Synaptotagmin-2 Is Essential for Survival and Contributes to Ca²⁺ Triggering of Neurotransmitter Release in Central and Neuromuscular Synapses

Biochemical and genetic data suggest that synaptotagmin-2 functions as a Ca²⁺ sensor for fast neurotransmitter release in caudal brain regions, but animals and/or synapses lacking synaptotagmin-2 have not been examined. We have now generated mice in which the 5’ end of the synaptotagmin-2 gene was replaced by lacZ. Using β-galactosidase as a marker, we show that, consistent with previous studies, synaptotagmin-2 is widely expressed in spinal cord, brainstem, and cerebellum, but is additionally present in selected forebrain neurons, including most striatal neurons and some hypothalamic, cortical, and hippocampal neurons. Synaptotagmin-2-deficient mice were indistinguishable from wild-type littermates at birth, but subsequently developed severe motor dysfunction, and perished at ~3 weeks of age. Electrophysiological studies in cultured striatal neurons revealed that the synaptotagmin-2 deletion slowed the kinetics of evoked neurotransmitter release without altering the total amount of release. In contrast, synaptotagmin-2-deficient neuromuscular junctions (NMJs) suffered from a large reduction in evoked release and changes in short-term synaptic plasticity. Furthermore, in mutant NMJs, the frequency of spontaneous miniature release events was increased both at rest and during stimulus trains. Viewed together, our results demonstrate that the synaptotagmin-2 deficiency causes a lethal impairment in synaptic transmission in selected synapses. This impairment, however, is less severe than that produced in forebrain neurons by deletion of synaptotagmin-1, presumably because at least in NMJs, synaptotagmin-1 is coexpressed with synaptotagmin-2, and both together mediate fast Ca²⁺-triggered release. Thus, synaptotagmin-2 is an essential synaptotagmin isoform that functions in concert with other synaptotagmins in the Ca²⁺ triggering of neurotransmitter release

Diagnosis of invasive aspergillus tracheobronchitis facilitated by endobronchial ultrasound-guided transbronchial needle aspiration: a case report

<p>Abstract</p> <p>Introduction</p> <p>Invasive pulmonary aspergillosis is the most common form of infection by <it>Aspergillus species </it>among immunocompromised patients. Although this infection frequently involves the lung parenchyma, it is unusual to find it limited to the tracheobronchial tree, a condition known as invasive aspergillus tracheobronchitis.</p> <p>Case presentation</p> <p>A 65 year-old Hispanic man from Bolivia with a history of chronic lymphocytic leukemia developed cough and malaise eight months after having an allogenic stem cell transplant. A computed tomography of the chest revealed an area of diffuse soft tissue thickening around the left main stem bronchus, which was intensely fluorodeoxyglucose-avid on positron emission tomography scanning. An initial bronchoscopic exam revealed circumferential narrowing of the entire left main stem bronchus with necrotic and friable material on the medial wall. Neither aspirates from this necrotic area nor bronchial washing were diagnostic. A second bronchoscopy with endobronchial ultrasound evidenced a soft tissue thickening on the medial aspect of the left main stem bronchus underlying the area of necrosis visible endoluminally. Endobronchial ultrasound-guided transbronchial needle aspiration performed in this area revealed multiple fungal elements suggestive of <it>Aspergillus species</it>.</p> <p>Conclusion</p> <p>We describe the first case of invasive aspergillus tracheobronchitis in which the diagnosis was facilitated by the use of endobronchial ultrasound guided trans-bronchial needle aspiration. To the best of our knowledge, we are also presenting the first positron emission tomography scan images of this condition in the literature. We cautiously suggest that endobronchial ultrasound imaging may be a useful tool to evaluate the degree of invasion and the involvement of vascular structures in these patients prior to bronchoscopic manipulation of the affected areas in an effort to avoid potentially fatal hemorrhage.</p

Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by∼50%. Homozygous mutant mice were not viable, but heterozygotes showed a ∼50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxiswas also reduced by ∼50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identifyMunc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs

Transcranial direct current stimulation (tDCS) reverts behavioral alterations and brainstem BDNF level increase induced by neuropathic pain model: Long-lasting effect

AbstractIntroductionNeuropathic pain (NP) is a chronic pain modality that usually results of damage in the somatosensory system. NP often shows insufficient response to classic analgesics and remains a challenge to medical treatment. The transcranial direct current stimulation (tDCS) is a non-invasive technique, which induces neuroplastic changes in central nervous system of animals and humans. The brain derived neurotrophic factor plays an important role in synaptic plasticity process. Behavior changes such as decreased locomotor and exploratory activities and anxiety disorders are common comorbidities associated with NP.ObjectiveEvaluate the effect of tDCS treatment on locomotor and exploratory activities, and anxiety-like behavior, and peripheral and central BDNF levels in rats submitted to neuropathic pain model.MethodsRats were randomly divided: Ss, SsS, SsT, NP, NpS, and NpT. The neuropathic pain model was induced by partial sciatic nerve compression at 14days after surgery; the tDCS treatment was initiated. The animals of treated groups were subjected to a 20minute session of tDCS, for eight days. The Open Field and Elevated Pluz Maze tests were applied 24h (phase I) and 7days (phase II) after the end of tDCS treatment. The serum, spinal cord, brainstem and cerebral cortex BDNF levels were determined 48h (phase I) and 8days (phase II) after tDCS treatment by ELISA.ResultsThe chronic constriction injury (CCI) induces decrease in locomotor and exploratory activities, increases in the behavior-like anxiety, and increases in the brainstem BDNF levels, the last, in phase II (one-way ANOVA/SNK, P<0.05 for all). The tDCS treatment already reverted all these effects induced by CCI (one-way ANOVA/SNK, P<0.05 for all). Furthermore, the tDCS treatment decreased serum and cerebral cortex BDNF levels and it increased these levels in the spinal cord in phase II (one-way ANOVA/SNK, P<0.05).ConclusiontDCS reverts behavioral alterations associated to neuropathic pain, indicating possible analgesic and anxiolytic tDCS effects. tDCS treatment induces changes in the BDNF levels in different regions of the central nervous system (CNS), and this effect can be attributed to different cellular signaling activations

Interstitial Lung Abnormalities After Hospitalization for Covid-19 in Patients With Cancer: A Prospective Cohort Study

INTRODUCTION: Survivors of SARS-CoV-2 pneumonia often develop persistent respiratory symptom and interstitial lung abnormalities (ILAs) after infection. Risk factors for ILA development and duration of ILA persistence after SARS-CoV-2 infection are not well described in immunocompromised hosts, such as cancer patients. METHODS: We conducted a prospective cohort study of 95 patients at a major cancer center and 45 patients at a tertiary referral center. We collected clinical and radiographic data during the index hospitalization for COVID-19 pneumonia and measured pneumonia severity using a semi-quantitative radiographic score, the Radiologic Severity Index (RSI). Patients were evaluated in post-COVID-19 clinics at 3 and 6 months after discharge and underwent comprehensive pulmonary evaluations (symptom assessment, chest computed tomography, pulmonary function tests, 6-min walk test). The association of clinical and radiological factors with ILAs at 3 and 6 months post-discharge was measured using univariable and multivariable logistic regression. RESULTS: Sixty-six (70%) patients of cancer cohort had ILAs at 3 months, of whom 39 had persistent respiratory symptoms. Twenty-four (26%) patients had persistent ILA at 6 months after hospital discharge. In adjusted models, higher peak RSI at admission was associated with ILAs at 3 (OR 1.5 per 5-point increase, 95% CI 1.1-1.9) and 6 months (OR 1.3 per 5-point increase, 95% CI 1.1-1.6) post-discharge. Fibrotic ILAs (reticulation, traction bronchiectasis, and architectural distortion) were more common at 6 months post-discharge. CONCLUSIONS: Post-COVID-19 ILAs are common in cancer patients 3 months after hospital discharge, and peak RSI and older age are strong predictors of persistent ILAs

Kupffer Cells Hasten Resolution of Liver Immunopathology in Mouse Models of Viral Hepatitis

Kupffer cells (KCs) are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV)-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1) protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology

Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis