Metabolic Disturbances in the Gut-brain Axis of a Mouse Model of MPTP-induced Parkinsonism Evaluated by Nuclear Magnetic Resonance.

Parkinson's Disease is a synucleinopathy that primarily affects the dopaminergic cells of the central nervous system, leading to motor and gastrointestinal disturbances. However, intestinal peripheral neurons undergo a similar neurodegeneration process, marked by α-synuclein (αSyn) accumulation and loss of mitochondrial homeostasis. We investigated the metabolic alterations in different biometrics that compose the gut-brain axis (blood, brain, large intestine, and feces) in an MPTP-induced mouse model of sporadic Parkinson's Disease. Animals received escalating administration of MPTP. Tissues and fecal pellets were collected, and the metabolites were identified through the untargeted Nuclear Magnetic Resonance spectroscopic (1H NMR) technique. We found differences in many metabolites from all the tissues evaluated. The differential expression of metabolites in these samples mainly reflects inflammatory aspects, cytotoxicity, and mitochondrial impairment (oxidative stress and energy metabolism) in the animal model used. The direct evaluation of fecal metabolites revealed changes in several classes of metabolites. This data reinforces previous studies showing that Parkinson's disease is associated with metabolic perturbation not only in brain-related tissues, but also in periphery structures such as the gut. In addition, the evaluation of the microbiome and metabolites from gut and feces emerge as promising sources of information for understanding the evolution and progression of sporadic Parkinson's Disease.

Calcium signaling in chemotherapy-induced neuropathy.

Alterations in calcium (Ca2+) signaling is a major mechanism in the development of chemotherapy-induced peripheral neuropathy (CIPN), a side effect caused by multiple chemotherapy regimens. CIPN is associated with numbness and incessant tingling in hands and feet which diminishes quality of life during treatment. In up to 50% of survivors, CIPN is essentially irreversible. There are no approved, disease-modifying treatments for CIPN. The only recourse for oncologists is to modify the chemotherapy dose, a situation that can compromise optimal chemotherapy and impact patient outcomes. Here we focus on taxanes and other chemotherapeutic agents that work by altering microtubule assemblies to kill cancer cells, but also have off-target toxicities. There have been many molecular mechanisms proposed to explain the effects of microtubule-disrupting drugs. In neurons, an initiating step in the off-target effects of treatment by taxane is binding to neuronal calcium sensor 1 (NCS1), a sensitive Ca2+ sensor protein that maintains the resting Ca2+ concentration and dynamically enhances responses to cellular stimuli. The taxane/NCS1 interaction causes a Ca2+ surge that starts a pathophysiological cascade of consequences. This same mechanism contributes to other conditions including chemotherapy-induced cognitive impairment. Strategies to prevent the Ca2+ surge are the foundation of current work.

Protocol for rapid obtention and fractionation of anaerobic bacterial conditioned media to study calcium signaling in enteroendocrine cells.

Gut microbiota influences neurodevelopment, behavior and contributes to neurodegenerative disorders. One possible mechanism is the direct modulation of calcium (Ca2+) signaling and protein homeostasis in enteroendocrine cells (EECs), a component of the gut epithelium. Here, we present a protocol to isolate fractions of conditioned media (CM) from the anaerobic bacteria Akkermansia muciniphila and the utilization of this CM to monitor Ca2+ fluctuation in EECs by imaging. This protocol can be adapted and applied to various bacterial cultures and cell types. For complete details on the use and execution of this protocol, please refer to Amorim Neto et al. (2022).

Boosting antitumor response with PSMA-targeted immunomodulatory VLPs, harboring costimulatory TNFSF ligands and GM-CSF cytokine.

Therapeutic strategies based on immunomodulation have improved cancer therapy. Most approaches target co-stimulatory pathways or the inhibition of immunosuppressive mechanisms, to enhance immune response and overcome the immune tolerance of tumors. Here, we propose a novel platform to deliver targeted immunomodulatory signaling, enhancing antitumor response. The platform is based on virus-like particles derived from lentiviral capsids. These particles may be engineered to harbor multifunctional ligands on the surface that drive tropism to the tumor site and deliver immunomodulatory signaling, boosting the antitumor response. We generated virus-like particles harboring a PSMA-ligand, TNFSF co-stimulatory ligands 4-1BBL or OX40L, and a membrane-anchored GM-CSF cytokine. The virus-like particles are driven to PSMA-expressing tumors and deliver immunomodulatory signaling from the TNFSF surface ligands and the anchored GM-CSF, inducing T cell proliferation, inhibition of regulatory T cells, and potentiating elimination of tumor cells. The PSMA-targeted particles harboring immunomodulators enhanced antitumor activity in immunocompetent challenged mice and may be explored as a potential tool for cancer immunotherapy.

Akkermansia muciniphila induces mitochondrial calcium overload and α -synuclein aggregation in an enteroendocrine cell line.

The gut microbiota influence neurodevelopment, modulate behavior, and contribute to neurodegenerative disorders. Several studies have consistently reported a greater abundance of Akkermansia muciniphila in Parkinson disease (PD) fecal samples. Therefore, we investigated whether A.muciniphila-conditioned medium (CM) could initiate α-synuclein (αSyn) misfolding in enteroendocrine cells (EEC) - a component of the gut epithelium featuring neuron-like properties. We found that A. muciniphila CM composition is influenced by the ability of the strain to degrade mucin. Our in vitro experiments showed that the protein-enriched fraction of mucin-free CM induces RyR-mediated Ca2+ release and increased mitochondrial Ca2+ uptake leading to ROS generation and αSyn aggregation. Oral administration of A. muciniphila cultivated in the absence of mucin to mice led to αSyn aggregation in cholecystokinin (CCK)-positive EECs but no motor deficits were observed. Noteworthy, buffering mitochondrial Ca2+ reverted the damaging effects observed. These molecular insights offer evidence that bacterial proteins can induce αSyn aggregation in EECs.

Transcellular propagation of fibrillar α-synuclein from enteroendocrine to neuronal cells requires cell-to-cell contact and is Rab35-dependent.

Parkinson's disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca2+) oscillations on an extracellular Ca2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS.

Mass spectrometry-based proteomics of 3D cell culture: A useful tool to validate culture of spheroids and organoids.

Worldwide obesity, defined as abnormal or excessive fat accumulation that may result in different comorbidities, is considered a pandemic condition that has nearly tripled in the last 45 years. Most studies on obesity use animal models or adipocyte monolayer cell culture to investigate adipose tissue. However, besides monolayer cell culture approaches do not fully recapitulate the physiology of living organisms, there is a growing need to reduce or replace animals in research. In this context, the development of 3D self-organized structures has provided models that better reproduce the in vitro aspects of the in vivo physiology in comparison to traditional monolayer cell culture. Besides, recent advances in omics technologies have allowed us to characterize these cultures at the proteome, metabolome, transcription factor, DNA-binding and transcriptomic levels. These two combined approaches, 3D culture and omics, have provided more realistic data about determined conditions. Thereby, here we focused on the development of an obesity study pipeline including proteomic analysis to validate adipocyte-derived spheroids. Through the combination of collected mass spectrometry data from differentiated 3T3-L1 spheroids and from murine white adipose tissue (WAT), we identified 1732 proteins in both samples. By using a comprehensive proteomic analysis, we observed that the in vitro 3D culture of differentiated adipocytes shares important molecular pathways with the WAT, including expression of proteins involved in central metabolic process of the adipose tissue. Together, our results show a combination of an orthogonal method and an image-based analysis that constitutes a useful pipeline to be applied in 3D adipocyte culture.

Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X.

Current studies estimate that 1-3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.

Alkaloid and acetogenin-rich fraction from Annona crassiflora fruit peel inhibits proliferation and migration of human liver cancer HepG2 cells.

Plant species from Annonaceae are commonly used in traditional medicine to treat various cancer types. This study aimed to investigate the antiproliferative potential of an alkaloid and acetogenin-rich fraction from the fruit peel of Annona crassiflora in HepG2 cells. A liquid-liquid fractionation was carried out on the ethanol extract of A. crassiflora fruit peel in order to obtain an alkaloid and acetogenin-rich fraction (AF-Ac). Cytotoxicity, proliferation and migration were evaluated in the HepG2 cells, as well as the proliferating cell nuclear antigen (PCNA), vinculin and epidermal growth factor receptor (EGFR) expression. In addition, intracellular Ca2+ was determined using Fluo4-AM and fluorescence microscopy. First, 9 aporphine alkaloids and 4 acetogenins that had not yet been identified in the fruit peel of A. crassiflora were found in AF-Ac. The treatment with 50 µg/mL AF-Ac reduced HepG2 cell viability, proliferation and migration (p < 0.001), which is in accordance with the reduced expression of PCNA and EGFR levels (p < 0.05). Furthermore, AF-Ac increased intracellular Ca2+ in the HepG2 cells, mobilizing intracellular calcium stores, which might be involved in the anti-migration and anti-proliferation capacities of AF-Ac. Our results support the growth-inhibitory potential of AF-Ac on HepG2 cells and suggest that this effect is triggered, at least in part, by PCNA and EGFR modulation and mobilization of intracellular Ca2+. This study showed biological activities not yet described for A. crassiflora fruit peel, which provide new possibilities for further in vivo studies to assess the antitumoral potential of A. crassiflora, especially its fruit peel.

Oxygen Generation via Water Splitting by a Novel Biogenic Metal Ion-Binding Compound.

Methanobactins (MBs) are small (<1,300-Da) posttranslationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some being reduced after binding, e.g., Cu2+ reduced to Cu+. Other metal ions, however, are bound but not reduced, e.g., K+. The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H218O, we show that when MBs from Methylocystis sp. strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au3+, Cu2, or Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed in the presence of either MB-SB2 or MB-OB3b alone, gold alone, copper alone, or silver alone or when K+ or Mo2+ was incubated with MB-SB2. In contrast to MB-OB3b, MB-SB2 binds Fe3+ with an N2S2 coordination and will also reduce Fe3+ to Fe2+. Iron reduction was also found to be coupled to the oxidation of 2H2O and the generation of O2. MB-SB2 will also couple Hg2+, Ni2+, and Co2+ reduction to the oxidation of 2H2O and the generation of O2, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions. To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate that O2 generation from metal ion reduction by MB-OB3b can support methane oxidation. IMPORTANCE The discovery that MB will couple the oxidation of H2O to metal ion reduction and the release of O2 suggests that methanotrophs expressing MB may be able to maintain their activity under hypoxic/anoxic conditions through the "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere.

Illuminating the Brain With X-Rays: Contributions and Future Perspectives of High-Resolution Microtomography to Neuroscience.

The assessment of three-dimensional (3D) brain cytoarchitecture at a cellular resolution remains a great challenge in the field of neuroscience and constant development of imaging techniques has become crucial, particularly when it comes to offering direct and clear obtention of data from macro to nano scales. Magnetic resonance imaging (MRI) and electron or optical microscopy, although valuable, still face some issues such as the lack of contrast and extensive sample preparation protocols. In this context, x-ray microtomography (µCT) has become a promising non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens. It is a new supplemental method to be explored for deciphering the cytoarchitecture and connectivity of the brain. This review aims to bring together published works using x-ray µCT in neurobiology in order to discuss the achievements made so far and the future of this technique for neuroscience.

Inositol 1,4,5-trisphosphate receptor type 3 plays a protective role in hepatocytes during hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury is seen in a variety of clinical conditions, including hepatic thrombosis, systemic hypotension, and liver transplantation. Calcium (Ca2+) signaling mediates several pathophysiological processes in the liver, but it is not known whether and how intracellular Ca2+ channels are involved in the hepatocellular events secondary to ischemia-reperfusion. Using an animal model of hepatic ischemia-reperfusion injury, we observed a progressive increase in expression of the type 3 isoform of the inositol trisphosphate receptor (ITPR3), an intracellular Ca2+ channel that is not normally expressed in healthy hepatocytes. ITPR3 expression was upregulated, at least in part, by a combination of demethylation of the ITPR3 promoter region and the increased transcriptional activity of the nuclear factor of activated T-cells (NFAT). Additionally, expression of pro-inflammatory interleukins and necrotic surface area were less pronounced in livers of control animals compared to liver-specific ITPR3 KO mice subjected to hepatic damage. Corroborating these findings, ITPR3 expression and activation of NFAT were observed in hepatocytes of liver biopsies from patients who underwent liver ischemia caused by thrombosis after organ transplant. Together, these results are consistent with the idea that ITPR3 expression in hepatocytes plays a protective role during hepatic injury induced by ischemia-reperfusion.

Molecular Mechanism for Protection Against Liver Failure in Human Yellow Fever Infection.

Yellow fever (YF) is a viral hemorrhagic fever that typically involves the liver. Brazil recently experienced its largest recorded YF outbreak, and the disease was fatal in more than a third of affected individuals, mostly because of acute liver failure. Affected individuals are generally treated only supportively, but during the recent Brazilian outbreak, selected patients were treated with liver transplant. We took advantage of this clinical experience to better characterize the clinical and pathological features of YF-induced liver failure and to examine the mechanism of hepatocellular injury in YF, to identify targets that would be amenable to therapeutic intervention in preventing progression to liver failure and death. Patients with YF liver failure rapidly developed massive transaminase elevations, with jaundice, coagulopathy, thrombocytopenia, and usually hepatic encephalopathy, along with pathological findings that included microvesicular steatosis and lytic necrosis. Hepatocytes began to express the type 3 isoform of the inositol trisphosphate receptor (ITPR3), an intracellular calcium (Ca2+) channel that is not normally expressed in hepatocytes. Experiments in an animal model, isolated hepatocytes, and liver-derived cell lines showed that this new expression of ITPR3 was associated with increased nuclear Ca2+ signaling and hepatocyte proliferation, and reduced steatosis and cell death induced by the YF virus. Conclusion: Yellow fever often induces liver failure characterized by massive hepatocellular damage plus steatosis. New expression of ITPR3 also occurs in YF-infected hepatocytes, which may represent an endogenous protective mechanism that could suggest approaches to treat affected individuals before they progress to liver failure, thereby decreasing the mortality of this disease in a way that does not rely on the costly and limited resource of liver transplantation.

The influence of hypertensive environment on adipose tissue remodeling measured by fluorescence lifetime imaging in spontaneously hypertensive rats.

There is a lack of information correlating low adiposity with hypertension experienced by Spontaneous Hypertensive Rats (SHR) or overweight and normotension in Wistar-Kyoto (WKY). We aimed to investigate this lipodystrophy phenomenon by measuring fluorescence lifetime (FLIM), optical redox ratio (ORR), serum levels of hypothalamic-pituitary-adrenal (HPA) and/or hypothalamic-pituitary-thyroid (HPT) hormones axes between Wistar, WKY and SHR before and after establishment of hypertension. Under high blood pressure, we evaluated serum adipokines. Brown adipose tissue was characterized as lower ORR and shorter FLIM compared to white adipose tissue. HPT axis showed a crucial role in the SHR adipose tissue configuration by attenuating whitening. The increased adiposity in WKY may act as a preventive agent for hypertension, since SHR, with low adiposity, establishes the disease. The hypertensive environment can highlight key adipokines that may result in new therapeutic approaches to the treatment of adiposity dysfunctions and hypertension.

Decreased nuclear distribution nudE-like 1 enzyme activity in an animal model with dysfunctional disrupted-in-schizophrenia 1 signaling featuring aberrant neurodevelopment and amphetamine-supersensitivity.

BACKGROUND: Interaction of nuclear-distribution element-like 1 with disrupted-in-schizophrenia 1 protein is crucial for neurite outgrowth/neuronal migration, and this interaction competitively inhibits nuclear-distribution element-like 1 peptidase activity. Nuclear-distribution element-like 1 activity is reduced in antipsychotic-naïve first-episode psychosis and in medicated chronic schizophrenia, with even lower activity in treatment-resistant schizophrenia. AIMS: The purpose of this study was to investigate in a rat model overexpressing human non-mutant disrupted-in-schizophrenia 1, with consequent dysfunctional disrupted-in-schizophrenia 1 signaling, the relation of nuclear-distribution element-like 1 activity with neurodevelopment and dopamine-related phenotypes. METHODS: We measured cell distribution in striatum and cortex by histology and microtomography, and quantified the basal and amphetamine-stimulated locomotion and nuclear-distribution element-like 1 activity (in blood and brain) of transgenic disrupted-in-schizophrenia 1 rat vs wild-type littermate controls. RESULTS: 3D assessment of neuronal cell body number and spatial organization of mercury-impregnated neurons showed defective neuronal positioning, characteristic of impaired cell migration, in striatum/nucleus accumbens, and prefrontal cortex of transgenic disrupted-in-schizophrenia 1 compared to wild-type brains. Basal nuclear-distribution element-like 1 activity was lower in the blood and also in several brain regions of transgenic disrupted-in-schizophrenia 1 compared to wild-type. Locomotion and nuclear-distribution element-like 1 activity were both significantly increased by amphetamine in transgenic disrupted-in-schizophrenia 1, but not in wild-type. CONCLUSIONS: Our findings in the transgenic disrupted-in-schizophrenia 1 rat allow us to state that decreased nuclear-distribution element-like 1 activity reflects both a trait (neurodevelopmental phenotype) and a state (amphetamine-induced dopamine release). We thus define here a role for decreased nuclear-distribution element-like 1 peptidase activity both for the developing brain (the neurodevelopmental phenotype) and for the adult (interaction with dopaminergic responses), and present nuclear-distribution element-like 1 activity in a novel way, as unifying neurodevelopmental with dysfunctional dopamine response phenotypes.

Desconhecido perigoso: a relação entre o adolescente ofensor sexual e sua mãe / Dangerous uknown: the relationship between the adolescent sexual offender and his mother

Este texto tem objetivo descritivo e exploratório sobre a relação entre o adolescente ofensor sexual e sua mãe, visto que o funcionamento familiar tem grande importância no comportamento ofensivo e deve ser foco de avaliação e intervenção. A partir do referencial teórico sistêmico, foi realizado um estudo de caso qualitativo por meio de análise documental, tendo como base os registros e materiais produzidos em um atendimento terapêutico familiar. Foram observadas dinâmicas familiares transgeracionais, vinculação afetiva distante e de rejeição entre a mãe e filho, inexistência da figura paterna, experiências traumáticas vividas pelo adolescente e uma estrutura familiar rígida e desmembrada. Os resultados apresentaram relação com a produção científica e corroboram a necessidade de intervenção tanto com o adolescente que comete abuso sexual, como com sua família.

This text has a descriptive and exploratory objective on the relationship between the adolescent sexual offender and his mother, since the familiar functioning is very important in the offensive behavior and should be focus of evaluation and intervention. From the systemic theoretical framework, a qualitative case study was carried out based on the records and materials produced in a family therapy service. Transgenerational family dynamics, distant affective attachment and rejection between mother and child, lack of paternal figure, traumatic experiences experienced by the adolescent and a rigid and dismembered family structure were observed. The results show the relationship with the scientific production and corroborate the need for intervention both with the adolescent who commits sexual abuse, and with his family.

Comparative analysis of sample preparation protocols of soft biological tissues for morphometric studies using synchrotron-based X-ray microtomography.

The spread of microtomography as a tool for visualization of soft tissues has had a significant impact on a better understanding of complex biological systems. This technique allows a detailed three-dimensional quantitative view of the specimen to be obtained, correlating its morphological organization with its function, providing valuable insights on the functionality of the tissue. Regularly overlooked, but of great importance, proper sample mounting and preparation are fundamental for achieving the highest possible image quality even for the high-resolution imaging systems currently under development. Here, a quantitative analysis compares some of the most common sample-mounting strategies used for synchrotron-based X-ray microtomography of soft tissues: alcoholic-immersion, paraffin-embedding and critical-point drying. These three distinct sample-mounting strategies were performed on the same specimen in order to investigate their impact on sample morphology regardless of individual sample variation. In that sense, the alcoholic-immersion strategy, although causing less shrinkage to the tissue, proved to be the most unsuitable approach for a high-throughput high-resolution imaging experiment due to sample drifting. Also, critical-point drying may present some interesting advantages regarding image quality but is also incompatible with a high-throughput experiment. Lastly, paraffin-embedding is shown to be the most suitable strategy for current soft tissue microtomography experiments. Such detailed analysis of biological sample-mounting strategies for synchrotron-based X-ray microtomography are expected to offer valuable insights on the best approach for using this technique for 3D imaging of soft tissues and following morphometric analysis.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Expression of the type 3 InsP3 receptor is a final common event in the development of hepatocellular carcinoma.

BACKGROUND & OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC. DESIGN: Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice. RESULTS: ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis. CONCLUSIONS: These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.