RESUMO
Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs.
Assuntos
Citosol/metabolismo , Exossomos/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Sequência de Aminoácidos , Citometria de Fluxo , Células HEK293 , Humanos , Metiltransferases/química , Microscopia Imunoeletrônica , RNA Mensageiro/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína 1 de Ligação a Y-Box/químicaRESUMO
Site-directed spin labeling (SDSL) is widely applied for structural studies of biopolymers by electron paramagnetic resonance (EPR). However, SDSL of long RNA sequences still remains a challenging task. Here, we propose a novel SDSL approach potentially suitable for long natural RNAs, which is based on the attachment of a linker containing an aliphatic amino group to the target nucleotide residue followed by selective coupling of a spin label to this amino group. Such a linker can be attached to the desired RNA residue via a sequence-specific reaction with the derivatives of oligodeoxyribonucleotides. To verify this approach, we applied it to model RNA duplex with known structure and expected distance between corresponding residues. A new 2,5-bis(spirocyclohexane)-substituted spin label with advanced stability and relaxation properties has been used, and the distance distribution measured using Q-band (34 GHz) pulsed double electron-electron resonance corresponds well to the expected one. We have additionally validated the obtained results by studying a similar RNA duplex, where the linker with the aliphatic amino group was introduced via solid-phase synthesis. Although this novel SDSL approach does not provide an advantage in precision of molecular distance measurements, we believe that its applicability to long RNAs is a crucial benefit for future structural studies using pulse EPR.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , RNA/química , Marcadores de Spin , Alquilação , Sequência de Bases , DNA Complementar/genética , Elétrons , Dados de Sequência Molecular , Conformação de Ácido NucleicoAssuntos
Calcinose/cirurgia , Cistos/cirurgia , Endoscopia Gastrointestinal/métodos , Obstrução Intestinal/cirurgia , Doenças do Colo Sigmoide/cirurgia , Calcinose/complicações , Cistos/complicações , Humanos , Obstrução Intestinal/etiologia , Masculino , Pessoa de Meia-Idade , Doenças do Colo Sigmoide/complicaçõesAssuntos
Doenças do Colo/parasitologia , Equinococose Hepática/complicações , Obstrução Intestinal/parasitologia , Calcinose/complicações , Doenças do Colo/diagnóstico , Doenças do Colo/cirurgia , Colonoscopia , Diagnóstico Diferencial , Humanos , Obstrução Intestinal/diagnóstico , Obstrução Intestinal/cirurgia , Litotripsia , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVE: To improve the quality of clinical care for women with severe pre-eclampsia. METHODS: A criteria-based audit was conducted in a large government hospital in Uganda. Management practices were evaluated against standards developed by an expert panel by retrospectively evaluating 43 case files. Results of the audit were presented, and recommendations developed and implemented. A re-audit was conducted 6 months later. RESULTS: The initial audit showed that most standards were rarely achieved. Reasons were discussed. Guidelines were produced, additional supplies were purchased following a fundraising effort, labor ward procedures were streamlined, and staffing was increased. In the re-audit there were significant improvements in diagnosis, monitoring, and treatment. CONCLUSION: Criteria-based audit can improve the quality of maternity care in countries with limited resources.
Assuntos
Serviços de Saúde Materna/normas , Unidade Hospitalar de Ginecologia e Obstetrícia/normas , Obstetrícia/normas , Pré-Eclâmpsia/terapia , Feminino , Humanos , Auditoria Médica , Obstetrícia/métodos , Gravidez , Cuidado Pré-Natal , Garantia da Qualidade dos Cuidados de Saúde , UgandaRESUMO
PROBLEM: Maternal mortality in Uganda has remained unchanged at 500/100 000 over the past 10 years despite concerted efforts to improve the standard of maternity care. It is especially difficult to improve standards in rural areas, where there is little money for improvements. Furthermore, staff may be isolated, poorly paid, disempowered, lacking in morale, and have few skills to bring about change. DESIGN: Training programme to introduce criteria based audit into rural Uganda. SETTING: Makerere University Medical School, Mulago Hospital (large government teaching hospital in Kampala), and Mpigi District (rural area with 10 small health centres around a district hospital). STRATEGIES FOR CHANGE: Didactic teaching about criteria based audit followed by practical work in own units, with ongoing support and follow up workshops. EFFECTS OF CHANGE: Improvements were seen in many standards of care. Staff showed universal enthusiasm for the training; many staff produced simple, cost-free improvements in their standard of care. LESSONS LEARNT: Teaching of criteria based audit to those providing health care in developing countries can produce low cost improvements in the standards of care. Because the method is simple and can be used to provide improvements even without new funding, it has the potential to produce sustainable and cost effective changes in the standard of health care. Follow up is needed to prevent a waning of enthusiasm with time.
Assuntos
Mortalidade Materna , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Feminino , Pesquisa sobre Serviços de Saúde , Hospitais Rurais , Humanos , Auditoria Médica , Projetos Piloto , Gravidez , Garantia da Qualidade dos Cuidados de Saúde/normas , Uganda/epidemiologiaRESUMO
Total large bowel evaluation remains an essential step in the treatment of patients with colorectal cancer (CRC). Colonoscopy is the gold standard in the evaluation of the colon for colorectal tumors, but may be incomplete due to tumor obstruction, which is a frequent event in distal cancers. Double-contrast barium enema has a lower accuracy and is not ideal in the presence of signs of obstruction. In theory, intraoperative colonoscopy is a valid alternative, but its routine use is impeded by various practical limitations. Preoperative survey of the colon in 521 consecutive patients treated for CRC in our department was based on colonoscopy (92.5%). Our series was characterised by a high percentage of distal lesions (76.4%) and therefore by a high percentage of incomplete preoperative colonoscopies (50.4%) due to tumor obstruction. In the presence of an incomplete preoperative colonoscopy, we evaluated the entire colon with a double-contrast barium enema in selected cases (36.7%) and with a postoperative colonoscopy within 3 months of surgery in almost all patients (93.4%). The overall rate of complete endoscopic evaluation, either pre- or postoperatively, was 96.7%. The incidence of adenomas was significantly higher in the preoperative examinations as compared to early postoperative colonoscopy. This means that in distal cancer the vast majority of polyps will be located in the distal colon and therefore included in a standard resection. Four patients (0.8%), required a second operation for treatment of a missed lesion (2 benign and 2 malignant). The need for a repeat surgery apparently did not affect the therapeutic results in these four patients. On the basis of our experience, intraoperative colonoscopy would not appear to be a mandatory procedure in all cases of incomplete preoperative evaluation of the colon. However, in the absence of prospective, randomised trials comparing intraoperative vs early postoperative colonoscopy, the dilemma as to the strategy of choice remains.
Assuntos
Adenoma/diagnóstico , Carcinoma/diagnóstico , Colonoscopia , Neoplasias Colorretais/diagnóstico , Adenoma/diagnóstico por imagem , Adenoma/cirurgia , Sulfato de Bário , Carcinoma/diagnóstico por imagem , Carcinoma/cirurgia , Pólipos do Colo/diagnóstico , Pólipos do Colo/diagnóstico por imagem , Pólipos do Colo/cirurgia , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/cirurgia , Enema , Humanos , Período Intraoperatório , Período Pós-Operatório , Cuidados Pré-Operatórios , Radiografia , Estudos RetrospectivosRESUMO
UNLABELLED: Colonoscopic screening has been recommended in asymptomatic first-degree relatives of patients with colorectal cancer. In fact this population is believed to have an increased risk in developing colorectal neoplasia. The purpose of this study is to report the impact of colonoscopy in a series of completely asymptomatic first-degree relatives of patients operated on for colorectal cancer at our institution. A total of 480 individuals was requested to participate in a screening program based on faecal occult blood testing (FOBT) and colonoscopy in those with positive FOBT. Colonoscopy was also suggested to persons with negative FOBT. After the first 195 examined relatives, FOBT was abandoned because of continuously increasing acceptance of colonoscopy. RESULTS: Two hundred fifty four subjects (52.4%) accepted to participate at the screening program. After the first 195 examined relatives, FOBT was abandoned because of continuously increasing acceptance of colonoscopy. A total of 142 colonoscopies was performed. Colonoscopy was completed in 112 relatives (78.9%). Thirty-three subjects (23.2%) had a positive colonoscopy: one had invasive adenocarcinoma, one had large villous adenoma and 31 had 54 polyps. Twenty-three lesions (40.3%) were located proximal to the splenic flexure. Of the 54 polyps, 45 (83.3%) were less than 1 cm in size. These findings confirm the utility of colonoscopic screening in asymptomatic first-degree relatives of patients with colorectal cancer. However, its feasibility with the current endoscopic facilities remains an unsolved question.
Assuntos
Colonoscopia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/prevenção & controle , Programas de Rastreamento/métodos , Vigilância da População , Adenocarcinoma/diagnóstico , Adenocarcinoma/prevenção & controle , Adenoma Viloso/diagnóstico , Adenoma Viloso/prevenção & controle , Adulto , Idoso , Idoso de 80 Anos ou mais , Pólipos do Colo/diagnóstico , Pólipos do Colo/prevenção & controle , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Sangue Oculto , Aceitação pelo Paciente de Cuidados de Saúde , Estudos RetrospectivosRESUMO
The Authors report a clinical case of a patient endoscopically treated for recurrent oesophageal obstruction by a foreign body. A review of the recent medical literature confirms that immediate endoscopic treatment is the best approach in the oesophageal retention of foreign bodies.
Assuntos
Estenose Esofágica/etiologia , Esôfago , Corpos Estranhos/complicações , Estenose Esofágica/diagnóstico , Estenose Esofágica/cirurgia , Esofagoscopia , Seguimentos , Corpos Estranhos/diagnóstico , Corpos Estranhos/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Fatores de TempoRESUMO
The use of the polymerase chain reaction (PCR) to detect the presence of Epstein-Barr virus (EBV) DNA in oral mucosa in the absence of specific lesions gives rise to the problem of identifying the real viral replication sites. To verify whether the detection of EBV is due to salivary contamination or its true replicative capacity in oral mucosa, saliva samples and exfoliated cells from four different oral mucosa sites were taken from 40 renal transplant patients and 20 normal subjects for examination by PCR using two pairs of primers specific for the BamHI-L and BamHI-K genomic regions. EBV-specific sequences were detected in one or more of the oral mucosa samples from 29 transplant patients (72.5%) and six healthy controls (30%), and in the saliva samples of 16 transplant patients (40%) and three healthy controls (15%). A total of 89 oral mucosa smears from 29 transplant patients, and 13 from healthy subjects, were EBV-positive. The positive samples were also investigated by means of in situ hybridization in order to confirm the intracellular presence of the viral genome, and by means of immunofluorescence testing with monoclonal antibodies to assess the possible expression of viral antigens. Hybridization with the EBV-specific probe was observed in 40/ 89 and 2/13 samples, respectively. Latent antigens (with or without lytic antigens) were detected in only 23 of the 40 samples (collected from eight different transplant patients) that were positive by in situ hybridization. Our data show that EBV is more frequently present in the oral mucosa of immunodeficient patients (where it can efficiently replicate) than in normal subjects.
Assuntos
Antígenos Virais/análise , DNA Viral/análise , Herpesvirus Humano 4/genética , Transplante de Rim , Mucosa Bucal/virologia , Adolescente , Adulto , Anticorpos Monoclonais , Southern Blotting , Relação CD4-CD8 , Feminino , Imunofluorescência , Genoma Viral , Antígenos HLA-DR/análise , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/fisiologia , Humanos , Hospedeiro Imunocomprometido , Imunoglobulina A/análise , Imunoglobulina G/análise , Hibridização In Situ , Leucoplasia Pilosa/virologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Saliva/virologia , Análise de Sequência de DNA , Replicação ViralRESUMO
The authors report on the use of nimodipine in 36 patients for coma due to severe cerebral (20 haemorrhagic, 2 ischaemic, 9 post-anoxic, 4 traumatic, 1 neoplastic) lesions. In addition to resuscitative therapy, all patients were given nimodipine per os, 60 mg every 4 hrs for 21 days. In all patients, survival, duration of the coma and degree of disability, according G.O.S., were evaluated. Decrease of mortality was highly significant, decrease of disability of surviving patients was significant if compared with comatose patients suffering from similar diseases, but not treated with nimodipine.
Assuntos
Transtornos Cerebrovasculares/tratamento farmacológico , Coma/tratamento farmacológico , Nimodipina/uso terapêutico , Doença Aguda , Adolescente , Adulto , Idoso , Transtornos Cerebrovasculares/complicações , Transtornos Cerebrovasculares/mortalidade , Criança , Coma/etiologia , Coma/mortalidade , Avaliação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RessuscitaçãoRESUMO
Free radicals, because of their marked chemical activity, have often been found to be involved in many human diseases. Enzymatic antioxidant systems, namely glutathione-reductase, present both in red blood cells and in serum, have been demonstrated to play a key role as free radicals scavengers. The present work has been carried out in order to evaluate the possible role played by free radicals in the demineralization process of the bone matrix. Glutathione-reductase activity, assayed by a slightly modified Horn's method, was related to bone density measurements. All the subjects with reduced densitometric values showed reduced glutathione-reductase levels. Our results seem to support the hypothesis of a strict relationship between low activity of antioxidant systems and demineralization process of the bone, in consequence of enhanced free radical levels.
Assuntos
Densidade Óssea , Sequestradores de Radicais Livres , Glutationa Redutase/sangue , Idoso , Reabsorção Óssea/enzimologia , Feminino , Glutationa Redutase/deficiência , Humanos , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/enzimologiaRESUMO
The Authors report a new case of chondrosarcoma of the mandible. After stressing the rarity of the tumour in the facial bones, they discuss the treatment problem. They propose a surgical approach as long as metastases are absent and recommend radio and chemotherapy just for palliation of advanced cases.