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In the field of artificial intelligence, a combination of scale in data and model capacity enabled by unsupervised learning has led to major advances in representation learning and statistical generation. In the life sciences, the anticipated growth of sequencing promises unprecedented data on natural sequence diversity. Protein language modeling at the scale of evolution is a logical step toward predictive and generative artificial intelligence for biology. To this end, we use unsupervised learning to train a deep contextual language model on 86 billion amino acids across 250 million protein sequences spanning evolutionary diversity. The resulting model contains information about biological properties in its representations. The representations are learned from sequence data alone. The learned representation space has a multiscale organization reflecting structure from the level of biochemical properties of amino acids to remote homology of proteins. Information about secondary and tertiary structure is encoded in the representations and can be identified by linear projections. Representation learning produces features that generalize across a range of applications, enabling state-of-the-art supervised prediction of mutational effect and secondary structure and improving state-of-the-art features for long-range contact prediction.
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Análisis de Secuencia de Proteína/métodos , Aprendizaje Automático no Supervisado , Aminoácidos/química , Conformación Proteica , Homología de Secuencia de AminoácidoAsunto(s)
Proteína 9 Asociada a CRISPR/genética , Técnicas de Inactivación de Genes/métodos , Biblioteca Genómica , Mutación con Pérdida de Función , ARN Guía de Kinetoplastida/genética , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , HumanosRESUMEN
Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with â¼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.
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The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.
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Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Meninges/citología , Meninges/inmunología , Animales , Encéfalo/citología , Encéfalo/inmunología , Agregación Celular , Criopreservación , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/virología , Femenino , Antígenos Comunes de Leucocito/metabolismo , Ratones , Adhesión en Parafina , Médula Espinal/citología , Médula Espinal/inmunología , Theilovirus/fisiología , Fijación del TejidoRESUMEN
Neuronal hyperactivity is one of the earliest events observed in Alzheimer's disease (AD). Moreover, alterations in the expression of glutamate transporters have been reported to exacerbate amyloid pathology and cognitive deficits in transgenic AD mouse models. However, the molecular links between these pathophysiological changes remain largely unknown. Here, we report novel interaction between presenilin 1 (PS1), the catalytic component of the amyloid precursor protein-processing enzyme, γ-secretase, and a major glutamate transporter-1 (GLT-1). Our data demonstrate that the interaction occurs between PS1 and GLT-1 expressed at their endogenous levels in vivo and in vitro, takes place in both neurons and astrocytes, and is independent of the PS1 autoproteolysis and γ-secretase activity. This intriguing discovery may shed light on the molecular crosstalk between the proteins linked to the maintenance of glutamate homeostasis and Aß pathology.
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Enfermedad de Alzheimer/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Presenilina-1/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Células Cultivadas , Transportador 2 de Aminoácidos Excitadores/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Humanos , Ratones , Neuronas/metabolismo , Neuronas/patología , Presenilina-1/genética , ProteolisisRESUMEN
Six of 6-week-old NZ rabbits underwent ultrasound treatment using a therapeutic dose (0.5 W/cm(2)) and other six were treated with a higher dose (2.2 W/cm(2)) to the lateral aspect of the left knee joint for 20 min per day and a total of six weeks. The right knee joint served as a control. The goal of this study is to see if the therapeutic dose and high dose (approximately 45-fold therapeutic dose) will have toxic effects on the physis. Histological review appeared normal growth plate in the therapeutic group. In the high dosage group three of six cases displayed flattening of the distal femoral epiphysis and wedging of the proximal tibial plateau and indistinct growth plate lines. It is of interest to note that there are opening radiolucent area in the lateral aspect on the femoral metaphysis in five of six cases, where bone resorption has taken place. Histological results showed that there are disordered arrays of the cartilaginous cells in the proliferative zone. The height of the lateral physis in the high dose group is not only greater than that in the therapeutic dose (1083.8 vs. 500.3 micro m), but also greater than that in their contralateral control (530.7 micro m) (P<0.05). This short-term study demonstrates that high dose ultrasound has profound pathologic effects in growing bone. Therapeutic doses of ultrasound do not have an adverse effect on bone growth in the short-term follow-up.
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Placa de Crecimiento/efectos de la radiación , Ultrasonido , Animales , Condrocitos/patología , Condrocitos/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Epífisis/diagnóstico por imagen , Epífisis/patología , Epífisis/efectos de la radiación , Fémur/diagnóstico por imagen , Fémur/patología , Fémur/efectos de la radiación , Placa de Crecimiento/diagnóstico por imagen , Placa de Crecimiento/patología , Articulación de la Rodilla/diagnóstico por imagen , Articulación de la Rodilla/patología , Articulación de la Rodilla/efectos de la radiación , Conejos , Radiografía , Terapia por UltrasonidoRESUMEN
Objectives To update the Massachusetts General Hospital (MGH) and Massachusetts Eye and Ear Infirmary (MEEI) experience in the management of esthesioneuroblastoma (ENB) with multimodality therapy and to reassess treatment outcomes and complications in a larger cohort with longer follow-up times. Design A retrospective chart review. Setting A tertiary referral center. Participants All patients presenting with ENB and managed at the MGH and MEEI from 1997 to 2013. Main Outcome Measures Disease-free and overall survival. Results Twenty-two patients were identified with an average follow-up of 73 months. Ten patients presented with Kadish stage B disease and 12 with stage C disease. A total of six patients (27%) developed regional metastases. Treatment for all patients included craniofacial resection (CFR) followed by proton beam irradiation with or without chemotherapy. The 5-year disease-free and overall survival rates were 86.4% and 95.2%, respectively, by Kaplan-Meier analysis. Negative margins were a significant factor in disease-free survival. One patient experienced severe late-radiation toxicity. Conclusions ENB is safely and effectively treated with CFR followed by proton beam irradiation. The high incidence of regional metastases warrants strong consideration for elective neck irradiation. Proton beam radiation is associated with lower rates of severe late-radiation toxicity than conventional radiotherapy.
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The expansion of endoscopic skull base surgery has resulted in the creation of large defects that must be repaired to ensure separation of the cranial vault from the nasal cavity. The workhorse of anterior skull base reconstruction remains the nasoseptal or Hadad-Bassagasteguy flap. Despite its success, the nasoseptal flap is limited in its ability to reach extremely anterior defects including those involving the frontal break, posterior frontal table, and anterior cribiform plate. Alternative approaches utilizing anteriorly pedicled flaps have been described which exploit the vascular supply to the anterior septum and lateral nasal wall. The diminutive nature of the anterior septal blood supply has led to the elaboration of a bipedicled anterior septal flap which is capable of reliable reconstruction of both the frontal beak as well as posterior frontal table defects. Similarly anteriorly based inferior turbinate flaps pedicled largely on anterior ethmoid arborizations have been used successfully to reconstruct anterior defects. In light of the limitations of the nasoseptal flap, the addition of anteriorly pedicled flaps to the clinical armamentarium offers the opportunity to provide vascularized mucosal coverage of virtually any region of the skull base which can be reached endoscopically.
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Endoscopía/métodos , Procedimientos de Cirugía Plástica/métodos , Base del Cráneo/cirugía , Colgajos Quirúrgicos/irrigación sanguínea , Humanos , Cavidad Nasal , Neoplasias de la Base del Cráneo/cirugíaRESUMEN
The field of endoscopic skull base surgery has evolved considerably in recent years fueled largely by advances in both imaging and instrumentation. While the indications for these approaches continue to be extended, the ability to reconstruct the resultant defects has emerged as a rate-limiting obstacle. Postoperative failures with current multilayer grafting techniques remain significant and may increase as the indications for endoscopic resections continue to expand. Laser tissue welding represents a novel method of wound repair in which laser energy is applied to a chromophore doped biologic solder at the wound edge to create a laser weld (fig. 1). These repairs are capable of withstanding forces far exceeding those exerted by intracranial pressure with negligible collateral thermal tissue injury. Recent clinical trials have demonstrated the safety and feasibility of endoscopic laser welding while exposing the limitations of first generation hyaluronic acid based solders. Novel supersaturated gel based solders are currently being tested in clinical trials and appear to possess significantly improved viscoelastic properties. While laser tissue welding remains an experimental technique, continued success with these novel solder formulations may catalyze the widespread adoption of this technique for skull base repair in the near future.
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Endoscopía/tendencias , Terapia por Láser/tendencias , Procedimientos de Cirugía Plástica/tendencias , Base del Cráneo/cirugía , Endoscopía/métodos , Humanos , Terapia por Láser/métodos , Procedimientos de Cirugía Plástica/métodosAsunto(s)
Servicios de Salud del Niño/provisión & distribución , Gastroenteritis/epidemiología , Cefalea/epidemiología , Necesidades y Demandas de Servicios de Salud , Infecciones del Sistema Respiratorio/epidemiología , Dolor Abdominal/diagnóstico , Enfermedad Aguda , Niño , Femenino , Gastroenteritis/diagnóstico , Haití/epidemiología , Cefalea/diagnóstico , Necesidades y Demandas de Servicios de Salud/estadística & datos numéricos , Humanos , Masculino , Infecciones del Sistema Respiratorio/diagnósticoRESUMEN
The importance of lymphotoxin (LT) betaR (LTbetaR) as a regulator of lymphoid organogenesis is well established, but its role in host defense has yet to be fully defined. In this study, we report that mice deficient in LTbetaR signaling were highly susceptible to infection with murine CMV (MCMV) and early during infection exhibited a catastrophic loss of T and B lymphocytes, although the majority of lymphocytes were themselves not directly infected. Moreover, bone marrow chimeras revealed that lymphocyte survival required LTalpha expression by hemopoietic cells, independent of developmental defects in lymphoid tissue, whereas LTbetaR expression by both stromal and hemopoietic cells was needed to prevent apoptosis. The induction of IFN-beta was also severely impaired in MCMV-infected LTalpha(-/-) mice, but immunotherapy with an agonist LTbetaR Ab restored IFN-beta levels, prevented lymphocyte death, and enhanced the survival of these mice. IFN-alphabetaR(-/-) mice were also found to exhibit profound lymphocyte death during MCMV infection, thus providing a potential mechanistic link between type 1 IFN induction and lymphocyte survival through a LTalphabeta-dependent pathway important for MCMV host defense.