MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

p17 from HIV induces brain endothelial cell angiogenesis through EGFR-1-mediated cell signalling activation

© 2018, United States & Canadian Academy of Pathology. HIV-associated neurocognitive disorder in HIV patients substantially reduces their quality of life. We previously showed that the HIV matrix protein, p17 could stimulate lymph-angiogenesis in vitro potentially contributing to lymphoma tumour growth and in addition is associated with vascular activation in neuro-degenerating brain tissue; here, therefore, we have investigated the detailed molecular mechanisms of this action. We performed in vitro cell culture, angiogenesis experiments, phospho-protein microarrays and Western blotting to identify cellular signalling induced by p17 within human brain endothelial cells (HbMEC), and inhibitor studies to block p17-induced vascular growth. We also characterised the effects of hippocampal CA1 injection of p17 on epidermal growth factor receptor-1 (EGFR1) expression linked to our murine model of dementia. p17 strongly induced angiogenesis of HbMEC (migration, tube formation and spheroid growth). p17 concomitantly increased phosphorylation of EGFR1 as well as down-stream intermediates ERK1/2, FAK, PLC-γ and PKC-β whilst an inhibitor peptide of EGFR, blocked cell signalling and angiogenesis. Finally, Mice that showed reduced cognitive function and behavioural deficiencies after p17 injection, demonstrated that p17 localised in cortical microvessels and also neurones many of which stained positive for p-EGFR1 by histology/IHC. This work provides strong support that p17 may be involved in initiating and/or perpetuating vascular tissue pathophysiology associated with comorbidity in HIV patients

Detection of a diastolic heart sound in children and young adults using high-frame-rate echocardiography

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Colin"s Kids, Inc. PO Box 93 Hopewell, NJ 08525 Introduction The auscultatory finding of a fourth heart sound (S4) is clinically associated with a pathologic decrease in left ventricular compliance, but may be seen in healthy children by phonocardiography. High-frame-rate difference imaging (HFRDI) has demonstrated several propagating myocardial phenomena in the left ventricular (LV) lateral wall throughout the cardiac cycle, including during diastole. We hypothesize that these visualized wave phenomena correlate with physiologic and pathologic heart sounds. Purpose Using HFRDI, we sought to define the characteristics of a LV lateral wall late diastolic wave in healthy children and young adults. Methods HFRDI was obtained from the apical 4-chamber view with synchronized electrocardiogram (ECG) on the T5 system (Phased Array Research Scanner) acquiring images for this study between 587 and 1174 frames per second (depending on participant imaging characteristics). In addition, tissue Doppler images were acquired with synchronized ECG and phonocardiography (PCG) on a commercial ultrasound machine (Figure 1). Timing events were measured from the QRS onset on the ECG tracing. An S4 wave correlate was defined by meeting the following criteria: 1) occurring after the P wave on ECG; 2) at or after the onset of a’ on TDI; 3) occurring during the PCG S4; and 4) occurring prior to the S1 wave complex. The timing of the S4 wave in HFRDI was measured from the onset of QRS to the first appearance of the wave, and the wave velocity was calculated by measuring the length of the lateral wall and counting temporally calibrated image frames while the wave was visible. Results Nine healthy volunteers completed the imaging protocol with a median age of 20 years (range 6 – 22 years). A single, distinct late diastolic HFRDI wave was seen in all participants propagating from apex to base of the lateral LV free wall. This wave met the defined criteria to be a visual correlate of S4 in all participants. Measurements of correlated events are presented in Table 1. S4 propagating velocity was 2.96 ± 1.8 m/sec and occurred 25 ± 18 msec prior to the QRS onset. There is a strong positive correlation between age and S4 velocity [r(7) = 0.76, p = 0.019]. Conclusion The S4 heart sound can be reliably visualized by high frame rate imaging and has similar characteristics in healthy children and young adults. Further definition of normal and abnormal S4 wave characteristics may lead to a novel tool in defining LV diastolic function. Abstract Table 1  Abstract Figure 1 </jats:sec

Conservative Tests under Satisficing Models of Publication Bias

Publication bias leads consumers of research to observe a selected sample of statistical estimates calculated by producers of research. We calculate critical values for statistical significance that could help to adjust after the fact for the distortions created by this selection effect, assuming that the only source of publication bias is file drawer bias. These adjusted critical values are easy to calculate and differ from unadjusted critical values by approximately 50%-rather than rejecting a null hypothesis when the t-ratio exceeds 2, the analysis suggests rejecting a null hypothesis when the t-ratio exceeds 3. Samples of published social science research indicate that on average, across research fields, approximately 30% of published t-statistics fall between the standard and adjusted cutoffs