RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Células Madre Pluripotentes Inducidas , Antígeno Ventral Neuro-Oncológico , Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Antígeno Ventral Neuro-Oncológico/genética , Antígeno Ventral Neuro-Oncológico/metabolismo , Proteínas Nucleares/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genéticaRESUMEN
The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.
RESUMEN
The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.
RESUMEN
Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule-positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Hipocampo/metabolismo , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Corteza Prefrontal/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Animales , Gránulos Citoplasmáticos/patología , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Femenino , Hipocampo/patología , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Masculino , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Neuronas/patología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Corteza Prefrontal/patología , Transporte de Proteínas/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genéticaRESUMEN
Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress. Furthermore, the product of one of these enzymes, AdoMet, is a regulator of SG assembly and composition. Decreases in AdoMet levels increase SG formation, while chronic elevation of AdoMet produces SG remnants lacking proteins associated with the 5' end of transcripts. Interestingly, acute elevation of AdoMet blocks SG formation in yeast and motor neurons. Treatment of ALS-derived motor neurons with AdoMet also suppresses the formation of TDP-43-positive SGs, a hallmark of ALS. Together, these results argue that AdoMet is an evolutionarily conserved regulator of SG composition and assembly with therapeutic potential in neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Gránulos Citoplasmáticos/metabolismo , Metabolismo Energético , Neuronas Motoras/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , S-Adenosilmetionina/farmacología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts, including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR-Cas9 screening with microraft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; CRISPR-based microRaft followed by guide RNA identification). By isolating microrafts that contain genetic clones harboring individual guide RNAs (gRNA), we identify RNA-binding proteins (RBPs) that influence the formation of stress granules, the punctate protein-RNA assemblies that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Microscopía Confocal/métodos , Estrés Oxidativo/genética , ARN Guía de Kinetoplastida/genética , Proteínas de Unión al ARN/genética , Análisis de Matrices Tisulares/métodos , Sistemas CRISPR-Cas/genética , Citoplasma/metabolismo , Humanos , Aprendizaje Automático , Agregado de Proteínas/genéticaRESUMEN
In our daily lives, we consume foods that have been transported, stored, prepared, cooked, or otherwise processed by ourselves or others. Food storage and preparation have drastic effects on the chemical composition of foods. Untargeted mass spectrometry analysis of food samples has the potential to increase our chemical understanding of these processes by detecting a broad spectrum of chemicals. We performed a time-based analysis of the chemical changes in foods during common preparations, such as fermentation, brewing, and ripening, using untargeted mass spectrometry and molecular networking. The data analysis workflow presented implements an approach to study changes in food chemistry that can reveal global alterations in chemical profiles, identify changes in abundance, as well as identify specific chemicals and their transformation products. The data generated in this study are publicly available, enabling the replication and re-analysis of these data in isolation, and serve as a baseline dataset for future investigations.
Asunto(s)
Bebidas/análisis , Análisis de los Alimentos , Manipulación de Alimentos , Espectrometría de Masas , Metabolómica , Fermentación , Flujo de TrabajoRESUMEN
Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Demencia Frontotemporal/metabolismo , Neuronas Motoras/efectos de los fármacos , Agregación Patológica de Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Estrés Fisiológico/efectos de los fármacos , Línea Celular , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas , Proteínas Intrínsecamente Desordenadas , Neuronas Motoras/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteína FUS de Unión a ARN/metabolismoRESUMEN
Glial cells regulate multiple aspects of synaptogenesis. In the absence of Schwann cells, a peripheral glial cell, motor neurons initially innervate muscle but then degenerate. Here, using a genetic approach, we show that neural activity-regulated negative factors produced by muscle drive neurodegeneration in Schwann cell-deficient mice. We find that thrombin, the hepatic serine protease central to the hemostatic coagulation cascade, is one such negative factor. Trancriptomic analysis shows that expression of the antithrombins serpin C1 and D1 is significantly reduced in Schwann cell-deficient mice. In the absence of peripheral neuromuscular activity, neurodegeneration is completely blocked, and expression of prothrombin in muscle is markedly reduced. In the absence of muscle-derived prothrombin, neurodegeneration is also markedly reduced. Together, these results suggest that Schwann cells regulate NMJs by opposing the effects of activity-regulated, muscle-derived negative factors and provide the first genetic evidence that thrombin plays a central role outside of the coagulation system.
Asunto(s)
Antitrombina III/genética , Cofactor II de Heparina/genética , Unión Neuromuscular/genética , Protrombina/genética , Sinapsis/genética , Animales , Perfilación de la Expresión Génica , Ratones , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Músculo Esquelético/metabolismo , Degeneración Nerviosa/genética , Neuroglía , Unión Neuromuscular/crecimiento & desarrollo , Células de Schwann/metabolismo , Trombina/genéticaRESUMEN
Sporadic amyotrophic lateral sclerosis (sALS) is the most common form of ALS, however, the molecular mechanisms underlying cellular damage and motor neuron degeneration remain elusive. To identify molecular signatures of sALS we performed genome-wide expression profiling in laser capture microdissection-enriched surviving motor neurons (MNs) from lumbar spinal cords of sALS patients with rostral onset and caudal progression. After correcting for immunological background, we discover a highly specific gene expression signature for sALS that is associated with phosphorylated TDP-43 (pTDP-43) pathology. Transcriptome-pathology correlation identified casein kinase 1ε (CSNK1E) mRNA as tightly correlated to levels of pTDP-43 in sALS patients. Enhanced crosslinking and immunoprecipitation in human sALS patient- and healthy control-derived frontal cortex, revealed that TDP-43 binds directly to and regulates the expression of CSNK1E mRNA. Additionally, we were able to show that pTDP-43 itself binds RNA. CK1E, the protein product of CSNK1E, in turn interacts with TDP-43 and promotes cytoplasmic accumulation of pTDP-43 in human stem-cell-derived MNs. Pathological TDP-43 phosphorylation is therefore, reciprocally regulated by CK1E activity and TDP-43 RNA binding. Our framework of transcriptome-pathology correlations identifies candidate genes with relevance to novel mechanisms of neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Quinasa de la Caseína I/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Transcriptoma , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neuronas Motoras/patología , Fosforilación , Médula Espinal/patologíaRESUMEN
PURPOSE: Brachial plexus birth injuries with multiple nerve root avulsions present a particularly difficult reconstructive challenge because of the limited availability of donor nerves. The contralateral C7 has been described for brachial plexus reconstruction in adults but has not been well-studied in the pediatric population. We present our technique and results for retropharyngeal contralateral C7 nerve transfer to the lower trunk for brachial plexus birth injury. METHODS: We performed a retrospective review. Any child aged less than 2 years was included. Charts were analyzed for patient demographic data, operative variables, functional outcomes, complications, and length of follow-up. RESULTS: We had a total of 5 patients. Average nerve graft length was 3 cm. All patients had return of hand sensation to the ulnar nerve distribution as evidenced by a pinch test, unprompted use of the recipient limb without mirror movement, and an Active Movement Scale (AMS) of at least 2/7 for finger and thumb flexion; one patient had an AMS of 7/7 for finger and thumb flexion. Only one patient had return of ulnar intrinsic hand function with an AMS of 3/7. Two patients had temporary triceps weakness in the donor limb and one had clinically insignificant temporary phrenic nerve paresis. No complications were related to the retropharyngeal nerve dissection in any patient. Average follow-up was 3.3 years. CONCLUSIONS: The retropharyngeal contralateral C7 nerve transfer is a safe way to supply extra axons to the severely injured arm in brachial plexus birth injuries with no permanent donor limb deficits. Early functional recovery in these patients, with regard to hand function and sensation, is promising. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.
Asunto(s)
Neuropatías del Plexo Braquial/cirugía , Plexo Braquial/cirugía , Transferencia de Nervios/métodos , Traumatismos del Nacimiento/complicaciones , Traumatismos del Nacimiento/cirugía , Plexo Braquial/lesiones , Neuropatías del Plexo Braquial/etiología , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Recuperación de la Función , Estudios Retrospectivos , Nervio Cubital/cirugíaRESUMEN
OBJECTIVES: Early warning scores use vital signs to identify patients at risk of critical illness. The current study examines the Hamilton Early Warning Score (HEWS) at emergency department (ED) triage among patients who experienced a critical event during their hospitalization. HEWS was also evaluated as a predictor of sepsis. METHODS: The study population included admissions to two hospitals over a 6-month period. Cases experienced a critical event defined by unplanned intensive care unit admission, cardiopulmonary resuscitation, or death. Controls were randomly selected from the database in a 2-to-1 ratio to match cases on the burden of comorbid illness. Receiver operating characteristic (ROC) curves were used to evaluate HEWS as a predictor of the likelihood of critical deterioration and sepsis. RESULTS: The sample included 845 patients, of whom 270 experienced a critical event; 89 patients were excluded because of missing vitals. An ROC analysis indicated that HEWS at ED triage had poor discriminative ability for predicting the likelihood of experiencing a critical event 0.62 (95% CI 0.58-0.66). HEWS had a fair discriminative ability for meeting criteria for sepsis 0.77 (95% CI 0.72-0.82) and good discriminative ability for predicting the occurrence of a critical event among septic patients 0.82 (95% CI 0.75-0.90). CONCLUSION: This study indicates that HEWS at ED triage has limited utility for identifying patients at risk of experiencing a critical event. However, HEWS may allow earlier identification of septic patients. Prospective studies are needed to further delineate the utility of the HEWS to identify septic patients in the ED.
Asunto(s)
Enfermedad Crítica , Servicio de Urgencia en Hospital/estadística & datos numéricos , Sepsis/diagnóstico , Triaje/estadística & datos numéricos , Anciano , Femenino , Estudios de Seguimiento , Humanos , Masculino , Proyectos Piloto , Pronóstico , Curva ROC , Estudios Retrospectivos , Índice de Severidad de la EnfermedadRESUMEN
The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion.
Asunto(s)
Proteínas Fúngicas/genética , Factor de Apareamiento/genética , Péptidos/genética , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Peroxidasa de Rábano Silvestre/genética , Peroxidasa de Rábano Silvestre/metabolismo , Lipasa/genética , Lipasa/metabolismo , Factor de Apareamiento/química , Factor de Apareamiento/metabolismo , Modelos Moleculares , Mutación , Péptidos/química , Péptidos/metabolismo , Pichia/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de SecuenciaRESUMEN
The importance of the nasal complex cannot be overstated from a functional, social, and psychological perspective. The goal of reconstruction is to restore the trilaminar composition of the nose. This is accomplished by recreating the nasal lining and providing a cartilaginous framework to simultaneously support a patent airway and project the defining features to the overlying soft tissue. The columella is one of the smallest subunits of the nose, but the loss of this structure has important esthetic and structural implications. The ideal operation for an isolated defect of the columella remains elusive. The ideal reconstruction would match the pigmentation and texture of the surrounding nasal skin and provide a convex contour with underlying structural support. In addition, the donor site would not create a secondary deformity by disrupting normal anatomy. This report describes a novel 2-stage technique for reconstruction of the columella and reviews the current literature.
Asunto(s)
Nariz/lesiones , Rinoplastia/métodos , Niño , Cartílago Auricular/trasplante , Estética , Humanos , Masculino , Tabique Nasal/cirugía , Colgajos QuirúrgicosRESUMEN
PURPOSE: To summarize current evidence on outcomes and complications of prostatic artery embolization as a treatment for patients with lower urinary tract symptoms secondary to benign prostatic hyperplasia. METHODS AND MATERIALS: A database search of MEDLINE, Embase, Web of Science, and Cochrane Library was performed for published literature up to August 2015 concerning PAE in the treatment of BPH. Inclusion and exclusion criteria were applied by two independent reviewers, and disagreements were resolved by consensus. Peer-reviewed studies concerning PAE with BPH with a sample size >10 and at least one measured parameter were included. RESULTS: The search yielded 193 articles, of which ten studies representing 788 patients, with a mean age of 66.97 years, were included. Patients had LUTS ranging from moderate to severe. At 6 months following procedure, PV, PVR, Qmax, IPSS, and QoL were significantly improved (P < 0.05), while for PSA there was no significant change. At 12 and 24 months, PV, PSA, PVR, Qmax, IPSS, and QoL were significantly improved (P < 0.05). IIEF was unchanged at 6 and 12 months but was significantly reduced at 24 months. CONCLUSION: This suggests that PAE is effective in treating LUTS in the short and intermediate term.
Asunto(s)
Embolización Terapéutica/métodos , Hiperplasia Prostática/terapia , Anciano , Humanos , Síntomas del Sistema Urinario Inferior/complicaciones , Síntomas del Sistema Urinario Inferior/terapia , Masculino , Próstata/irrigación sanguínea , Próstata/diagnóstico por imagen , Hiperplasia Prostática/complicaciones , Resultado del TratamientoRESUMEN
The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to â¼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.
Asunto(s)
Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteína FUS de Unión a ARN/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Regiones no Traducidas 3'/genética , Animales , Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibroblastos , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células Madre Pluripotentes Inducidas , Intrones/genética , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Factores Asociados con la Proteína de Unión a TATA/metabolismoRESUMEN
BACKGROUND: The authors previously described the generation of vascularized bone in a pig model, using a hemimandibular allograft scaffold, adipose-derived stem cells, recombinant human bone morphogenetic protein-2, and periosteum. This study tests the hypothesis that this "allograft revitalization" technique is as effective as vascularized autograft for repairing critical bony defects. METHODS: Three groups of pigs had 3-cm defects created in their bilateral tibial diaphyses for repair using rigid fixation and one of three modalities. Negative control tibias were repaired with allograft tibia alone. To simulate repair using vascularized autograft, the osteotomized bone in positive control animals was left in situ, with the posterior periosteum intact. Experimental animals' defects were repaired with allograft tibia packed with autologous adipose-derived stem cells and recombinant human bone morphogenetic protein-2, with native periosteum intact. After 8 weeks, unilateral midgraft osteotomies were performed to assess graft healing potential. Serial radiographs and terminal micro-computed tomography and histology enabled evaluation of healing. RESULTS: At week 7 after ostectomy, no negative control tibias had healed (zero of six) whereas most positive control (five of six) and all experimental tibias (six of six) had healed. Unilateral midgraft osteotomies were performed at 8 weeks to assess graft ability to heal. As expected, no negative control tibias (three of three) had radiographic union 7 weeks later. However, all positive control (two of two; p = 0.05) and experimental (three of three; p = 0.01) tibias had healed their repeated osteotomies by this time. CONCLUSION: Similar to vascularized autograft, revitalized allograft successfully repaired a critical tibial defect, including after refracture, suggesting that this technique may be an alternative to osseous free flaps.
Asunto(s)
Proteína Morfogenética Ósea 2/uso terapéutico , Trasplante Óseo/métodos , Fijación Interna de Fracturas/métodos , Trasplante de Células Madre Mesenquimatosas , Periostio/trasplante , Tibia/trasplante , Fracturas de la Tibia/cirugía , Factor de Crecimiento Transformador beta/uso terapéutico , Animales , Femenino , Osteotomía , Proteínas Recombinantes/uso terapéutico , Porcinos , Tibia/lesiones , Tibia/cirugía , Trasplante Homólogo , Resultado del Tratamiento , Cicatrización de HeridasRESUMEN
Despite contributing a small percentage to the total body surface area, hands are the most commonly burned body part and are involved in over 90% of severe burns. Although the mortality of isolated hand burns is negligible, morbidity can be substantial given our need for functioning hands when performing activities of daily living. The greatest challenges of treating hand burns are 2-fold. First, determining the depth of injury can be difficult even for the most experienced surgeon, but despite many diagnostic options, clinical examination remains the gold standard. Second, appropriate postoperative hand therapy is crucial and requires a multidisciplinary approach with an experienced burn surgeon, hand surgeon, and hand therapist. Ultimately, the goals of treatment should include preservation of function and aesthetics. In this review, we present an approach to the management of the acutely burned hand with discussion of both conservative and surgical options. Regardless of the initial treatment decision, subsequent care for this subset of patients should be aimed at preventing debilitating postburn scar contractures that can severely limit hand function and ultimately require reconstructive surgery.
Asunto(s)
Quemaduras/terapia , Deformidades Adquiridas de la Mano/terapia , Traumatismos de la Mano/terapia , Actividades Cotidianas , Humanos , Procedimientos de Cirugía PlásticaRESUMEN
Pediatric frontal sinus fractures are a rare clinical entity. Owing to the large amount of force required to fracture the frontal sinus, it is often associated with severe intracranial and craniofacial injuries. The treatment of frontal sinus fractures is controversial, with many different established algorithms based mainly on the adult population. The authors present their experience with pediatric frontal sinus fractures; they also present a treatment algorithm. A retrospective review of the Cincinnati Children's Hospital Medical Center trauma database was performed. From 1998 to 2010, the authors identified patients between the ages of 0 and 18 with frontal sinus fractures and analyzed demographics, fracture pattern, associated injuries, methods of treatment, and complications. Descriptive statistics and univariate analyses were performed.A total of 39 patients were included in the study with a mean follow-up of 31.2 months. Fractures of the anterior and posterior table with displacement greater than one table width were significantly associated with higher hospital costs, higher velocity mechanism of injuries, lower Glasgow Coma Scale scores, nasofrontal outflow tract (NFOT) involvement, and cerebrospinal fluid leak. There were no differences in short- and long-term complications. Additionally, these patients were more likely to be treated surgically in the form of obliteration or cranialization.Patients without NFOT involvement can be managed with observation only. Patients with NFOT involvement or persistent cerebrospinal fluid leak should be treated with obliteration or cranialization, respectively, to reduce the risk of severe complications.
Asunto(s)
Algoritmos , Seno Frontal/lesiones , Seno Frontal/cirugía , Fracturas Craneales/cirugía , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Masculino , Complicaciones Posoperatorias/etiología , Estudios Retrospectivos , Fracturas Craneales/diagnósticoRESUMEN
The diagnosis of childhood neurological disorders remains challenging given the overlapping clinical presentation across subgroups and heterogeneous presentation within subgroups. To determine the underlying genetic cause of a severe neurological disorder in a large consanguineous Pakistani family presenting with severe scoliosis, anarthria and progressive neuromuscular degeneration, we performed genome-wide homozygosity mapping accompanied by whole-exome sequencing in two affected first cousins and their unaffected parents to find the causative mutation. We identified a novel homozygous splice-site mutation (c.3512+1G>A) in the ALS2 gene (NM_020919.3) encoding alsin that segregated with the disease in this family. Homozygous loss-of-function mutations in ALS2 are known to cause juvenile-onset amyotrophic lateral sclerosis (ALS), one of the many neurological conditions having overlapping symptoms with many neurological phenotypes. RT-PCR validation revealed that the mutation resulted in exon-skipping as well as the use of an alternative donor splice, both of which are predicted to cause loss-of-function of the resulting proteins. By examining 216 known neurological disease genes in our exome sequencing data, we also identified 9 other rare nonsynonymous mutations in these genes, some of which lie in highly conserved regions. Sequencing of a single proband might have led to mis-identification of some of these as the causative variant. Our findings established a firm diagnosis of juvenile ALS in this family, thus demonstrating the use of whole exome sequencing combined with linkage analysis in families as a powerful tool for establishing a quick and precise genetic diagnosis of complex neurological phenotypes.