The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation in Escherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activates csrB transcription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreased csrB transcription but did not affect csrA-lacZ expression. The uvrY defect was severalfold more severe than that of barA. Both csrA and uvrY were required for optimal barA expression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression of uvrY suppressed the csrB-lacZ expression defects caused by uvrY, csrA, or barA mutations; csrA suppressed csrA or barA defects; and barA complemented only the barA mutation. Purified UvrY protein stimulated csrB-lacZ expression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY on csrB transcription. Disruption of sdiA, which encodes a LuxR homologue, decreased the expression of uvrY-lacZ and csrB-lacZ fusions but did not affect csrA-lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression of uvrY stimulated biofilm formation by a csrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate that uvrY resides downstream from csrA in a signaling pathway for csrB and that CsrA stimulates UvrY-dependent activation of csrB expression by BarA-dependent and -independent mechanisms.

BackgroundEarly recognition is a key factor to achieve improved outcomes for septic patients. Combinations of biomarkers, as opposed to single ones, may improve timely diagnosis and survival. We investigated the performance characteristics of sepsis biomarkers, alone and in combination, for diagnosis of verified bacterial sepsis using Sepsis-2 and Sepsis-3 criteria, respectively.MethodsProcalcitonin (PCT), neutrophil-lymphocyte count ratio (NLCR), C-reactive protein (CRP), and lactate were determined in a total of 1,572 episodes of adult patients admitted to the emergency department on suspicion of sepsis. All sampling were performed prior to antibiotic administration. Discriminant analysis was used to construct two composite biomarkers consisting of linear combinations of the investigated biomarkers, one including three selected biomarkers (i.e., NLCR, CRP, and lactate), and another including all four (i.e., PCT, NLCR, CRP, and lactate). The diagnostic performances of the composite biomarkers as well as the individual biomarkers were compared using the area under the receiver operating characteristic curve (AUC).ResultsFor diagnosis of bacterial sepsis based on Sepsis-3 criteria, the AUC for PCT (0.68; 95% CI 0.65–0.71) was comparable to the AUCs for the both composite biomarkers. Using the Sepsis-2 criteria for bacterial sepsis diagnosis, the AUC for the NLCR (0.68; 95% CI 0.65–0.71) but not for the other single biomarkers, was equal to the AUCs for the both composite biomarkers. For diagnosis of severe bacterial sepsis or septic shock based on the Sepsis-2 criteria, the AUCs for both composite biomarkers were significantly greater than those of the single biomarkers (0.85; 95% CI 0.82–0.88 for the composite three-biomarker, and 0.86; 95% CI 0.83–0.89 for the composite four-biomarker).ConclusionsCombinations of biomarkers can improve the diagnosis of verified bacterial sepsis in the most critically ill patients, but in less severe septic conditions either the NLCR or PCT alone exhibit equivalent performance.

BarA is a membrane-associated protein that belongs to a subclass of tripartite sensors of the two-component signal transduction system family. In this study, we report that UvrY is the cognate response regulator for BarA of Escherichia coli.

The volume of point of care (POC) testing continues to grow steadily due to the increased availability of easy-to-use devices, thus making it possible to deliver less costly care closer to the patient site in a shorter time relative to the central laboratory services. A novel class of molecules called microRNAs have recently gained attention in healthcare management for their potential as biomarkers for human diseases. The increasing interest of miRNAs in clinical practice has led to an unmet need for assays that can rapidly and accurately measure miRNAs at the POC. However, the most widely used methods for analyzing miRNAs, including Northern blot-based platforms, in situ hybridization, reverse transcription qPCR, microarray, and next-generation sequencing, are still far from being used as ideal POC diagnostic tools, due to considerable time, expertize required for sample preparation, and in terms of miniaturizations making them suitable platforms for centralized labs. In this review, we highlight various existing and upcoming technologies for miRNA amplification and detection with a particular emphasis on the POC testing industries. The review summarizes different miRNA targets and signals amplification-based assays, from conventional methods to alternative technologies, such as isothermal amplification, paper-based, oligonucleotide-templated reaction, nanobead-based, electrochemical signalingbased, and microfluidic chip-based strategies. Based on critical analysis of these technologies, the possibilities and feasibilities for further development of POC testing for miRNA diagnostics are addressed and discussed.

The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.