RESUMEN
We describe a severe congenital myasthenic syndrome (CMS) caused by two missense mutations in the gene encoding the muscle specific receptor tyrosine kinase (MUSK). The identified MUSK mutations M605I and A727V are both located in the kinase domain of MuSK. Intracellular microelectrode recordings and microscopy studies of the neuromuscular junction conducted in an anconeus muscle biopsy revealed decreased miniature endplate potential amplitudes, reduced endplate size and simplification of secondary synaptic folds, which were consistent with postsynaptic deficit. The study also showed a striking reduction of the endplate potential quantal content, consistent with additional presynaptic failure. Expression studies in MuSK deficient myotubes revealed that A727V, which is located within the catalytic loop of the enzyme, caused severe impairment of agrin-dependent MuSK phosphorylation, aggregation of acetylcholine receptors (AChRs) and interaction of MuSK with Dok-7, an essential intracellular binding protein of MuSK. In contrast, M605I, resulted in only moderate impairment of agrin-dependent MuSK phosphorylation, aggregation of AChRs and interaction of MuSK with Dok-7. There was no impairment of interaction of mutants with either the low-density lipoprotein receptor-related protein, Lrp4 (a co-receptor of agrin) or with the mammalian homolog of the Drosophila tumorous imaginal discs (Tid1). Our findings demonstrate that missense mutations in MUSK can result in a severe form of CMS and indicate that the inability of MuSK mutants to interact with Dok-7, but not with Lrp4 or Tid1, is a major determinant of the pathogenesis of the CMS caused by MUSK mutations.
Asunto(s)
Proteínas Musculares/metabolismo , Síndromes Miasténicos Congénitos/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Colinérgicos/genética , Agrina/metabolismo , Animales , Línea Celular , Femenino , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiología , Mutación Missense , Síndromes Miasténicos Congénitos/metabolismo , Síndromes Miasténicos Congénitos/patología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Estructura Secundaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Adulto JovenRESUMEN
We describe a severe form of congenital myasthenic syndrome (CMS) caused by two heteroallelic mutations: a nonsense and a missense mutation in the gene encoding agrin (AGRN). The identified mutations, Q353X and V1727F, are located at the N-terminal and at the second laminin G-like (LG2) domain of agrin, respectively. A motor-point muscle biopsy demonstrated severe disruption of the architecture of the neuromuscular junction (NMJ), including: dispersion and fragmentation of endplate areas with normal expression of acetylcholinesterase; simplification of postsynaptic membranes; pronounced reduction of the axon terminal size; widening of the primary synaptic cleft; and, collection of membranous debris material in the primary synaptic cleft and in the subsynaptic cytoplasm. Expression studies in heterologous cells revealed that the Q353X mutation abolished expression of full-length agrin. Moreover, the V1727F mutation decreased agrin-induced clustering of the acetylcholine receptor (AChR) in cultured C2 muscle cells by >100-fold, and phosphorylation of the MuSK receptor and AChR beta subunit by ~tenfold. Surprisingly, the V1727F mutant also displayed increased binding to α-dystroglycan but decreased binding to a neural (z+) agrin-specific antibody. Our findings demonstrate that agrin mutations can associate with a severe form of CMS and cause profound distortion of the architecture and function of the NMJ. The impaired ability of V1727F agrin to activate MuSK and cluster AChRs, together with its increased affinity to α-dystroglycan, mimics non-neural (z-) agrin and are important determinants of the pathogenesis of the disease.
Asunto(s)
Agrina/genética , Codón sin Sentido , Mutación Missense , Síndromes Miasténicos Congénitos/genética , Acetilcolinesterasa/metabolismo , Adulto , Agrina/química , Agrina/metabolismo , Secuencia de Bases , Línea Celular , Distroglicanos/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Síndromes Miasténicos Congénitos/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Linaje , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Análisis de Secuencia de ADNRESUMEN
Not all infants carry specialized gut microbes, meaning they cannot digest human milk oligosaccharides and therefore do not receive complete benefits from human milk. B. infantis EVC001 is equipped to convert the full array of complex oligosaccharides into compounds usable by the infant, making it an ideal candidate to stabilize gut function and improve nutrition in preterm infants. A prospective, open-label study design was used to evaluate the tolerability of B. infantis EVC001 and its effects on the fecal microbiota in preterm infants in a Neonatal Intensive Care Unit. Thirty preterm infants <1,500 g and/or <33 weeks gestation at birth were divided into two matched groups, and control infants were enrolled and discharged prior to enrolling EVC001 infants to prevent cross-colonization of B. infantis: (1) fifteen control infants received no EVC001, and (2) fifteen infants received once-daily feedings of B. infantis EVC001 (8.0 x 109 CFU) in MCT oil. Clinical information regarding medications, growth, nutrition, gastrointestinal events, diagnoses, and procedures was collected throughout admission. Infant stool samples were collected at baseline, Study Days 14 and 28, and 34-, 36-, and 38-weeks of gestation. Taxonomic composition of the fecal microbiota, functional microbiota analysis, B. infantis, and human milk oligosaccharides (HMOs) in the stool were determined or quantified using 16S rRNA gene sequencing, metagenomic sequencing, qPCR, and mass spectrometry, respectively. No adverse events or tolerability issues related to EVC001 were reported. Control infants had no detectable levels of B. infantis. EVC001 infants achieved high levels of B. infantis (mean = 9.7 Log10 CFU/µg fecal DNA) by Study Day 14, correlating with less fecal HMOs (ρ = -0.83, P < 0.0001), indicating better HMO utilization in the gut. In this study, B. infantis EVC001 was shown to be safe, well-tolerated, and efficient in colonizing the preterm infant gut and able to increase the abundance of bifidobacteria capable of metabolizing HMOs, resulting in significantly improved utilization of human milk. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT03939546, identifier: NCT03939546.
RESUMEN
The gut microbiome plays an important role in early life, protecting newborns from enteric pathogens, promoting immune system development and providing key functions to the infant host. Currently, there are limited data to broadly assess the status of the US healthy infant gut microbiome. To address this gap, we performed a multi-state metagenomic survey and found high levels of bacteria associated with enteric inflammation (e.g. Escherichia, Klebsiella), antibiotic resistance genes, and signatures of dysbiosis, independent of location, age, and diet. Bifidobacterium were less abundant than generally expected and the species identified, including B. breve, B. longum and B. bifidum, had limited genetic capacity to metabolize human milk oligosaccharides (HMOs), while B. infantis strains with a complete capacity for HMOs utilization were found to be exceptionally rare. Considering microbiome composition and functional capacity, this survey revealed a previously unappreciated dysbiosis that is widespread in the contemporary US infant gut microbiome.
Asunto(s)
Bifidobacterium/genética , Microbioma Gastrointestinal , Metagenómica/métodos , Bifidobacterium/aislamiento & purificación , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Bases de Datos Factuales , Dieta , Farmacorresistencia Bacteriana/genética , Disbiosis , Heces/microbiología , Humanos , Lactante , Recién Nacido , Leche Humana/metabolismo , Oligosacáridos/metabolismo , Estados UnidosRESUMEN
OBJECTIVE: To evaluate sensitivity and specificity of echocardiography for diagnosis of cardiac masses in dogs with pericardial effusion. DESIGN: Retrospective case series. ANIMALS: 107 dogs with pericardial effusion. PROCEDURES: Records of dogs with pericardial effusion examined at the University of California-Davis Veterinary Medical Teaching Hospital from 1985 to 2006 were reviewed. Dogs were included when echocardiography and pericardectomy or necropsy were performed. Sensitivity, specificity, and metastatic rates were calculated for various causes of pericardial effusion. RESULTS: 107 dogs with pericardial effusion were evaluated by surgery (n = 48 dogs), necropsy (44), or both (15). Echocardiography revealed no mass (n = 41 dogs), a right atrial (RA) mass (38), a heart base (HB) mass (23), a pericardial mass (2), an HB and an RA mass (2), and a right ventricular mass (1). Sensitivity and specificity were 82% and 100%, respectively, for detection of a cardiac mass; 82% and 99%, respectively, for detection of an RA mass; and 74% and 98%, respectively, for detection of an HB mass. Most HB masses were neuroendocrine or ectopic thyroid gland tissue, but 3 were hemangiosarcomas and 4 were mesotheliomas. Most RA masses were hemangiosarcomas, but this group also included a neuroendocrine tumor, ectopic thyroid gland tissue, mesothelioma, lymphosarcoma, and sarcoma. Metastatic rates did not differ (50% to 66%) among neoplastic causes. CONCLUSIONS AND CLINICAL RELEVANCE: Echocardiography had high sensitivity and specificity for diagnosis and differentiation of RA or HB masses in dogs with pericardial effusion. There was a high rate of metastasis for cardiac masses of all causes.