Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12.

The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS ("sensitive" genes), and genes whose expression changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS.IMPORTANCE The sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses.

Enhanced and Differential Capture of Circulating Tumor Cells from Lung Cancer Patients by Microfluidic Assays Using Aptamer Cocktail.

Collecting circulating tumor cells (CTCs) shed from solid tumor through a minimally invasive approach provides an opportunity to solve a long-standing oncology problem, the real-time monitoring of tumor state and analysis of tumor heterogeneity. However, efficient capture and detection of CTCs with diverse phenotypes is still challenging. In this work, a microfluidic assay is developed using the rationally-designed aptamer cocktails with synergistic effect. Enhanced and differential capture of CTCs for nonsmall cell lung cancer (NSCLC) patients is achieved. It is also demonstrated that the overall consideration of CTC counts obtained by multiple aptamer combinations can provide more comprehensive information in treatment monitoring.

Evolutionary dynamics of light-independent protochlorophyllide oxidoreductase genes in the secondary plastids of cryptophyte algae.

Plastid genes encoding light-independent protochlorophyllide oxidoreductase (LIPOR) subunits were isolated from cryptophyte algae, the first example of such genes in plastids of secondary endosymbiotic origin. The presence of functional and nonfunctional copies of LIPOR genes in cryptophytes suggests that light-independent chlorophyll biosynthesis is a nonessential pathway in these organisms.

Impact of a clinical pharmacy consult service on guideline adherence and management of gabapentin for neuropathic pain.

OBJECTIVE: Our objectives were to (1) determine whether a computerized clinical pharmacy approval and follow-up consult process for ordering new prescriptions for gabapentin for the treatment of neuropathic pain decreased the number of patients without documented treatment benefit while increasing follow-up and documentation of effectiveness, and (2) describe gabapentin use patterns at a Veterans Affairs (VA) Medical Center, including the use of first-line therapies prior to gabapentin therapy for neuropathic pain. METHODS: The clinical pharmacy intervention included review of (1) the indication for gabapentin; (2) the required use and failure or contraindication of 3 first-line therapies: nonsteroidal anti-inflammatory drugs (NSAIDs), tricyclic antidepressants (TCAs), and capsaicin cream; (3) the initial pain assessment; and (4) patient follow-up in 4 to 6 weeks, with repeat pain assessment. A retrospective chart review was performed for all patients who received a new prescription for gabapentin from October 2002 to April 2003 at the Portland VA Medical Center (PVAMC). The outcomes of interest for the provider group versus the clinical pharmacy managed group included follow-up at 6 weeks or less versus follow-up at more than 6 weeks, documentation of treatment benefit, how many of the 3 first-line therapies were tried before gabapentin, and whether the gabapentin therapy was discontinued. RESULTS: There were 237 patients who received a new prescription for gabapentin between October 2002 and April 2003. Of these gabapentin prescriptions, 61% (n=144) were prescribed for neuropathic pain. Of the new gabapentin prescriptions for neuropathic pain, 61% (n=88) were made from approved clinical pharmacy consults, 38% (n=54) were ordered without a clinical pharmacy consult, and 1% (n=2) were not included because the patient received the drug despite denial by the clinical pharmacy consult. The rate of follow-up to assess documentation of benefit of therapy with gabapentin was 87% (n=62) in the clinical pharmacy consult group compared with 51% (n=27) in the provider-managed group (chi2=18.07, P<0.001). Of the patients who were assessed by follow-up, 89% (n=55) of the clinical pharmacy consult group received follow-up within 6 weeks versus 52% (n=14) of the provider-managed group (chi2=12.63, P <0.001). Compared with the patients managed by clinical pharmacists, 43% (n=23) of the gabapentin patients in the provider-managed group had no evidence of prior use of any of the 3 agents required by the gabapentin neuropathic pain guideline, 55% (n=29) had evidence of prior use of 1 or 2 first-line agents, and only 2% (n=1) had evidence of prior use of all 3 required first-line agents, versus 100% (n=71) of the patients managed by clinical pharmacy consult. There was no difference in the rate of continuation of gabapentin therapy in the group of patients who received clinical pharmacy consults (65%) compared with the provider-managed group (68%, chi2=0.11, P=0.718). Of the 148 pharmacy consults for new gabapentin prescriptions that were completed during the 7-month period from October 2002 through April 2003, 60 (40%) were denied, which resulted in the lack of gabapentin use in these 60 patients. CONCLUSIONS: A clinical pharmacy intervention as part of the management of a treatment guideline for appropriate gabapentin use promotes documentation of drug therapy effectiveness in neuropathic pain and prevention of gabapentin use prior to a trial with alternative first-line therapies.

Programming thermoresponsiveness of NanoVelcro substrates enables effective purification of circulating tumor cells in lung cancer patients.

Unlike tumor biopsies that can be constrained by problems such as sampling bias, circulating tumor cells (CTCs) are regarded as the "liquid biopsy" of the tumor, providing convenient access to all disease sites, including primary tumor and fatal metastases. Although enumerating CTCs is of prognostic significance in solid tumors, it is conceivable that performing molecular and functional analyses on CTCs will reveal much significant insight into tumor biology to guide proper therapeutic intervention. We developed the Thermoresponsive NanoVelcro CTC purification system that can be digitally programmed to achieve an optimal performance for purifying CTCs from non-small cell lung cancer (NSCLC) patients. The performance of this unique CTC purification system was optimized by systematically modulating surface chemistry, flow rates, and heating/cooling cycles. By applying a physiologically endurable stimulation (i.e., temperature between 4 and 37 °C), the mild operational parameters allow minimum disruption to CTCs' viability and molecular integrity. Subsequently, we were able to successfully demonstrate culture expansion and mutational analysis of the CTCs purified by this CTC purification system. Most excitingly, we adopted the combined use of the Thermoresponsive NanoVelcro system with downstream mutational analysis to monitor the disease evolution of an index NSCLC patient, highlighting its translational value in managing NSCLC.

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Insight into the diversity and evolution of the cryptomonad nucleomorph genome.

The cryptomonads are an enigmatic group of marine and freshwater unicellular algae that acquired their plastids through the engulfment and retention of a eukaryotic ("secondary") endosymbiont. Together with the chlorarachniophyte algae, the cryptomonads are unusual in that they have retained the nucleus of their endosymbiont in a miniaturized form called a nucleomorph. The nucleomorph genome of the cryptomonad Guillardia theta has been completely sequenced and with only three chromosomes and a total size of 551 kb, is a model of nuclear genome compaction. Using this genome as a reference, we have investigated the structure and content of nucleomorph genomes in a wide range of cryptomonad algae. In this study, we have sequenced nine new cryptomonad nucleomorph 18S ribosomal DNA (rDNA) genes and four heat shock protein 90 (hsp90) gene fragments, and using pulsed-field gel electrophoresis and Southern hybridizations, have obtained nucleomorph genome size estimates for nine different species. We also used long-range polymerase chain reaction to obtain nucleomorph genomic fragments from Hanusia phi CCMP325 and Proteomonas sulcata CCMP704 that are syntenic with the subtelomeric region of nucleomorph chromosome I in G. theta. Our results indicate that (1) the presence of three chromosomes is a common feature of the nucleomorph genomes of these organisms, (2) nucleomorph genome size varies dramatically in the cryptomonads examined, (3) unidentified cryptomonad species CCMP1178 has the largest nucleomorph genome identified to date at approximately 845 kb, (4) nucleomorph genome size reductions appear to have occurred multiple times independently during cryptomonad evolution, (5) the relative positions of the 18S rDNA, ubc4, and hsp90 genes are conserved in three different cryptomonad genera, and (6) interchromosomal recombination appears to be rapidly changing the size and sequence of a repetitive subtelomeric region of the nucleomorph genome between the 18S rDNA and ubc4 loci. These results provide a glimpse into the genetic diversity of nucleomorph genomes in cryptomonads and set the stage for more comprehensive sequence-based studies in closely and distantly related taxa.