In vitro effects of G-Protein Coupled Receptor 120 agonist on osteoblast differentiation and activity in osteosarcoma cells

National Research Foundationhttps://drive.google.com/file/d/1dE7RR62sc4KkBLHMIpNW6ScUoYkEZjUJ/view?usp=sharinghttps://drive.google.com/drive/folders/1zHnJYGbKy6ehN1aYaCerFl1_SodEBYoz?usp=sharinghttps://drive.google.com/drive/folders/1QfaeMDpZYaV-hPXmra01zIsR0NfpDPPJ?usp=sharin

Titin circular RNAs create a back-splice motif essential for SRSF10 splicing

Background: Titin (TTN), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study we show that the backsplice junction formed by this class of circRNAs creates a unique motif, which binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. Methods: We calculated genetic constraint of the identified motif in 125.748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the backsplice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Results: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in hiPSC-CM induced structural abnormalities, apoptosis and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets like MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. Conclusions: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the backsplice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome, but provides a yet unrecognized source for functional sequence variation

The web-based simulation and information service for multi-hazard impact chains. Design document.

The overall objective of the PARATUS project and the platform is the co-development of a web-based simulation and information service for first and second responders and other stakeholders to evaluate the impact chains of multi-hazard events with particular emphasis on cross-border and cascading impacts. This deliverable provides a first impression of the platform and its components. A central theme in the PARATUS project is the co-development of the tools with stakeholders. The central stakeholders within the four applications case studies are therefore full project partners. They will be directly involved in the development of the platform. We foresee that the PARATUS Platform will have two major blocks: an information service that provides static information (or regularly updated information) and simulation service, which is a dynamic component where stakeholders can interactively work with the tools in the platform. The PARATUS will further make sure that documentation (e.g., software accompanying documentation) is also publicly available via the project website1 and other trusted repositories. The deliverable 4.1 was submitted to the European Commission on 31/07/2023 and is waiting for approval by the Research Executive Agency. Therefore, this current version may not represent the final version of the deliverable

BRCA1/2 mutation testing in breast cancer patients: a prospective study of the long-term psychological impact of approach during adjuvant radiotherapy

This study assessed psychological distress during the first year after diagnosis in breast cancer patients approached for genetic counseling at the start of adjuvant radiotherapy and identified those vulnerable to long-term high distress. Of the approached patients some chose to receive a DNA test result (n = 58), some were approached but did not fulfill criteria for referral (n = 118) and some declined counseling and/or testing (n = 44). The comparative group consisted of patients not eligible for genetic counseling (n = 182) and was therefore not approached. Patients actively approached for genetic counseling showed no more long-term distress than patients not eligible for such counseling. There were no differences between the subgroups of approached patients. Predictors for long-term high distress or an increase in distress over time were pre-existing high distress and a low quality of life, having children, and having no family members with breast cancer. It is concluded that breast cancer patients can be systematically screened and approached for genetic counseling during adjuvant radiotherapy without imposing extra psychological burden. Patients vulnerable to long-term high distress already displayed high distress shortly after diagnosis with no influence of their medical treatment on their level of distress at long-term

Seasonal Variation in Vitamin D3 Levels Is Paralleled by Changes in the Peripheral Blood Human T Cell Compartment

It is well-recognized that vitamin D3 has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D3 status. Here, we investigated if and to what extent seasonality of vitamin D3 levels are associated with changes in T cell numbers and phenotypes. Every three months during the course of the entire year, human PBMC and whole blood from 15 healthy subjects were sampled and analyzed using flow cytometry. We observed that elevated serum 25(OH)D3 and 1,25(OH)2D3 levels in summer were associated with a higher number of peripheral CD4+ and CD8+ T cells. In addition, an increase in naïve CD4+CD45RA+ T cells with a reciprocal drop in memory CD4+CD45RO+ T cells was observed. The increase in CD4+CD45RA+ T cell count was a result of heightened proliferative capacity rather than recent thymic emigration of T cells. The percentage of Treg dropped in summer, but not the absolute Treg numbers. Notably, in the Treg population, the levels of forkhead box protein 3 (Foxp3) expression were increased in summer. Skin, gut and lymphoid tissue homing potential was increased during summer as well, exemplified by increased CCR4, CCR6, CLA, CCR9 and CCR7 levels. Also, in summer, CD4+ and CD8+ T cells revealed a reduced capacity to produce pro-inflammatory cytokines. In conclusion, seasonal variation in vitamin D3 status in vivo throughout the year is associated with changes in the human peripheral T cell compartment and may as such explain some of the seasonal variation in immune status which has been observed previously. Given that the current observations are limited to healthy adult males, larger population-based studies would be useful to validate these findings

A model of the PI cycle reveals the regulating roles of lipid-binding proteins and pitfalls of using mosaic biological data

The phosphatidylinositol (PI) cycle is central to eukaryotic cell signaling. Its complexity, due to the number of reactions and lipid and inositol phosphate intermediates involved makes it difficult to analyze experimentally. Computational modelling approaches are seen as a way forward to elucidate complex biological regulatory mechanisms when this cannot be achieved solely through experimental approaches. Whilst mathematical modelling is well established in informing biological systems, many models are often informed by data sourced from multiple unrelated cell types (mosaic data) or from purified enzyme data. In this work, we develop a model of the PI cycle informed by experimental and omics data taken from a single cell type, namely platelets. We were able to make a number of predictions regarding the regulation of PI cycle enzymes, the importance of the number of receptors required for successful GPCR signaling and the importance of lipid- and protein-binding proteins in regulating second messenger outputs. We then consider how pathway behavior differs, when fully informed by data for HeLa cells and show that model predictions remain consistent. However, when informed by mosaic experimental data model predictions greatly vary illustrating the risks of using mosaic datasets from unrelated cell types

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

Frizzled 7 and PIP₂ binding by syntenin PDZ₂ domain supports Frizzled 7 trafficking and signalling

PDZ domain-containing proteins work as intracellular scaffolds to control spatio-temporal aspects of cell signalling. This function is supported by the ability of their PDZ domains to bind other proteins such as receptors, but also phosphoinositide lipids important for membrane trafficking. Here we report a crystal structure of the syntenin PDZ tandem in complex with the carboxy-terminal fragment of Frizzled 7 and phosphatidylinositol 4,5-bisphosphate (PIP₂). The crystal structure reveals a tripartite interaction formed via the second PDZ domain of syntenin. Biophysical and biochemical experiments establish co-operative binding of the tripartite complex and identify residues crucial for membrane PIP₂-specific recognition. Experiments with cells support the importance of the syntenin–PIP₂ interaction for plasma membrane targeting of Frizzled 7 and c-jun phosphorylation. This study contributes to our understanding of the biology of PDZ proteins as key players in membrane compartmentalization and dynamics

PIPKIγ Regulates Focal Adhesion Dynamics and Colon Cancer Cell Invasion

Focal adhesion assembly and disassembly are essential for cell migration and cancer invasion, but the detailed molecular mechanisms regulating these processes remain to be elucidated. Phosphatidylinositol phosphate kinase type Iγ (PIPKIγ) binds talin and is required for focal adhesion formation in EGF-stimulated cells, but its role in regulating focal adhesion dynamics and cancer invasion is poorly understood. We show here that overexpression of PIPKIγ promoted focal adhesion formation, whereas cells expressing either PIPKIγK188,200R or PIPKIγD316K, two kinase-dead mutants, had much fewer focal adhesions than those expressing WT PIPKIγ in CHO-K1 cells and HCT116 colon cancer cells. Furthermore, overexpression of PIPKIγ, but not PIPKIγK188,200R, resulted in an increase in both focal adhesion assembly and disassembly rates. Depletion of PIPKIγ by using shRNA strongly inhibited formation of focal adhesions in HCT116 cells. Overexpression of PIPKIγK188,200R or depletion of PIPKIγ reduced the strength of HCT116 cell adhesion to fibronection and inhibited the invasive capacities of HCT116 cells. PIPKIγ depletion reduced PIP2 levels to ∼40% of control and PIP3 to undetectable levels, and inhibited vinculin localizing to focal adhesions. Taken together, PIPKIγ positively regulates focal adhesion dynamics and cancer invasion, most probably through PIP2-mediated vinculin activation

Vitamin D and HIV Progression among Tanzanian Adults Initiating Antiretroviral Therapy

Background: There is growing evidence of an association between low vitamin D and HIV disease progression; however, no prospective studies have been conducted among adults receiving antiretroviral therapy (ART) in sub-Saharan Africa. Methods Serum 25-hydroxyvitamin D (25(OH)D) levels were assessed at ART initiation for a randomly selected cohort of HIV-infected adults enrolled in a trial of multivitamins (not including vitamin D) in Tanzania during 2006–2010. Participants were prospectively followed at monthly clinic visits for a median of 20.6 months. CD4 T-cell measurements were obtained every 4 months. Proportional hazard models were utilized for mortality analyses while generalized estimating equations were used for CD4 T-cell counts. Results: Serum 25(OH)D was measured in 1103 adults 9.2% were classified as vitamin D deficient (30 ng/mL). After multivariate adjustment, vitamin D deficiency was significantly associated with increased mortality as compared to vitamin D sufficiency (HR: 2.00; 95% CI: 1.19–3.37; p = 0.009), whereas no significant association was found for vitamin D insufficiency (HR: 1.24; 95% CI: 0.87–1.78; p = 0.24). No effect modification by ART regimen or change in the associations over time was detected. Vitamin D status was not associated with change in CD4 T-cell count after ART initiation. Conclusions: Deficient vitamin D levels may lead to increased mortality in individuals receiving ART and this relationship does not appear to be due to impaired CD4 T-cell reconstitution. Randomized controlled trials are needed to determine the safety and efficacy of vitamin D supplementation for individuals receiving ART