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1.
J Transl Med ; 22(1): 644, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38982507

RESUMO

BACKGROUND: Genetic disorders often manifest as abnormal fetal or childhood development. Copy number variations (CNVs) represent a significant genetic mechanism underlying such disorders. Despite their importance, the effectiveness of clinical exome sequencing (CES) in detecting CNVs, particularly small ones, remains incompletely understood. We aimed to evaluate the detection of both large and small CNVs using CES in a substantial clinical cohort, including parent-offspring trios and proband only analysis. METHODS: We conducted a retrospective analysis of CES data from 2428 families, collected from 2018 to 2021. Detected CNV were categorized as large or small, and various validation techniques including chromosome microarray (CMA), Multiplex ligation-dependent probe amplification assay (MLPA), and/or PCR-based methods, were employed for cross-validation. RESULTS: Our CNV discovery pipeline identified 171 CNV events in 154 cases, resulting in an overall detection rate of 6.3%. Validation was performed on 113 CNVs from 103 cases to assess CES reliability. The overall concordance rate between CES and other validation methods was 88.49% (100/113). Specifically, CES demonstrated complete consistency in detecting large CNV. However, for small CNVs, consistency rates were 81.08% (30/37) for deletions and 73.91% (17/23) for duplications. CONCLUSION: CES demonstrated high sensitivity and reliability in CNV detection. It emerges as an economical and dependable option for the clinical CNV detection in cases of developmental abnormalities, especially fetal structural abnormalities.


Assuntos
Variações do Número de Cópias de DNA , Sequenciamento do Exoma , Doenças Genéticas Inatas , Humanos , Variações do Número de Cópias de DNA/genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Reprodutibilidade dos Testes , Feminino , Valor Preditivo dos Testes , Masculino , Estudos Retrospectivos
2.
Environ Res ; 251(Pt 1): 118573, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38431070

RESUMO

Anaerobically digested sludge supernatant, characterized by its high ammonia and low biodegradable chemical oxygen demand (COD) content, has raised concerns when returned to mainstream treatment lines due to potential impacts on effluent quality. Addressing this, an aerobic granular sludge (AGS) reactor adopted nitritation/denitritation with external COD addition was utilized and achieved a considerable nitrogen treatment capacity of 4.2 kg N/m3/d, reaching over 90% removal efficiencies for both ammonia and total inorganic nitrogen. This study applied progressively increased nitrogen loading to select for a microbial community that exhibited high nitrogen oxidation and reduction rates, demonstrating peak rates of 0.5 g N/g VSS/d and 3 g N/g VSS/d, respectively. The enrichment of highly efficient microbial community was achieved along with the increased biomass density peaked at 17 g/L MLVSS, with the system retaining small-sized granular sludge at 0.5 mm. The primary ammonia oxidizing bacteria was Nitrosomonas, while Thauera was the dominated denitrifiers. Quantitative polymerase chain reaction analyses reinforced the enhanced nitrogen removal capacity based on the progressively increased abundance of nitrogen cycling functional genes. The high nitrogen treatment capacity, synergistic attributes of high specific microbial activities and the substantial biomass retention, suggest the AGS's efficacy and capacity in ammonia rich wastewater treatment.


Assuntos
Amônia , Reatores Biológicos , Nitrogênio , Esgotos , Reatores Biológicos/microbiologia , Esgotos/microbiologia , Amônia/metabolismo , Nitrogênio/metabolismo , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias/microbiologia , Águas Residuárias/química , Aerobiose , Bactérias/metabolismo , Bactérias/genética , Poluentes Químicos da Água/metabolismo
3.
Int J Mol Sci ; 25(6)2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38542256

RESUMO

This study aimed to evaluate the impact of Candida albicans on subgingival biofilm formation on dental implant surfaces. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to compare biofilm structure and microbial biomass in the presence and absence of the fungus after periods of 24, 48, and 72 h. Quantitative polymerase chain reaction (qPCR) was used to quantify the number of viable and total micro-organisms for each of the biofilm-forming strains. A general linear model was applied to compare CLSM and qPCR results between the control and test conditions. The biofilm developed with C. albicans at 72 h had a higher bacterial biomass and a significantly higher cell viability (p < 0.05). After both 48 and 72 h of incubation, in the presence of C. albicans, there was a significant increase in counts of Fusobacterium nucleatum and Porphyromonas gingivalis and in the cell viability of Streptococcus oralis, Aggregatibacter actinomycetemcomitans, F. nucleatum, and P. gingivalis. Using a dynamic in vitro multispecies biofilm model, C. albicans exacerbated the development of the biofilm grown on dental implant surfaces, significantly increasing the number and cell viability of periodontal bacteria.


Assuntos
Candida albicans , Implantes Dentários , Sobrevivência Celular , Biofilmes , Porphyromonas gingivalis
4.
J Cell Biochem ; 124(1): 118-126, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36436137

RESUMO

Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases in the world and has a strong genetic predisposition. At present, there is still no effective method for the early diagnosis and prevention of AD. Accumulating evidence shows the association of several loci with AD risk, such as apolipoprotein E (APOE) and translocase of outer mitochondrial membrane 40 (TOMM40). However, for routine disease diagnosis in clinics, genotype detection methods based on gene sequencing technology are time-consuming and excessively costly. Thus, in this study, we developed a high-sensitivity, low-cost, and convenient single nucleotide polymorphism (SNP) detection assay method based on allele-specific quantitative polymerase chain reaction (AS-qPCR) technology, which can be used to determine the SNP genotype in APOE and TOMM40. A total of 40 patients were recruited from the outpatient department of the memory clinic of Dongzhimen Hospital, Beijing University of Chinese Medicine. The SNP detection assay method includes three steps. First, positive plasmids with different genotypes (TT/CC/TC) in APOE rs429358, rs7412, and TOMM40 rs11556505 were prepared. Second, 3'-T/3'-C primers were designed to amplify these positive plasmids for each SNP site. Finally, we calculated the log10 of the copy number ratio for each positive plasmid, and the genotype interpretation interval was established. Based on this method, we investigated whether the SNPs in 40 patients could be accurately calculated using AS-qPCR technology. The accuracy of SNP detection was verified by PCR-Pooling sequencing. The results showed that SNP genotypes assessed by AS-qPCR technology corresponded perfectly to the results obtained by conventional DNA sequencing. We have developed a genotype detection method for AD based on AS-qPCR, which can be performed easily, rapidly, accurately, and at low cost. The method will contribute to the early diagnosis of patients with late-onset Alzheimer's and the detection of large clinical samples in the future.


Assuntos
Doença de Alzheimer , Polimorfismo de Nucleotídeo Único , Humanos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Alelos , Predisposição Genética para Doença , Genótipo , Apolipoproteínas E/genética
5.
Electrophoresis ; 44(21-22): 1714-1724, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37847880

RESUMO

Correctly inferring the tissue origin types of forensic-relevant body fluids left at a crime scene is beneficial for reconstructing a crime scene. However, it is still a challenge to accurately identify different kinds of body fluids at a crime scene. Shorter sequence length and anti-degradation microRNA (miRNA) can be used to infer the tissue sources of biological fluid traces, but a limited number of miRNAs are tissue specific. The application of messenger RNA (mRNA) has been confirmed by different studies based on its high tissue specificity. According to the differential expression features of mRNA or miRNA in forensically relevant body fluids, this study developed a simultaneously reversed mRNA and miRNA system and then used these two types of RNAs for the determinations of five common kinds of body fluids. Compared with previously reported single kind of mRNA or miRNA assay, the combined mRNA and miRNA system showed good advantages for human body fluid identifications, especially it could be applied in mixed samples. In conclusion, the obtained results indicated that this combined mRNA and miRNA system might provide a scientific and accurate reference for body fluid identifications.


Assuntos
Líquidos Corporais , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/análise , Saliva/química , RNA Mensageiro/genética , Sêmen/química , Sêmen/metabolismo , Genética Forense/métodos , Menstruação , Líquidos Corporais/química
6.
Int J Legal Med ; 137(5): 1353-1360, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37306739

RESUMO

Teeth are frequently used for human identification from burnt remains, as the structure of a tooth is resilient against heat exposure. The intricate composition of hydroxyapatite (HA) mineral and collagen in teeth favours DNA preservation compared to soft tissues. Regardless of the durability, the integrity of the DNA structure in teeth can still be disrupted when exposed to heat. Poor DNA quality can negatively affect the success of DNA analysis towards human identification. The process of isolating DNA from biological samples is arduous and costly. Thus, an informative pre-screening method that could aid in selecting samples that can potentially yield amplifiable DNA would be of excellent value. A multiple linear regression model to predict the DNA content in incinerated pig teeth was developed based on the colourimetry, HA crystallite size and quantified nuclear and mitochondrial DNA. The chromaticity a* was found to be a significant predictor of the regression model. This study outlines a method to predict the viability of extracting nuclear and mitochondrial DNA from pig teeth that were exposed to a wide range of temperatures (27 to 1000 °C) with high accuracy (99.5-99.7%).


Assuntos
DNA Mitocondrial , Dente , Humanos , Suínos , Animais , DNA Mitocondrial/análise , Dente/química , Colorimetria , Núcleo Celular , Temperatura Alta
7.
BMC Infect Dis ; 23(1): 413, 2023 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-37337136

RESUMO

BACKGROUND: Rapid and accurate identification of carbapenemase-producing organism (CPO) intestinal carriers is essential for infection prevention and control. Molecular diagnostic methods can produce results in as little as 1 h, but require special instrumentation and are expensive. Therefore, it is urgent to find an alternative method. The broth enrichment-multiplex lateral flow immunochromatographic assay was recently reported, but using it to directly detect CPO intestinal carriers in rectal swabs still requires the evaluation of many samples. The aim of this study was to compare the performance of these two methods, and to explore the control measures of CPO infection. METHODS: Through CPO selective culture, PCR and DNA sequencing, 100 rectal swabs confirmed to be CPO-positive and 100 rectal swabs with negative results were collected continuously. After eluting the rectal swabs with saline, three aliquots were used: one for counting, one for detection by Xpert Carba-R, and one for culture in broth for 0 h, 1 h, 2 h, 3 h and 4 h, followed by NG-Test CARBA 5 assessment. The sensitivity and specificity of the NG-Test CARBA 5 method after different incubation times were calculated. The limit of detection (LoD) of this assay after 4 h broth incubation was estimated by examining the bacterial suspensions and simulated faecal suspensions prepared with CPOs producing different types of carbapenemases. RESULTS: Xpert Carba-R demonstrated a combined sensitivity of 99.0% and specificity of 98.0%. The sensitivity and specificity were higher than 90.0% for the different enzyme types. The specificities of five common carbapenemases detected by the broth enrichment NG-Test CARBA 5 combined method after different incubation times were 100%. The sensitivities increased with increasing incubation time. At 4 h, the Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase (OXA) -48 detection sensitivities were 93.0%, 96.3%, 100%, 100% and 85.7%, respectively. The LoDs were between 102 and 104 CFU/mL for all five enzymes after 4 h of incubation. CONCLUSIONS: This investigation highlighted that the broth enrichment-multiplex lateral flow immunochromatographic assay can be used as a new method for screening CPOs in rectal swabs.


Assuntos
Proteínas de Bactérias , beta-Lactamases , Humanos , Suspensões , Proteínas de Bactérias/genética , Proteínas de Bactérias/análise , beta-Lactamases/genética , beta-Lactamases/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Imunoensaio
8.
BMC Vet Res ; 19(1): 163, 2023 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-37723566

RESUMO

BACKGROUND: Dental plaque microbes play a key role in the development of periodontal disease. Numerous high-throughput sequencing studies have generated understanding of the bacterial species associated with both canine periodontal health and disease. Opportunities therefore exist to utilise these bacterial biomarkers to improve disease diagnosis in conscious-based veterinary oral health checks. Here, we demonstrate that molecular techniques, specifically quantitative polymerase chain reaction (qPCR) can be utilised for the detection of microbial biomarkers associated with canine periodontal health and disease. RESULTS: Over 40 qPCR assays targeting single microbial species associated with canine periodontal health, gingivitis and early periodontitis were developed and validated. These were used to quantify levels of the respective taxa in canine subgingival plaque samples collected across periodontal health (PD0), gingivitis (PD1) and early periodontitis (PD2). When qPCR outputs were compared to the corresponding high-throughput sequencing data there were strong correlations, including a periodontal health associated taxa, Capnocytophaga sp. COT-339 (rs =0.805), and two periodontal disease associated taxa, Peptostreptococcaceae XI [G-4] sp. COT-019 (rs=0.902) and Clostridiales sp. COT-028 (rs=0.802). The best performing models, from five machine learning approaches applied to the qPCR data for these taxa, estimated 85.7% sensitivity and 27.5% specificity for Capnocytophaga sp. COT-339, 74.3% sensitivity and 67.5% specificity for Peptostreptococcaceae XI [G-4] sp. COT-019, and 60.0% sensitivity and 80.0% specificity for Clostridiales sp. COT-028. CONCLUSIONS: A qPCR-based approach is an accurate, sensitive, and cost-effective method for detection of microbial biomarkers associated with periodontal health and disease. Taken together, the correlation between qPCR and high-throughput sequencing outputs, and early accuracy insights, indicate the strategy offers a prospective route to the development of diagnostic tools for canine periodontal disease.


Assuntos
Doenças do Cão , Gengivite , Doenças Periodontais , Periodontite , Animais , Cães , Estudos Prospectivos , Periodontite/diagnóstico , Periodontite/veterinária , Doenças Periodontais/diagnóstico , Doenças Periodontais/veterinária , Gengivite/diagnóstico , Gengivite/veterinária , Doenças do Cão/diagnóstico , Aprendizado de Máquina
9.
BMC Ophthalmol ; 23(1): 177, 2023 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-37098507

RESUMO

BACKGROUND: A microfluidic real-time polymerase chain reaction (PCR) system can rapidly detect the viral DNA in specimens. Detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA in tears is a useful diagnostic tool for herpes simplex virus keratitis (HSK) and herpes zoster ophthalmicus (HZO). METHODS: In total, 20 patients were included in this cross-sectional study. Among them, 8 patients with infectious epithelial HSK and 12 patients with HZO were included in HSK and HZO groups, respectively. In addition, 8 patients with non-herpetic keratitis and 4 healthy individuals without keratitis were included in the control group. Numbers of HSV and VZV DNA copies in tears of all patients and individuals were evaluated using a microfluidic real-time PCR system. Regarding HSV/VZV DNA test, tear specimens were collected by filter paper method using Schirmer's test paper, and subsequently, DNA was extracted from the filter paper using an automated nucleic acid extractor. Afterward, quantitative PCR was performed using a microfluidic real-time PCR system. RESULTS: From tear collection to real-time PCR result determination, the HSV/VZV DNA test took approximately 40 min. In the HSK group, the sensitivity and specificity of the HSV DNA tests were 100% each. The median value (range) of number of HSV DNA copies for affected eyes was 3.4 × 105 copies/µL (under a lower detection limit of 7.6). In the HZO group, the sensitivity and specificity of the VZV DNA tests were 100% each. The median value (range) of number of VZV DNA copies for affected eyes was 5.3 × 105 copies/µL (under a lower detection limit of 5.6 × 10-2). CONCLUSION: In conclusion, quantitative PCR for HSV and VZV DNA in tears using a microfluidic real-time PCR system is useful for diagnosing and monitoring HSK and HZO.


Assuntos
Herpes Simples , Herpesvirus Humano 1 , Ceratite Herpética , Humanos , Herpesvirus Humano 3/genética , Estudos Transversais , Microfluídica , Herpesvirus Humano 1/genética , Ceratite Herpética/diagnóstico , Herpes Simples/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Viral/análise
10.
J Dairy Sci ; 106(11): 7477-7485, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37641239

RESUMO

Bifidobacterium animalis, one of the predominant bacteria in the intestines of humans and other mammals, is widely added to dairy products. We employed RNA sequencing to analyze gene expression variance on a genome-wide scale and found stable reference genes (RG) in B. animalis. A total of 1,665 genes were identified by analyzing the data from the transcriptome under 4 different conditions, and 13 probable candidate RG with variation coefficient values <0.1 were validated using reverse-transcription quantitative PCR (RT-qPCR). The amplification efficiency of candidate RG were ranging from 94.16% to 126.25%. We integrated the analysis results of BestKeeper, geNorm, NormFinder, and RefFinder algorithms and revealed that rplD and atpA comprehensive ranked 1.68 and 2.82, respectively, which were more stable than traditional RG. Compared with plate count (1.58 × 106 cfu/mL), the concentrations of B. animalis AR668 by RT-qPCR using rplD, atpA, and 16S rRNA as RG were 2.27 × 106, 2.24 × 106, and 6.66 × 106 cfu/mL, respectively, after 10 h of fermentation in fermented skim milk. It suggested that rplD and atpA as RG can be accurate for colony counting of B. animalis. Our study provides the foundation for more accurate analysis of colony counting by RT-qPCR of B. animalis in dairy foods.

11.
Int J Mol Sci ; 24(20)2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37894839

RESUMO

Mesenchymal stem cells derived from rheumatoid arthritis patients (RA-MSCs) provide an understanding of a variety of cellular and immunological responses within the inflammatory milieu. Sustained exposure of MSCs to inflammatory cytokines is likely to exert an influence on genetic variations, including reference genes (RGs). The sensitive effect of cytokines on the reference genes of RA-SF-MSCs may be a variation factor affecting patient-derived MSCs as well as the accuracy and reliability of data. Here, we comparatively evaluated the stability levels of nine RG candidates, namely GAPDH, ACTB, B2M, EEF1A1, TBP, RPLP0, PPIA, YWHAZ, and HPRT1, to find the most stable ones. Alteration of the RG expression was evaluated in MSCs derived from the SF of healthy donors (H-SF-MSCs) and in RA-SF-MSCs using the geNorm and NormFinder software programs. The results showed that TBP, PPIA, and YWHAZ were the most stable RGs for the normalization of H-SF-MSCs and RA-SF-MSCs using RT-qPCR, whereas ACTB, the most commonly used RG, was less stable and performed poorly. Additionally, the sensitivity of RG expression upon exposure to proinflammatory cytokines (TNF-α and IL-1ß) was evaluated. RG stability was sensitive in the H-SF-MSCs exposed to TNF-α and IL-1ß but insensitive in the RA-SF-MSCs. Furthermore, the normalization of IDO expression using ACTB falsely diminished the magnitude of biological significance, which was further confirmed with a functional analysis and an IDO activity assay. In conclusion, the results suggest that TBP, PPIA, and YWHAZ can be used in SF-MSCs, regardless of their exposure to inflammatory cytokines.


Assuntos
Artrite Reumatoide , Células-Tronco Mesenquimais , Humanos , Citocinas/genética , Citocinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Líquido Sinovial , Reprodutibilidade dos Testes , Perfilação da Expressão Gênica/métodos , Células-Tronco Mesenquimais/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Padrões de Referência , Reação em Cadeia da Polimerase em Tempo Real/métodos
12.
J Environ Manage ; 325(Pt B): 116669, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36335700

RESUMO

Stormwater control measures such as detention basins are used to mitigate the negative effects of urban stormwater resulting from watershed development. In this study, the performance of a detention basin in mitigating nitrogen pollution was examined and the abundance of N-cycling genes (amoA, nirK, nosZ, hzsB and Ntsp-amoA) present in the soil media of the basin was measured using quantitative PCR. Results showed a net export of nitrogen from the basin, however, differences between in- and outflow concentrations were not significant. Furthermore, the quantitative PCR showed that nirK (denitrification gene) was more abundant in the winter season, whereas amoA (nitrification gene) was more abundant in the summer season. The abundance of nirK, Ntsp-amoA and hzsB genes also varied with the sampling depth of soil and based on 16S rRNA gene sequencing of soil samples, Actinobacteria and Proteobacteria were the most dominant phyla. Species diversity appeared higher in summer, while the top and bottom layer of soil clustered separately based on the bacterial community structure. These results underline the importance of understanding nitrogen dynamics and microbial processes within stormwater control measures to enhance their design and performance.


Assuntos
Microbiota , Nitrogênio , Nitrogênio/análise , Desnitrificação/genética , RNA Ribossômico 16S/genética , Nitrificação , Solo/química , Microbiologia do Solo
13.
J Helminthol ; 97: e15, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36722449

RESUMO

This meta-analysis was designed to assess the effect of the addition of a bead-beating (BB) step during DNA extraction to effectively isolate Trichuris trichiura DNA from stool samples for quantitative polymerase chain reaction (qPCR)-based diagnosis. qPCR-based molecular studies comparing the inclusion of a bead-beating step during the DNA extraction from stool samples with extraction without the step were included in the analysis. Studies using real patient samples in community settings were included. The PubMed database and Google search engine were searched in December 2019. Risk of bias and applicability were assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 checklist. Odds ratios (ORs) for individual studies were combined to estimate the random effects model OR. A total of six independent sub-studies were gathered from two published original articles. The division of the two major studies into six sub-studies was indispensable due to the nature of the study carried out. 128 of the total 192 samples (in all studies) were positive for T. trichiura when BB was used during DNA extraction compared to 108/192 when BB was excluded. The combined OR was 1.66 (95% confidence interval: 1.059 to 2.602). Though only two articles were included in the study, six exclusive individual sub-studies were analyzed. Inherent differences in the background prevalence of helminths in the study population could impact the sensitivity of qPCR. It was found that the inclusion of the BB step during DNA extraction significantly increased the sensitivity of the test. This study was not registered in any database.


Assuntos
Lista de Checagem , Trichuris , Animais , Humanos , Trichuris/genética , Ferramenta de Busca , Reação em Cadeia da Polimerase
14.
Br J Haematol ; 197(1): 52-62, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34997766

RESUMO

The clinical outcome of chronic myeloid leukaemia patients has vastly improved since the introduction of tyrosine kinase inhibitor treatment, with a significant proportion of patients able to achieve treatment-free remission. However, studies have shown that patients with the e13a2 transcript were less likely to achieve major molecular response compared to those with e14a2 transcripts. Most quantitative polymerase chain reaction (PCR) assays for detection of the BCR-ABL1 fusion gene do not differentiate between the two transcripts and we therefore hypothesised that technical bias linked to the qPCR assay could partially explain the discrepancy in outcomes. We designed an e14a2-specific assay and identified no difference in results compared to an e13a2 standard assay. We then demonstrated that the commercial e14a2 standards were causing a significant overestimation of the e13a2 transcripts. Finally, we reviewed patient management after the qPCR values were corrected, using our new evaluation. We concluded that despite statistically significant differences in qPCR results, there was no impact on patient management or outcome. We conclude that, at least in our institution, it would be inappropriate to perform separate assays for patients with e13a2 or e14a2.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Reação em Cadeia da Polimerase em Tempo Real , Tecnologia
15.
J Pediatr ; 241: 154-161, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34619114

RESUMO

OBJECTIVE: To determine changes in mitochondrial DNA (mtDNA) copy number in peripheral blood in Rett syndrome caused by methyl-CpG-binding protein-2 (MECP2) variants and explore the mechanism of mitochondrial dysfunction in Rett syndrome. STUDY DESIGN: Female patients who were diagnosed with Rett syndrome and had an MECP2 variant (n = 142) were recruited in this study, along with the same number of age- and sex-matched healthy controls. MtDNA copy number was quantified by real-time quantitative polymerase chain reaction with TaqMan probes. The differences in mtDNA copy number between the Rett syndrome group and the control group were analyzed using the independent-samples t test. Linear regression, biserial correlation analysis, and one-way ANOVA were applied for the correlations between mtDNA copy number and age, clinical severity, variant types, functional domains, and hot-spot variants. RESULTS: MtDNA copy number was found to be significantly increased in the patients with Rett syndrome with MECP2 gene variants compared with the control subjects. Age, clinical severity, variant types, functional domains, and hot-spot variants were not related to mtDNA copy number in patients with Rett syndrome. CONCLUSIONS: MtDNA copy number is increased significantly in patients with Rett syndrome, suggesting that changes in mitochondrial function in Rett syndrome trigger a compensatory increase in mtDNA copy number and providing new possibilities for treating Rett syndrome, such as mitochondria-targeted therapies.


Assuntos
Variações do Número de Cópias de DNA , DNA Mitocondrial , Proteína 2 de Ligação a Metil-CpG/genética , Mitocôndrias/genética , Síndrome de Rett/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos Transversais , Feminino , Marcadores Genéticos , Humanos , Lactente , Modelos Lineares , Gravidade do Paciente , Síndrome de Rett/fisiopatologia , Adulto Jovem
16.
Transpl Int ; 35: 10728, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36187462

RESUMO

The impact of T cell-mediated rejection (TCMR) after liver transplantation (LT) on the alterations in the gut microbiota (GM) and associated intestinal environment represented by fecal organic acids (OAs) require further elucidation. A rat allogeneic LT model was prepared without immunosuppressants or antibiotics, and a syngeneic model was used as a control. Qualitative and quantitative analyses of fecal samples at fixed time points were performed. Correlation analyses were also performed between liver function and GMs and OA levels. In the allogeneic TCMR group, the number of predominant obligate anaerobes decreased as liver function declined. Clostridioides difficile, Enterobacteriaceae, Enterococcus, Streptococcus, and Staphylococcus were significantly increased. Regarding fecal OA concentration, short-chain fatty acid (SCFA) concentrations were depleted as liver function declined. In contrast, in the syngeneic group, GM and OAs exhibited only slight, transient, and reversible disturbances. In addition, alanine aminotransferase and total bilirubin were positively correlated with the number of Enterobacteriaceae and Enterococcus, and negatively correlated with the fecal concentration of SCFAs. The allogeneic TCMR model demonstrated distinct dysbiosis and depletion of fecal OAs as TCMR progressed after LT. The degree of graft injury was closely related to the number of specific bacterial strains and the concentrations of fecal SCFAs.


Assuntos
Disbiose , Transplante de Fígado , Alanina Transaminase , Animais , Antibacterianos , Bilirrubina , Disbiose/microbiologia , Ácidos Graxos Voláteis/análise , Imunossupressores , Transplante de Fígado/efeitos adversos , Ratos
17.
Mol Biol Rep ; 49(6): 4999-5016, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35184269

RESUMO

MicroRNAs (microRNAs) have been implicated to play crucial roles in various liver diseases. Hepatic microRNAs are released in to the circulation in a systematic fashion, and are, therefore, being extensively explored for their role as prognostic or diagnostic markers or therapeutic targets for numerous hepatic diseases. Advantages such as disease- or tissue-specific expression, and ease of detection have implicated circulating microRNAs (c-microRNAs) as the most desirable candidate for biomarker studies. Although several studies have explored c-microRNAs in serum or plasma, consistency of detection of microRNAs remains demanding because of many biological and methodological challenges. Lack of methodological consensus has limited the universal use of c-microRNAs as potential prognostic or diagnostic disease-specific biomarkers. In this review, we have discussed pre-analytical and analytical factors that might affect c-miRNA expression and selection of appropriate data normalization strategy by incorporating endogenous reference microRNAs. This review will aid designing of better approaches and protocols for future diagnostic research on hepatic c-microRNAs.


Assuntos
MicroRNA Circulante , Hepatopatias , MicroRNAs , Biomarcadores , MicroRNA Circulante/genética , Humanos , Hepatopatias/diagnóstico , Hepatopatias/genética , MicroRNAs/genética
18.
Indoor Air ; 32(1): e12975, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34897813

RESUMO

Fluorescent aerosol cytometry (FAC) was compared to concurrent recovery of airborne ß-N-acetylhexosaminidase (NAHA) and quantitative polymerase chain reaction (qPCR) for the respective ability of these methods to detect significant changes in airborne fungal loads in response to building renovations. Composite, site-randomized indoor aerosol samples for airborne fungi measurements were acquired from more than 70 occupied classrooms in 26 different public schools in the Colorado Rocky Mountain Front Range region of the United States. As judged by ANOVA and Pearson's correlation test, statistically significant associations were observed between real-time FAC and airborne NAHA levels, which detected significant reductions in airborne fungal loads immediately following building rehabilitations. With lower confidence, a statistically significant association was also resolved between fluorescing aerosols, NAHA levels, and the recovery of fungal 18S rRNA gene copies by qPCR from simultaneous, collocated aerosol samples. Quantitative differences encountered between the recovery of common genomic markers for airborne fungi and that of optical and biochemical methods are attributed to the variance in 18S rRNA target gene copies that different fungal species can host.


Assuntos
Microbiologia do Ar , Poluição do Ar em Ambientes Fechados , Aerossóis , Poluição do Ar em Ambientes Fechados/análise , Fluorescência , Fungos/genética , Genômica , Hexosaminidases , Instituições Acadêmicas , beta-N-Acetil-Hexosaminidases
19.
Chem Eng Sci ; 251: 117430, 2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35043022

RESUMO

Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 µL reaction at isothermal 58℃ within 40 min. Importantly, the primers for LAMP assay locate at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to be a promising test in COVID-19 surveillance.

20.
J Infect Dis ; 223(9): 1590-1600, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32877517

RESUMO

BACKGROUND: Limited information is available on pneumococcal colonization among adults. We studied pneumococcal carriage dynamics in healthy adults using high-sensitivity approaches. METHODS: Eighty-seven adults (25-50 years old) were followed for 6 months in Portugal. Nasopharyngeal, oropharyngeal, and saliva samples were obtained monthly; pneumococcal carriers were also sampled weekly. Carriage was investigated by quantitative polymerase chain reaction (targeting lytA and piaB) and culture. Positive samples were serotyped. RESULTS: Approximately 20% of the adults were intermittent carriers; 10% were persistent carriers (>4 months). Pneumococcal acquisition and clearance rates were 16.5 (95% confidence interval [CI], 11.2-24.2) and 95.9 (95% CI, 62.3-145.0) cases/1000 person-weeks, respectively. Living with children increased pneumococcal acquisition (hazard ratio, 9.7 [95% CI, 2.6-20.5]; P < .001). Median duration of carriage was 7 weeks and did not depend on regular contact with children. CONCLUSIONS: The pneumococcal carrier state in healthy adults is more dynamic than generally assumed: Acquisition is frequent and duration of carriage is often long. This suggests that some adults may act as reservoirs of pneumococci and hence, depending on the social structure of a community, the magnitude of herd effects potentially attainable through children vaccination may vary. These findings are important when designing strategies to prevent pneumococcal disease in adults.


Assuntos
Portador Sadio , Infecções Pneumocócicas , Adulto , Portador Sadio/epidemiologia , Humanos , Pessoa de Meia-Idade , Nasofaringe/microbiologia , Orofaringe/microbiologia , Infecções Pneumocócicas/epidemiologia , Portugal/epidemiologia , Saliva/microbiologia , Estrutura Social , Streptococcus pneumoniae
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