conserved hydrophobic residues from both monomers. The smaller size of active site is consistent with its substrate specificity for a pyrimidine, whereas a much more open active site, as in RCL might be required to accommodate a larger purine ring. In addition, BlsM confers its substrate specificity for a ribosyl-nucleotide through a key residue, Phe19. When mutated to a tyrosine, F19Y reverses its substrate preference. While significantly impaired in its hydrolytic capability, F19Y exhibited a pronounced deaminase activity on CMP, presumably due to an altered substrate orientation as a result of a steric clash between the 2'-hydroxyl of CMP and the ζ-OH group of F19Y. Finally, Glu105 appears to be critical for the dual function of BlsM.

Insights into the mechanism of paraoxonase-1: Comparing the reactivity of the six-bladed ß-propeller hydrolases.

The mammalian protein paraoxonase-1 (PON1) has been explored as a promising bioscavenger treatment for organophosphorus (OP) agent poisoning, but it is not active enough to protect against many agents. Engineering is limited because PON1's catalytic mechanism is poorly understood; moreover, its native activity and substrate are unknown. PON1 is a calcium-bound six-bladed ß-propeller hydrolase that shares high structural homology, a conserved metal-coordinating active site, and substrate specificity overlap with other members of a superfamily that includes squid diisopropylfluorophosphatase (DFPase), bacterial drug responsive protein 35 (Drp35), and mammalian senescence marker protein 30 (SMP30). We hypothesized that, by examining the reactivity of all four hydrolases using a common set of conservative mutations, we could gain further insight into the catalytic mechanism of PON1. We chose a set of mutations to examine conserved Asp and Glu residues in the hydrolase active sites, as well as the ligation sphere around the catalytic calcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant hydrolases were assayed against a set of lactones, aryl esters, and OPs that PON1 is known to hydrolyze. Surprisingly, some mutations of Ca2+ coordinating residues, previously thought to be essential for turnover, resulted in significant activity toward all substrate classes examined. Additionally, merely maintaining WT-like charge in the active site of PON1 was insufficient for high activity. Finally, the H115-H134 dyad does not appear to be essential for catalysis against any substrate. Therefore, previously proposed mechanisms must be re-evaluated.

Constitutive optimized production of streptokinase in Saccharomyces cerevisiae utilizing glyceraldehyde 3-phosphate dehydrogenase promoter of Pichia pastoris.

A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions.

RCL hydrolyzes 2'-deoxyribonucleoside 5'-monophosphate via formation of a reaction intermediate.

RCL is an enzyme that catalyzes the N-glycosidic bond cleavage of purine 2'-deoxyribonucleoside 5'-monophosphates. Recently, the structures of both free wild type and GMP-bound mutant complex have been determined by multidimensional NMR, revealing a doubly wound α/ß protein existing in a symmetric homodimer. In this work, we investigated the catalytic mechanism by rational site-directed mutagenesis, steady-state and pre-steady-state kinetics, ITC binding analysis, methanolysis, and NMR study. First, we provide kinetic evidence in support of the structural studies that RCL functions in a dimeric form, with an apparent dissociation constant around 0.5 µM in the presence of substrate dGMP. Second, among the eight residues under investigation, the highly conserved Glu93 is absolutely critical and Tyr13 is also important likely contributing to the chemical step, whereas Ser117 from the neighboring subunit and Ser87 could be the key residues for the phosphate group recognition. Lastly, we demonstrate by methanolysis study that the catalytic reaction proceeds via the formation of a reaction intermediate, which is subsequently hydrolyzed by solvent nucleophile resulting in the formation of normal product deoxyribose monophosphate (dR5P) or methoylated-dR5P. In conclusion, the current study provides mechanistic insights into a new class of nucleotide hydrolase, which resembles nucleoside 2'-deoxyribosyltransferases structurally and functionally but also possesses clear distinction.

Characterization of a transient unfolding intermediate in a core mutant of γS-crystallin.

In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T(2)-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.

Solution structure of Gfi-1 zinc domain bound to consensus DNA.

Gfi-1 is a crucial transcriptional repressor for the precise regulation of cell proliferation and differentiation in hematopoiesis. Recently, this protein has also been demonstrated to be capable of restricting the proliferation of hematopoietic stem cells, a process that appears to be vital for the long-term competency of hematopoietic stem cells. These two seemingly opposite outcomes of regulation are likely to arise from its interactions with a variety of cellular partners. Such interactions can directly affect the genes that Gfi-1 recognizes through its DNA binding zinc-finger domain. In this work, we report the determination of the solution structure of Gfi-1 zinc fingers 3-5 in complex with a 16-mer consensus DNA using multidimensional NMR method. Unlike a proposed minor-groove binding model based on methylation interference experiments, our structure clearly shows that Gfi-1 zinc fingers 3-5 bind into the major groove of the target DNA reminiscent of canonical C(2)H(2) zinc-finger domains. The fourth and fifth zinc fingers recognize the AATC core sequence by forming base-specific hydrogen bonds between the side chains of Asn382, Gln379, and Asp354 and the bases of the invariant adenines and cytosine. Overall, the current work provides valuable insight into the structural determinants for DNA binding specificity, in particular for the TCA triplet that has not been observed in any other structures of zinc finger-DNA complexes, as well as molecular rationales for a naturally occurring mutation that causes acute myeloid leukemia.

Solution structure of RCL, a novel 2'-deoxyribonucleoside 5'-monophosphate N-glycosidase.

RCL is an enzyme that catalyzes the N-glycosidic bond cleavage of purine 2'-deoxyribonucleoside 5'-monophosphates, a novel enzymatic reaction reported only recently. In this work, we determined the solution structure by multidimensional NMR and provide a structural framework to elucidate its mechanism with computational simulation. RCL is a symmetric homodimer, with each monomer consisting of a five-stranded parallel beta-sheet sandwiched between five alpha-helices. Three of the helices form the dimer interface, allowing two monomers to pack side by side. The overall architecture featuring a Rossmann fold is topologically similar to that of deoxyribosyltransferases, with major differences observed in the putative substrate binding pocket and the C-terminal tail. The latter is seemingly flexible and projecting away from the core structure in RCL, but loops back and is positioned at the bottom of the neighboring active site in the transferases. This difference may bear functional implications in the context of nucleobase recognition involving the C-terminal carboxyl group, which is only required in the reverse reaction by the transferases. It was also noticed that residues around the putative active site show significant conformational variation, suggesting that protein dynamics may play an important role in the enzymatic function of apo-RCL. Overall, the work provides invaluable insight into the mechanism of a novel N-glycosidase from the structural point of view, which in turn will allow rational anti-tumor and anti-angiogenesis drug design.

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus.

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28kDa, pH and temperature optima for this protease were 10 and 60 degrees C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu(2+) ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.

Single and double incompatibility at vWA and D8S1179/D21S11 loci between mother and child: implications in kinship analysis.

BACKGROUND: Two cases of paternity dispute, examined with 17 autosomal short tandem repeats signified a possible single and double maternal mismatch at vWA and D8S1179/D21S11 loci in the children under investigation. METHODS: Seventeen autosomal STR loci were analyzed using AmpFlSTR Identifiler, PowerPlex 16 kits. Six STR markers on X chromosome were amplified and analyzed. Mutated alleles were amplified, cloned in pCR(R)II-Topo vector, sequenced and investigated. RESULTS: In case S1 the vWA locus indicated an allele mismatch with the mother. All the vWA alleles on amplification, cloning and sequencing depicted an increase of 2 repeats in the child. In case D1 maternal child inconsistency at D8S1179 and D21S11 loci was observed. The alleles were amplified, cloned and sequenced to analyze the repeat structure. Increase of 1 repeat in D8S1179 locus and an insertion mutation in D21S11 locus between the mother and questioned child were confirmed. A complete match with the 17 autosomal loci of the father and 6 X chromosome STR loci of the mother was observed in both the cases. CONCLUSION: This is the first report of a maternally transmitted single mismatch at vWA locus and double mismatch at D8S1179 and D21S11 loci due to increase/mutation of the repeat in the paternity DNA testing. The results of nucleotide sequencing and STR analyses convincingly established that the suspected father and the mother are undeniably the biological parents of the questioned child.

Purification and characterization of an alkaline keratinase from Streptomyces sp.

A protease producing bacterial culture ('S7') was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Streptomyces sp. on the basis of biochemical properties and 16S rRNA gene sequencing. Purification of the protease present in the culture medium supernatant on sephacryl S-100 indicated that it contains a keratinase with 67% recovery, 2.5-fold purification and an estimated molecular mass of approximately 44,000 Da. Keratinase showed an optimal activity at 45 degrees C and pH 11. Keratinase activity increased substantially in presence of Ca(2+) and was inhibited in presence of PMSF and EDTA identifying it as a serine metalloprotease. Stability in the presence of detergents, surfactants and solvents make this keratinase extremely useful for biotechnological process involving keratin hydrolysis or in the leather industry.

Optimization of keratinase production and enzyme activity using response surface methodology with Streptomyces sp7.

A two-step response surface methodology (RSM) study was conducted for the optimization of keratinase production and enzyme activity from poultry feather by Streptomyces sp7. Initially different combinations of salts were screened for maximal production of keratinase at a constant pH of 6.5 and feather meal concentration of 5 g/L. A combination of K2HPO4, KH2PO4, and NaCl gave a maximum yield of keratinase (70.9 U/mL) production. In the first step of the RSM study, the selected five variables (feather meal, K2HPO4, KH2PO4, NaCl, and pH) were optimized by a 25 full-factorial rotatable central composite design (CCD) that resulted in 95 U/mL of keratinase production. The results of analysis of variance and regression of a second-order model showed that the linear effects of feather meal concentration (p<0.005) and NaCl (p<0.029) and the interactive effects of all variables were more significant and that values of the quadratic effects of feather meal (p<1.72e-5), K2HPO4 (p<4.731e-6), KH2PO4 (p<1.01e-10), and pH (p 7.63e-7) were more significant than the linear and interactive effects of the process variables. In the second step, a 23 rotatable full-factorial CCD and response surface analysis were used for the selection of optimal process parameters (pH, temperature, and rpm) for keratinase enzyme activity. These optima were pH 11.0, 45 degrees C, and 300 rpm.

Microsatellite mutation in the maternally/paternally transmitted D18S51 locus: two cases of allele mismatch in the child.

BACKGROUND: We investigated 2 cases of paternity dispute with 17 autosomal short tandem repeats (STR), that indicated a mismatch to the maternally and paternally inherited allele at D18S51 locus in children under inquiry. METHODS: 17 autosomal and Y STR loci were analyzed using AmpFlSTR Identifiler, PowerPlex 16, AmpFlSTR(R)Y-filertrade mark kits. The mitochondrial DNA hypervariable regions HV1 and HV2 and 6 STR markers on X chromosome were amplified and sequenced. RESULTS: In case M1, allelic representation in the mother, questioned child and suspected father was 14/19, 12/20 and 12/14 respectively. A complete match with the mother at 6 X STR loci and mitochondrial hypervariable regions was observed. In case F1, allelic representation was 13/14, 14/20 and 16/18 respectively. A complete match with the father at 17 Y chromosome STR loci was observed. D18S51 sequence analysis indicates the expansion of 1 repeat in M1 and 2 repeats in F1 leading to allele mismatch in the child. CONCLUSION: The probability of maternity and paternity were 0.999999 and 0.999999 respectively. This is the first report of a maternally/paternally transmitted D18S51 mutations in the paternity DNA testing. These results conclusively determined that the mother and suspected father are the biological parents of the questioned children in both the cases.