Papers by Patrick R Gonzales
Blood clotting proceeds through the sequential proteolytic activation of a series of serine prote... more Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.
Genetics in Medicine, 2007
Clinical testing using various array comparative genomic hybridization platforms is being incorpo... more Clinical testing using various array comparative genomic hybridization platforms is being incorporated rapidly into cytogenetic testing algorithms. Comprehensive validation of these complex assays presents unique challenges and very few studies reporting the validation of commercially available array platforms have been published.
Clinical Chemistry, 2006
Background: Glycerol kinase deficiency (GKD) is an X-linked recessive disorder that presents in b... more Background: Glycerol kinase deficiency (GKD) is an X-linked recessive disorder that presents in both isolated and complex forms. The contiguous deletion that leads to GKD also commonly affects NR0B1 (DAX1), the gene associated with adrenal hypoplasia congenita, and DMD, the Duchenne muscular dystrophy gene. Molecular testing to delineate this deletion is expensive and has only limited availability. Methods: We designed a multiplex PCR assay for the detection and mapping of a contiguous deletion potentially affecting the IL1RAPL1, NR0B1, GK, and DMD genes in a 29-month-old male with GKD. Results: Multiplex PCR detected a contiguous deletion that involves the IL1RAPL1, NR0B1, GK, and DMD genes. Although the patient had a creatine kinase concentration within the reference interval, further mapping with PCR revealed that exon 74 was the last intact exon at the 3 end of the DMD gene. Conclusions: Multiplex PCR is an effective and inexpensive way to detect and map the contiguous deletion in cases of complex GKD. The extension of a deletion to include DMD exon 75 in a patient with a creatine kinase concentration within the reference interval suggests that this region of the gene may not be essential for protein function.
American Journal of Medical Genetics Part A, 2007
Microarray-based comparative genomic hybridization (aCGH) allows for simultaneous high-resolution... more Microarray-based comparative genomic hybridization (aCGH) allows for simultaneous high-resolution analysis of multiple genomic loci. Recently, focused aCGH platforms have emerged allowing for analysis of numerous clinically relevant chromosome loci. The purpose of our study was to evaluate the Spectral Genomics Constitutional Chip TM 1.0 (CC) for use in the clinical laboratory. The CC consisted of 429 BAC clones for 41 known genetic deletion/duplication syndromes, subtelomeric regions, and chromosomal backbone clones. We conducted a blinded study of 48 samples including 46 patients (one sample was run in triplicate) with previously determined constitutional chromosome anomalies and two negative controls. Patient samples included 31 microdeletions, four duplications, three derivative chromosomes, three trisomies, and five sex chromosome aneuploidies. Our results show that the CC identified the expected gains and/or losses in 46 of 48 samples. The two negative controls were considered to be normal and the three replicates of the same patient sample were concordant. Two samples yielded false-negative results; however, repeat analysis produced acceptable results for one of them. One sample ultimately had an insufficient amount of DNA precluding aCGH analysis. While promising, the results suggest that further studies are needed to reduce protocol variability and to establish standard analysis and interpretation criteria. Further, this study verifies the importance of extensive validation studies prior to clinical implementation of new clinically available methodologies. ß 2007 Wiley-Liss, Inc.
The American Journal of Human Genetics, 2010
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resista... more Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide, exhibiting increasing resistance to the latest antibiotic therapies. Here we show that the triple β-lactam combination meropenem-piperacillin-tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA subspecies N315 and 72 other clinical MRSA isolates in vitro and clears MRSA N315 infection in a mouse model. ME/PI/TZ suppresses evolution of resistance in MRSA via reciprocal collateral sensitivity of its constituents. We demonstrate that these activities also extend to other carbapenem-penicillin–β-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to β-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein-2a (PBP2a) via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing in vivo activity similar to that of linezolid, ME/PI/TZ demonstrates that combinations of older β-lactam antibiotics could be effective against MRSA infections in humans.
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Papers by Patrick R Gonzales