ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis

The double‐membrane‐bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl‐inositol‐3‐phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E‐Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E‐Syt‐containing domains during autophagy and that inhibition of E‐Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E‐Syts are essential for autophagy‐associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy.     

Collagen type XV and the ‘osteogenic status’

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo‐induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo‐induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as ‘mineralized MSCs’, during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium ‘per se’, negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early‐phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.     

Characterisation of yeast microbiota,chemical and sensory properties of organic and biodynamic Sangiovese red wines

Wine quality is closely linked to the fermentation step, which is driven by the microbial ecology of grape and the use of selected microbial strains as well. The microbial species developing during fermentation determines the type and concentration of many substances, which contribute to the sensory properties of wine and its safety. In this view, the present work aims to characterise the yeast microbiota, chemical and sensory properties of Sangiovese red wines obtained from both biodynamic and organic agriculture. The natural yeast populations of grape musts and their evolution during spontaneous were monitored and investigated. In addition, the volatile composition, physico-chemical and safety features (ethyl-carbamate) and sensory properties of wines were evaluated. The results showed that the yeast population was mostly related to the grape management, i.e. organic or biodynamic, while the wine composition was mainly affected by the winemaking process, and then by the grape management.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Bioclimatic transect networks: Powerful observatories of ecological change

Transects that traverse substantial climate gradients are important tools for climate change research and allow questions on the extent to which phenotypic variation associates with climate, the link between climate and species distributions, and variation in sensitivity to climate change among biomes to be addressed. However, the potential limitations of individual transect studies have recently been highlighted. Here, we argue that replicating and networking transects, along with the introduction of experimental treatments, addresses these concerns. Transect networks provide cost‐effective and robust insights into ecological and evolutionary adaptation and improve forecasting of ecosystem change. We draw on the experience and research facilitated by the Australian Transect Network to demonstrate our case, with examples, to clarify how population‐ and community‐level studies can be integrated with observations from multiple transects, manipulative experiments, genomics, and ecological modeling to gain novel insights into how species and systems respond to climate change. This integration can provide a spatiotemporal understanding of past and future climate‐induced changes, which will inform effective management actions for promoting biodiversity resilience.     

Potential advantages of acute kidney injury management by mesenchymal stem cells

Mesenchymal stem cells are currently considered as a promising tool for therapeutic application in acute kidney injury (AKI) management. AKI is characterized by acute tubular injury with rapid loss of renal function. After AKI, inflammation, oxidative stress and excessive deposition of extracellular matrix are the molecular events that ultimately cause the end-stage renal disease. Despite numerous improvement of supportive therapy, the mortality and morbidity among patients remain high. Therefore, exploring novel therapeutic options to treat AKI is mandatory. Numerous evidence in animal models has demonstrated the capability of mesenchymal stem cells (MSCs) to restore kidney function after induced kidney injury. After infusion, MSCs engraft in the injured tissue and release soluble factors and microvesicles that promote cell survival and tissue repairing. Indeed, the main mechanism of action of MSCs in tissue regeneration is the paracrine/endocrine secretion of bioactive molecules. MSCs can be isolated from several tissues, including bone marrow, adipose tissue, and blood cord; pre-treatment procedures to improve MSCs homing and their paracrine function have been also described. This review will focus on the application of cell therapy in AKI and it will summarize preclinical studies in animal models and clinical trials currently ongoing about the use of mesenchymal stem cells after AKI.     

Evidence of post-pollination barriers among three colour morphs of the deceptive orchid Dactylorhiza sambucina (L.) Soó

Floral-colour polymorphism in rewardless orchids has been hypothesized to be maintained by means of naïve insects, which after visiting a flower without reward will tend to fly elsewhere, looking for a flower of a different colour. In this study, levels of male and female reproductive success were monitored in Southern Italy populations of the deceptive orchid Dactylorhiza sambucina, through field observations over 3 years. These populations were characterized by the presence of a rare pink morph which is sympatric with the more frequent yellow and red morphs. In addition, final plant fertility was evaluated through percentages of embryo-containing seeds produced in both natural conditions and hand-pollination experiments. Results showed that pollinator preferences were independent of the morph frequencies and thus do not promote the predicted negative frequency-dependent selection. Although yellow and pink morphs showed significantly higher male reproductive success (RS), we found comparable levels of female RS, which suggest that pollinator behaviour cannot be the main mechanism which maintains this polymorphism. Interestingly, we found different percentages of embryo-containing seeds in fruits set under natural conditions as well as in those obtained from experimental crosses. In particular, pink morph showed a very low intrinsic fertility. Moreover, fertility in intra- was higher than in inter-morph crosses. To our knowledge, this is the first study pointing out the occurrence in the orchid family of post-pollination reproductive barriers. Findings are discussed in light of present hypothesis on the evolution and maintenance of colour polymorphism in deceptive orchids and other angiosperms.     

Study of Adhesion and Survival of Lactobacilli and Bifidobacteria on Table Olives with the Aim of Formulating a New Probiotic Food

With the aim of developing new functional foods, a traditional product, the table olive, was used as a vehicle for incorporating probiotic bacterial species. Survival on table olives of Lactobacillus rhamnosus (three strains), Lactobacillus paracasei (two strains), Bifidobacterium bifidum (one strain), and Bifidobacterium longum (one strain) at room temperature was investigated. The results obtained using a selected olive sample demonstrated that bifidobacteria and one strain of L. rhamnosus (Lactobacillus GG) showed a good survival rate, with a recovery of about 10⁶ CFU g⁻¹ after 30 days. The Lactobacillus GG population remained unvaried until the end of the experiment, while a slight decline (to about 10⁵ CFU g⁻¹) was observed for bifidobacteria. High viability, with more than 10⁷ CFU g⁻¹, was observed throughout the 3-month experiment for L. paracasei IMPC2.1. This strain, selected for its potential probiotic characteristics and for its lengthy survival on olives, was used to validate table olives as a carrier for transporting bacterial cells into the human gastrointestinal tract. L. paracasei IMPC2.1 was recovered from fecal samples in four out of five volunteers fed 10 to 15 olives per day carrying about 10⁹ to 10¹⁰ viable cells for 10 days.