KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing.

Histone modifying enzymes play a central role in maintaining cell identity by establishing a conducive chromatin environment for lineage specific transcription factor activity. Pluripotent embryonic stem cell (ESC) identity is characterized by a lower abundance of gene repression associated histone modifications that enables rapid response to differentiation cues. The KDM3 family of histone demethylases removes the repressive histone H3 lysine 9 dimethylation (H3K9me2). Here we uncover a surprising role for the KDM3 proteins in the maintenance of the pluripotent state through post-transcriptional regulation. We find that KDM3A and KDM3B interact with RNA processing factors such as EFTUD2 and PRMT5. Acute selective degradation of the endogenous KDM3A and KDM3B proteins resulted in altered splicing independent of H3K9me2 status or catalytic activity. These splicing changes partially resemble the splicing pattern of the more blastocyst-like ground state of pluripotency and occurred in important chromatin and transcription factors such as Dnmt3b, Tbx3 and Tcf12. Our findings reveal non-canonical roles of histone demethylating enzymes in splicing to regulate cell identity.

Composite haemangioendothelioma with neuroendocrine marker differentiation presenting as a pink-brown nodule.

We report a case of a 55-year-old female with an asymptomatic pink-brown nodule. Histological examination demonstrated a composite haemangioendothelioma with positive synaptophysin staining.

Quantifying Deaths from Aspergillosis in HIV Positive People.

Aspergillus spp. are ubiquitous and cause severe infections in immunocompromised patients. Less is known about its incidence and prognosis in patients with HIV/AIDS. We reviewed the mortality of invasive aspergillosis in HIV/AIDS patients. Pubmed, Embase and Medline databases were used to search for articles. Studies were excluded if they contained other aspergillosis risk factors, no original or patient survival data or were not in English. From 747 articles published, 54 studies and case reports were identified following reading, published between 1985 and 2021, with 54% papers prior to 2000 reporting 853 patients from 16 countries, none from Africa. 707 (83%) patients died with an average time from diagnosis to death of 77.5 days. Postmortem diagnosis was seen in 21% of deaths recorded. A national series from France of 242 cases of invasive aspergillosis diagnosed in life recorded a 3 month mortality of 68% pre-ART, falling to 31% after introduction of ART and voriconazole. CD4 count was recorded in 39 studies and ranged from 2 to >1000 cells/mm3; only 8 patients (1.8%) had a CD4 > 100 cells/mm3. Aspergillosis occurs in patients with HIV/AIDS and associated with high mortality but its slow progression should allow diagnosis and treatment with improved outcome.

A conserved role for sleep in supporting Spatial Learning in Drosophila.

Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. Here we use the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a "thermal maze" consisting of a 5 × 5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains-Cs and 2U, validating our design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, we found that flies exhibited a dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, we find that Spatial Learning requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. Our results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging.

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila.

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. Here, we describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents >95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function.

Sleep restores place learning to the adenylyl cyclase mutant rutabaga.

Sleep plays an important role in regulating plasticity. In Drosophila, the relationship between sleep and learning and memory has primarily focused on mushroom body dependent operant-learning assays such as aversive phototaxic suppression and courtship conditioning. In this study, sleep was increased in the classic mutant rutabaga (rut2080) and dunce (dnc1) by feeding them the GABA-A agonist gaboxadol (Gab). Performance was evaluated in each mutant in response to social enrichment and place learning, tasks that do not require the mushroom body. Gab-induced sleep did not restore behavioral plasticity to either rut2080 or dnc1 mutants following social enrichment. However, increased sleep restored place learning to rut2080 mutants. These data extend the positive effects of enhanced sleep to place learning and highlight the utility of Gab for elucidating the beneficial effects of sleep on brain functioning.

Social determinants of mental disorders and the Sustainable Development Goals: a systematic review of reviews.

Mental health has been included in the UN Sustainable Development Goals. However, uncertainty exists about the extent to which the major social determinants of mental disorders are addressed by these goals. The aim of this study was to develop a conceptual framework for the social determinants of mental disorders that is aligned with the Sustainable Development Goals, to use this framework to systematically review evidence regarding these social determinants, and to identify potential mechanisms and targets for interventions. We did a systematic review of reviews using a conceptual framework comprising demographic, economic, neighbourhood, environmental events, and social and culture domains. We included 289 articles in the final Review. This study sheds new light on how the Sustainable Development Goals are relevant for addressing the social determinants of mental disorders, and how these goals could be optimised to prevent mental disorders.

Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes.

Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.

The tetraspanin CD63 is required for efficient IgE-mediated mast cell degranulation and anaphylaxis.

Mast cell (MC) activation through the high-affinity IgE receptor FcεRI leads to the release of mediators involved in immediate-type allergic reactions. Although Abs against the tetraspanins CD63 and CD81 inhibit FcεRI-induced MC degranulation, the intrinsic role of these molecules in FcεRI-induced MC activation is unknown. In MCs, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). In this study, we investigated the role of CD63 in MC using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo MC numbers and tissue distribution. Bone marrow-derived MC developed normally in the absence of CD63 protein. However, CD63-deficient bone marrow-derived MC showed a significant decrease in FcεRI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by ß-hexosaminidase release assays. The secretion of TNF-α, which is both released from granules and synthesized de novo upon MC activation, was also decreased. IL-6 secretion and production of the lipid mediator leukotriene C4 were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression, FcεRI-induced global tyrosine phosphorylation, and Akt phosphorylation. Finally, local reconstitution in genetically MC-deficient Kit(w/w-v) mice was unaffected by the absence of CD63. However, the sites reconstituted with CD63-deficient MC developed significantly attenuated cutaneous anaphylactic reactions. These findings demonstrate that the absence of CD63 results in a significant decrease of MC degranulation, which translates into a reduction of acute allergic reactions in vivo, thus identifying CD63 as an important component of allergic inflammation.

Vesicle-mediated secretion of human eosinophil granule-derived major basic protein.

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

Vascular bed-specific endothelium-dependent vasomomotor relaxation in the hagfish, Myxine glutinosa.

The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates that lived some time more than 500 million years ago. Features that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. We recently reported that hagfish endothelium displays phenotypic heterogeneity in ultrastructure, lectin binding, and mechanisms of leukocyte adhesion. Thus, phenotypic cell heterogeneity evolved as an early feature of the endothelium. In the present study, we wanted to extend these observations by determining whether hagfish endothelium plays a role in mediating vasomotor tone. Response of mesenteric and skeletal muscle arteries to a variety of mediators was assayed by videomicroscopy. Phenylephrine and acetylcholine induced vasoconstriction of mesenteric and skeletal muscle arteries. Bradykinin (BK) and ADP promoted vasorelaxation in precontracted mesenteric arteries but not those from skeletal muscle. BK- and ADP-mediated vasorelaxation of the mesenteric artery was abrogated by mechanical denudation of the endothelium but was unaffected by N(G)-nitro-L-arginine methyl ester. Indomethacin significantly inhibited the vasodilatory response to ADP but not BK. The nitric oxide donor sodium nitroprusside resulted in endothelium-independent relaxation of both mesenteric and skeletal muscle arteries. Together, these data suggest that site-specific endothelium-dependent vasorelaxation is an evolutionarily conserved property of this cell lineage.

Phenotypic heterogeneity is an evolutionarily conserved feature of the endothelium.

Mammalian endothelial cells (ECs) display marked phenotypic heterogeneity. Little is known about the evolutionary mechanisms underlying EC heterogeneity. The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates, which lived some time more than 500 million years ago. Features of ECs that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. The goal of this study was to determine whether the hagfish endothelium displays phenotypic heterogeneity. Electron microscopy of the aorta, dermis, heart, and liver revealed ultrastructural heterogeneity of the endothelium. Immunofluorescent studies demonstrated marked differences in lectin binding between vascular beds. Intravital microscopy of the dermis revealed histamine-induced adhesion of leukocytes in capillaries and postcapillary venules, but no such adhesion in arterioles. Together, these data suggest that structural, molecular, and functional heterogeneity of the endothelium evolved as an early feature of this cell lineage.

Loss of histochemical identity in mast cells lacking carboxypeptidase A.

Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa-/- mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa-/- peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa-/- peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa-/- mice. The Mc-cpa-/- mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.

Basophils produce IL-4 and accumulate in tissues after infection with a Th2-inducing parasite.

Using mice in which the eGfp gene replaced the first exon of the Il4 gene (G4 mice), we examined production of interleukin (IL)-4 during infection by the intestinal nematode Nippostrongylus brasiliensis (Nb). Nb infection induced green fluorescent protein (GFP)pos cells that were FcepsilonRIpos, CD49bbright, c-kitneg, and Gr1neg. These cells had lobulated nuclei and granules characteristic of basophils. They were found mainly in the liver and lung, to a lesser degree in the spleen, but not in the lymph nodes. Although some liver basophils from naive mice express GFP, Nb infection enhanced GFP expression and increased the number of tissue basophils. Similar basophil GFP expression was found in infected Stat6-/- mice. Basophils did not increase in number in infected Rag2-/- mice; Rag2-/- mice reconstituted with CD4 T cells allowed significant basophil accumulation, indicating that CD4 T cells can direct both tissue migration of basophils and enhanced IL-4 production. IL-4 production was immunoglobulin independent and only partially dependent on IL-3. Thus, infection with a parasite that induces a "Th2-type response" resulted in accumulation of tissue basophils, and these cells, stimulated by a non-FcR cross-linking mechanism, are a principal source of in vivo IL-4 production.

The case for extending storage and secretion functions of human mast cell granules to include synthesis.

Ultrastructural studies using standard procedures have for years indicated close associations of ribosomes and secretory granules in human mast cells. These descriptive studies have informed new studies, using established and new ultrastructural methods based on different principles, designed to investigate the possible role of RNA metabolism in secretory granules of human mast cells. In aggregate, these studies indicate human mast cell secretory granule associations with ribosomes, the protein synthetic machine of cells, with ribosomal proteins, with RNA, with poly(A)-positive mRNA and with various long-lived, or short-lived, uridine-rich, and poly(A)-poor RNA species with key roles in RNA processing and splicing. These studies indicate that secretory-storage granules in human mast cells may also be synthetic granules.