Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

Development of micro-electrospray mass spectrometry for ultra high sensitivity and application in the study of drugs of abuse on endogenous \lbrack Met\rbrack $\sp5$-enkephalin release

A micro-electrospray interface was developed specifically for the neurobiological applications described in this dissertation. Incorporation of a unique nano-flow liquid chromatography micro-electrospray "needle" into the micro-electrospray interface (micro-ES/MS) increased the sensitivity of the mass spectrometric assay by $\sim$1000 fold and thus permitted the first analysis of specific neuroactive compounds in brain extracellular fluid collected by in vivo microdialysis (Md).^ Initial in vivo data presented deals with the pharmacodynamics of a novel GABA$\sb{\rm B}$ antagonist and the availability of the compound in its parent (unmetabolized) form to the brain of the anesthetized rat. Next, the first structurally specific endogenous release of (Met) $\sp5$-enkephalin was demonstrated in unanesthetized freely-moving animals (release of $\sim$6.5 fmole of (Met) $\sp5$-enkephalin into the dialysate by direct neuronal depolarization). The Md/micro-ES/MS system was used to test the acute effects of drugs of abuse on the endogenous release of (Met) $\sp5$-enkephalin from the globus pallidus/ventral pallidum brain region in rats. Four drugs known to be abused by man (morphine, cocaine, methamphetamine and diazepam) were tested. Morphine and cocaine both elicited a two-fold or more increase in the release of (Met) $\sp5$-enkephalin over vehicle controls. Diazepam elicited a small decrease in (Met) $\sp5$-enkephalin levels and methamphetamine showed no significant effect on (Met) $\sp5$-enkephalin. These results imply that (Met) $\sp5$-enkephalin may be involved in the reward pathway of certain drugs of abuse. ^

High-resolution microarray analysis of chromosome 20q in human colon cancer metastasis model systems

Amplification of human chromosome 20q DNA is the most frequently occurring chromosomal abnormality detected in sporadic colorectal carcinomas and shows significant correlation with liver metastases. Through comprehensive high-resolution microarray comparative genomic hybridization and microarray gene expression profiling, we have characterized chromosome 20q amplicon genes associated with human colorectal cancer metastasis in two in vitro metastasis model systems. The results revealed increasing complexity of the 20q genomic profile from the primary tumor-derived cell lines to the lymph node and liver metastasis derived cell lines. Expression analysis of chromosome 20q revealed a subset of over expressed genes residing within the regions of genomic copy number gain in all the tumor cell lines, suggesting these are Chromosome 20q copy number responsive genes. Bases on their preferential expression levels in the model system cell lines and known biological function, four of the over expressed genes mapping to the common intervals of genomic copy gain were considered the most promising candidate colorectal metastasis-associated genes. Validation of genomic copy number and expression array data was carried out on these genes, with one gene, DNMT3B, standing out as expressed at a relatively higher levels in the metastasis-derived cell lines compared with their primary-derived counterparts in both the models systems analyzed. The data provide evidence for the role of chromosome 20q genes with low copy gain and elevated expression in the clonal evolution of metastatic cells and suggests that such genes may serve as early biomarkers of metastatic potential. The data also support the utility of the combined microarray comparative genomic hybridization and expression array analysis for identifying copy number responsive genes in areas of low DNA copy gain in cancer cells. ^

School screening for tuberculosis in high risk areas of Texas

Screening for latent tuberculosis infection (LTBI) is an integral component of an effective tuberculosis control strategy, but one that is often relegated to the lowest priority. In a state with higher than national average rates of tuberculosis, due consideration should be given to LTBI screening. Recent large scale contact investigations in the middle school of Del Rio, Texas, raised questions about the status of school screening for LTBI. An evidence based approach was used to evaluate school screening in high risk areas of Texas. A review of the literature revealed that the current recommendations for LTBI screening in children is based on administration of a risk factor questionnaire that should be based on the four main risk factors for LTBI in children that have been identified. Six representative areas in Texas were identified for evaluation of the occurrence of contact investigations in schools for the period of 2006 to 2009 and any use of school screening programs. Of the five reporting areas that responded, only one utilized a school screening program; this reporting area had the lowest percentage of contact investigations occurring in schools. Contact investigations were most common in middle schools and least common in elementary schools. In metropolitan areas, colleges represented up to 42.9% of contact investigations. The number of contact investigations has increased from 2006 to 2008. This report represents a small sample, and further research into the frequency, distribution and risk for contact investigations in schools and the efficacy of screening programs should be done. ^

Bench-scale evaluation of the activated sludge process for treatment of a high -strength chemical plant wastewater

A bench-scale treatability study was conducted on a high-strength wastewater from a chemical plant to develop an alternative for the existing waste stabilization pond treatment system. The objective of this study was to determine the treatability of the wastewater by the activated sludge process and, if treatable, to determine appropriate operating conditions, and to evaluate the degradability of bis(2-chloroethyl)ether (Chlorex) and benzene in the activated sludge system. Four 4-L Plexi-glass, complete mixing, continuous flow activated sludge reactors were operated in parallel under different operating conditions over a 6-month period. The operating conditions examined were hydraulic retention time (HRT), sludge retention time (SRT), nutrient supplement, and Chlorex/benzene spikes. Generally the activated sludge system treating high-strength wastewater was stable under large variations of organic loading and operating conditions. At an HRT of 2 days, more than 90% removal efficiency with good sludge settleability was achieved when the organic loading was less than 0.4 g BOD$\sb5$/g MLVSS/d or 0.8 g COD/g MLVSS/d. At least 20 days of SRT was required to maintain steady operation. Phosphorus addition enhanced the performance of the system especially during stressed operation. On the average, removals of benzene and Chlorex were 73-86% and 37-65%, respectively. In addition, the low-strength wastewater was treatable by activated sludge process, showing more than 90% BOD removal at a HRT of 0.5 days. In general, the sludge had poor settling characteristics. The aerated lagoon process treating high-strength wastewater also provided significant organic reduction, but did not produce an acceptable effluent concentration. ^