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Magnitudes or quantities of the different dimensions that define a stimulus (e.g., space, speed or numerosity) influence the perceived duration of that stimulus, a phenomenon known as (temporal) interference effects. This complicates studying the neurobiological foundation of the perception of time, as any signatures of temporal processing are tainted by interfering dimensions. In earlier work, in which judgements on either time or numerosity were made while EEG was recorded, we used Maximum Likelihood Estimation (MLE) to estimate, for each participant separately, the influence of temporal and numerical information on making duration or numerosity judgements. We found large individual differences in the estimated magnitudes, but ML-estimates allowed us to partial out interference effects. However, for such analyses, it is essential that estimates are meaningful and stable. Therefore, in the current study, we examined the reliability of the MLE procedure by comparing the interference magnitudes estimated in two sessions, spread a week apart. In addition to the standard paradigm, we also presented task variants in which the interfering dimension was manipulated, to assess which aspects of the numerosity dimension exert the largest influence on temporal processing. The results indicate that individual interference magnitudes are stable, both between sessions and over tasks. Further, the ML-estimates of the time-numerosity judgement tasks were predictive of performance in a standard temporal judgement task. Thus, how much temporal information participants use in time estimations tasks seems to be a stable trait that can be captured with the MLE procedure. ML-estimates are, however, not predictive of performance in other interference-tasks, here operationalized by a numerical Stroop task. Taken together, the MLE procedure is a reliable tool to quantify individual differences in magnitude interference effects and can therefore reliably inform the analysis of neuroimaging data when contrasts are needed between the accumulation of a temporal and an interfering dimension.

AIMS:In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE. MATERIAL AND METHODS:Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM). RESULTS:Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified. CONCLUSION:The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may result in improved outcomes.

Aims/hypothesisDiabetic voiding dysfunction has been reported in epidemiological dimension of individuals with diabetes mellitus. Animal models might provide new insights into the molecular mechanisms of this dysfunction to facilitate early diagnosis and to identify new drug targets for therapeutic interventions.MethodsThirty male Sprague-Dawley rats received either chow or high-fat diet for eleven weeks. Proteomic alterations were comparatively monitored in both groups to discover a molecular fingerprinting of the urinary bladder remodelling/dysfunction. Results were validated by ELISA, Western blotting and immunohistology.ResultsIn the proteome analysis 383 proteins were identified and canonical pathway analysis revealed a significant up-regulation of acute phase reaction, hypoxia, glycolysis, β-oxidation, and proteins related to mitochondrial dysfunction in high-fat diet rats. In contrast, calcium signalling, cytoskeletal proteins, calpain, 14-3-3η and eNOS signalling were down-regulated in this group. Interestingly, we found increased ubiquitin proteasome activity in the high-fat diet group that might explain the significant down-regulation of eNOS, 14-3-3η and calpain.Conclusions/interpretationThus, high-fat diet is sufficient to induce significant remodelling of the urinary bladder and alterations of the molecular fingerprint. Our findings give new insights into obesity related bladder dysfunction and identified proteins that may indicate novel pathophysiological mechanisms and therefore constitute new drug targets.

Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways.HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1.Saturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain cytokines might counteract the palmitate-induced downregulation of cell proliferation and vitality. This could be an important link to clinical findings of increased risk of metabolic related bladder diseases such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).

BACKGROUND: The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta1 (TGFβ1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF). METHODOLOGY/PRINCIPAL FINDINGS: HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFβ1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC. CONCLUSIONS/SIGNIFICANCE: Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.

Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC).Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kappaB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kappaB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kappaB dependent pathways in a concentration- and time-dependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kappaB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted.Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.