The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity

Neuroprotective Effects of Human Amniotic Fluid Stem Cells-derived Secretome in an Ischemia/reperfusion Model

Stem cells offer the basis for the promotion of robust new therapeutic approaches for a variety of human disorders. There are still many limitations to be overcome before clinical therapeutic application, including a better understanding of the mechanism by which stem cell therapies may lead to enhanced recovery. In vitro investigations are necessary to dissect the mechanisms involved and to support the potential development in stem cell-based therapies. In spite of growing interest in human amniotic fluid stem cells, not much is known about the characteristics of their secretome and regarding the potential neuroprotective mechanism in different pathologies, including stroke. To get more insight on amniotic fluid cells therapeutic potential, signal transduction pathways activated by human amniotic fluid stem cells (hAFSCs)-derived secretome in a stroke in vitro model (ischemia/reperfusion [I/R] model) were investigated by Western blot. Moreover, miRNA expression in the exosomal fraction of the conditioned medium was analyzed. hAFSCs-derived secretome was able to activate pro-survival and anti-apoptotic pathways. MicroRNA analysis in the exosomal component revealed a panel of 16 overexpressed miRNAs involved in the regulation of coherent signaling pathways. In particular, the pathways of relevance in ischemia/reperfusion, such as neurotrophin signaling, and those related to neuroprotection and neuronal cell death, were analyzed. The results obtained strongly point toward the neuroprotective effects of the hAFSCs-conditioned medium in the in vitro stroke model here analyzed. This can be achieved by the modulation and activation of pro-survival processes, at least in part, due to the activity of secreted miRNAs

Neuroprotective Effects of Human Amniotic Fluid Stem Cells-derived Secretome in an Ischemia/reperfusion Model

Stem cells offer the basis for the promotion of robust new therapeutic approaches for a variety of human disorders. There are still many limitations to be overcome before clinical therapeutic application, including a better understanding of the mechanism by which stem cell therapies may lead to enhanced recovery. In vitro investigations are necessary to dissect the mechanisms involved and to support the potential development in stem cell-based therapies. In spite of growing interest in human amniotic fluid stem cells, not much is known about the characteristics of their secretome and regarding the potential neuroprotective mechanism in different pathologies, including stroke. To get more insight on amniotic fluid cells therapeutic potential, signal transduction pathways activated by human amniotic fluid stem cells (hAFSCs)-derived secretome in a stroke in vitro model (ischemia/reperfusion [I/R] model) were investigated by Western blot. Moreover, miRNA expression in the exosomal fraction of the conditioned medium was analyzed. hAFSCs-derived secretome was able to activate pro-survival and anti-apoptotic pathways. MicroRNA analysis in the exosomal component revealed a panel of 16 overexpressed miRNAs involved in the regulation of coherent signaling pathways. In particular, the pathways of relevance in ischemia/reperfusion, such as neurotrophin signaling, and those related to neuroprotection and neuronal cell death, were analyzed. The results obtained strongly point toward the neuroprotective effects of the hAFSCs-conditioned medium in the in vitro stroke model here analyzed. This can be achieved by the modulation and activation of pro-survival processes, at least in part, due to the activity of secreted miRNAs

Large-Scale Functional Genomics Screen to Identify Modulators of Human β-Cell Insulin Secretion

Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide. Impaired function of pancreatic β-cells is both a hallmark of T2D and an underlying factor in the pathophysiology of the disease. Understanding the cellular mechanisms regulating appropriate insulin secretion has been of long-standing interest in the scientific and clinical communities. To identify novel genes regulating insulin secretion we developed a robust arrayed siRNA screen measuring basal, glucose-stimulated, and augmented insulin secretion by EndoC-βH1 cells, a human β-cell line, in a 384-well plate format. We screened 521 candidate genes selected by text mining for relevance to T2D biology and identified 23 positive and 68 negative regulators of insulin secretion. Among these, we validated ghrelin receptor (GHSR), and two genes implicated in endoplasmic reticulum stress, ATF4 and HSPA5. Thus, we have demonstrated the feasibility of using EndoC-βH1 cells for large-scale siRNA screening to identify candidate genes regulating β-cell insulin secretion as potential novel drug targets. Furthermore, this screening format can be adapted to other disease-relevant functional endpoints to enable large-scale screening for targets regulating cellular mechanisms contributing to the progressive loss of functional β-cell mass occurring in T2D

Mode of birth in women with low-lying placenta: protocol for a prospective multicentre 1:3 matched case-control study in Italy (the MODEL-PLACENTA study)

Introduction: The term placenta praevia defines a placenta that lies over the internal os, whereas the term low-lying placenta identifies a placenta that is partially implanted in the lower uterine segment with the inferior placental edge located at 1-20 mm from the internal cervical os (internal-os-distance). The most appropriate mode of birth in women with low-lying placenta is still controversial, with the majority of them undergoing caesarean section. The current project aims to evaluate the rate of vaginal birth and caesarean section in labour due to bleeding by offering a trial of labour to all women with an internal-os-distance >5 mm as assessed by transvaginal sonography in the late third trimester. Methods and analysis: The MODEL-PLACENTA is a prospective, multicentre, 1:3 matched case-control study involving 17 Maternity Units across Lombardy and Emilia-Romagna regions, Italy. The study includes women with a placenta located in the lower uterine segment at the second trimester scan. Women with a normally located placenta will be enrolled as controls. A sample size of 30 women with an internal-os-distance >5 mm at the late third trimester scan is needed at each participating Unit. Since the incidence of low-lying placenta decreases from 2% in the second trimester to 0.4% at the end of pregnancy, 150 women should be recruited at each centre at the second trimester scan. A vaginal birth rate ≥60% in women with an internal-os-distance >5 mm will be considered appropriate to start routinely admitting to labour these women. Ethics and dissemination: Ethical approval for the study was given by the Brianza Ethics Committee (No 3157, 2019). Written informed consent will be obtained from study participants. Results will be disseminated by publication in peer-reviewed journals and presentation in international conferences. Trial registration number: NCT04827433 (pre-results stage)

Adult stem cell activity in naked mole rats for long-term tissue maintenance

Abstract The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5 + ) in their native in vivo state, we find an expanded pool of Lgr5 + cells in NMRs, and these cells specifically at the crypt base (Lgr5 +CBC ) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5 +CBC cells. Instead of entering quiescence (G0), NMR Lgr5 +CBC cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone

Adult stem cell activity in naked mole rats for long-term tissue maintenance

The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5+) in their native in vivo state, we find an expanded pool of Lgr5+ cells in NMRs, and these cells specifically at the crypt base (Lgr5+CBC) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5+CBC cells. Instead of entering quiescence (G0), NMR Lgr5+CBC cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone

Diagnostic issues faced by a rare disease healthcare network during Covid-19 outbreak: data from the Campania Rare Disease Registry

Background: The aims of this study were: to investigate the capacity of the rare disease healthcare network in Campania to diagnose patients with rare diseases during the outbreak of Covid-19; and to shed light on problematic diagnoses during this period. Methods: To describe the impact of the Covid-19 pandemic on the diagnosis of patients with rare diseases, a retrospective analysis of the Campania Region Rare Disease Registry was performed. A tailored questionnaire was sent to rare disease experts to investigate major issues during the emergency period. Results: Prevalence of new diagnoses of rare disease in March and April 2020 was significantly lower than in 2019 (117 versus 317, P < 0.001 and 37 versus 349, P < 0.001, respectively) and 2018 (117 versus 389, P < 0.001 and 37 versus 282, P < 0.001, respectively). Eighty-two among 98 rare disease experts completed the questionnaire. Diagnostic success (95%), access to diagnosis (80%) and follow-up (72%), lack of Personal Protective Equipment (60%), lack of Covid-19 guidelines (50%) and the need for home therapy (78%) were the most important issues raised during Covid-19 outbreak. Conclusions: This study describes the effects of the Covid-19 outbreak on the diagnosis of rare disease in a single Italian region and investigates potential issues of diagnosis and management during this period