RESUMO
mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.
Assuntos
COVID-19 , Humanos , Indicadores e Reagentes , Espectrometria de Massas/métodos , Cromatografia Líquida/métodos , Padrões de Referência , Isótopos , Marcação por Isótopo/métodosRESUMO
Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.
Assuntos
Pseudouridina/análise , Processamento Pós-Transcricional do RNA , RNA Ribossômico/análise , RNA Ribossômico/metabolismo , RNA Nuclear Pequeno/análise , RNA Nuclear Pequeno/metabolismo , Animais , Linhagem Celular , Deutério/química , Humanos , Marcação por Isótopo , Espectrometria de Massas , Camundongos , Complexos Multienzimáticos/química , Orotato Fosforribosiltransferase/química , Orotidina-5'-Fosfato Descarboxilase/química , Pseudouridina/química , RNA Ribossômico/química , RNA Nuclear Pequeno/químicaRESUMO
BACKGROUND: The gold standard for diagnosis of cutaneous sporotrichosis involves the isolation of the fungus, Sporothrix, by a culture test. Generally, the sampling for the culture test is performed at the same time as skin biopsy under local anaesthesia. However, the culture test may occasionally return a false negative result. OBJECTIVE: The aim of our study was to investigate the diagnostic value of a molecular method for diagnosing cutaneous sporotrichosis from formalin-fixed and paraffin-embedded (FFPE) tissues. METHODS: Over a 30-year period, we collected 52 cases of cutaneous sporotrichosis from biopsied specimens that had been positively diagnosed by a culture test. A nested PCR specific for Sporothrix detection was applied using FFPE tissue as template. The results were compared with control samples from 79 patients diagnosed with other cutaneous diseases according to histopathological, clinical findings and a cutler test. RESULTS: Of the 52 patients who were tested positive on the culture test, all cutaneous diseases were detected by PCR. Of the 59 patients in the control group, 58 tested negative by PCR. Under our conditions, the calculated sensitivity of this method was 100%, the specificity was 98.7% and the kappa coefficient was 0.984 (95% CI: 0.953-1.000). CONCLUSIONS: The specific PCR assay used appears to be a useful tool for the prompt and accurate diagnosis of sporotrichosis. Using this method, it would be possible to diagnose cutaneous sporotrichosis for patients who were suspected of cutaneous sporotrichosis but tested negative on culturing, and for pathologically suspected cutaneous sporotrichosis patients for whom the culture test was not undertaken.
Assuntos
Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/normas , Pele/microbiologia , Sporothrix/isolamento & purificação , Esporotricose/diagnóstico , Idoso , Biópsia , DNA Fúngico/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Sensibilidade e EspecificidadeRESUMO
Rheumatoid arthritis (RA) is a disease of unknown aetiology that causes irreversible joint destruction and has been known to present with not only various extra-articular symptoms, but also various autoimmune disorders. Mucous membrane pemphigoid (MMP) is a chronic autoimmune disease characterised by inflamed and eroded mucosa. The prognosis of MMP can be poor, so early diagnosis and prompt initiation of therapy are necessary for optimal management. Here, we report a rare case of RA complicated with MMP involving a 68-year-old woman admitted to our hospital because of hoarseness and symmetrical narrowing of the eye fissures. She presented with bilateral coxalgia and had been diagnosed with RA 24 years earlier. Oral methotrexate was prescribed, but subsequently discontinued, and this was followed by treatment with tocilizumab 3 years earlier. Tocilizumab was discontinued because of financial distress 5 months earlier, after which her RA disease activity worsened. She presented to our hospital after further worsening of her eye-opening difficulty. Physical and laboratory examinations led to a diagnosis of MMP. Her sputum, cough, throat discomfort, conjunctival congestion, mucous erosion, and blistering promptly disappeared after treatment with rituximab (500 mg per week for 4 weeks). She subsequently recovered her vocalisation ability, and her hoarseness, dysphagia, and eye-opening difficulty gradually improved, but not completely. This case suggests that that RA and MMP share common immunological mechanisms. Therefore, MMP should be considered when encountering patients with RA who present with systemic membrane mucous disorders.
Assuntos
Artrite Reumatoide , Penfigoide Mucomembranoso Benigno , Idoso , Artrite Reumatoide/diagnóstico , Feminino , Humanos , Penfigoide Mucomembranoso Benigno/complicaçõesRESUMO
Potassium iodide (KI), initially derived from seaweed in the early 19th century, is used for treating sporotrichosis in dermatological practice. KI has also been used to treat several noninfectious inflammatory skin diseases. However, the mechanisms underlying the improvement in such skin diseases remain unknown, and KI is not used widely. Thus, although KI is an old drug, physicians may not prescribe it frequently because they lack knowledge about it. Although KI is very inexpensive and causes few side effects, it has been superseded by new powerful and expensive drugs, such as biological agents. We applied 3% KI topically to areas of inflammation induced by SDS in mice. The levels of IL-1 and TNF-α gene expression were reduced, whereas that of IL-10 gene expression was increased. Small interfering RNA that was designed to reduce IL-10 gene expression levels was injected into the same mice, and the anti-inflammatory effects of KI were not observed. Thus, the pharmacologic action of KI is based on its anti-inflammatory effects caused by the increase in IL-10 levels. This information would increase dermatologists' awareness of KI as an efficacious and cost-effective treatment.
Assuntos
Anti-Inflamatórios/farmacologia , Dermatite/tratamento farmacológico , Iodeto de Potássio/farmacologia , Animais , Citocinas/genética , Dermatite/imunologia , Dermatite/patologia , Feminino , Interleucina-10/fisiologia , Interleucinas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Iodeto de Potássio/uso terapêutico , Dodecilsulfato de Sódio/farmacologiaRESUMO
OBJECTIVE: Rebamipide tablets, which are used in the treatment of patients with gastric ulcers or gastritis, can be difficult to administer in subjects with reduced swallowing ability or impaired swallowing. The granule formulation may be more easily administered in these patients. The bioequivalence between rebamipide granules (20%/0.5g) and tablets (100mg) was determined in healthy male adult volunteers, in accordance with the Partially Revised Guidelines for Bioequivalence Studies of Generic Products. STUDY DESIGN: In a randomised, nonblind, crossover design, 28 individuals were allocated into two groups of 14 to receive either rebamipide granules or rebamipide tablets. Each individual, under fasting conditions, was administered a single oral dose of rebamipide 100mg followed by a 7-day washout period. Individuals then received a single oral dose of the other rebamipide formulation. Blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 hours. Plasma rebamipide concentrations were measured by validated high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The plasma concentration-time profiles and pharmacokinetic parameters of rebamipide after administration of the granule formulation were similar to those of the tablet in 27 healthy male volunteers. Following administration of the granule formulation, the area under the plasma concentration-time curve from time 0-24 hours (AUC(24h)) was 912.82 mug/L . h, the maximum plasma concentration (C(max)) was 241.82 mug/L, time to maximum plasma concentration (t(max)) was 2.5 hours, and plasma elimination half-life (t((1/2))) was 1.97 hours. Corresponding values for the tablet formulation were 873.55 microg/L . h, 216.19 mug/L, 2.4 hours, and 1.94 hours. The difference in mean log values was 1.01 for AUC(24h) and 1.09 for C(max) after granule and tablet administration. The 90% confidence interval of this difference in mean log value was 0.93-1.10 for AUC(24h), and 0.97-1.21 for C(max). This satisfies the criteria for bioequivalence in the guidelines [within log (0.8) to log (1.25)]. CONCLUSIONS: Rebamipide granules (20%/0.5g) and tablet (100mg) were bioequivalent. Rebamipide granules may therefore be a more practical treatment option in patients with gastric ulcers or gastritis who have difficulty swallowing tablets.
Assuntos
Autoanticorpos/sangue , Dermatomiosite/diagnóstico , Helicase IFIH1 Induzida por Interferon/imunologia , Autoanticorpos/imunologia , Criança , Dermatomiosite/sangue , Dermatomiosite/imunologia , Humanos , Pulmão/diagnóstico por imagem , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/imunologia , Masculino , Pele/patologia , Tomografia Computadorizada por Raios XRESUMO
Although the majority of hepatitis E virus (HEV) infections in Japan are 'domestic' due to the presence of indigenous strains, there are still 'imported' cases as well. Among 83 patients with non-A, non-B and non-C acute liver diseases admitted to Saitama Medical University Hospital, 7 (8.4%) were positive for serum HEV-RNA, of whom 2 had a recent history of traveling to China, one to Xian and another to Shanghai. We determined the full-genome sequences of HEV from these 2 patients (isolate names are JKO-ChiSai98c and JYI-ChiSai01c, genotype 4 in both) for phylogenetic analyses. Initially, when compared only to the 13 full-genome sequences of genotype 4 so far reported, our 2 isolates were thought to be novel strains because they showed a significant genetic difference from the sequences known to date. However, when we included a set of short sequences (150 nt) recently reported from China in the comparison, we found that our 2 isolates represent a subgroup of genotype 4, which seems to be restricted to eastern China. In conclusion, the 2 HEV isolates reported here could serve as full-genome prototypes for an eastern China-indigenous subgroup of the genotype 4 HEV.
Assuntos
Genoma Viral/genética , Vírus da Hepatite E/genética , Hepatite E/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , China , Feminino , Genótipo , Hepatite E/patologia , Vírus da Hepatite E/classificação , Vírus da Hepatite E/isolamento & purificação , Histocitoquímica , Humanos , Japão , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , RNA Viral/sangue , Análise de Sequência de DNA , Homologia de Sequência , ViagemRESUMO
Little is understood about the usefulness of sulfur isotopic ratios (sigma 34S) in tree rings because the sulfur content in rings is generally insufficient for analysis using conventional methods. We present sigma 34S values of the water-soluble and the organically bound sulfur fractions in rings of coniferous trees grown in Japan, analyzed using a large-volume oxygen bomb. Comparing the sigma 34S values of the organically bound fraction in tree rings with past atmospheric sulfur concentrations and with those of their sources, we find clear evidence that the sigma 34S values of the organically bound fraction in the rings are dependent upon the values of the atmospheric sulfur sources. The evidence suggests that the sigma 34S values in tree rings are a useful chronological proxy for evaluating possible causes of past atmospheric sulfur pollution.