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Amyotrophic lateral sclerosis (ALS) is an adult-onset incurable motor neuron (MN) disease. The reasons for selective MN vulnerability in ALS are unknown. Axonal pathology is among the earliest signs of ALS. We searched for novel modulatory genes in human MN axon shortening affected by TARDBP mutations. In transcriptome analysis of RNA present in the axon compartment of human-derived induced pluripotent stem cell (iPSC)-derived MNs, PHOX2B (paired-like homeobox protein 2B) showed lower expression in TARDBP mutant axons, which was consistent with axon qPCR and in situ hybridization. PHOX2B mRNA stability was reduced in TARDBP mutant MNs. Furthermore, PHOX2B knockdown reduced neurite length in human MNs. Finally, phox2b knockdown in zebrafish induced short spinal axons and impaired escape response. PHOX2B is known to be highly express in other types of neurons maintained after ALS progression. Collectively, TARDBP mutations induced loss of axonal resilience, which is an important ALS-related phenotype mediated by PHOX2B downregulation.
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Esclerosis Amiotrófica Lateral/metabolismo , Axones/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Factores de Transcripción/metabolismo , Pez Cebra/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Proteínas de Homeodominio/genética , Humanos , Mutación , Fenotipo , Factores de Transcripción/genética , TranscriptomaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an inherited muscle disease caused by misexpression of the DUX4 gene in skeletal muscle. DUX4 is a transcription factor, which is normally expressed in the cleavage-stage embryo and regulates gene expression involved in early embryonic development. Recent studies revealed that DUX4 also activates the transcription of repetitive elements such as endogenous retroviruses (ERVs), mammalian apparent long terminal repeat (LTR)-retrotransposons and pericentromeric satellite repeats (Human Satellite II). DUX4-bound ERV sequences also create alternative promoters for genes or long non-coding RNAs, producing fusion transcripts. To further understand transcriptional regulation by DUX4, we performed nanopore long-read direct RNA sequencing (dRNA-seq) of human muscle cells induced by DUX4, because long reads show whole isoforms with greater confidence. We successfully detected differential expression of known DUX4-induced genes and discovered 61 differentially expressed repeat loci, which are near DUX4-ChIP peaks. We also identified 247 gene-ERV fusion transcripts, of which 216 were not reported previously. In addition, long-read dRNA-seq clearly shows that RNA splicing is a common event in DUX4-activated ERV transcripts. Long-read analysis showed non-LTR transposons including Alu elements are also transcribed from LTRs. Our findings revealed further complexity of DUX4-induced ERV transcripts. This catalogue of DUX4-activated repetitive elements may provide useful information to elucidate the pathology of FSHD. Also, our results indicate that nanopore dRNA-seq has complementary strengths to conventional short-read complementary DNA sequencing.
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Proteínas de Homeodominio/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Nanoporos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ARN/métodos , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Células Musculares/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Isoformas de Proteínas/genética , Isoformas de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN/estadística & datos numéricosRESUMEN
BACKGROUND: Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of mammalian germline cells. A large proportion of ERVs lose their open reading frames (ORFs), while others retain them and become exapted by the host species. However, it remains unclear what proportion of ERVs possess ORFs (ERV-ORFs), become transcribed, and serve as candidates for co-opted genes. RESULTS: We investigated characteristics of 176,401 ERV-ORFs containing retroviral-like protein domains (gag, pro, pol, and env) in 19 mammalian genomes. The fractions of ERVs possessing ORFs were overall small (~ 0.15%) although they varied depending on domain types as well as species. The observed divergence of ERV-ORF from their consensus sequences showed bimodal distributions, suggesting that a large proportion of ERV-ORFs either recently, or anciently, inserted themselves into mammalian genomes. Alternatively, very few ERVs lacking ORFs were found to exhibit similar divergence patterns. To identify candidates for ERV-derived genes, we estimated the ratio of non-synonymous to synonymous substitution rates (dN/dS) for ERV-ORFs in human and non-human mammalian pairs, and found that approximately 42% of the ERV-ORFs showed dN/dS < 1. Further, using functional genomics data including transcriptome sequencing, we determined that approximately 9.7% of these selected ERV-ORFs exhibited transcriptional potential. CONCLUSIONS: These results suggest that purifying selection operates on a certain portion of ERV-ORFs, some of which may correspond to uncharacterized functional genes hidden within mammalian genomes. Together, our analyses suggest that more ERV-ORFs may be co-opted in a host-species specific manner than we currently know, which are likely to have contributed to mammalian evolution and diversification.
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BACKGROUND: The characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. METHOD: Two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference (p.H517D) in the fused in sarcoma (FUS) were constructed. By combining MN reporter lentivirus, MN specific phenotype was analyzed. Moreover, RNA profiling of isolated axons were conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo. The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology. FINDINGS: We identified aberrant increasing of axon branchings in FUS-mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. We identified increased level of Fos-B mRNA, the binding target of FUS, in FUS-mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo. The commonality of those phenotypes was further confirmed with other ALS causative mutation than FUS. INTERPRETATION: Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS-mutant axon branching. FUND: Japan Agency for Medical Research and development; Japanese Ministry of Education, Culture, Sports, Science and Technology Clinical Research, Innovation and Education Center, Tohoku University Hospital; Japan Intractable Diseases (Nanbyo) Research Foundation; the Kanae Foundation for the Promotion of Medical Science; and "Inochi-no-Iro" ALS research grant.
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Esclerosis Amiotrófica Lateral/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteína FUS de Unión a ARN/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Axones/metabolismo , Axones/patología , Diferenciación Celular/genética , Línea Celular , Edición Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lentivirus/genética , Neuronas Motoras/metabolismo , Mutación , Neurogénesis/genética , Fenotipo , ARN Interferente Pequeño/genéticaRESUMEN
Tandemly repeated DNA is highly mutable and causes at least 31 diseases, but it is hard to detect pathogenic repeat expansions genome-wide. Here, we report robust detection of human repeat expansions from careful alignments of long but error-prone (PacBio and nanopore) reads to a reference genome. Our method is robust to systematic sequencing errors, inexact repeats with fuzzy boundaries, and low sequencing coverage. By comparing to healthy controls, we prioritize pathogenic expansions within the top 10 out of 700,000 tandem repeats in whole genome sequencing data. This may help to elucidate the many genetic diseases whose causes remain unknown.
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Epilepsias Mioclónicas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Secuencias Repetidas en Tándem , Secuenciación Completa del Genoma/métodos , Adulto , Algoritmos , Biología Computacional/métodos , Predisposición Genética a la Enfermedad , HumanosRESUMEN
The C-terminal Ig-domain of lamin A plays critical roles in cell function via interaction with proteins, DNA, and chromatin. Mutations in this domain are known to cause various diseases including Emery-Dreifuss muscular dystrophy (EDMD) and familial partial lipodystrophy (FPLD). Here we examined the biophysical and biochemical properties of mutant Ig-domains identified in patients with EDMD and FPLD. EDMD-related mutant Ig-domain showed decreased stability to heat and denaturant. This result was also confirmed by experiments using full-length mutant lamin A, although the decrease in melting temperature was much less than that of the mutant Ig-domain alone. The unstable EDMD Ig-domain disrupted the proper assembly of lamin A, resulting in abnormal paracrystal formation and decreased viscosity. In contrast, FPLD-related mutant Ig-domains were thermally stable, although they lost DNA binding function. Alanine substitution experiments revealed a functional domain of DNA binding in the Ig-domain. Thus, the overall biophysical property of Ig-domains is closely associated with clinical phenotype.
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Lamina Tipo A/química , Distrofia Muscular de Emery-Dreifuss/metabolismo , Sustitución de Aminoácidos , Fenómenos Biofísicos , ADN/química , ADN/metabolismo , Humanos , Técnicas In Vitro , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/metabolismo , Microscopía Electrónica de Transmisión , Modelos Moleculares , Distrofia Muscular de Emery-Dreifuss/genética , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios Proteicos , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
We present a plasmid-based system in which upstream trans-splicing efficiently generates mRNAs that encode head-to-tail protein multimers. In this system, trans-splicing occurs between one of two downstream splice donors in the sequence encoding a C-terminal V5 epitope tag and an upstream splice acceptor in the 5' region of the pCS2(+) host plasmid. Using deletion and fusion constructs of the DUX4 protein as an example, we found that this system produced trans-spliced mRNAs in which coding regions from independent transcripts were fused in phase such that covalent head-to-tail protein multimers were translated. For a cDNA of ~450 bp, about half of the expressed proteins were multimeric, with the efficiency of trans-splicing and extent of multimer expression decreasing as cDNA length increased. This system generated covalent heterodimeric proteins upon co-transfections of plasmids encoding separate proteins and did not require a long complementary binding domain to position mRNAs for trans-splicing. This plasmid-based trans-splicing system is adaptable to multiple gene delivery systems, and it presents new opportunities for investigating molecular mechanisms of trans-splicing, generating covalent protein multimers with novel functions within cells, and producing mRNAs encoding large proteins from split precursors.
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Ingeniería Genética , Plásmidos/genética , ARN Mensajero , Trans-Empalme , Células HEK293 , Células HeLa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Plásmidos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Facioscapulohumeral dystrophy type 1 (FSHD-1) is the most common autosomal dominant form of muscular dystrophy with a prevalence of â¼1 in 8000 individuals. It is considered a late-onset form of muscular dystrophy and leads to asymmetric muscle weakness in the facial, scapular, trunk and lower extremities. The prevalent hypothesis on disease pathogenesis is explained by misexpression of a germ line, primate-specific transcription factor DUX4-fl (double homeobox 4, full-length isoform) linked to the chromosome 4q35. In vitro and in vivo studies have demonstrated that very low levels of DUX4-fl expression are sufficient to induce an apoptotic and/or lethal phenotype, and therefore modeling of the disease has proved challenging. In this study, we expand upon our previously established injection model of DUX4 misexpression in zebrafish and describe a DUX4-inducible transgenic zebrafish model that better recapitulates the expression pattern and late onset phenotype characteristic of FSHD patients. We show that an induced burst of DUX4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable. We also utilize our injection model to study long-term consequences of DUX4 expression in those that fail to show a developmental phenotype. Herein, we introduce a hypothesis that DUX4 expression during developmental stages is sufficient to induce FSHD-like phenotypes in later adulthood. Our findings point to a developmental role of DUX4 misexpression in the pathogenesis of FSHD and should be factored into the design of future therapies.
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Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Contracción Muscular , Músculo Esquelético/embriología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal , Distrofia Muscular Facioescapulohumeral/embriología , Distrofia Muscular Facioescapulohumeral/etiología , Distrofia Muscular Facioescapulohumeral/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Aberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites.This article has an associated First Person interview with the first author of the paper.
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INTRODUCTION: Single-incision laparoscopic cholecystectomy (SILC) is widely used as a treatment option for gallbladder disease. However, obesity has been considered a relative contraindication to this approach due to more advanced technical difficulties. The aim of this report was to review our experience with SILC to evaluate the impact of body mass index (BMI) on the surgical outcome. PATIENTS AND METHODS: Between May 2009 and February 2013, 237 patients underwent SILC at our institute. Pre- and post-operative data of the 17 obese patients (O-group) (BMI ≥30 kg/m2) and 220 non-obese patients (NO-group) (BMI <29.9 kg/m2) were compared retrospectively. SILC was performed under general anaesthesia, using glove technique. Indications for surgery included benign gallbladder disease, except for emergent surgeries. RESULTS: Mean age of patients was significantly higher in the NO-group than O-group (58.9 ± 13.5 years vs. 50.8 ± 14.0 years, P = 0.025). SILC was successfully completed in 233 patients (98.3%). Four patients (1.7%) in the NO-group required an additional port, and one patient was converted to an open procedure. The median operative time was 70 ± 25 min in the NO-group and 75.2 ± 18.3 min in the O-group. All complications were minor, except for one case in the NO-group that suffered with leakage of the cystic duct stump, for which endoscopic nasobiliary drainage was need. CONCLUSION: Our findings show that obesity, intended as a BMI ≥30 kg/m2, does not have an adverse impact on the technical difficulty and post-operative outcomes of SILC. Obesity-related comorbidities did not increase the risks for SILC.
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Subtelomeric macrosatellite repeats are difficult to sequence using conventional sequencing methods owing to the high similarity among repeat units and high GC content. Sequencing these repetitive regions is challenging, even with recent improvements in sequencing technologies. Among these repeats, a haplotype carrying a particular sequence and shortening of the D4Z4 array on human chromosome 4q35 causes one of the most prevalent forms of muscular dystrophy with autosomal-dominant inheritance, facioscapulohumeral muscular dystrophy (FSHD). Here, we applied a nanopore-based ultra-long read sequencer to sequence a BAC clone containing 13 D4Z4 repeats and flanking regions. We successfully obtained the whole D4Z4 repeat sequence, including the pathogenic gene DUX4 in the last D4Z4 repeat. The estimated sequence accuracy of the total repeat region was 99.8% based on a comparison with the reference sequence. Errors were typically observed between purine or between pyrimidine bases. Further, we analyzed the D4Z4 sequence from publicly available ultra-long whole human genome sequencing data obtained by nanopore sequencing. This technology may be a new tool for studying D4Z4 repeats and pathomechanism of FSHD in the future and has the potential to widen our understanding of subtelomeric regions.
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Cromosomas Humanos Par 4/genética , Proteínas de Homeodominio/genética , Distrofia Muscular Facioescapulohumeral/genética , Nanoporos , Secuencias Repetitivas de Aminoácido , Análisis de Secuencia de ADN/métodos , HumanosRESUMEN
In order to facilitate reliable and efficient determination of both the particle number concentration (PNC) and the size of nanoparticles (NPs) by single-particle ICP-MS (spICP-MS) without the need to correct for the particle transport efficiency (TE, a possible source of bias in the results), a total-consumption sample introduction system consisting of a large-bore, high-performance concentric nebulizer and a small-volume on-axis cylinder chamber was utilized. Such a system potentially permits a particle TE of 100 %, meaning that there is no need to include a particle TE correction when calculating the PNC and the NP size. When the particle TE through the sample introduction system was evaluated by comparing the frequency of sharp transient signals from the NPs in a measured NP standard of precisely known PNC to the particle frequency for a measured NP suspension, the TE for platinum NPs with a nominal diameter of 70 nm was found to be very high (i.e., 93 %), and showed satisfactory repeatability (relative standard deviation of 1.0 % for four consecutive measurements). These results indicated that employing this total consumption system allows the particle TE correction to be ignored when calculating the PNC. When the particle size was determined using a solution-standard-based calibration approach without an NP standard, the particle diameters of platinum and silver NPs with nominal diameters of 30-100 nm were found to agree well with the particle diameters determined by transmission electron microscopy, regardless of whether a correction was performed for the particle TE. Thus, applying the proposed system enables NP size to be accurately evaluated using a solution-standard-based calibration approach without the need to correct for the particle TE.
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This study was aimed to assess the feasibility and short-term outcomes of adjuvant systemic chemotherapy with either S-1/oxaliplatin (SOX) or mFOLFOX6 (FOLFOX)after curative resection of distant metastases from colorectal cancer. We retrospectively examined 16 patients who underwent R0 resection of colorectal metastases, including the liver (n=6), lung (n=5), lymph node (n=3), and peritoneum (n=2), followed by chemotherapy with SOX (n=7) or FOLFOX (n=9) until disease progression. The mean recurrence-free survival was 13.2 months in the SOX group and 16.9 months in the FOLFOX group. The mean overall survival was 17.9 and 22.9 months, respectively. The number of given courses were 6.5 and 11.0, respectively. Although sensory neuropathy was observed in 38% of the patients, relative dose intensity was higher than 80%. Adjuvant chemotherapy with SOX or FOLFOX was feasible and effective. Further randomized prospective trials are warranted to confirm these results.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Recto/tratamiento farmacológico , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Quimioterapia Adyuvante , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Combinación de Medicamentos , Femenino , Fluorouracilo/uso terapéutico , Humanos , Leucovorina/uso terapéutico , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Compuestos Organoplatinos/administración & dosificación , Compuestos Organoplatinos/uso terapéutico , Oxaliplatino , Ácido Oxónico/administración & dosificación , Neoplasias del Recto/patología , Neoplasias del Recto/cirugía , Recurrencia , Estudios Retrospectivos , Tegafur/administración & dosificaciónRESUMEN
This data article tested whether polymorphisms within the dopamine D4 receptor (DRD4) gene promoter can lead to differences in the promoter activity. The variants, a 120-bp variable number tandem repeat (VNTR), -906 T/C, -809 G/A, -616G/C, and -521C/T, were introduced into the DRD4 promoter and the promoter activity was measured in a neural cell line using the luciferase assay. However, no differences were detected among the haplotypes investigated, and the in vitro data obtained from our protocol could not support the involvement of DRD4 promoter polymorphisms in heritable human traits.
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The demand for precious metals has increased in recent years. However, low concentrations of precious metals dissolved in wastewater are yet to be recovered because of high operation costs and technical problems. The unicellular red alga, Galdieria sulphuraria, efficiently absorbs precious metals through biosorption. In this study, over 90% of gold and palladium could be selectively recovered from aqua regia-based metal wastewater by using G. sulphuraria. These metals were eluted from the cells into ammonium solutions containing 0.2M ammonium salts without other contaminating metals. The use of G. sulphuraria is an eco-friendly and cost-effective way of recovering low concentrations of gold and palladium discarded in metal wastewater.
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Oro/aislamiento & purificación , Paladio/aislamiento & purificación , Rhodophyta , Aguas Residuales/química , Purificación del Agua/métodos , IonesRESUMEN
Expression of chloride channel 1 (CLCN1/ClC-1) in skeletal muscle is driven by alternative splicing, a process regulated in part by RNA-binding protein families MBNL and CELF. Aberrant splicing of CLCN1 produces many mRNAs, which were translated into inactive proteins, resulting in myotonia in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. This increase in abnormal splicing variants containing exons 6B, 7A or the insertion of a TAG stop codon just before exon 7 leads to a decrease in expression of the normal splice pattern. The majority of studies examining splicing in CLCN1 have been performed using mouse Clcn1, as have investigations into the activation and suppression of normal splicing variant expression by MBNL1-3 and CELF3-6, respectively. In contrast, examinations of human CLCN1 have been less common due to the greater complexity of splicing patterns. Here, we constructed a minigene containing CLCN1 exons 5-7 and established a novel assay system to quantify the expression of the normal splicing variant of CLCN1 using real-time RT-PCR. Antisense oligonucleotides could promote normal CLCN1 alternative splicing but the effective sequence was different from that of Clcn1. This result differs from previous reports using Clcn1, highlighting the effect of differences in splicing patterns between mice and humans.
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Myotonic dystrophy type 1 (DM1) is a genetic disorder in which multiple genes are aberrantly spliced. Sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) is one of these genes, and it encodes a P-type ATPase. SERCA1 transports Ca(2+) from the cytosol to the lumen, and is involved in muscular relaxation. It has two splice variants (SERCA1a and SERCA1b) that differ in the last eight amino acids, and the contribution of these variants to DM1 pathology is unclear. Here, we show that SERCA1b protein is highly expressed in DM1 muscle tissue, mainly localised at fast twitch fibres. Additionally, when SERCA1a and SERCA1b were overexpressed in cells, we found that the ATPase and Ca(2+) uptake activity of SERCA1a was almost double that of SERCA1b. Although the affinity for both ATP and Ca(2+) was similar between the two variants, SERCA1b was more sensitive to the inner microsomal environment. Thus, we hypothesise that aberrant expression of SERCA1b in DM1 patients is the cause of abnormal intracellular Ca(2+) homeostasis.
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CHRNA1 encodes the α subunit of nicotinic acetylcholine receptors (nAChRs) and is expressed at the neuromuscular junction. Moreover, it is one of the causative genes of Congenital Myasthenic Syndromes (CMS). CHRNA1 undergoes alternative splicing to produce two splice variants: P3A(-), without exon P3A, and P3A(+), with the exon P3A. Only P3A(-) forms functional nAChR. Aberrant alternative splicing caused by intronic or exonic point mutations in patients leads to an extraordinary increase in P3A(+) and a concomitant decrease in P3A(-). Consequently this resulted in a shortage of functional receptors. Aiming to restore the imbalance between the two splice products, antisense oligonucleotides (AONs) were employed to induce exon P3A skipping. Three AON sequences were designed to sterically block the putative binding sequences for splicing factors necessary for exon recognition. Herein, we show that AON complementary to the 5' splice site of the exon was the most effective at exon skipping of the minigene with causative mutations, as well as endogenous wild-type CHRNA1. We conclude that single administration of the AON against the 5' splice site is a promising therapeutic approach for patients based on the dose-dependent effect of the AON and the additive effect of combined AONs. This conclusion is favorable to patients with inherited diseases of uncertain etiology that arise from aberrant splicing leading to a subsequent loss of functional translation products because our findings encourage the option of AON treatment as a therapeutic for these prospectively identified diseases.
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Exones , Síndromes Miasténicos Congénitos/terapia , Oligonucleótidos Antisentido/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , Receptores Nicotínicos/genética , Células HEK293 , Humanos , Empalme del ARNRESUMEN
According to the amyloid hypothesis, amyloid ß accumulates in brains with Alzheimer's disease (AD) and triggers cell death and memory deficit. Previously, we developed a rice Aß vaccine expressing Aß, which reduced brain Aß levels in the Tg2576 mouse model of familial AD. We used senescence-accelerated SAMP8 mice as a model of sporadic AD and investigated the relationship between Aß and oxidative stress. Insoluble Aß and 4-hydroxynonenal (4-HNE) levels tended to be reduced in SAMP8 mice-fed the rice Aß vaccine. We attempted to clarify the relationship between oxidative stress and Aß in vitro. Addition of Aß peptide to the culture medium resulted in an increase in 4-HNE levels in SH-SY5Y cells. Tg2576 mice, which express large amounts of Aß in their brain, also exhibited increased 4-HNE levels; this increase was inhibited by the Aß vaccine. These results indicate that Aß induces oxidative stress in cultured cells and in the mouse brain.
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Envejecimiento , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Estrés Oxidativo , Fragmentos de Péptidos/metabolismo , Aldehídos/metabolismo , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Encéfalo/metabolismo , Tampones (Química) , Humanos , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Transgénicos , Oryza/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Solubilidad , Vacunas/genéticaRESUMEN
OBJECTIVES: Emerging concepts of "reduced port surgery" have gained considerable attention from laparoscopic surgeons, including the field of liver resection. To date, 86 cases of single-incision laparoscopic hepatectomy (SILH) have been reported, with commercially available access devices being used in most of these cases. We report herein a use of homemade transumbilical glove port for SILH. METHODS: A 39-year-old woman represented giant hepatic hemangioma (9-cm in size) located at the left lateral segment (S2/3). Partial hepatectomy was performed by the glove method via single port access with conventional laparoscopic bipolar forceps, grasper and scissors without the need of any single-port specific devices. RESULTS: The operative time was 77 minutes, and intraoperative blood loss was 50 mL. The postoperative course was uneventful. CONCLUSIONS: Glove method not only has significant advantages in terms of cost, but also is superior in its versatility, allowing wider range of movements compared to conventional access devices. Taking in consideration its cost effectiveness and versatility, glove method may be a good option for SILH.