Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome

Recurrent urinary tract infections (UTIs) pose a significant burden on the health care system. Underlying mechanisms predisposing children to UTIs and associated changes in the urinary proteome are not well understood. We aimed to investigate the urinary proteome of a subset of children who have vesicoureteral reflux (VUR) and recurrent UTIs because of their risk of developing infection-related renal damage. Improving diagnostic modalities to identify UTI risk factors would significantly alter the clinical management of children with VUR. We profiled the urinary proteomes of 22 VUR patients with low grade VUR (1-3 out of 5), a history of recurrent UTIs, and renal scarring, comparing them to those obtained from 22 age-matched controls. Urinary proteins were analyzed by mass spectrometry followed by protein quantitation based on spectral counting. Of the 2,551 proteins identified across both cohorts, 964 were robustly quantified, as defined by meeting criteria with spectral count (SC) \u3e /=2 in at least 7 patients in either VUR or control cohort based on optimization of signal-to-noise ratio. Eighty proteins had differential expression between the two cohorts, with 44 proteins significantly upregulated and 36 downregulated (q \u3c 0.075, |FC| \u3e 1.2). Urinary proteins involved in inflammation, acute phase response (APR), modulation of extracellular matrix (ECM), and carbohydrate metabolism were overrepresented among the study cohort

Identification of a Lactate-Quinone Oxidoreductase in Staphylococcus aureus that is Essential for Virulence

Staphylococcus aureus is an important human pathogen commonly infecting nearly every host tissue. The ability of S. aureus to resist innate immunity is critical to its success as a pathogen, including its propensity to grow in the presence of host nitric oxide (NO·). Upon exogenous NO· exposure, S. aureus immediately excretes copious amounts of L-lactate to maintain redox balance. However, after prolonged NO·-exposure, S. aureus reassimilates L-lactate specifically and in this work, we identify the enzyme responsible for this L-lactate-consumption as a L-lactate-quinone oxidoreductase (Lqo, SACOL2623). Originally annotated as Mqo2 and thought to oxidize malate, we show that this enzyme exhibits no affinity for malate but reacts specifically with L-lactate (KM = ∼330 μM). In addition to its requirement for reassimilation of L-lactate during NO·-stress, Lqo is also critical to respiratory growth on L-lactate as a sole carbon source. Moreover, Δlqo mutants exhibit attenuation in a murine model of sepsis, particularly in their ability to cause myocarditis. Interestingly, this cardiac-specific attenuation is completely abrogated in mice unable to synthesize inflammatory NO· (iNOS−/−). We demonstrate that S. aureus NO·-resistance is highly dependent on the availability of a glycolytic carbon sources. However, S. aureus can utilize the combination of peptides and L-lactate as carbon sources during NO·-stress in an Lqo-dependent fashion. Murine cardiac tissue has markedly high levels of L-lactate in comparison to renal or hepatic tissue consistent with the NO·-dependent requirement for Lqo in S. aureus myocarditis. Thus, Lqo provides S. aureus with yet another means of replicating in the presence of host NO·

Morphological and immunohistochemical characteristics of human trigeminal ganglion neurons in the prenatal period of development

Introduction: Data related to the amount, size and morphological characteristics of cell elements of sensory ganglia at different stages of prenatal development has not been fully elucidated in recent scientific publications. At the same time publications considering the study of cell structure of trigeminal ganglion in the postnatal period confirm heterogeneity of its neurons. The aim of the research was to study morphological and immunohistochemical characteristics of human trigeminal ganglion neurons at 12-14 weeks of prenatal development. Material and Methods: The study was made on 24 trigeminal ganglions of 12 human fetuses at 12 to 14 weeks of prenatal development after abortion made on social and medical indications. Results: At the studied period of the intrauterine development nerve cells of the trigeminal ganglion significantly differed in size, tinctorial properties and degree of argentophility of the perikaryon. At the same time, the number of small nerve cells with an average diameter of less than 15 pm prevailed. Immunohistochemical study allowed detecting the apparent Bd-2 expression in the overwhelming number of small neurons; the expression of this marker has been observed in 50% of cells of the medium-sized neurons. No Bd-2 expression has been found in most of the large neurons. Almost all the neurons, regardless of the size, showed moderate Ki-67 expression, protein S-100. VEGF expression has also occurred in the vast majority of the nerve cells of all size groups. Conclusions: 1. Human trigeminal ganglion neurons both at 12-14 weeks of prenatal development and in postnatal period are represented by heterogeneous population. 2. Polymorphism of trigeminal ganglion neurons has been found by all applied techniques. 3. Detected polymorphism is the evidence of processes of maturation and differentiation of neurons in human trigeminal ganglion at 12-14 weeks of prenatal developmen

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity

Activation of heme biosynthesis by a small molecule that is toxic to fermenting Staphylococcus aureus

Staphylococcus aureus is a significant infectious threat to global public health. Acquisition or synthesis of heme is required for S. aureus to capture energy through respiration, but an excess of this critical cofactor is toxic to bacteria. S. aureus employs the heme sensor system (HssRS) to overcome heme toxicity; however, the mechanism of heme sensing is not defined. Here, we describe the identification of a small molecule activator of HssRS that induces endogenous heme biosynthesis by perturbing central metabolism. This molecule is toxic to fermenting S. aureus, including clinically relevant small colony variants. The utility of targeting fermenting bacteria is exemplified by the fact that this compound prevents the emergence of antibiotic resistance, enhances phagocyte killing, and reduces S. aureus pathogenesis. Not only is this small molecule a powerful tool for studying bacterial heme biosynthesis and central metabolism; it also establishes targeting of fermentation as a viable antibacterial strategy

Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn=3200 g mol-1) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca2+ and DNA were studied using molecular dynamics simulations (MDS), Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (>0.5%) inhibited biofilm formation, demonstrating a significant reduction in both biomass and biofilm height (17.8 vs. 5.5 µm; P <0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of sugar residues, and extracellular (e)DNA (P <0.05) with a corresponding increase in nanoparticle diffusion (P<0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca2+ evident in FTIR and MDS. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca2+-DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections

Medical-record review of potential donor pool in the Czech Republic suggests a possible increase to more than double the number of donors

The objective of this study was the investigation of the maximal potential donor pool and causes of non-realized organ donation. On-site retrospective medical-record review was performed for all cases of patient death (n=1608) occurring in 1999 at 34 (83% of all) intensive care units (ICUs) in the region of the transplant center in Prague, Czech Republic. Two hundred and eighty-eight (18%) patients died with clinical signs of brain death. ICU physicians considered 111 of them as being potential donors at the time; 63 became donors and 48 did not. The remaining 177 patients with clinical signs of brain death were, in retrospect, assessed as being unsuitable (n=105) or suitable (n=72) for donation. The maximal potential donor rate was 55.7 per million population (pmp), with a more conservative estimate of 37.4 pmp. The actual donor rate was 18.1 pmp. For the maximal level to be approached, further educational efforts are warranted, targeting ICU physicians and concentrating on the identification of potential donors

Efficacy and safety of enteric-coated mycophenolate sodium (myfortic) in de novo renal transplant recipients: results of a 12-month multicenter, open-label, prospective study

Enteric-coated mycophenolate sodium (EC-MPS) has been developed as an alternative formulation of mycophenolate acid aiming for improved gastrointestinal (GI) tolerability. This 12-month, open-label, multicenter, prospective study investigated the efficacy and tolerability of EC-MPS (720 mg twice a day) given in combination with cyclosporine microemulsion (CsA-ME) in de novo renal transplant recipients (n=140). The efficacy evaluation was the incidence of treatment failure (defined as biopsy-proven acute rejection [BPAR], graft loss, or death) after 6 and 12 months of treatment. The incidences of treatment failure, BPAR, and graft loss were comparable at 6 and 12 months (18.6% vs 22.1%, 15.7% vs 19.3%, and 1.4% vs 2.1%, respectively). Renal function at 6 and 12 months (creatinine clearance) was 60.6+/-19.8 mL/min and 63.2+/-27.1 mL/min, respectively. EC-MPS was generally well tolerated; 95.9% of the reported GI adverse events (AEs) were rated as mild or moderate. The rate of EC-MPS dose reduction was 26.4%; 4.3% were due to GI AEs. The rate of EC-MPS dose interruption was 10%; 2.1% were due to GI AEs. In summary, EC-MPS given in combination with CsA-ME demonstrates good efficacy and tolerability in de novo renal transplant recipients

Estimated and measured donor creatinine clearance are poor predictors of long-term renal graft function and survival

The objective of this study was to evaluate estimated and measured donor renal function in predicting graft function long-term and to identify donor criteria associated with nonacceptable graft prognosis. In 200 consecutive cadaver donors creatinine clearance was measured at explantation and estimated using the Cockcroft formula on admission serum creatinine. Graft function was evaluated in recipients (n = 387) by 24-h creatinine clearance regularly during 3years after transplantation. Measured creatinine clearance correlated to some extent with long-term graft function, while Cockcroft estimation was slightly superior and similar to using donor age only. Kidneys from donors with intra-operative creatinine clearance less than or equal to555mL/min (median 50mL/min) produced acceptable recipient graft function of 48mL/min at 3years and 76% 3-year graft survival. Donor age greater than or equal to60years resulted in clearance at 3years of 29mL/min and 78% 3-year graft survival; adding the criteria of admission Cockcroft less than or equal to60mL/min, graft function at 3years (28mL/min) and 3-year graft survival (76%) were similar. In conclusion, creatinine-based estimates of the functional capacity of the donor kidney, calculated or intra-operatively measured, do little to improve the ability of donor age alone to predict long-term allograft function after renal transplantation, and nonacceptable donors are not discriminated