BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.

PRMT1 acts as a suppressor of MHC-I and anti-tumor immunity.

Cancer immunotherapies have demonstrated remarkable success; however, the majority of patients do not respond or develop resistance. Here, we conduct epigenetic gene-targeted CRISPR-Cas9 screens to identify epigenomic factors that limit CD8+ T cell-mediated anti-tumor immunity. We identify that PRMT1 suppresses interferon gamma (Ifnγ)-induced MHC-I expression, thus dampening CD8+ T cell-mediated killing. Indeed, PRMT1 knockout or pharmacological targeting of type I PRMT with the clinical inhibitor GSK3368715 enhances Ifnγ-induced MHC-I expression through elevated STAT1 expression and activation, while re-introduction of PRMT1 in PRMT1-deficient cells reverses this effect. Importantly, loss of PRMT1 enhances the efficacy of anti-PD-1 immunotherapy, and The Cancer Genome Atlas analysis reveals that PRMT1 expression in human melanoma is inversely correlated with expression of human leukocyte antigen molecules, infiltration of CD8+ T cells, and overall survival. Taken together, we identify PRMT1 as a negative regulator of anti-tumor immunity, unveiling clinical type I PRMT inhibitors as immunotherapeutic agents or as adjuncts to existing immunotherapies.

High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production.

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3Δ/Δ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.

The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production.

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine-rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

ISFM guidelines on population management and welfare of unowned domestic cats (Felis catus).

UNLABELLED: GUIDELINES RATIONALE: Cats are among the most commonly kept domestic pets, and coexist with humans in a variety of different circumstances. Cats are sentient beings and, as such, humans have a responsibility for cat welfare where humans and cats coexist. Because cats reproduce efficiently, measures to control populations are frequently needed, but these should be based on ethical and humane approaches. FRAMEWORK: These consensus guidelines from the International Society of Feline Medicine's Welfare Advisory Panel provide a framework for the approach to welfare and population control measures, primarily among unowned cats and those going through a homing programme.

AAFP and ISFM feline environmental needs guidelines.

GUIDELINES RATIONALE: A cat's level of comfort with its environment is intrinsically linked to its physical health, emotional wellbeing and behavior. Having a basic understanding of the cat's species-specific environmental needs and how cats interact with their environment will provide a foundation for addressing these fundamental requirements. ENVIRONMENTAL NEEDS: Addressing environmental needs is essential (not optional) for optimum wellbeing of the cat. Environmental needs include those relating not only to the cat's physical surroundings (indoors or outdoors; in the home environment or at the veterinary practice) but also those affecting social interaction, including responses to human contact. FIVE 'PILLARS' FRAMEWORK: The authorship panel has organized the Guidelines around five primary concepts ('pillars') that provide the framework for a healthy feline environment. Understanding these principles and the unique environmental needs of the cat will help veterinarians, cat owners and care-givers to reduce stress, the incidence of stress-related disorders, and unwanted behavior in their feline patients and pets. The recommendations in the Guidelines apply to all pet cats, regardless of lifestyle.

The location and cellular composition of the hemopoietic stem cell niche.

While it is accepted that hemopoietic stem cells (HSC) are located in a three-dimensional microenvironment, termed a niche, the cellular and extracellular composition, as well as the multifaceted effects the components of the niche have on HSC regulation, remains undefined. Over the past four decades numerous advances in the field have led to the identification of roles for some cell types and propositions of potentially a number of HSC niches. We present evidence supporting the roles of multiple cell types and extracellular matrix molecules in the HSC niche, as well as discuss the potential significant overlap and intertwining of previously proposed distinct HSC niches.

The relationship between bone, hemopoietic stem cells, and vasculature.

A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly expressed on the blood vessel endothelium in the metaphysis. Analysis of the early stages of homing and the spatial dis-tribution of transplanted HSPCs at the single-cell level in mice devoid of Has3-synthesized HA, provides evidence for a previously undescribed role for HA expressed on endothelial cells in directing the homing of HSPCs to the metaphysis.

Environmental enrichment: practical strategies for improving feline welfare.

PRACTICAL RELEVANCE: The clinical application of evidence-based enrichment strategies for the domestic cat housed in a variety of confined environments, ranging from the veterinary cage to the domestic home, is of particular importance - both in relation to providing opportunity for appropriate feline behaviour, and in the prevention and treatment of behavioural and associated health problems (eg, feline lower urinary tract disease associated with negative emotional states such as generalised anxiety). Environmental enrichment has gained particular relevance in the light of current animal welfare legislation. For example, in the UK, the Animal Welfare Act 2006 stipulates that owners/keepers have a duty of care to their animal(s) that includes allowing the animal to exhibit normal behaviour patterns. EVIDENCE BASE: Research into environmental enrichment as a means of improving animal welfare is still very much in its infancy, particularly in relation to the domestic cat. Thus, evidence-based studies are somewhat sparse and more are needed to validate current recommended enrichment practices. AUDIENCE: This article aims to assist general veterinary practitioners to recognise how cats respond to confinement, and to understand what constitutes environmental enrichment, to help them implement or advise on appropriate enrichment strategies for cats confined in a hospital cage, home environment (particularly an indoor-only home), or cattery or rescue shelter, based on published evidence to date.

An innovative triple immunogold labeling method to investigate the hemopoietic stem cell niche in situ.

The ultrastructural study of rare cells within their niche in situ is very difficult. We have developed a method for locating individual transplanted cells and simultaneously identifying and analyzing the molecules and cellular phenotypes surrounding them in situ using transmission electron microscopy. This innovative method involves triple immunogold labeling combined with serial ultrathin sectioning. We demonstrate the validity of this approach by examining the niche of individual transplanted cells from a population highly enriched for hemopoietic stem cells and the ultrastructural expression of two key stem cell regulatory molecules, hyaluronic acid and osteopontin. In addition, we describe the phenotypes of the surrounding cells.