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1.
Proc Natl Acad Sci U S A ; 121(42): e2409636121, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39374398

RESUMO

The Ca2+ sensor synaptotagmin-1 (Syt1) triggers neurotransmitter release together with the neuronal sensitive factor attachment protein receptor (SNARE) complex formed by syntaxin-1, SNAP25, and synaptobrevin. Moreover, Syt1 increases synaptic vesicle (SV) priming and impairs spontaneous vesicle release. The Syt1 C2B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of Syt1 and the mechanism underlying Ca2+ triggering of release are unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: Binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca2+-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of Syt1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of Syt1 in vesicle priming and clamping of spontaneous release and, importantly, show that Ca2+ triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with biophysical studies presented in [K. Jaczynska et al., bioRxiv [Preprint] (2024). https://doi.org/10.1101/2024.06.17.599417 (Accessed 18 June 2024)], our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast SV fusion.


Assuntos
Cálcio , Neurotransmissores , Proteínas SNARE , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Cálcio/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Neurotransmissores/metabolismo , Sintaxina 1/metabolismo , Sintaxina 1/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Ratos , Ligação Proteica , Transmissão Sináptica
2.
Can Vet J ; 64(7): 659-665, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37397695

RESUMO

Objective: To report on operative technique and outcomes following prophylactic total laparoscopic gastropexy (PTLG) using a novel knotless tissue control device (KTCD) in dogs. Animals: This study included 44 dogs. Procedure: Medical records were reviewed, and perioperative data were collected. Right-sided incisional gastropexy was performed using 2 strands of KTCD introduced through a 12-millimeter cannula in a single-incision multi-channeled port. Dog owners were contacted to obtain outcome data. Results: Median age and weight of dogs were 17 mo (6 to 60 mo) and 48.5 kg (14 to 73.3 kg). Median surgical and anesthesia times were 90 min (60 to 150 min) and 195 min (135 to 270 min). Major intraoperative complications were not reported. Follow-up data were available for 40/44 (91%) dogs. Median follow-up time was 522 d (43 to 983 d). Gastric dilatation volvulus (GDV) was not reported in any dog. One dog developed suspected colonic entrapment around the gastropexy that required surgical revision. All owners were satisfied with the procedure and indicated they would repeat the procedure with future pets. Conclusion: The PTLG procedure using novel KTCD in this cohort of dogs was effective at preventing GDV for the duration of follow-up and was associated with low perioperative complication rate and high owner satisfaction. Clinical relevance: This retrospective study reports on operative technique and outcomes associated with KTCD use in PTLG. Our findings warrant prospective evaluation of KTCD use in PTLG.


Caractéristiques peropératoires et résultats à long terme après une gastropexie laparoscopique totale prophylactique à l'aide d'un nouveau dispositif de contrôle des tissus sans nœuds chez 44 chiens. Objectif: Rendre compte de la technique opératoire et des résultats après une gastropexie laparoscopique totale prophylactique (PTLG) à l'aide d'un nouveau dispositif de contrôle des tissus sans nœuds (KTCD) chez le chien. Animaux: Cette étude a inclus 44 chiens. Procédure: Les dossiers médicaux ont été examinés et les données peropératoires ont été recueillies. La gastropexie incisionnelle du côté droit a été réalisée à l'aide de 2 brins de KTCD introduits par une canule de 12 millimètres dans un orifice multicanal à incision unique. Les propriétaires de chiens ont été contactés pour obtenir des données sur les résultats. Résultats: L'âge et le poids médians des chiens étaient de 17 mois (6 à 60 mois) et 48,5 kg (14 à 73,3 kg). Les temps chirurgicaux et anesthésiques médians étaient de 90 min (60 à 150 min) et 195 min (135 à 270 min). Des complications peropératoires majeures n'ont pas été rapportées. Des données de suivi étaient disponibles pour 40/44 (91 %) chiens. La durée médiane de suivi était de 522 jours (43 à 983 jours). Le volvulus de dilatation gastrique (GDV) n'a été signalé chez aucun chien. Un chien a développé une suspicion de piégeage du côlon autour de la gastropexie qui a nécessité une reprise chirurgicale. Tous les propriétaires étaient satisfaits de la procédure et ont indiqué qu'ils répéteraient la procédure avec de futurs animaux de compagnie. Conclusion: La procédure PTLG utilisant le nouveau KTCD dans cette cohorte de chiens a été efficace pour prévenir le GDV pendant la durée du suivi et a été associée à un faible taux de complications peropératoires et à une satisfaction élevée des propriétaires. Pertinence clinique: Cette étude rétrospective rend compte de la technique opératoire et des résultats associés à l'utilisation de KTCD dans le PTLG. Nos résultats justifient une évaluation prospective de l'utilisation de KTCD dans le PTLG.(Traduit par Dr Serge Messier).


Assuntos
Doenças do Cão , Dilatação Gástrica , Gastropexia , Laparoscopia , Volvo Gástrico , Cães , Animais , Gastropexia/veterinária , Gastropexia/métodos , Estudos Retrospectivos , Doenças do Cão/prevenção & controle , Doenças do Cão/cirurgia , Dilatação Gástrica/veterinária , Volvo Gástrico/prevenção & controle , Volvo Gástrico/cirurgia , Volvo Gástrico/veterinária , Laparoscopia/veterinária
3.
Plant Cell Physiol ; 57(5): 919-32, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26903527

RESUMO

The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs. GDC H- and P-proteins were undetectable in leaves of gdch lines. Plants exhibited a photorespiratory-deficient phenotype with stunted growth, accelerated leaf senescence, reduced chlorophyll, soluble protein and sugars, and increased glycine accumulation in leaves. Gas exchange measurements indicated an impaired ability to regenerate ribulose 1,5-bisphosphate in photorespiratory conditions. In addition, MCs of gdch lines exhibited a significant reduction in chloroplast area and coverage of the cell wall when grown in air, traits that occur during the later stages of C4 evolution. The presence of these two traits important for C4 photosynthesis and the non-lethal, down-regulation of the photorespiratory C2 cycle positively contribute to efforts to produce a C4 rice prototype.


Assuntos
Regulação da Expressão Gênica de Plantas , Complexo Glicina Descarboxilase/metabolismo , Oryza/genética , Fotossíntese , Ciclo do Carbono , Respiração Celular , Cloroplastos/metabolismo , Técnicas de Silenciamento de Genes , Complexo Glicina Descarboxilase/genética , Luz , MicroRNAs/genética , Oryza/enzimologia , Oryza/fisiologia , Oryza/efeitos da radiação , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo
4.
Elife ; 122024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38512129

RESUMO

The SNARE proteins are central in membrane fusion and, at the synapse, neurotransmitter release. However, their involvement in the dual regulation of the synchronous release while maintaining a pool of readily releasable vesicles remains unclear. Using a chimeric approach, we performed a systematic analysis of the SNARE domain of STX1A by exchanging the whole SNARE domain or its N- or C-terminus subdomains with those of STX2. We expressed these chimeric constructs in STX1-null hippocampal mouse neurons. Exchanging the C-terminal half of STX1's SNARE domain with that of STX2 resulted in a reduced RRP accompanied by an increased release rate, while inserting the C-terminal half of STX1's SNARE domain into STX2 leads to an enhanced priming and decreased release rate. Additionally, we found that the mechanisms for clamping spontaneous, but not for Ca2+-evoked release, are particularly susceptible to changes in specific residues on the outer surface of the C-terminus of the SNARE domain of STX1A. Particularly, mutations of D231 and R232 affected the fusogenicity of the vesicles. We propose that the C-terminal half of the SNARE domain of STX1A plays a crucial role in the stabilization of the RRP as well as in the clamping of spontaneous synaptic vesicle fusion through the regulation of the energetic landscape for fusion, while it also plays a covert role in the speed and efficacy of Ca2+-evoked release.


Assuntos
Fusão de Membrana , Vesículas Sinápticas , Sintaxina 1 , Animais , Camundongos , Constrição , Camundongos Knockout , Neurotransmissores , Proteínas SNARE , Sintaxina 1/genética
5.
bioRxiv ; 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38948868

RESUMO

The Ca2+ sensor synaptotagmin-1 triggers neurotransmitter release together with the neuronal SNARE complex formed by syntaxin-1, SNAP25 and synaptobrevin. Moreover, synaptotagmin-1 increases synaptic vesicle priming and impairs spontaneous vesicle release. The synaptotagmin-1 C2B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of synaptotagmin-1, and the mechanism underlying Ca2+-triggering of release is unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca2+-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of synaptotagmin-1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of synaptotagmin-1 in vesicle priming and clamping of spontaneous release, and, importantly, show that Ca2+-triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with modeling and biophysical studies presented in the accompanying paper, our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast synaptic vesicle fusion.

6.
Elife ; 112022 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-35638903

RESUMO

SNAREs are undoubtedly one of the core elements of synaptic transmission. Contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations in cultured mouse hippocampal neurons: (1) elongation of STX1A's JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; (2) charge reversal mutations in STX1A's JMD; and (3) palmitoylation deficiency mutations in STX1A's TMD. We found that both JMD elongations and charge reversal mutations have position-dependent differential effects on Ca2+-evoked and spontaneous neurotransmitter release. Importantly, we show that STX1A's JMD regulates the palmitoylation of STX1A's TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1A's JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.


Assuntos
Lipoilação , Fusão de Membrana , Animais , Mamíferos/metabolismo , Fusão de Membrana/fisiologia , Camundongos , Neurônios/fisiologia , Proteínas SNARE/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo
7.
Elife ; 102021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34427183

RESUMO

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex through two major binding modes: through STX1's N-peptide and through STX1's closed conformation driven by its Habc- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.


Assuntos
Proteínas Munc18/metabolismo , Neurônios/metabolismo , Peptídeos/metabolismo , Sinapses/metabolismo , Sintaxina 1/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Fusão de Membrana , Camundongos , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Conformação Proteica , Sinapses/genética , Transmissão Sináptica , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo , Sintaxina 1/química , Sintaxina 1/genética
8.
J Clin Med ; 9(10)2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977606

RESUMO

This study aimed to build an easily applicable prognostic model based on routine clinical, radiological, and laboratory data available at admission, to predict mortality in coronavirus 19 disease (COVID-19) hospitalized patients. METHODS: We retrospectively collected clinical information from 1968 patients admitted to a hospital. We built a predictive score based on a logistic regression model in which explicative variables were discretized using classification trees that facilitated the identification of the optimal sections in order to predict inpatient mortality in patients admitted with COVID-19. These sections were translated into a score indicating the probability of a patient's death, thus making the results easy to interpret. RESULTS: Median age was 67 years, 1104 patients (56.4%) were male, and 325 (16.5%) died during hospitalization. Our final model identified nine key features: age, oxygen saturation, smoking, serum creatinine, lymphocytes, hemoglobin, platelets, C-reactive protein, and sodium at admission. The discrimination of the model was excellent in the training, validation, and test samples (AUC: 0.865, 0.808, and 0.883, respectively). We constructed a prognostic scale to determine the probability of death associated with each score. CONCLUSIONS: We designed an easily applicable predictive model for early identification of patients at high risk of death due to COVID-19 during hospitalization.

9.
Neuron ; 100(5): 1097-1115.e15, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30392800

RESUMO

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.


Assuntos
Polaridade Celular , Córtex Cerebral/crescimento & desenvolvimento , MicroRNAs/fisiologia , Neurônios/fisiologia , Fatores de Transcrição SOX9/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Axônios/fisiologia , Córtex Cerebral/citologia , Dendritos/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Knockout , MicroRNAs/genética , Neurônios/citologia , Fatores de Transcrição SOX9/genética , Ubiquitina-Proteína Ligases/genética
10.
J Neurosci Methods ; 291: 113-121, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28782628

RESUMO

BACKGROUND: Generation of different neuronal subtypes during neocortical development is the most important step in the establishment of cortical cytoarchitecture. The transcription factor Satb2 is expressed in neocortical projection neurons that send their axons intracortically as opposed to Satb2-negative neurons that preferentially project to subcortical targets. NEW METHOD: In this report, we present a novel method to carry out large scale screening for molecules that control cell fate in the developing neocortex. It is based on a Satb2Cre/+ mouse strain that expresses Cre recombinase from the Satb2 locus. RESULTS: By transfecting neuronould determine the proportion of cells that become al progenitors with a Cre-inducible reporter construct by nucleofection or in utero electroporation, we cSatb2-positive. COMPARISON WITH EXISTING METHODS: Compared to genetic tracing or lineage analysis, this method offers a fast, easy-to-perform, and reliable way of determining cell fate of newly born neurons. CONCLUSIONS: We demonstrate that the Satb2Cre/+ mouse can be applied to study factors, such as small molecule inhibitors, sh-RNAs or overexpression constructs, that can alter the proportion of Satb2-positive cells and thus play key roles in differentiation and acquisition of cell fate.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Modelos Animais , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Eletroporação , Citometria de Fluxo , Imuno-Histoquímica , Integrases/genética , Integrases/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos Transgênicos , Neocórtex/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Transfecção , Proteínas Supressoras de Tumor/metabolismo
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