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1.
BMC Res Notes ; 14(1): 345, 2021 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-34479650

RESUMEN

OBJECTIVE: Species identification of Shigella isolates are so prominent for epidemiological studies and infection prevention strategies. We developed and evaluated RAPD and ERIC-PCR coupled with HRM for differentiation of non-dysenteriae Shigella species as potential alternative methods. After isolation of eighteen Shigella strains from faecal specimens collected from children under 2 years of age with diarrhea (n = 143), the species of the isolates were identified by slide agglutination assay. Also, species were identified using developed RAPD-PCR-HRM and ERIC-PCR-HRM techniques. Differentiation of the data sets was measured by principal component analysis as a dimension reduction method. Then, sensitivity and specificity of the methods were evaluated. RESULTS: We found RAPD-PCR-HRM method with high sensitivity and specificity (100 and 85% respectively) to identify non-dysenteriae Shigella species in clinical specimens. However, sensitivity and specificity of ERIC-PCR-HRM were evaluated 33 and 46% respectively and significantly lower than that of RAPD-PCR-HRM assay. Regardless of inherent poor reproducibility of DNA fingerprinting-based methods, RAPD-PCR-HRM assay can be considered as a potential alternative method to identify non-dysenteriae species of Shigella in clinical specimens. As we observed in the current study, HRM technique is more rapid, inexpensive, and sensitive than gel electrophoresis method to characterize PCR amplicons.


Asunto(s)
Shigella , Niño , Dermatoglifia del ADN , ADN Bacteriano , Humanos , Lactante , Reacción en Cadena de la Polimerasa , Técnica del ADN Polimorfo Amplificado Aleatorio , Reproducibilidad de los Resultados , Shigella/genética
2.
Nihon Ronen Igakkai Zasshi ; 58(3): 470-475, 2021.
Artículo en Japonés | MEDLINE | ID: mdl-34483175

RESUMEN

The coronavirus disease 2019 (COVID-19) infection has spread worldwide, with no sign of its control in Japan yet. Eight elderly COVID-19 patients over 90 years of age were treated at our hospital. We herein report three cases with characteristic progression. Case 1 was a 91-year-old female patient diagnosed with bacterial pneumonia previously who did not show improvement with medication; thus, she was transferred to our hospital 16 days after the onset. She was diagnosed with COVID-19 using the SARS-CoV-2 polymerase chain reaction (PCR) test. Favipiravir, methylprednisolone, and unfractionated heparin were administered, but she only tested negative 68 days after the onset, at which point she was discharged. However, she was transferred back to our hospital 80 days after the onset since she tested positive again. She was transferred to another hospital 110 days after the onset without testing negative. Case 2 was a 102-year-old female. Despite being a mild case, it took 32 days to obtain negative PCR findings, leading to a decline in the activities of daily living. Case 3 was a 90-year-old male patient treated with favipiravir, dexamethasone, and unfractionated heparin, but his condition deteriorated. He never tested negative for PCR and ultimately died 20 days after the onset. Reports suggest that PCR positivity does not necessarily indicate infectivity, but there are no clear criteria for lifting a quarantine. Therefore, PCR negativity is often sought for "peace of mind." In the current situation where hospitals are fully occupied, clear criteria for lifting the quarantine should be promptly determined. After the completion of treatment, it is more important to monitor symptoms and take standard precautions, such as daily health monitoring, wearing a mask, and keeping an appropriate distance from others, than to obtain a negative PCR result.


Asunto(s)
COVID-19 , Actividades Cotidianas , Anciano de 80 o más Años , COVID-19/diagnóstico , COVID-19/tratamiento farmacológico , Prueba de Ácido Nucleico para COVID-19 , Femenino , Humanos , Masculino , Alta del Paciente , Reacción en Cadena de la Polimerasa
3.
Rev Assoc Med Bras (1992) ; 67(4): 522-528, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-34495055

RESUMEN

OBJECTIVE: We retrospectively assessed whether there was a relationship between lung complications and some easily accessible markers to predict the presence of pulmonary consolidation in patients with coronavirus disease 2019 (COVID-19). METHODS: According to the polymerase chain reaction and chest computerized tomography results, the study was categorized into three groups. Group 1 (n=87) included the patients with polymerase chain reaction (+), group 2 (n=55) included the patients with polymerase chain reaction (-) and chest computerized tomography (+), and group 3 (n=77) included the patients with polymerase chain reaction (-) and chest computerized tomography (-), respectively. RESULTS: High-sensitivity C-reactive protein and increased age were associated with higher computerized tomography (CT) scores. CONCLUSION: Increased age and C-reactive protein (CRP) may suggest pulmonary infiltration on chest CT in patients with COVID-19.


Asunto(s)
COVID-19 , Humanos , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , SARS-CoV-2 , Tomografía Computarizada por Rayos X
4.
Blood Adv ; 5(17): 3322-3332, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34477813

RESUMEN

IKZF1 deletions (ΔIKZF1) are commonly detected in B-precursor acute lymphoblastic leukemia (ALL; B-ALL) and are widely assumed to have a significant impact on outcome. We compared the ability of multiplex ligand-dependent probe amplification (MLPA) and polymerase chain reaction (PCR) to detect ΔIKZF1 and to determine the impact on event-free survival of patients with precursor B-ALL aged 23 to 65 years recruited to the completed trial UKALL14 (ISRCTN 66541317). From 655 recruits with BCR-ABL1+ and BCR-ABL1- B-ALL, all available diagnostic DNA samples (76% of the recruited population) were screened by multiplex end point PCR covering 4 deletions: dominant-negative (DN) Δ4-7 or the loss of function Δ2-7, Δ4-8, and Δ2-8 (n = 498), MLPA (n = 436), or by both (n = 420). Although patients with BCR-ABL1- ΔIKZF1 were more likely to have minimal residual disease at the end of induction, we did not find any impact of ΔIKZF1 (including subgroup analysis for DN or loss-of-function lesions) or the IKZF1plus genotype on event-free, overall survival, or relapse risk by univariable or multivariable analyses. Consistent with the technical approach, MLPA not only detected a wider range of deletions than PCR but also failed to detect some PCR-detected lesions. The main difference between our study and others reporting an association between ΔIKZF1 and outcome is the older age of participants in our population. The impact of ΔIKZF1 in ALL may be less marked in an older population of patients. Our study underscores the need for analyses in large, harmonized data sets. This trial was registered at www.clinicaltrials.gov as #NCT01085617.


Asunto(s)
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Anciano , Proteínas de Fusión bcr-abl/genética , Humanos , Factor de Transcripción Ikaros/genética , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética
5.
Environ Pollut ; 287: 117651, 2021 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-34426396

RESUMEN

Antibiotic resistance in aquatic ecosystems presents an environmental health issue worldwide. Urban recipient water quality is susceptible to effluent discharges with antibiotic resistance contaminants and needs to be protected, particularly for those as sources of drinking water production. Knowledge on aquatic resistome profiles in downstream of wastewater treatment plants allows a better understanding of the extent to which antibiotic resistance contaminants emerge and spread in recipient waters, but such information remains very limited in Sweden. The key objective of this study was to determine the resistome profiles of numerous antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and other genes in urban recipient water systems connected to Sweden's major drinking water reservoir. This was achieved through analysis of surface water samples for 296 genes using high-throughput quantitative PCR arrays. A total of 167 genes were detected in at least one of the samples, including 150 ARGs conferring resistance to 11 classes of antibiotics, 7 integrase MGEs and 9 other genes. There was a spatial difference in the resistome profiles with the greatest average relative abundance of resistance genes observed in the water body of Västerås followed by Uppsala, Stockholm and Eskilstuna, as similar to the general pattern of the antibiotic sales for these regions. ARGs against ß-lactams and sulfonamides showed the highest average relative abundance in the studied water bodies, while vancomycin resistance genes were only found in the Uppsala water environment. Generally, the recipient water bodies were detected with higher numbers of genes and greater relative abundances as compared to the upstream sites. Anthropogenic pollution, i.e., wastewater discharge, in the recipient water was also reflected by the finding of intI, sul1 and crAssphage. Overall, this study provided the first quantitative assessment of aquatic environmental resistomes in Sweden, highlighting the widespread of antibiotic resistance contaminants in urban recipient waters.


Asunto(s)
Antibacterianos , Genes Bacterianos , Ecosistema , Reacción en Cadena de la Polimerasa , Suecia , Aguas Residuales
6.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-34360884

RESUMEN

Colorectal cancer (CRC) is the third most common cause of cancer-related deaths worldwide. Human papillomaviruses (HPVs) and Epstein-Barr virus (EBV) have been reported to be present in different types of human cancers, including CRCs, where they can play a key role in the onset and/or progression of these cancers. Thus, we herein explored the prevalence of high-risk HPVs and EBV in a cohort of 94 CRC tissue samples and 13 colorectal normal tissues from the Lebanese population using polymerase chain reaction, immunohistochemistry, and tissue microarray methodologies. We found that high-risk HPVs are present in 64%, while EBV is present in 29% of our CRC samples. Additionally, our data showed that high-risk HPV types (16, 18, 35, 58, 51, 45, 52, 31, and 33) are the most frequent in CRC in the Lebanese cohort, respectively. Our data point out that HPVs and EBV are copresent in 28% of the samples. Thus, this study clearly suggests that high-risk HPVs and EBV are present/copresent in CRCs, where they could play an important role in colorectal carcinogenesis. Nevertheless, further investigations using a larger cohort are needed to elucidate the possible cooperation between these oncoviruses in the development of CRC.


Asunto(s)
Alphapapillomavirus/genética , Neoplasias Colorrectales/epidemiología , Infecciones por Virus de Epstein-Barr/epidemiología , Herpesvirus Humano 4/genética , Infecciones por Papillomavirus/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Preescolar , Estudios de Cohortes , Neoplasias Colorrectales/virología , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/virología , Femenino , Humanos , Inmunohistoquímica , Líbano/epidemiología , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa , Prevalencia , Factores de Riesgo , Adulto Joven
8.
Postepy Biochem ; 67(1): 59-63, 2021 03 31.
Artículo en Polaco | MEDLINE | ID: mdl-34378898

RESUMEN

High resolution melting (HRM) is a method based on the identification of differences in the denaturation of PCR reaction products in the presence of fluorescent dyes. It allows for a detailed analysis of the genetic profiles. In addition this analysis is low-cost, single-step, closed-tube and has high sensitivity. HRM found applications in diagnostics, laboratory and clinical researches. This article is a literature review of the applications of HRM analysis in medicine.


Asunto(s)
Reacción en Cadena de la Polimerasa , Humanos
9.
Postepy Biochem ; 67(1): 54-58, 2021 03 31.
Artículo en Polaco | MEDLINE | ID: mdl-34378899

RESUMEN

DNA denaturation with High Resolution Melting PCR-HRM is a method based on the identification of differences in the denaturation of PCR reaction products in the presence of fluorescent dyes. It is used to detect genetic variation in nucleic acid sequences in many branches of science, medicine and industry. This article is a review of the current literature of the methodology, applications and development of HRM analysis, which, thanks to its advantages such as speed, low cost, flexibility and simplicity, has found many applications, and its spectrum is still expanding.


Asunto(s)
Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa
10.
Methods Mol Biol ; 2327: 119-137, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34410643

RESUMEN

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.


Asunto(s)
Genoma Viral , SARS-CoV-2/genética , Saliva/virología , Secuenciación Completa del Genoma/métodos , Biología Computacional , ADN Complementario/genética , Humanos , Sondas Moleculares/genética , Reacción en Cadena de la Polimerasa/métodos , SARS-CoV-2/aislamiento & purificación , Manejo de Especímenes/métodos , Estreptavidina , Secuenciación Completa del Genoma/instrumentación
11.
BMC Res Notes ; 14(1): 316, 2021 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-34404471

RESUMEN

OBJECTIVE: We investigated the discrepancy between clinical and PCR-based diagnosis of COVID-19. We compared results of ten patients with mild to severe COVID-19. Respiratory samples from all cases were tested on the Roche SARS-CoV-2 (Cobas) assay, Filmarray RP2.1 (bioMereiux) and TaqPath™ COVID19 (Thermofisher) PCR assays. RESULTS: Laboratory records of ten patients with mild to severe COVID-19 were examined. Initially, respiratory samples from the patients were tested as negative on the SARS-CoV-2 Roche® assay. Further investigation using the BIOFIRE® Filmarray RP2.1 assay identified SARS-CoV-2 as the pathogen in all ten cases. To investigate possible discrepancies between PCR assays, additional testing was conducted using the TaqPath™ COVID19 PCR. Eight of ten samples were positive for SARS-CoV-2 on the TaqPath assay. Further, Spike gene target failures (SGTF) were identified in three of these eight cases. Discrepancy between the three PCR assays could be due to variation in PCR efficiencies of the amplification reactions or, variation at primer binding sites. Strains with SGTF indicate the presence of new SARS-CoV-2 variant strains. Regular modification of gene targets in diagnostic assays may be necessary to maintain robustness and accuracy of SARS-CoV-2 diagnostic assays to avoid reduced case detection, under-surveillance, and missed opportunities for control.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad
12.
In Vivo ; 35(5): 2947-2949, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34410992

RESUMEN

BACKGROUND/AIM: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been affecting Hokkaido, Japan since late February 2020 until present. The aim of this study was to report the relationship between anti-SARS-CoV-2 antibody-positive and SARS-CoV-2 PCR-positive cases by analyzing anti-SARS-CoV-2 antibodies (IgG and total-Ig). PATIENTS AND METHODS: Serum samples were collected from care workers and nurses in two nursing homes and two hospitals which underwent virus outbreak. All people were confirmed to be SARS-CoV-2-positive by RT-qPCR and their sera was analyzed for anti-SARS-CoV-2 antibodies (IgG and total-Ig). RESULTS: Although 34 out of 43 samples (79.1%) showed enough amount of anti-SARS-CoV-2 antibodies, 9 RT-qPCR -positive samples (20.9%) showed absence of anti-SARS-CoV-2 antibodies in their sera. CONCLUSION: The results that 20.9% of RT-qPCR-positive samples with SARS-CoV-2 showed absence of anti-SARS-CoV-2 antibodies provides a possibility that the innate immune reaction could eliminate the virus without activating adaptive immune reaction.


Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Humanos , Inmunoglobulina G , Reacción en Cadena de la Polimerasa
13.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 33(7): 779-785, 2021 Jul.
Artículo en Chino | MEDLINE | ID: mdl-34412744

RESUMEN

OBJECTIVE: To verify the specific differentiated subsets of monocytes in sepsis, and to screen and construct the differential gene set of monocytes used for early diagnosis of sepsis. METHODS: Patients with sepsis admitted to Guangdong Provincial People's Hospital from June 2020 to March 2021 were enrolled, and peripheral blood mononuclear cells (PBMC) were extracted. Single-cell sequencing technology and pseudo-time analysis were used to verify the differential subsets of monocytes. Bioinformatics methods were used to analyze the expression of genes in differential subsets of monocytes and screen out differential genes for the preliminary construction of a candidate differential gene set. The digital polymerase chain reaction (PCR) technology was used to verify the candidate differential genes in PBMC of sepsis patients and sepsis human myeloid leukemia mononuclear cells (THP-1) models, and the Venn diagram was used to construct the final differential gene set of monocytes. Gene Expression Omnibus (GEO) database was used to validate the differential gene set of monocytes. RESULTS: (1) The results of cell annotation and pseudo-time analysis showed that the differentiation of NEAT1+CD163+ monocyte occurred in the early stage of sepsis was significantly different from other subsets, which validated that NEAT1+CD163+ monocyte was the characteristic subset in the pathological process of sepsis. (2) Twenty-two differential genes related to sepsis were screened out from the gene expression of NEAT1+CD163+ monocyte. After further verification by digital PCR, basic leucine zipper ATF-like transcription factor (BATF), JUNB proto-oncogene, carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4), chromosome 9 open reading frame 95 (C9orf95), G protein subunit alpha 15 (GNA15), complement C3a receptor 1 (C3AR1), transforming growth factor beta 1 (TGFB1) and mitochondrial carrier homolog 1 (MTCH1) were screened out to construct the final differential gene set of monocytes. (3) The external validation results showed that C9orf95 gene had no data in GSE154918 and GSE133822 from GEO, it was excluded during validation. In GSE154918, the expressions of BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1 in the sepsis group were significantly higher than those in the healthy control group (log2expression level: BATF was 12.78±0.08 vs. 11.39±0.35, JUNB was 16.88±0.07 vs. 16.04±0.03, CEACAM4 was 14.73±0.08 vs. 13.77±0.05, GNA15 was 13.16±0.06 vs. 12.30±0.04, C3AR1 was 14.62±0.13 vs. 12.87±0.05, TGFB1 was 16.95±0.05 vs. 16.57±0.36, MTCH1 was 14.80±0.02 vs. 14.61±0.15, all P < 0.05). In GSE133822, the expressions of BATF, CEACAM4, GNA15, and C3AR1 in the sepsis group were significantly higher than those in the health control group (log2expression level: BATF was 8.66±0.16 vs. 7.92±0.14, CEACAM4 was 9.20±0.16 vs. 8.36±0.20, GNA15 was 10.66±0.18 vs. 10.13±0.16, C3AR1 was 11.49±0.27 vs. 10.48±0.16, all P < 0.05), while the expressions of JUNB, TGFB1, and MTCH1 were not statistically different between two groups. The results of gene set variation analysis (GSVA) showed that the enrichment scores of monocytes differential gene set of sepsis group were significantly higher than those of the healthy control group in both GSE154918 (0.38±0.04 vs. -0.44±0.02) and GSE133822 (0.56±0.02 vs. 0.20±0.05, both P < 0.01). Receiver operator characteristic curve (ROC curve) analysis showed that the differential gene set of monocytes had a reliable diagnostic value for early sepsis with the area under ROC curve (AUC) of 0.993 [95% confidence interval (95%CI) was 0.980-1.000] in GSE154918 and 0.944 (95%CI was 0.873-1.000) in GSE133822. CONCLUSIONS: A differential gene set of monocytes (BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1) screened out by single-cell sequencing and digital PCR technology has a reliable diagnostic value for the early sepsis, and may provide a new idea for the early diagnosis of sepsis.


Asunto(s)
Monocitos , Sepsis , Diagnóstico Precoz , Humanos , Leucocitos Mononucleares , Reacción en Cadena de la Polimerasa , Sepsis/diagnóstico , Sepsis/genética , Tecnología
14.
Pan Afr Med J ; 38: 358, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34367437

RESUMEN

Gonorrhea is all diseases caused by Neisseria gonorrhoeae. Prepubertal child is more susceptible to N. gonorrhoeae infection because the vagina is alkaline and contains no estrogen. Gonorrhea vaginitis is the most common form of gonorrhoea in prepubertal children beyond neonatal period. Transmission in child can be through sexual contact (abuse) or non-sexual contact. Gonorrhea vaginitis in children more often asymptomatic, with clinical manifestation such as mucopurulent discharge, vaginal pruritus and vulval erythema. Supporting examination comprise of gram staining from vaginal discharge, culture and nucleic acid amplification testing (NAAT). Ceftriaxone is drug of choice gonorrhea without complication in children. We report a case of 4 year and 9-month female girl that was diagnosed by history taking and supporting examination from gram staining and polymerase chain reaction (PCR) from vaginal discharge, and then treated with single dose ceftriaxone 125 mg intramuscular that gave clinical improvement.


Asunto(s)
Antibacterianos/administración & dosificación , Gonorrea/diagnóstico , Neisseria gonorrhoeae/aislamiento & purificación , Vaginosis Bacteriana/diagnóstico , Ceftriaxona/administración & dosificación , Preescolar , Femenino , Gonorrea/tratamiento farmacológico , Humanos , Reacción en Cadena de la Polimerasa , Vaginosis Bacteriana/tratamiento farmacológico , Vaginosis Bacteriana/microbiología
15.
J Biomed Nanotechnol ; 17(7): 1284-1292, 2021 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-34446132

RESUMEN

This study aimed to introduce nano-gold PCR for detection of TERT methylation, and explore the correlation between TERT methylation and prognosis of hepatocellular carcinoma (HCC). From March 2016 to March 2018, 154 HBV carriers treated in our hospital were enrolled in the study and divided into HCC (68 cases), cirrhosis (45 cases) and chronic hepatitis (CH) groups (41 cases) based on clinical disease. HCC patients were further divided into methylation (30 cases) and non-methylation (38 cases) subgroup based on methylation status of the TERT. TERT methylation of HCC specimens were 44.12% and 35.24% by nano-PCR and conventional PCR, respectively. The TERT methylation and TERT expression in HCC specimens were higher than for cirrhosis and CH specimens. A significant positive correlation was observed between TERT methylation and TERT expression. AFP, Edmondson classification, tumor size, hilar lymph node and intrahepatic metastasis, and TNM staging in the methylation group were higher than in non-methylation group. Further, overall survival and progression-free survival were significantly shorter. Nano-gold PCR is more sensitive in detecting TERT methylation. As CHB progresses, TERT methylation increases. Greater methylation of the gene is associated with worse prognosis in HCC patients.


Asunto(s)
Carcinoma Hepatocelular , Hepatitis B Crónica , Hepatitis B , Neoplasias Hepáticas , Telomerasa , Carcinoma Hepatocelular/genética , Metilación de ADN , Oro , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Humanos , Neoplasias Hepáticas/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Telomerasa/genética
16.
Methods Mol Biol ; 2351: 25-39, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34382182

RESUMEN

Post-transcriptional processing strongly affects the stability and the relative quantification of RNA molecules, so that steady-state levels of mature RNA, such as mRNAs, rarely reflect accurately the rate of in situ transcription in nuclei by RNA polymerases (RNAPs). The "Global Run-on Sequencing (GRO-Seq)" method, developed in 2008, combines the nuclear run-on assay with next-generation deep sequencing to detect nascent RNA levels to annotate the positions, the relative levels and the orientation of transcriptionally engaged RNA polymerase II (RNAPII) molecules genome-wide. Thus, GRO-Seq is a powerful method to infer mechanistic insights into the multiple levels of transcriptional regulation such as promoter-proximal pausing of RNAP, bidirectional transcription, and enhancer activity. Here, we describe a protocol for mammalian cells that can reliably detect low abundant nascent RNA from both coding and noncoding genomic regions. This protocol can easily be adapted for most mammalian cells to define the transcriptionally active regions of the genome and to measure dynamic transcriptional responses with high sensitivity upon external stimuli.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Polimerasa II/metabolismo , Análisis de Secuencia de ARN/métodos , Transcripción Genética , Elementos de Facilitación Genéticos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Control de Calidad , ARN/genética , ARN/aislamiento & purificación , ARN no Traducido/genética
17.
Methods Mol Biol ; 2351: 41-65, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34382183

RESUMEN

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.


Asunto(s)
Elementos de Facilitación Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , Transcripción Genética , Animales , Células Cultivadas , Cromatina/genética , Biología Computacional/métodos , ADN , Biblioteca de Genes , Humanos , Reacción en Cadena de la Polimerasa , ARN , ARN Polimerasa II/metabolismo , Programas Informáticos
18.
Anal Chem ; 93(33): 11488-11496, 2021 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34383461

RESUMEN

Polymerase chain reaction (PCR) technology has become the cornerstone of DNA analysis. However, special samples (e.g., forensic samples, soil, food, and mineral medicine) may contain powerful PCR inhibitors. High levels of inhibitors can hardly be sufficiently removed by conventional DNA extraction approaches and may result in the complete failure of PCR. In this work, the removal of PCR inhibitors by electromembrane extraction (EME) was investigated for the first time. To demonstrate the universality of the approach, EME formats with and without supported membranes (termed parallel-EME and µ-EME, respectively) were employed, and both anionic [humic acid (HA)] and cationic (Ca2+) PCR inhibitors were used as models. During EME, charged inhibitors in the sample migrate into the liquid membrane in the presence of an electric field and might further leech into the waste solution, while PCR analytes remain in the sample. After EME, the clearance values for HA at 0.2 and 2.5 mg mL-1 were 94 and 85%, respectively, and that for Ca2+ (275 mM) was 63%. Forensic PCR-short tandem repeat (PCR-STR) genotyping showed that EME significantly reduced the interference by HA in PCR-STR analysis and displayed a higher HA purge capability compared to existing methods. Furthermore, by combining EME with liquid-liquid extraction or solid-phase extraction, satisfactory STR profiles were obtained from HA-rich blood samples. In addition, false-negative reports of bacterial detection in mineral medicine and shrimps were avoided after the removal of Ca2+ by µ-EME. Our research demonstrates the great potential of EME for the purification of DNA samples containing high-level PCR inhibitors and opens up a new application direction for EME.


Asunto(s)
Electricidad , Membranas Artificiales , Aniones , Cationes , Reacción en Cadena de la Polimerasa
19.
N Engl J Med ; 385(8): 707-719, 2021 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-34347949

RESUMEN

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).


Asunto(s)
Azoospermia/genética , Exorribonucleasas/genética , Infertilidad Masculina/genética , Meiosis/fisiología , Mutación , ARN Interferente Pequeño/metabolismo , Testículo/patología , Adulto , Azoospermia/fisiopatología , Biopsia , Expresión Génica , Humanos , Masculino , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/ultraestructura , Análisis de Secuencia de ARN , Testículo/metabolismo , Secuenciación del Exoma Completo
20.
Pan Afr Med J ; 39: 22, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34394813

RESUMEN

Introduction: the spread of enterobacteria producing extended-broad-spectrum beta-lactamases (ESBL) is a global public health-problem. In a study carried in 2003-2005 at the Pasteur Institute in Bangui, 450 enterobacteria were identified in clinical isolates, of which 17 were ESBL (prevalence: 3.78%). The aim of this study was to update this data. Methods: from May 2018 to April 2019, a total of 941 enterobacteria were isolated and identified under identical conditions of recruitment and with the same techniques used in the previous study: phenotypic identification using Api 20E strips (bioMérieux SA, Marcy-l'Etoile, France) and antimicrobial drug susceptibility using the disk diffusion method (Bio-Rad antibiotic discs, Marnes la Coquette, France). Resistance genes were identified by polymerase chain reaction (PCR) and sequencing. Results: from May 2018 to April 2019, a total of 941 enterobacteria were isolated of which 478 were ESBL, thus amounting to a prevalence of 50.80%. The genetic profiles of the bla CTX-M resistance genes exhibited the emergence of the CTX-M28 variant (CTX-M1 group) and variants of the M2 and M9 groups. There was also a notable increase, from 35 to 64%, in the ESBL with a bla SHV gene. Conclusion: this study documents a 13 fold increase in the prevalence of ESBL derived from clinical isolates of the bacteriology laboratory of the Institute Pasteur in Bangui, by comparing its data with that of the publication by Frank et al. 2006. Together with this increase a significant diversification of the circulating CTX-M resistance genes was noticed.


Asunto(s)
Antibacterianos/farmacología , Infecciones por Enterobacteriaceae/epidemiología , Enterobacteriaceae/aislamiento & purificación , beta-Lactamasas/genética , Proteínas Bacterianas/genética , República Centroafricana/epidemiología , Pruebas Antimicrobianas de Difusión por Disco , Farmacorresistencia Bacteriana/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/enzimología , Infecciones por Enterobacteriaceae/microbiología , Humanos , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos
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