Public Health Research Institute Center

Jersey City, NJ, United States

Public Health Research Institute Center

Jersey City, NJ, United States
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Briley Jr K.,Rutgers University | Briley Jr K.,University of Pennsylvania | Prepiak P.,Public Health Research Institute Center | Dias M.J.,Public Health Research Institute Center | And 2 more authors.
Molecular Microbiology | Year: 2011

Transformable (competent) cells of Bacillus subtilis are blocked in cell division because the traffic ATPase ComGA prevents the formation of FtsZ rings. Although ComGA-deficient cells elongate and form FtsZ rings, cell division remains blocked at a later stage and the cells become mildly filamented. Here we show that the highly conserved protein Maf is synthesized predominantly in competent cells under the direct control of the transcription factor ComK and is solely responsible for the later block in cell division. In vivo and in vitro data show that Maf binds to both ComGA and DivIVA. A point mutation in maf that interferes with Maf binding to DivIVA also interferes with the ability of Maf to inhibit cell division. Based on these findings, we propose that Maf and ComGA mediate mechanisms for the inhibition of cell division in competent cells with Maf acting downstream of ComGA. We further suggest that Maf must interact with DivIVA to inhibit cell division. © 2011 Blackwell Publishing Ltd.

Zhao Y.,Public Health Research Institute Center | Petraitiene R.,Cornell University | Walsh T.J.,Cornell University | Perlin D.S.,Public Health Research Institute Center
Journal of Clinical Microbiology | Year: 2013

A species-specific molecular beacon real-time PCR assay was developed for rapid diagnosis of Exserohilum rostratum infection. As low as 100 fg of E. rostratum DNA can be reliably detected in the presence of 50 ng of human DNA, with a dynamic linear quantification range from 20 ng to 200 fg. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Johnson L.M.,University of Wisconsin - Madison | Mortenson D.E.,University of Wisconsin - Madison | Yun H.G.,University of Wisconsin - Madison | Horne W.S.,University of Wisconsin - Madison | And 4 more authors.
Journal of the American Chemical Society | Year: 2012

We report a new method for preorganization of α/β-peptide helices, based on the use of a dense array of acidic and basic side chains. Previously we have used cyclically constrained β residues to promote α/β-peptide helicity; here we show that an engineered ion pair array can be comparably effective, as indicated by mimicry of the CHR domain of HIV protein gp41. The new design is effective in biochemical and cell-based infectivity assays; however, the resulting α/β-peptide is susceptible to proteolysis. This susceptibility was addressed via introduction of a few cyclic β residues near the cleavage site, to produce the most stable, effective α/β-peptide gp41 CHR analogue identified. Crystal structures of an α- and α/β-peptide (each involved in a gp41-mimetic helix bundle) that contain the dense acid/base residue array manifest disorder in the ionic side chains, but there is little side-chain disorder in analogous α- and α/β-peptide structures with a sparser ionic side-chain array. These observations suggest that dense arrays of complementary acidic and basic residues can provide conformational stabilization via Coulombic attractions that do not require entropically costly ordering of side chains. © 2012 American Chemical Society.

Parashar V.,UMDNJ New Jersey Medical School | Mirouze N.,Public Health Research Institute Center | Dubnau D.A.,UMDNJ New Jersey Medical School | Dubnau D.A.,Public Health Research Institute Center | Neiditch M.B.,UMDNJ New Jersey Medical School
PLoS Biology | Year: 2011

Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic "switch" residue to an internal position when the β4-α4 loop adopts an active-site proximal conformation. © 2011 Parashar et al.

Metzler K.,University of Saskatchewan | Drlica K.,Public Health Research Institute Center | Blondeau J.M.,University of Saskatchewan | Blondeau J.M.,Royal University
Journal of Antimicrobial Chemotherapy | Year: 2013

Background: Previous work showed a higher prevalence of macrolide/azalide resistance in provinces of Canada where azithromycin was the major treatment for Streptococcus pneumoniae as compared with regions where clarithromycin was the dominant treatment. These data provided a way to test the mutant selection window hypothesis, which predicts that the serum drug concentration (AUC24) relative to the mutant prevention concentration (MPC) would be higher for clarithromycin than for azithromycin. Methods: The MIC and MPC were determined for 191 penicillin/macrolide-susceptible clinical isolates of S. pneumoniae with azithromycin, clarithromycin and erythromycin using agar plate assays. Results: The MIC50/90 (mg/L) and MPC50/90 (mg/L), respectively, were as follows: azithromycin 0.13/0.25 and 1/4; clarithromycin 0.031/0.063 and 0.13/0.5; erythromycin 0.063/0.13 and 0.25/2. We calculated from published pharmacokinetic values that the AUC24/MPC90 for azithromycin was 0.85; for clarithromycin it was 96, and for erythromycin base and estolate it was 4 and 10, respectively. Thus the AUC. 24/MPC. 90 was about 50 times higher for clarithromycin than for azithromycin. Conclusions: The elevated prevalence of azithromycin resistance may derive in part from a low value of AUC24/MPC90 and/or time above MPC, since previous work indicates that the number of prescriptions per person was similar in the geographical regions examined. © The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Liu Y.,Harbin Medical University | Liu X.,Harbin Medical University | Qu Y.,Harbin Medical University | Wang X.,Harbin Medical University | And 2 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2012

Perturbation of hydroxyl radical accumulation by subinhibitory concentrations of 2,2′-bipyridyl plus thiourea protects Escherichia coli from being killed by 3 lethal antimicrobial classes. Here, we show that 2,2′-bipyridyl plus thiourea delays and/or reduces antimicrobial killing of Staphylococcus aureus by daptomycin, moxifloxacin, and oxacillin. While the protective effect of 2,2′-bipyridyl plus thiourea varied among strains and compounds, the data support the hypothesis that hydroxyl radical enhances antimicrobial lethality. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Rooklin D.W.,New York University | Lu M.,Public Health Research Institute Center | Zhang Y.,New York University
Journal of the American Chemical Society | Year: 2012

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) represents a new family of apyrase enzymes that catalyze the hydrolysis of nucleotide di- and triphosphates, thereby modulating extracellular purinergic and pyrimidinergic signaling. Among well-characterized phosphoryl transfer enzymes, hSCAN-1 is unique not only in its unusual calcium-dependent activation, but also in its novel phosphate-binding motif. Its catalytic site does not utilize backbone amide groups to bind phosphate, as in the common P-loop, but contains a large cluster of acidic ionizable side chains. By employing a state-of-the-art computational approach, we have revealed a previously uncharacterized catalytic calcium-binding site in hSCAN-1, which elucidates the unusual calcium-dependence of its apyrase activity. In a high-order coordination shell, the newly identified calcium ion organizes the active site residues to mediate nucleotide binding, to orient the nucleophilic water, and to facilitate the phosphoryl transfer reaction. From ab initio QM/MM molecular dynamics simulations with umbrella sampling, we have characterized a reverse protonation catalytic mechanism for hSCAN-1 and determined its free energy reaction profile. Our results are consistent with available experimental studies and provide new detailed insight into the structure-function relationship of this novel calcium-activated phosphoryl transfer enzyme. © 2012 American Chemical Society.

Mindich L.,Public Health Research Institute Center
Advances in Experimental Medicine and Biology | Year: 2012

Several families of viruses have segmented genomes with 3-12 chromosomes. They are capable of packaging these segments in a precise manner so that each virus particle contains one each of the genomic segments. The Cystoviridae are a family of bacteriophages that contain three genomic segments of dsRNA. During infection, the virus produces empty dodecahedral core particles that have the ability to specifically package plus strand transcripts of the genomic segments. The program of packaging makes use of the conformational changes in the surface of the particle as each transcript is packaged. The particles have complexes of a hexameric NTPase that serve as motors to bring the transcripts into the particle, and they have polymerase molecules in the interior that synthesize minus and plus strand copies of the genomic segments. © 2012 Springer Science+Business Media, LLC.

Zhao Y.,Public Health Research Institute Center | Perlin D.S.,Public Health Research Institute Center
Methods in Molecular Biology | Year: 2013

Rapid and quantitative detection of Aspergillus from clinical samples may facilitate an early diagnosis of invasive pulmonary aspergillosis (IPA). As nucleic acid-based detection is a viable option, we demonstrate that Aspergillus burdens can be rapidly and accurately detected by a novel real-time nucleic acid assay other than qPCR by using the combination of nucleic acid sequence-based amplification (NASBA) and the molecular beacon (MB) technology. Here, we detail a real-time NASBA assay to determine quantitative Aspergillus burdens in lungs and bronchoalveolar lavage (BAL) fl uids of rats with experimental IPA. © Springer Science+Business Media New York 2013.

Zhao Y.,Public Health Research Institute Center | Stensvold C.R.,Statens Serum Institute | Perlin D.S.,Public Health Research Institute Center | Arendrup M.C.,Statens Serum Institute
Journal of Antimicrobial Chemotherapy | Year: 2013

Objectives: Triazole resistance in Aspergillus fumigatus has been increasing. We explored the A. fumigatus azole resistance profiles in bronchoalveolar lavage (BAL) fluid samples from Danish patients examined for aspergillosis. Methods: A total of 94 BAL samples from 87 patients were evaluated by galactomannan (GM) test and A. fumigatus CYP51A profiling by PCR. Results: Aspergillus spp. were isolated from 27/48 (56.3%) cultured samples, including 23 A. fumigatus with one resistant strain (4.3%). Samples were classified into GM-positive (≥3.0), GM-intermediate (0.5 to <3.0) and GM-negative (<0.5) groups, where the CYP51A PCR was positive in 81.8% (36/44), 56.3% (18/32) and 38.9% (7/18) of samples, respectively. Nine CYP51A PCR-positive samples (9/61, 14.8%) were found to have mutations resulting in amino acid substitutions. M220V was detected from a sample culture positive for susceptible A. fumigatus and P216L was found in a culture-negative BAL sample. Conversely, no mutation was found in one sample culture positive for azole-resistant A. fumigatus. The tandemrepeat/L98Hmutation was not detected. Conclusions: Our study shows that azole resistance in A. fumigatus can be cryptic and may go undiagnosed. The combination of improved culture/susceptibility tests and the direct molecular detection of resistance markers will facilitate prompt institution of appropriate antifungal therapy. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

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