How Treatment Effect Heterogeneity Works.

How Treatment Effect Heterogeneity WorksThis Stats, STAT! animated video explores the concept of treatment effect heterogeneity. Differences in the effectiveness of treatments across participants in a clinical trial is important to understand when deciding how to apply clinical trial results to clinical practice.

Threats to Blinding.

Blinding is a critical strategy used to limit certain types of bias in randomized controlled trials. This animated video explores the rationale and examines potential threats to keeping group allocation concealed - from study participants and investigators.

Analysis of the in planta transcriptome expressed by the corn pathogen Pantoea stewartii subsp. stewartii via RNA-Seq.

Pantoea stewartii subsp. stewartii is a bacterial phytopathogen that causes Stewart's wilt disease in corn. It uses quorum sensing to regulate expression of some genes involved in virulence in a cell density-dependent manner as the bacterial population grows from small numbers at the initial infection site in the leaf apoplast to high cell numbers in the xylem where it forms a biofilm. There are also other genes important for pathogenesis not under quorum-sensing control such as a Type III secretion system. The purpose of this study was to compare gene expression during an in planta infection versus either a pre-inoculum in vitro liquid culture or an in vitro agar plate culture to identify genes specifically expressed in planta that may also be important for colonization and/or virulence. RNA was purified from each sample type to determine the transcriptome via RNA-Seq using Illumina sequencing of cDNA. Fold gene expression changes in the in planta data set in comparison to the two in vitro grown samples were determined and a list of the most differentially expressed genes was generated to elucidate genes important for plant association. Quantitative reverse transcription PCR (qRT-PCR) was used to validate expression patterns for a select subset of genes. Analysis of the transcriptome data via gene ontology revealed that bacterial transporters and systems important for oxidation reduction processes appear to play a critical role for P. stewartii as it colonizes and causes wilt disease in corn plants.

Analyzing the Transcriptomes of Two Quorum-Sensing Controlled Transcription Factors, RcsA and LrhA, Important for Pantoea stewartii Virulence.

The Gram-negative proteobacterium Pantoea stewartii subsp. stewartii causes wilt disease in corn plants. Wilting is primarily due to bacterial exopolysaccharide (EPS) production that blocks water transport in the xylem during the late stages of infection. EsaR, the master quorum-sensing (QS) regulator in P. stewartii, modulates EPS levels. At low cell densities EsaR represses or activates expression of a number of genes in the absence of its acyl homoserine lactone (AHL) ligand. At high cell densities, binding of AHL inactivates EsaR leading to derepression or deactivation of its direct targets. Two of these direct targets are the key transcription regulators RcsA and LrhA, which in turn control EPS production and surface motility/adhesion, respectively. In this study, RNA-Seq was used to further examine the physiological impact of deleting the genes encoding these two second-tier regulators. Quantitative reverse transcription PCR (qRT-PCR) was used to validate the regulation observed in the RNA-Seq data. A GFP transcriptional fusion reporter confirmed the existence of a regulatory feedback loop in the system between LrhA and RcsA. Plant virulence assays carried out with rcsA and lrhA deletion and complementation strains demonstrated that both transcription factors play roles during establishment of wilt disease in corn. These efforts further define the hierarchy of the QS-regulated network controlling plant virulence in P. stewartii.

OpaR controls a network of downstream transcription factors in Vibrio parahaemolyticus BB22OP.

Vibrio parahaemolyticus is an emerging world-wide human pathogen that is associated with food-borne gastroenteritis when raw or undercooked seafood is consumed. Expression of virulence factors in this organism is modulated by the phenomenon known as quorum sensing, which permits differential gene regulation at low versus high cell density. The master regulator of quorum sensing in V. parahaemolyticus is OpaR. OpaR not only controls virulence factor gene expression, but also the colony and cellular morphology associated with growth on a surface and biofilm formation. Whole transcriptome Next Generation sequencing (RNA-Seq) was utilized to determine the OpaR regulon by comparing strains BB22OP (opaR+, LM5312) and BB22TR (∆opaR1, LM5674). This work, using the published V. parahaemolyticus BB22OP genome sequence, confirms and expands upon a previous microarray analysis for these two strains that used an Affymetrix GeneChip designed from the closely related V. parahaemolyticus RIMD2210633 genome sequence. Overall there was excellent correlation between the microarray and RNA-Seq data. Eleven transcription factors under OpaR control were identified by both methods and further confirmed by quantitative reverse transcription PCR (qRT-PCR) analysis. Nine of these transcription factors were demonstrated to be direct OpaR targets via in vitro electrophoretic mobility shift assays with purified hexahistidine-tagged OpaR. Identification of the direct and indirect targets of OpaR, including small RNAs, will enable the construction of a network map of regulatory interactions important for the switch between the nonpathogenic and pathogenic states.

Transcriptome-based analysis of the Pantoea stewartii quorum-sensing regulon and identification of EsaR direct targets.

Pantoea stewartii subsp. stewartii is a proteobacterium that causes Stewart's wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression of esaR itself as well as the genes for the capsular synthesis operon transcription regulator, rcsA, and a 2,5-diketogluconate reductase, dkgA. It simultaneously directly activates expression of genes for a putative small RNA, esaS, the glycerol utilization operon, glpFKX, and another transcriptional regulator, lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficient P. stewartii strains was performed to identify additional targets of EsaR. EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon of P. stewartii orchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response.