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Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.
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Encefalopatías , Proteínas de Transporte Vesicular , Animales , Ratas , Proteínas de Transporte Vesicular/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Fusión de Membrana/fisiología , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Neurotransmisores/metabolismo , Mamíferos/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.
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VIH-1 , Provirus , Desoxiguanosina/genética , Guanosina/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/fisiología , Provirus/genética , ARN Viral/genética , Secuencias Repetidas TerminalesRESUMEN
BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.
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Síndrome Coronario Agudo , Linfocitos T CD4-Positivos , Placa Aterosclerótica , Análisis de la Célula Individual , Humanos , Síndrome Coronario Agudo/inmunología , Síndrome Coronario Agudo/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Masculino , Persona de Mediana Edad , Femenino , Anciano , RNA-Seq , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Vasos Coronarios/inmunología , Vasos Coronarios/patología , Análisis de Secuencia de ARN , Enfermedad de la Arteria Coronaria/inmunología , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , FenotipoRESUMEN
Human T cell leukemia/T-lymphotropic virus type 1 (HTLV-1) infection occurs by cell-to-cell transmission and can induce fatal adult T cell leukemia. Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of 10 vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the 2 vaccinated macaques without NAb induction and 10 unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.
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Anticuerpos Neutralizantes , Infecciones por HTLV-I , Virus Linfotrópico T Tipo 1 Humano , Vacunación , Animales , Virus Linfotrópico T Tipo 1 Humano/inmunología , Infecciones por HTLV-I/inmunología , Infecciones por HTLV-I/prevención & control , Anticuerpos Neutralizantes/inmunología , Humanos , Macaca fascicularis , Carga Viral , Linfocitos T CD8-positivos/inmunología , Productos del Gen env/inmunología , Anticuerpos Antivirales/inmunología , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: SINE-VNTR-Alu (SVA) retrotransposons move from one genomic location to another in a 'copy-and-paste' manner. They continue to move actively and cause monogenic diseases through various mechanisms. Currently, disease-causing SVA retrotransposons are classified into human-specific young SVA_E or SVA_F subfamilies. In this study, we identified an evolutionarily old SVA_D retrotransposon as a novel cause of occipital horn syndrome (OHS). OHS is an X-linked, copper metabolism disorder caused by dysfunction of the copper transporter, ATP7A. METHODS: We investigated a 16-year-old boy with OHS whose pathogenic variant could not be detected via routine molecular genetic analyses. RESULTS: A 2.8 kb insertion was detected deep within the intron of the patient's ATP7A gene. This insertion caused aberrant mRNA splicing activated by a new donor splice site located within it. Long-read circular consensus sequencing enabled us to accurately read the entire insertion sequence, which contained highly repetitive and GC-rich segments. Consequently, the insertion was identified as an SVA_D retrotransposon. Antisense oligonucleotides (AOs) targeting the new splice site restored the expression of normal transcripts and functional ATP7A proteins. AO treatment alleviated excessive accumulation of copper in patient fibroblasts in a dose-dependent manner. Pedigree analysis revealed that the retrotransposon had moved into the OHS-causing position two generations ago. CONCLUSION: This is the first report of a human monogenic disease caused by the SVA_D retrotransposon. The fact that the evolutionarily old SVA_D is still actively transposed, leading to increased copy numbers may make a notable impact on rare genetic disease research.
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Rationale: General anesthesia and mechanical ventilation have negative impacts on the respiratory system, causing heterogeneous distribution of lung aeration, but little is known about the ventilation patterns of postoperative patients and their association with clinical outcomes. Objectives: To clarify the phenotypes of ventilation patterns along a gravitational direction after surgery by using electrical impedance tomography (EIT) and to evaluate their association with postoperative pulmonary complications (PPCs) and other relevant clinical outcomes. Methods: Adult postoperative patients at high risk for PPCs, receiving mechanical ventilation on ICU admission (N = 128), were prospectively enrolled between November 18, 2021 and July 18, 2022. PPCs were prospectively scored until hospital discharge, and their association with phenotypes of ventilation patterns was studied. The secondary outcomes were the times to wean from mechanical ventilation and oxygen use and the length of ICU stay. Measurements and Main Results: Three phenotypes of ventilation patterns were revealed by EIT: phenotype 1 (32% [n = 41], a predominance of ventral ventilation), phenotype 2 (41% [n = 52], homogeneous ventilation), and phenotype 3 (27% [n = 35], a predominance of dorsal ventilation). The median PPC score was higher in phenotype 1 and phenotype 3 than in phenotype 2. The median time to wean from mechanical ventilation was longer in phenotype 1 versus phenotype 2. The median duration of ICU stay was longer in phenotype 1 versus phenotype 2. The median time to wean from oxygen use was longer in phenotype 1 and phenotype 3 than in phenotype 2. Conclusions: Inhomogeneous ventilation patterns revealed by EIT on ICU admission were associated with PPCs, delayed weaning from mechanical ventilation and oxygen use, and a longer ICU stay.
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Impedancia Eléctrica , Complicaciones Posoperatorias , Respiración Artificial , Tomografía , Humanos , Masculino , Femenino , Impedancia Eléctrica/uso terapéutico , Persona de Mediana Edad , Anciano , Respiración Artificial/métodos , Estudios Prospectivos , Tomografía/métodos , Complicaciones Posoperatorias/fisiopatología , Tiempo de Internación/estadística & datos numéricos , Desconexión del Ventilador/métodos , Unidades de Cuidados Intensivos , AdultoRESUMEN
Hypoxemic respiratory failure is one of the leading causes of mortality in intensive care. Frequent assessment of individual physiological characteristics and delivery of personalized mechanical ventilation (MV) settings is a constant challenge for clinicians caring for these patients. Electrical impedance tomography (EIT) is a radiation-free bedside monitoring device that is able to assess regional lung ventilation and changes in aeration. With real-time tomographic functional images of the lungs obtained through a thoracic belt, clinicians can visualize and estimate the distribution of ventilation at different ventilation settings or following procedures such as prone positioning. Several studies have evaluated the performance of EIT to monitor the effects of different MV settings in patients with acute respiratory distress syndrome, allowing more personalized MV. For instance, EIT could help clinicians find the positive end-expiratory pressure that represents a compromise between recruitment and overdistension and assess the effect of prone positioning on ventilation distribution. The clinical impact of the personalization of MV remains to be explored. Despite inherent limitations such as limited spatial resolution, EIT also offers a unique noninvasive bedside assessment of regional ventilation changes in the ICU. This technology offers the possibility of a continuous, operator-free diagnosis and real-time detection of common problems during MV. This review provides an overview of the functioning of EIT, its main indices, and its performance in monitoring patients with acute respiratory failure. Future perspectives for use in intensive care are also addressed.
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Síndrome de Dificultad Respiratoria , Insuficiencia Respiratoria , Humanos , Impedancia Eléctrica , Tomografía Computarizada por Rayos X/métodos , Pulmón , Insuficiencia Respiratoria/diagnóstico por imagen , Insuficiencia Respiratoria/terapia , Tomografía/métodos , Síndrome de Dificultad Respiratoria/diagnóstico por imagen , Síndrome de Dificultad Respiratoria/terapiaRESUMEN
Lissencephaly is a rare brain malformation characterized by abnormal neuronal migration during cortical development. In this study, we performed a comprehensive genetic analysis using next-generation sequencing in 12 unsolved Japanese lissencephaly patients, in whom PAFAH1B1, DCX, TUBA1A, and ARX variants were excluded using the Sanger method. Exome sequencing (ES) was conducted on these 12 patients, identifying pathogenic variants in CEP85L, DYNC1H1, LAMC3, and DCX in four patients. Next, we performed genome sequencing (GS) on eight unsolved patients, and structural variants in PAFAH1B1, including an inversion and microdeletions involving several exons, were detected in three patients. Notably, these microdeletions in PAFAH1B1 could not to be detected by copy number variation (CNV) detection tools based on the depth of coverage methods using ES data. The density of repeat sequences, including Alu sequences or segmental duplications, which increase the susceptibility to structural variations, is very high in some lissencephaly spectrum genes (PAFAH1B1, TUBA1A, DYNC1H1). These missing CNVs were due to the limitations of detecting repeat sequences in ES-based CNV detection tools. Our study suggests that a combined approach integrating ES with GS can contribute to a higher diagnostic yield and a better understanding of the genetic landscape of the lissencephaly spectrum.
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Amorphous silica has been approved as a food and pharmaceutical additive. However, its potential to enhance the carcinogenicity of epithelial cells is incontrovertible. With their expanded surface area per unit mass and distinctive cellular incorporation, nano-sized silica particles (nSPs) exhibit heightened cytotoxicity compared to micrometer-sized counterparts. The precise effect of nSPs on the generation of small extracellular vesicles (sEVs) within endosomes after cellular uptake remains unclear. In the present study, we explored the secretion of sEVs from cells and their functional implications following exposure to nSPs. Our findings demonstrate that nSP50 exposure not only induced epithelial-mesenchymal transition (EMT) but also promoted the maturation of multivesicular endosomes (MVEs) along with the secretion of sEVs in A549 cells. Inhibition of sEV secretion using GW4869 and apoptosis activator 2 exacerbated nSP50-induced EMT, indicating that sEV secretion may suppress EMT. Analysis of the function of sEV in a cell-free system revealed that co-incubation of sEVs with nSP50 led to the formation of micrometer-sized aggregates, which exhibited limited uptake efficiency within A549 cells. These results strongly suggest that the secretion of sEVs plays a protective role against the cytotoxicity attributed to nSP50 exposure.
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OBJECTIVE: Degree of indication for epilepsy surgery is determined by taking multiple factors into account. This study aimed to investigate the usefulness of the Specific Consistency Score (SCS), a proposed score for focal epilepsy to rate the indication for epilepsy focal resection. METHODS: This retrospective cohort study included patients considered for resective epilepsy surgery in Kyoto University Hospital from 2011 to 2022. Plausible epileptic focus was tentatively defined. Cardinal findings were scored based on specificity and consistency with the estimated laterality and lobe. The total points represented SCS. The association between SCS and the following clinical parameters was assessed by univariate and multivariate analysis: (1) probability of undergoing resective epilepsy surgery, (2) good postoperative seizure outcome (Engel I and II or Engel I only), and (3) lobar concordance between the noninvasively estimated focus and intracranial electroencephalographic (EEG) recordings. RESULTS: A total of 131 patients were evaluated. Univariate analysis revealed higher SCS in the (1) epilepsy surgery group (8.4 [95% confidence interval (CI) = 7.8-8.9] vs. 4.9 [95% CI = 4.3-5.5] points; p < .001), (2) good postoperative seizure outcome group (Engel I and II; 8.7 [95% CI = 8.2-9.3] vs. 6.4 [95% CI = 4.5-8.3] points; p = .008), and (3) patients whose focus defined by intracranial EEG matched the noninvasively estimated focus (8.3 [95% CI = 7.3-9.2] vs. 5.4 [95% CI = 3.5-7.3] points; p = .004). Multivariate analysis revealed areas under the curve of .843, .825, and .881 for Parameters 1, 2, and 3, respectively. SIGNIFICANCE: SCS provides a reliable index of good indication for resective epilepsy surgery and can be easily available in many institutions not necessarily specializing in epilepsy.
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Selección de Paciente , Humanos , Femenino , Masculino , Adulto , Estudios Retrospectivos , Adulto Joven , Persona de Mediana Edad , Adolescente , Electroencefalografía/métodos , Epilepsia/cirugía , Epilepsia/diagnóstico , Resultado del Tratamiento , Niño , Estudios de Cohortes , Procedimientos Neuroquirúrgicos/métodos , Epilepsias Parciales/cirugía , Epilepsias Parciales/fisiopatología , Epilepsias Parciales/diagnósticoRESUMEN
Here, we describe two patients with juvenile xanthogranuloma (JXG) manifesting with Langerhans cell histiocytosis (LCH)-associated neurodegenerative disease (ND)-like radiological findings. One patient showed typical radiological abnormalities at onset, which worsened with progressing central nervous system symptoms 7 years after LCH-oriented chemotherapy. Another showed spontaneous regression of clinical symptoms, with a transient radiological change 1 year after salvage chemotherapy for recurrence of JXG. These data regarding JXG-associated ND will facilitate future investigation of the disease, as well as development of therapeutic interventions.
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Histiocitosis de Células de Langerhans , Enfermedades Neurodegenerativas , Xantogranuloma Juvenil , Niño , Humanos , Lactante , Masculino , Histiocitosis de Células de Langerhans/diagnóstico por imagen , Histiocitosis de Células de Langerhans/patología , Histiocitosis de Células de Langerhans/complicaciones , Histiocitosis de Células de Langerhans/tratamiento farmacológico , Imagen por Resonancia Magnética , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/complicaciones , Xantogranuloma Juvenil/diagnóstico por imagen , Xantogranuloma Juvenil/patologíaRESUMEN
PURPOSE: To assess the longitudinal changes of the posterior vortex veins (VVs) in highly myopic (HM) eyes. METHODS: The medical records of 1,730 consecutive HM eyes that had undergone indocyanine green angiography were studied. Eyes that had posterior VVs and had undergone at least two indocyanine green angiography examinations with a minimum interval of 3 years were selected from this group. RESULTS: Ninety-one eyes of 78 patients met the inclusion criteria. A total of 124 posterior VVs were identified. Over an average interval of 7.8 ± 5.0 years, 41 (33.1%) of the 124 posterior VVs had marked changes consisting mainly of an attenuation of vessels in 36 posterior VVs (87.8%) and alterations in the drainage course in 16 posterior VVs (39.0%). Fifteen posterior VVs had both types of changes. Most of the attenuations of the vessels occurred for smaller branches, but a complete loss of the entire trunk was seen in three eyes. Additionally, four eyes had posterior VV changes in association with changes of peripheral VVs. CONCLUSION: Posterior VV in highly myopic eyes can undergo changes with increasing time. The associated factors included the development and progression of myopic maculopathy lesions. In some cases, the blood drainage shifted from posterior VV to peripheral VV by forming anastomotic channels.
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Verde de Indocianina , Miopía , Humanos , Estudios Retrospectivos , Angiografía con Fluoresceína , Miopía/complicaciones , Miopía/diagnóstico , Coroides/irrigación sanguíneaRESUMEN
BACKGROUND: Atopic dermatitis (AD) is an inflammatory disorder characterized by dominant type 2 inflammation leading to chronic pruritic skin lesions, allergic comorbidities, and Staphylococcus aureus skin colonization and infections. S aureus is thought to play a role in AD severity. OBJECTIVES: This study characterized the changes in the host-microbial interface in subjects with AD following type 2 blockade with dupilumab. METHODS: Participants (n = 71) with moderate-severe AD were enrolled in a randomized (dupilumab vs placebo; 2:1), double-blind study at Atopic Dermatitis Research Network centers. Bioassays were performed at multiple time points: S aureus and virulence factor quantification, 16s ribosomal RNA microbiome, serum biomarkers, skin transcriptomic analyses, and peripheral blood T-cell phenotyping. RESULTS: At baseline, 100% of participants were S aureus colonized on the skin surface. Dupilumab treatment resulted in significant reductions in S aureus after only 3 days (compared to placebo), which was 11 days before clinical improvement. Participants with the greatest S aureus reductions had the best clinical outcomes, and these reductions correlated with reductions in serum CCL17 and disease severity. Reductions (10-fold) in S aureus cytotoxins (day 7), perturbations in TH17-cell subsets (day 14), and increased expression of genes relevant for IL-17, neutrophil, and complement pathways (day 7) were also observed. CONCLUSIONS: Blockade of IL-4 and IL-13 signaling, very rapidly (day 3) reduces S aureus abundance in subjects with AD, and this reduction correlates with reductions in the type 2 biomarker, CCL17, and measures of AD severity (excluding itch). Immunoprofiling and/or transcriptomics suggest a role for TH17 cells, neutrophils, and complement activation as potential mechanisms to explain these findings.
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Dermatitis Atópica , Infecciones Estafilocócicas , Humanos , Dermatitis Atópica/genética , Staphylococcus aureus , Anticuerpos Monoclonales Humanizados/uso terapéutico , Piel/metabolismo , Infecciones Estafilocócicas/tratamiento farmacológico , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.
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COVID-19 , Humanos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
Familial episodic pain syndrome (FEPS) is an early childhood onset disorder of severe episodic limb pain caused mainly by pathogenic variants of SCN11A, SCN10A, and SCN9A, which encode three voltage-gated sodium channels (VGSCs) expressed as key determinants of nociceptor excitability in primary sensory neurons. There may still be many undiagnosed patients with FEPS. A better understanding of the associated pathogenesis, epidemiology, and clinical characteristics is needed to provide appropriate diagnosis and care. For this study, nationwide recruitment of Japanese patients was conducted using provisional clinical diagnostic criteria, followed by genetic testing for SCN11A, SCN10A, and SCN9A. In the cohort of 212 recruited patients, genetic testing revealed that 64 patients (30.2%) harbored pathogenic or likely pathogenic variants of these genes, consisting of 42 (19.8%), 14 (6.60%), and 8 (3.77%) patients with variants of SCN11A, SCN10A, and SCN9A, respectively. Meanwhile, the proportions of patients meeting the tentative clinical criteria were 89.1%, 52.0%, and 54.5% among patients with pathogenic or likely pathogenic variants of each of the three genes, suggesting the validity of these clinical criteria, especially for patients with SCN11A variants. These clinical diagnostic criteria of FEPS will accelerate the recruitment of patients with underlying pathogenic variants who are unexpectedly prevalent in Japan.
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Pruebas Genéticas , Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.9/genética , Japón/epidemiología , Canal de Sodio Activado por Voltaje NAV1.8/genética , Masculino , Femenino , Pruebas Genéticas/métodos , Adulto , Adolescente , Niño , Predisposición Genética a la Enfermedad , Adulto Joven , Preescolar , Mutación , Dolor , Recto/anomalíasRESUMEN
BACKGROUND: We hypothesized that the high-dose opioid requirement in patients carrying the rs4680-GG variant in the COMT gene encoding catechol-O-methyltransferase would be greater for patients taking morphine than for those taking oxycodone, thus providing a much-needed biomarker to inform opioid selection for cancer pain. METHODS: A randomized, multicenter, open-label trial was conducted at a Japanese hospital's palliative care service. Patients with cancer pain treated with regular doses of nonsteroidal anti-inflammatory drugs or acetaminophen were enrolled and randomized (1:1) into morphine (group M) and oxycodone (group O) groups. The minimum standard dose of immediate-release (IR) oral opioids was repeatedly administered by palliative care physicians to achieve pain-reduction goals (Pain reductionâ ≥â 33% from baseline and up toâ ≤â 3 on a numerical rating scale). The primary endpoint was the proportion of subjects requiring high-dose opioids on day 0 with the GG genotype. RESULTS: Of 140 participants who developed cancer-related pain among 378 subjects registered and pre-screened for the genotype, 139 were evaluated in the current study. Among patients carrying a COMT rs4680-GG genotype, 48.3% required high-dose opioids in group M, compared with the 20.0% in group O (95% CI, 3.7%-50.8%; Pâ =â .029). Of those with the non-GG genotype, 41.5% treated with morphine and 23.1% with oxycodone required high-dose opioids (95% CI, 3.3%-38.3%; Pâ =â 0.098). CONCLUSION: Using the COMT rs4680 genotype alone is not recommended for selecting between morphine and oxycodone for pain relief.
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Dolor en Cáncer , Neoplasias , Humanos , Morfina/uso terapéutico , Oxicodona/uso terapéutico , Oxicodona/efectos adversos , Analgésicos Opioides/uso terapéutico , Analgésicos Opioides/efectos adversos , Dolor en Cáncer/tratamiento farmacológico , Dolor en Cáncer/genética , Catecol O-Metiltransferasa/genética , Catecol O-Metiltransferasa/uso terapéutico , Dolor/etiología , Dolor/genética , Genotipo , Biomarcadores , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
Scanning ion conductance microscopy (SICM) is a promising tool for visualizing the dynamics of nanoscale cell surface topography. However, there are still no guidelines for fabricating nanopipettes with ideal shape consisting of small apertures and thin glass walls. Therefore, most of the SICM imaging has been at a standstill at the submicron scale. In this study, we established a simple and highly reproducible method for the fabrication of nanopipettes with sub-20 nm apertures. To validate the improvement in the spatial resolution, we performed time-lapse imaging of the formation and disappearance of endocytic pits as a model of nanoscale time-lapse topographic imaging. We have also successfully imaged the localization of the hot spot and the released extracellular vesicles. The nanopipette fabrication guidelines for the SICM nanoscale topographic imaging can be an essential tool for understanding cell-cell communication.
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Vesículas Extracelulares , Microscopía , Cintigrafía , Comunicación Celular , Membrana Celular , IonesRESUMEN
OBJECTIVES: Patient-ventilator asynchrony is often observed during mechanical ventilation and is associated with higher mortality. We hypothesized that patient-ventilator asynchrony causes lung and diaphragm injury and dysfunction. DESIGN: Prospective randomized animal study. SETTING: University research laboratory. SUBJECTS: Eighteen New Zealand White rabbits. INTERVENTIONS: Acute respiratory distress syndrome (ARDS) model was established by depleting surfactants. Each group (assist control, breath stacking, and reverse triggering) was simulated by phrenic nerve stimulation. The effects of each group on lung function, lung injury (wet-to-dry lung weight ratio, total protein, and interleukin-6 in bronchoalveolar lavage), diaphragm function (diaphragm force generation curve), and diaphragm injury (cross-sectional area of diaphragm muscle fibers, histology) were measured. Diaphragm RNA sequencing was performed using breath stacking and assist control ( n = 2 each). MEASUREMENTS AND MAIN RESULTS: Inspiratory effort generated by phrenic nerve stimulation was small and similar among groups (esophageal pressure swing ≈ -2.5 cm H 2 O). Breath stacking resulted in the largest tidal volume (>10 mL/kg) and highest inspiratory transpulmonary pressure, leading to worse oxygenation, worse lung compliance, and lung injury. Reverse triggering did not cause lung injury. No asynchrony events were observed in assist control, whereas eccentric contractions occurred in breath stacking and reverse triggering, but more frequently in breath stacking. Breath stacking and reverse triggering significantly reduced diaphragm force generation. Diaphragmatic histology revealed that the area fraction of abnormal muscle was ×2.5 higher in breath stacking (vs assist control) and ×2.1 higher in reverse triggering (vs assist control). Diaphragm RNA sequencing analysis revealed that genes associated with muscle differentiation and contraction were suppressed, whereas cytokine- and chemokine-mediated proinflammatory responses were activated in breath stacking versus assist control. CONCLUSIONS: Breath stacking caused lung and diaphragm injury, whereas reverse triggering caused diaphragm injury. Thus, careful monitoring and management of patient-ventilator asynchrony may be important to minimize lung and diaphragm injury from spontaneous breathing in ARDS.
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Síndrome de Dificultad Respiratoria , Lesión Pulmonar Inducida por Ventilación Mecánica , Humanos , Animales , Conejos , Diafragma , Estudios Prospectivos , Pulmón , Volumen de Ventilación Pulmonar/fisiología , Lesión Pulmonar Inducida por Ventilación Mecánica/etiologíaRESUMEN
Viral protein U (Vpu) is an accessory protein encoded by human immunodeficiency virus type 1 (HIV-1) and certain simian immunodeficiency virus (SIV) strains. Some of these viruses were reported to use Vpu to overcome restriction by BST-2 of their natural hosts. Our own recent report revealed that Vpu of SIVgsn-99CM71 (SIVgsn71) antagonizes human BST-2 through two AxxxxxxxW motifs (A22W30 and A25W33), whereas antagonizing BST-2 of its natural host, greater spot-nosed monkey (GSN), involved only the A22W30 motif. Here, we show that residues A22, A25, W30, and W33 of SIVgsn71 Vpu are all essential to antagonize human BST-2, whereas a single mutation of either A22 or W30 did not affect the ability to antagonize GSN BST-2. Similar to A18, which is located in the middle of the A14xxxxxxxW22 motif in HIV-1 NL4-3 Vpu and is essential to antagonize human BST-2, A29, located in the middle of the A25W33 motif of SIVgsn71 Vpu was found to be necessary for antagonizing human but not GSN BST-2. Further mutational analyses revealed that residues L21 and K32 of SIVgsn71 Vpu were also essential for antagonizing human BST-2. On the other hand, the ability of SIVgsn71 Vpu to target GSN BST-2 was unaffected by single amino acid substitutions but required multiple mutations to render SIVgsn71 Vpu inactive against GSN BST-2. These results suggest additional requirements for SIVgsn71 Vpu antagonizing human BST-2, implying evolution of the bst-2 gene under strong selective pressure. IMPORTANCE Genes related to survival against life-threating pathogens are important determinants of natural selection in animal evolution. For instance, BST-2, a protein showing broad-spectrum antiviral activity, shows polymorphisms entailing different phenotypes even among primate species, suggesting that the bst-2 gene of primates has been subject to strong selective pressure during evolution. At the same time, viruses readily adapt to these evolutionary changes. Thus, we found that the Vpu of an SIVgsn isolate (SIVgsn-99CM71) can target BST-2 from humans as well as from its natural host, thus potentially facilitating zoonosis. Here, we mapped residues in SIVgsn71 Vpu potentially contributing to cross-species transmission. We found that the requirements for targeting human BST-2 are distinct from and more complex than those for targeting GSN BST-2. Our results suggest that the human bst-2 gene might have evolved to acquire more restrictive phenotype than GSN bst-2 against viral proteins after being derived from their common ancestor.
Asunto(s)
Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencias de Aminoácidos , Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Cercopithecus , Regulación hacia Abajo , Evolución Molecular , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , VIH-1/genética , VIH-1/metabolismo , Interacciones Microbiota-Huesped , Humanos , Mutación , Unión Proteica , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Especificidad de la Especie , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
INTRODUCTION/AIMS: In idiopathic inflammatory myopathies (IIMs), the change in muscle echogenicity and its histopathological basis are not well understood. We quantitatively measured muscle echogenicity in patients with IIMs and evaluated its correlation with disease activity and histopathological findings. METHODS: This study involved patients with IIMs who underwent both ultrasonography (US) and muscle biopsy, as well as age- and sex-matched rheumatoid arthritis patients as inflammatory disease controls. On US, axial images of the right biceps brachii and vastus medialis were obtained. Standardized histopathological scoring was used to quantitatively measure each pathological domain. RESULTS: Forty-two patients (17 with inclusion body myositis [IBM] and 25 with IIMs other than IBM) and 25 controls were included. The muscle echo intensity (EI) of patients with IIMs was significantly higher than that of controls. Muscle EI showed significant correlations with creatine kinase (r = 0.66, p < .001) and muscle strength (r = -0.73, p < .0001) in patients with non-IBM IIMs. In patients with IBM, moderate correlation was found between muscle EI and quadriceps muscle strength (r = -0.53, p = .028). Histopathologically, the number of infiltrating CD3+ inflammatory cells correlated with muscle EI in the non-IBM group (r = 0.56, p = .017), but not in the IBM group. DISCUSSION: Muscle EI may be useful as a surrogate marker of muscle inflammation in non-IBM IIM. Increased muscle EI may be difficult to interpret in patients with long-standing IBM, which has advanced and complex histopathology.