Cystatin B: mutation detection, alternative splicing and expression in progressive myclonus epilepsy of Unverricht-Lundborg type (EPM1) patients

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Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

The onset of depression in middle-aged presumed healthy Slovenian family practice attendees and its associations with genetic risk assessment, quality of life and health status

Despite depression being a major driver of morbidity and mortality, the majority of primary care patients remain undiagnosed, so this study aimed to assess the prevalence of depression and the association with demographic and clinical variables, genetic risk, and quality of life. The participants were presumably healthy model family medicine practice (MFMP) attendees between 30 and 65 years of age and recruited during a preventive check-up in 2019. Each of the 40 pre-selected MFMP pragmatically invited 30 attendees to voluntarily participate. They completed a questionnaire of demographic, clinical, and social determinants, as well as a three-generational family history. The results were analyzed using multivariable modelling to calculate the associations with signs of depression. A modified Scheuner method was used to calculate the level genetic risk level using family history. Of 968 participants, aged 42.8 ± 8.6 years, 627 (64.8%) were women. The prevalence of depression was 4.1%. Signs of depression were negatively associated with health-related quality of life score, in particular in the domains of self-care (p = 0.001) and anxiety/depression (p < 0.001). Depression was also associated with predicted high risk for comorbidities given the family history (p = 0.030). Primary care directed at improving patients’ quality of life should implement more widespread screening for mental health disorders. Family history for disease even beyond depression can be used by physicians as an important primary prevention tool

Expression of fibrosis-related genes in liver and kidney fibrosis in comparison to inflammatory bowel diseases

Fibrosis is an important feature of inflammatory bowel diseases (IBD), but its pathogenesis is incompletely understood. Our aim was to identify genes important for fibrosis in IBD by comparison with kidney and liver fibrosis. First, we performed bioinformatics analysis of Gene Expression Omnibus datasets of liver and kidney fibrosis and identified CXCL9, THBS2, MGP, PTPRC, CD52, GZMA, DPT and DCN as potentially important genes with altered expression in fibrosis. We then performed qPCR analysis of the selected genes’ expression on samples of fibrotic kidney, liver, Crohn’s disease (CD) with and without fibrosis and ulcerative colitis (UC), in comparison to corresponding normal tissue. We found significantly altered expression in fibrosis for all selected genes. A significant difference for some genes was observed in CD with fibrosis in comparison to CD without fibrosis and UC. We conclude that similar changes in the expression of selected genes in liver, kidney fibrosis and IBD provide further evidence that fibrosis in IBD might share common mechanisms with other organs, supporting the hypothesis that fibrosis is the common pathway in diseases of various organs. Some genes were already active in IBD with inflammation without fibrosis, suggesting the early activation of profibrotic pathways or overlapping function in fibrosis and inflammation

Expression of cytokine-coding genes BMP8B, LEFTY1 and INSL5 could distinguish between ulcerative colitis and Crohn\u27s disease

Ulcerative colitis (UC) and Crohn’s disease (CD) are characterized by an imbalance between pro-inflammatory and anti-inflammatory cytokines, interfering with the resolution of inflammation. Due to the crucial role of cytokines, new insights into their profiles in UC and CD would help to improve our understanding of pathogenesis and enable the development of new treatment modalities. We provide an expression profile of cytokines in UC and CD, using bioinformatics approach, and experimental validation of expression of the selected genes. We retrieved data and analyzed the cytokine gene expression profiles of UC and CD. From ten genes with inverse expression, common to CD and UC, BMP8B, LEFTY1 and INSL5 were selected for gene expression experimental validation. Experimentally, BMP8B and INSL5 were down-regulated in both CD and UC but followed the bioinformatics trend. The expression of genes LEFTY1 and BMP8B was statistically significant when comparing UC and CD in colon and the expression of gene LEFTY1 showed statistical significance when CD in ileum and colon were compared. Using the bioinformatics approach and experimental validation, we found differences in expression profiles between UC and CD for INSL5, LEFTY1 and BMP8B. These three promising candidate genes need to be further explored at different levels, such as DNA methylation and protein expression, to provide more evidence on their potential diagnostic role in CD and UC

Pathology of fibrosis in Crohn\u27s disease - contribution to understanding its pathogenesis

Background: Despite significant progress in the research of fibrosis in various organs, fibrosis remains a poorly understood complication of Crohn\u27s disease (CD). We analyzed pathologic features of fibrosis and inflammation in CD and compared them with the normal bowel, aiming to clarify whether fibrosis in CD pathogenetically resembles fibrosis in other organs. Methods: Resection specimens from 30 patients with CD were included. Normal bowel from resection specimens of colorectal carcinoma was used for comparison. Trichrome Masson staining, immunohistochemistry for α-smooth muscle actin, fibroblast activation protein, CD34 and erg, in situ hybridization for TGF-β1 and analysis of selected fibrosis-related microRNAs were performed. Results: In normal bowel, CD34-positive fibroblasts/pericytes were detected in the submucosa and subserosa, particularly around blood vessels. In CD, fibrosis prevailed in the submucosa and subserosa, together with proliferation of myofibroblasts and disappearance of CD34-positive fibroblasts/pericytes. TGF-β1 was present in the lamina propria in normal bowel and CD, and in deeper parts of the bowel wall in CD. MicroRNAs miR-29c, miR-155 miR-150, and miR-155, which have been demonstrated to contribute to fibrosis in various organs, showed significant deregulation in CD. Conclusions: Distribution of fibroblasts/pericytes in the submucosa and subserosa of normal bowel, their disappearance in fibrosis in CD, together with the appearance of myofibroblasts, suggest that fibroblasts/pericytes are the most likely source of myofibroblasts in CD. Furthemore, fibrosis-related microRNAs showed deregulation in fibrotic areas. Pathogenesis of fibrosis in CD is thus comparable to fibrosis in other organs, in which myofibroblasts are the key effector cells, and pericytes have emerged as the main origin of myofibroblasts. Fibrosis in CD should be regarded as a result of (over)response of the bowel wall to the presence of inflammation in deep structures of the bowel wall, presenting another example of a common pathogenetic pathway of fibrosis development

The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

<div><p>Toll-like receptor 3 (TLR3) is a dsRNA sensing receptor that is localized in the cellular compartments but also at the plasma membrane. Overexpression of UNC93B1 promoted localization of TLR3, but not other nucleic acid sensing TLRs, to the plasma membrane. Here we show that UNC93B1 itself is localized at the plasma membrane. We investigated the role of different domains of TLR3 on cell signaling by preparing chimeric receptors between TLR3 and TLR9 where each of the transmembrane segments or cytosolic domains has been exchanged. While the ectodomain completely governs ligand specificity and the cytosolic TIR domain determines the engagement of the signaling adapters as well as the potentiation of receptor activation by UNC93B1, the ectodomain but not transmembrane segment or cytosolic domain determines plasma membrane localization of TLR3. Nevertheless, TLR3 receptor and ligand endocytosis as well as endosomal acidification are important for the robust signaling of TLR3.</p></div

Localization of chimeric receptors with exchanged TM segments of TLR3 and TLR9.

<p>HEK293T cells were transiently transfected TLR3-9-3-mCer (A and B - cyan), TLR9-3-9-YFP (C and D - yellow), and with UNC93B1. Plasma membrane markers SynaptoRed and CTB-Alexa 555 are shown in magenta. (A) Localization of TLR3-9-3-mCer on plasma membrane in cells overexpressing UNC93B1. (B) Intracellular localization of TLR3-9-3-mCer in HEK293T without overexpression of UNC93B1. (C) Intracellular localization of TLR9-3-9-YFP in cells overexpressing UNC93B1. (D) Intracellular localization of TLR9-3-9-YFP in HEK293T without overexpression of UNC93B1. (A-D) Data are representative of three experiments. TLR membrane localization was evaluated from plots (bottom) of normalized fluorescence intensities of TLR and plasma membrane (PM) within 3 μm line profiles (n = 9). Three representative lines are marked on merged images. Images are selected from three independent experiments. Scale bars, 10 μm.</p

Poly(I:C) and TLR3 colocalize at the surface of plasma membrane.

<p>HEK293T cells were transfected with TLR3-mCer alone (A) or cotransfected with UNC93B1 (B). Cells were stimulated with rhodamine labeled poly(I:C) (poly(I:C)-R). (B) TLR3 and poly(I:C)-R colocalization was evaluated from plots (right) of fluorescence intensities within 3 μm line profiles (n = 3; i, ii, iii). Three representative speckles where cross-sections were analyzed are marked with the white arrows on the merged image. (C–E) HEK293 cells were transfected with TLR3 alone or with UNC93B1 encoding plasmid. Cells were cotransfected with IFN-β (left) or NF-κB (right) promoter reporter plasmid and <i>Renilla</i> reporter plasmid. Cells were simultaneously treated with poly(I:C) (10 μg/ml) and inhibitors. Cells were treated with increasing amounts (0.2–5 μM) of cytochalasin D (abbr. CD) (C), Dynasore (abbr. Dy) (20–80 μM) (D) or bafilomycin A (abbr. BA) (0.2–2 μM) (E). 8 h after treatment luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.</p