On butter testing

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Culture Negative Endocarditis: Advances in Diagnosis and Treatment

Culture-negative endocarditis (CNE) is a challenging clinical entity, both diagnostically and therapeutically. In this chapter, the changed epidemiology and microbiology of CNE are reviewed with cases highlighting typical pathogens in patients pre-treated with antibiotics, less common fastidious pathogens such as bacteria of the HACEK group, nutritionally deficient bacteria, Legionella spp. and Mycobacteria, “quintessential” CNE pathogens such as Bartonella spp., Coxiella burnetti and Tropheryma whipplei, as well as fungal CNE. Contemporary diagnostic methods are reviewed including polymerase chain reaction-based pathogen 16s RNA amplification coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Finally, treatment options per the recently updated 2015 American Heart Association and European Society for Cardiology guideline are presented

Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) <em>Pseudomonas aeruginosa</em>

Antibiotic resistance in non-lactose fermenting pathogens such as Pseudomonas aeruginosa (P. aeruginosa) is increasing, making these clinical pathogens more difficult to treat. Multiple resistance mechanisms exist within P. aeruginosa that affect all classes of antibiotics used in the clinic. New strategies and treatment targets within these MDR pathogens must be exploited. One heretofore untapped target is the family of cell wall enzymes known as lytic transglycosylases (Lts). Lts work in concert with penicillin binding proteins (PBPs) and other cell wall proteins such as amidases and peptidoglycan hydrolases to affect normal cell division, and during stress and programmed cell death. Lts are inhibited by natural products called bulgecins, produced by non-pathogenic Paraburkholderia and Burkholderia spp. New research describing the ability of Lt inhibition to restore susceptibility to β-lactams in MDR P. aeruginosa, as well as the structural biologic basis for the activity of bulgecins will be reviewed. Other targets and applications of bulgecins will also be discussed

Detailing renal hemodynamics and oxygenation in rats by a combined near-infrared spectroscopy and invasive probe approach

We hypothesize that combining quantitative near-infrared spectroscopy (NIRS) with established invasive techniques will enable advanced insights into renal hemodynamics and oxygenation in small animal models. We developed a NIRS technique to monitor absolute values of oxygenated and deoxygenated hemoglobin and of oxygen saturation of hemoglobin within the renal cortex of rats. This NIRS technique was combined with invasive methods to simultaneously record renal tissue oxygen tension and perfusion. The results of test procedures including occlusions of the aorta or the renal vein, hyperoxia, hypoxia, and hypercapnia demonstrated that the combined approach, by providing different but complementary information, enables a more comprehensive characterization of renal hemodynamics and oxygenation

Aldosterone and vasopressin affect α- and γ-ENaC mRNA translation

Vasopressin and aldosterone play key roles in the fine adjustment of sodium and water re-absorption in the nephron. The molecular target of this regulation is the epithelial sodium channel (ENaC) consisting of α-, β- and γ-subunits. We investigated mRNA-specific post-transcriptional mechanisms in hormone-dependent expression of ENaC subunits in mouse kidney cortical collecting duct cells. Transcription experiments and polysome gradient analysis demonstrate that both hormones act on transcription and translation. RNA-binding proteins (RBPs) and mRNA sequence motifs involved in translational control of γ-ENaC synthesis were studied. γ-ENaC–mRNA 3′-UTR contains an AU-rich element (ARE), which was shown by RNA affinity chromatography to interact with AU-rich element binding proteins (ARE-BP) like HuR, AUF1 and TTP. Some RBPs co-localized with γ-ENaC mRNA in polysomes in a hormone-dependent manner. Reporter gene co-expression experiments with luciferase γ-ENaC 3′-UTR constructs and ARE-BP expression plasmids demonstrate the importance of RNA–protein interaction for the up-regulation of γ-ENaC synthesis. We document that aldosterone and the V2 receptor agonist dDAVP act on synthesis of α- and γ-ENaC subunits mediated by RBPs as effectors of translation but not by mRNA stabilization. Immunoprecipitation and UV-crosslinking analysis of γ-ENaC–mRNA/HuR complexes document the significance of γ-ENaC–mRNA–3′-UTR/HuR interaction for hormonal control of ENaC synthesis

Dissecting the action of an evolutionary conserved non-coding region on renin promoter activity

Elucidating the mechanisms of the human transcriptional regulatory network is a major challenge of the post-genomic era. One important aspect is the identification and functional analysis of regulatory elements in non-coding DNA. Genomic sequence comparisons between related species can guide the discovery of cis-regulatory sequences. Using this technique, we identify a conserved region CNSmd of ∼775 bp in size, ∼14 kb upstream of the renin gene. Renin plays a pivotal role for mammalian blood pressure regulation and electrolyte balance. To analyse the cis-regulatory role of this region in detail, we perform 132 combinatorial reporter gene assays in an in vitro Calu-6 cell line model. To dissect the role of individual subregions, we fit several mathematical models to the experimental data. We show that a multiplicative switch model fits best the experimental data and that one subregion has a dominant effect on promoter activity. Mapping of the sub-sequences on phylogenetic conservation data reveals that the dominant regulatory region is the one with the highest multi-species conservation score

The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation

Deregulated translation plays an important role in human cancer. We previously reported decreased eukaryotic initiation factor 3 subunit f (eIF3f) expression in pancreatic cancer. Whether decreased eIF3f expression can transform normal epithelial cells is not known. In our current study, we found evidence that stable knockdown of eIF3f in normal human pancreatic ductal epithelial cells increased cell size, nuclear pleomorphism, cytokinesis defects, cell proliferation, clonogenicity, apoptotic resistance, migration, and formation of 3-dimensional irregular masses. Our findings support the tumor suppressive role of eIF3f in pancreatic cancer. Mechanistically, we found that eIF3f inhibited both cap-dependent and cap-independent translation. An increase in the ribosomal RNA (rRNA) level was suggested to promote the generation of cancer. The regulatory mechanism of rRNA degradation in mammals is not well understood. We demonstrated here that eIF3f promotes rRNA degradation through direct interaction with heterogeneous nuclear ribonucleoprotein (hnRNP) K. We showed that hnRNP K is required for maintaining rRNA stability: under stress conditions, eIF3f dissociates hnRNP K from rRNA, thereby preventing it from protecting rRNA from degradation. We also demonstrated that rRNA degradation occurred in non-P body, non-stress granule cytoplasmic foci that contain eIF3f. Our findings established a new mechanism of rRNA decay regulation mediated by hnRNP K/eIF3f and suggest that the tumor suppressive function of eIF3f may link to impaired rRNA degradation and translation

Left Ventricular Assist Device Infections

Left ventricular assist device (LVAD) infections are important causes of morbidity and mortality in patients who receive these mechanical circulatory supports as a bridge to transplantation (BTT) or as destination therapy (DT) (for individuals who are not candidates for cardiac transplant). Infections are more common among persons who received pulsatile flow LVADs as opposed to newer continuous flow (CF) devices. Other risk factors for infection include obesity, renal failure, depression and immunosuppression. An LVAD infection increases the risk of infections in persons who undergo cardiac transplantation. Infections include percutaneous site, driveline, pump pocket and pump/cannula infections; sepsis, bacteremia, mediastinitis and endocarditis. Diagnosis is achieved by monitoring LVAD flow parameters and observing typical clinical and laboratory manifestations of infection. Imaging such as PET-CT or SPECT-CT imaging can be helpful to establish a diagnosis of pump pocket infection. Echocardiography may aid in detecting native valve endocarditis and thrombus associated with the LVAD. The most common pathogens include Staphylococcus, Corynebacterium, Enterococcus, Pseudomonas and Candida spp. Treatment requires targeted antimicrobials plus surgical debridement of infected tissue and device components. In cases of pump/cannula/LVAD endocarditis, especially if fungal pathogens or Mycobacterium chimaera are involved, LVAD removal/reimplantation vs. transplant is necessary, combined with extended antimicrobial therapy