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Abstract Regnase-2 (Reg-2/MCPIP2/ZC3H12B) is uniquely expressed at a high level in the healthy brain and down-regulated in samples from patients with glioma, reaching the lowest level in high-grade glioblastoma multiforme (GBM). This RNase is involved in the regulation of neuroinflammation through the degradation of IL-6 and IL-1 mRNAs, key pro-inflammatory cytokines for GBM pathology. Reg-2 is a strong inhibitor of the proliferation of human glioblastoma cell lines and blocks their potential to form colonies. Here, we describe that overexpression of Reg-2 stalls glioblastoma cells in the G1 phase of the cell cycle and reduces the level of transcripts implicated in cell cycle progression. These newly identified targets include CCND1, CCNE1, CCNE2, CCNA2, CCNB1, and CCNB2, encoding the cyclins as well as AURKA and PLK1, encoding two important mitosis regulators. By RNA immunoprecipitation we confirmed the direct interaction of Reg-2 with the investigated transcripts. We also tested mRNA regions involved in their interaction with Reg-2 on the example of CCNE2. Reg-2 interacts with the 3’UTR of CCNE2 in a dose-dependent manner. In conclusion, our results indicate that Reg-2 controls key elements in GBM biology by restricting neuroinflammation and inhibiting cancer cell proliferation.

Abstract The genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats (atypical in males). The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XX SRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes (SRY, TAC3, CYP11B1 and LHCGR). Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 (modal), and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5′UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.

Abstract This study aims to determine the effects of differences in variety and irrigations levels on potato yield (appropriate humidity, temperature, shading the plants from the sun if necessary) on potato yield in greenhouse conditions. Functions of potato production with respect to water use were developed for five dates of measurements of plant growth, in relation to the various vegetation phases. On the basis of potato vegetation phases, the potato water demand was determined. An experiment was conducted using the randomized sub-block method. The first order factor were the two varieties of potato that were grown under drip irrigation with three water regimes as a second factor experiment: level 1 (pF 2.7), level 2 (pF 2.5) and level 3 (pF 2.2). The variety had a significant effect on the weight of potato tubers. The irrigation level had only a significant effect on the total potato biomass. The potato harvest date had a significant effect on both of the examined treatments. The growth dynamics of the aboveground part and potato tubers were the highest in conditions of constant level 1. Regardless of the variety studied, on level 3 caused a reduction in potato biomass production.The highest water consumption was observed during the tuber potato growth period to flowering. The values were varied from 0.39 l/pot day level 1 (in both investigated cultivars) to 0.99 l/ pot/day (humidity level 3 for Julinka cultivar) in July. The most intensive increase in water consumption was observed at the level 3. The average W index of the average daily water dose calculated for Denar cultivar amounted 0.40 l day−1 in the first period (O1) to 0.79 l day−1 in the fifth period (O5) and for Julinka cultivar 0.49 l day−1 (O1) to 0.92 l day−1 (O5). Stress due to water shortage and/or excessive levels of water in the soil negatively influenced the yield of potato tuber. Potato varieties reacted differently to soil water content.

Introduction: The Hopkins Symptom Checklist-25 (HSCL-25) is an effective, reliable, and ergonomic tool that can be used for depression diagnosis and monitoring in daily practice. To allow its broad use by family practice physicians (FPs), it was translated from English into nine European languages (Greek, Polish, Bulgarian, Croatian, Catalan, Galician, Spanish, Italian, and French) and the translation homogeneity was confirmed. This study describes this process.Methods: First, two translators (an academic translator and an FP researcher) were recruited for the forward translation (FT). A panel of English-speaking FPs that included at least 15 experts (researchers, teachers, and practitioners) was organized in each country to finalize the FT using a Delphi procedure.Results: One or two Delphi procedure rounds were sufficient for each translation. Then, a different translator, who did not know the original version of the HSCL-25, performed a backward translation in English. An expert panel of linguists compared the two English versions. Differences were listed and a multicultural consensus group determined whether they were due to linguistic problems or to cultural differences. All versions underwent cultural check.Conclusion: All nine translations were finalized without altering the original meaning.

Abstract Background General Practice (GP) seems to be perceived as less attractive throughout Europe. Most of the policies on the subject focused on negative factors. An EGPRN research team from eight participating countries was created in order to clarify the positive factors involved in appeals and retention in GP throughout Europe. The objective was to explore the positive factors supporting the satisfaction of General Practitioners (GPs) in clinical practice throughout Europe. Method Qualitative study, employing face-to-face interviews and focus groups using a phenomenological approach. The setting was primary care in eight European countries: France, Belgium, Germany, Slovenia, Bulgaria, Finland, Poland and Israel. A thematic qualitative analysis was performed following the process described by Braun and Clarke. Codebooks were generated in each country. After translation and back translation of these codebooks, the team clarified and compared the codes and constructed one international codebook used for further coding. Results A purposive sample of 183 GPs, providing primary care to patients in their daily clinical practice, was interviewed across eight countries. The international codebook included 31 interpretative codes and six themes. Five positive themes were common among all the countries involved across Europe: the GP as a person, special skills needed in practice, doctor-patient relationship, freedom in the practice and supportive factors for work-life balance. One theme was not found in Poland or Slovenia: teaching and learning. Conclusion This study identified positive factors which give GPs job satisfaction in their clinical practice. This description focused on the human needs of a GP. They need to have freedom to choose their working environment and to organize their practice to suit themselves. In addition, they need to have access to professional education so they can develop specific skills for General Practice, and also strengthen doctor-patient relationships. Stakeholders should consider these factors when seeking to increase the GP workforce.

Extracellular HMGB1 acts as an alarmin in multiple autoimmune diseases. While its release and functions have been extensively studied, there is a substantial lack of knowledge regarding HMGB1 regulation at the site of inflammation. Herein we show that enzymes present in arthritis-affected joints process HMGB1 into smaller peptides in vitro. Gel electrophoresis, Western blotting and mass spectrometry analyses indicate cleavage sites for human neutrophil elastase, cathepsin G, and matrix metalloproteinase 3 within the HMGB1 structure. While human neutrophil elastase and matrix metalloproteinase 3 might alter the affinity of HMGB1 to its receptors by cleaving the acidic C-terminal tail, cathepsin G rapidly and completely degraded the alarmin. Contrary to a previous report we demonstrate that HMGB1 is not a substrate for dipeptidyl peptidase IV. We also provide novel information regarding the presence of these proteases in synovial fluid of juvenile idiopathic arthritis patients. Correlation analysis of protease levels and HMGB1 levels in synovial fluid samples did not, however, reveal any direct relationship between the recorded levels. This study provides knowledge of proteolytic processing of HMGB1 relevant for the regulation of HMGB1 during inflammatory disease.

Abstract Background High mobility group box 1 (HMGB1) protein is a central endogenous inflammatory mediator contributing to the pathogenesis of several inflammatory disorders. HMGB1 interacts with toll-like receptors (TLRs) but contradictory evidence regarding its identity as a TLR2 ligand persists. The aim of this study was to investigate if highly purified HMGB1 interacts with TLR2 and if so, to determine the functional outcome. Methods Full length or C-terminal truncated (Δ30) HMGB1 was purified from E.coli. Binding to TLR2-Fc was investigated by direct-ELISA. For the functional studies, proteins alone or in complex with peptidoglycan (PGN) were added to human embryonic kidney (HEK) cells transfected with functional TLR2, TLR 1/2 or TLR 2/6 dimers, macrophages, whole blood or peripheral blood mononuclear cells (PBMCs). Cytokine levels were determined by ELISA. Results In vitro binding experiments revealed that Δ30 HMGB1, lacking the acidic tail domain, but not full length HMGB1 binds dose dependently to TLR2. Control experiments confirmed that the interaction was specific to TLR2 and could be inhibited by enzymatic digestion. Δ30 HMGB1 alone was unable to induce cytokine production via TLR2. However, full length HMGB1 and Δ30 HMGB1 formed complexes with PGN, a known TLR2 ligand, and synergistically potentiated the inflammatory response in PBMCs. Conclusions We have demonstrated that TLR2 is a receptor for HMGB1 and this binding is negatively regulated by the C-terminal tail. HMGB1 did not induce functional activation of TLR2 while both full length HMGB1 and Δ30 HMGB1 potentiated the inflammatory activities of the TLR2 ligand PGN. We hypothesize that Δ30 HMGB1 generated in vivo by enzymatic cleavage could act as an enhancer of TLR2-mediated inflammatory activities.

Abstract Objective From a systematic literature review (SLR), it became clear that a consensually validated tool was needed by European General Practitioner (GP) researchers in order to allow multi-centred collaborative research, in daily practice, throughout Europe. Which diagnostic tool for depression, validated against psychiatric examination according to the DSM, would GPs select as the best for use in clinical research, taking into account the combination of effectiveness, reliability and ergonomics? A RAND/UCLA, which combines the qualities of the Delphi process and of the nominal group, was used. GP researchers from different European countries were selected. The SLR extracted tools were validated against the DSM. The Youden index was used as an effectiveness criterion and Cronbach’s alpha as a reliability criterion. Ergonomics data were extracted from the literature. Ergonomics were tested face-to-face. Results The SLR extracted 7 tools. Two instruments were considered sufficiently effective and reliable for use: the Hospital Anxiety and Depression Scale and the Hopkins Symptoms Checklist-25 (HSCL-25). After testing face-to-face, HSCL-25 was selected. A multicultural consensus on one diagnostic tool for depression was obtained for the HSCL-25. This tool will provide the opportunity to select homogeneous populations for European collaborative research in daily practice.

The members of the ZC3H12/MCPIP/Regnase family of RNases have emerged as important regulators of inflammation. In contrast to Regnase-1, -2 and -4, a thorough characterization of Regnase-3 (Reg-3) has not yet been explored. Here we demonstrate that Reg-3 differs from other family members in terms of NYN/PIN domain features, cellular localization pattern and substrate specificity. Together with Reg-1, the most comprehensively characterized family member, Reg-3 shared IL-6, IER-3 and Reg-1 mRNAs, but not IL-1β mRNA, as substrates. In addition, Reg-3 was found to be the only family member which regulates transcript levels of TNF, a cytokine implicated in chronic inflammatory diseases including psoriasis. Previous meta-analysis of genome-wide association studies revealed Reg-3 to be among new psoriasis susceptibility loci. Here we demonstrate that Reg-3 transcript levels are increased in psoriasis patient skin tissue and in an experimental model of psoriasis, supporting the immunomodulatory role of Reg-3 in psoriasis, possibly through degradation of mRNA for TNF and other factors such as Reg-1. On the other hand, Reg-1 was found to destabilize Reg-3 transcripts, suggesting reciprocal regulation between Reg-3 and Reg-1 in the skin. We found that either Reg-1 or Reg-3 were expressed in human keratinocytes in vitro. However, in contrast to robustly upregulated Reg-1 mRNA levels, Reg-3 expression was not affected in the epidermis of psoriasis patients. Taken together, these data suggest that epidermal levels of Reg-3 are negatively regulated by Reg-1 in psoriasis, and that Reg-1 and Reg-3 are both involved in psoriasis pathophysiology through controlling, at least in part different transcripts.

Abstract Background Physicians’ clinical decision-making may be influenced by non‐analytical thinking, especially when perceiving uncertainty. Incidental gut feelings in general practice have been described, namely, as “a sense of alarm” and “a sense of reassurance”. A Dutch Gut Feelings Questionnaire (GFQ) was developed, validated and afterwards translated into English following a linguistic validation procedure. The aims were to translate the GFQ from English into French, German and Polish; to describe uniform elements as well as differences and difficulties in the linguistic validation processes; to propose a procedural scheme for future GFQ translations into other languages. Methods We followed a structured, similar and equivalent procedure. Forward and backward-translations, repeated consensus procedures and cultural validations performed in six steps. Exchanges between the several research teams, the authors of the Dutch GFQ, and the translators involved continued throughout the procedure. Results 12 translators, 52 GPs and 8 researchers in the field participated to the study in France, Germany, Switzerland and Poland. The collaborating research teams created three versions of the 10-item GFQ. Each research team found and agreed on compromises between comparability and similarity on one hand, and linguistic and cultural specificities on the other. Conclusions The gut feeling questionnaire is now available in five European languages: Dutch, English, French, German and Polish. The uniform procedural validation scheme presented, and agreed upon by the teams, can be used for the translation of the GFQ into other languages. Comparing results of research into the predictive value of gut feelings and into the significance of the main determinants in five European countries is now possible.