The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within eachrfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe toEscherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coliO157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.
Generating skeletal muscle in vitro is an attractive approach to overcome problems associated with autologous transfer of muscle and donor site morbidity during plastic surgery. Such tissue engineering requires application of biomaterials that selectively control the attachment, morphology, and proliferation of muscle progenitor ("satellite") cells. This study examined the initial attachment, morphological characteristics, and proliferative behavior of murine C2C12 myoblasts on glass substrata derivatized with self-assembled monolayers (SAMs) of the organosiloxanes N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane (EDA) and tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosil ane (13F). The fraction of myoblasts resisting detachment upon rinsing was greater on EDA than on 13F. Application of a quantitative moments-based analysis of cell morphology demonstrated that projected area and two size-invariant metrics of shape (extension and dispersion) for these cells were greater for EDA than for 13F. Myoblasts also proliferated faster on EDA than on 13F. These data indicate that EDA-derivatized glass provides a superior substratum for myoblast culture compared to 13F-derivatized glass. Understanding myoblast behavior on these biomaterials that promotes contrasting cellular responses is the first step toward using patterned SAMs to control myotube alignment for tissue engineering skeletal muscle.
BackgroundMore than a million diagnostic cardiac catheterizations are performed annually in the US for evaluation of coronary artery anatomy and the presence of atherosclerosis. Nearly half of these patients have no significant coronary lesions or do not require mechanical or surgical revascularization. Consequently, the ability to rule out clinically significant coronary artery disease (CAD) using low cost, low risk tests of serum biomarkers in even a small percentage of patients with normal coronary arteries could be highly beneficial.MethodsSerum from 359 symptomatic subjects referred for catheterization was interrogated for proteins involved in atherogenesis, atherosclerosis, and plaque vulnerability. Coronary angiography classified 150 patients without flow-limiting CAD who did not require percutaneous intervention (PCI) while 209 required coronary revascularization (stents, angioplasty, or coronary artery bypass graft surgery). Continuous variables were compared across the two patient groups for each analyte including calculation of false discovery rate (FDR ≤ 1%) and Q value (P value for statistical significance adjusted to ≤ 0.01).ResultsSignificant differences were detected in circulating proteins from patients requiring revascularization including increased apolipoprotein B100 (APO-B100), C-reactive protein (CRP), fibrinogen, vascular cell adhesion molecule 1 (VCAM-1), myeloperoxidase (MPO), resistin, osteopontin, interleukin (IL)-1β, IL-6, IL-10 and N-terminal fragment protein precursor brain natriuretic peptide (NT-pBNP) and decreased apolipoprotein A1 (APO-A1). Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity) while maintaining 95% sensitivity for patients requiring revascularization. Osteopontin (14 times) and resistin (10 times) were most frequently represented among these diagnostic signatures. The most efficacious protein signature in validation studies comprised osteopontin (OPN), resistin, matrix metalloproteinase 7 (MMP7) and interferon γ (IFNγ) as a four-marker panel while the addition of either CRP or adiponectin (ACRP-30) yielded comparable results in five protein signatures.ConclusionsProteins in the serum of CAD patients predominantly reflected (1) a positive acute phase, inflammatory response and (2) alterations in lipid metabolism, transport, peroxidation and accumulation. There were surprisingly few indicators of growth factor activation or extracellular matrix remodeling in the serum of CAD patients except for elevated OPN. These data suggest that many symptomatic patients without significant CAD could be identified by a targeted multiplex serum protein test without cardiac catheterization thereby eliminating exposure to ionizing radiation and decreasing the economic burden of angiographic testing for these patients.
Copy number variant (CNV) analysis was performed on renal cell carcinoma (RCC) specimens (chromophobe, clear cell, oncocytoma, papillary type 1, and papillary type 2) using high-resolution arrays (1.85 million probes). The RCC samples exhibited diverse genomic changes within and across tumor types, ranging from 106 to 2238 CNV segments in a clear-cell specimen and in a papillary type 2 specimen, respectively. Despite this heterogeneity, distinct CNV segments were common within each tumor classification: chromophobe (seven segments), clear cell (three segments), oncocytoma (nine segments), and papillary type 2 (two segments). Shared segments ranged from a 6.1-kb deletion (oncocytomas) to a 208.3-kb deletion (chromophobes). Among common tumor type-specific variations, chromophobes, clear-cell tumors, and oncocytomas were composed exclusively of noncoding DNA. No CNV regions were common to papillary type 1 specimens, although there were 12 amplifications and 12 deletions in five of six samples. Three microRNAs and 12 mRNA genes had a ≥98% coding region contained within CNV regions, including multiple gene families (chromophobe: amylases 1A, 1B, and 1C; oncocytoma: general transcription factors 2H2, 2B, 2C, and 2D). Gene deletions involved in histone modification and chromatin remodeling affected individual subtypes (clear cell: SFMBT and SETD2; papillary type 2: BAZ1A) and the collective RCC group (KDM4C). The genomic amplifications/deletions identified herein represent potential diagnostic and/or prognostic biomarkers.
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