Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum.
全长 5 'race 识别 HBV 感染的肝细胞和患者血清中的所有主要 HBV 转录本。
- 作者列表："Stadelmayer B","Diederichs A","Chapus F","Rivoire M","Neveu G","Alam A","Fraisse L","Carter K","Testoni B","Zoulim F
BACKGROUND AND AIMS:Covalently closed circular DNA (cccDNA) is the episomal form of the Hepatitis B virus (HBV) genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of six major viral RNAs, i.e. preC- pg-, preS1/2-, S- and HBx-RNA. All viral transcripts share the same 3' end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab. METHODS:We set-up the HBV full-length 5'RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. RESULTS:In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that Interferon β treatment leads to a strong reduction of preC- and pg-RNAs but only has a moderate effect on the other viral transcripts. We found pgRNA, one spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx. CONCLUSIONS:The HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may inform the development of novel therapies aimed at targeting cccDNA.
背景和目的: 共价闭合环状 DNA (cccDNA) 是乙型肝炎病毒 (HBV) 基因组的附加体形式，稳定驻留在感染肝细胞的细胞核中。CccDNA 是六种主要病毒 rna 转录的模板，即PreC-pg-、 preS1/2-、 S-和 HBx-RNA。所有病毒转录本共享相同的 3 '端，并且在不同程度上都是彼此的子集。特别是在感染条件下，很难深入研究不同病毒转录本的转录。因此，我们希望开发一种方法，使我们能够在任何实验室中轻松检测病毒 rna 的全谱。 方法: 我们建立了 HBV 全长 5 'race (cDNA 末端的快速扩增) 我们测量和表征细胞培养和慢性感染患者中病毒 rna 全谱的方法。 结果: 除了编码全长 X 的经典 HBx 转录本之外，我们还发现了可能编码短 X 蛋白的较短 HBx 转录本。我们发现干扰素 β 治疗导致 preC-和 pg-RNAs 的强烈减少，但对其他病毒转录本只有适度的影响。我们发现了 pgRNA，一种剪接的 pgRNA 变异体和多种与 HepAD38 细胞产生的病毒颗粒相关的 HBx 转录本。不同的 HBx rna 都是封顶的和非封顶的。最后，我们确定了慢性 HBV 感染患者循环 RNA 物种的 3 个主要类别: pgRNA，剪接 pgRNA 变异和 HBx。 结论: HBV 全长 5 'race 方法应显著有助于在感染和治疗过程中对 HBV 转录的理解，并可能告知旨在靶向 cccDNA 的新疗法的发展。
METHODS:BACKGROUND:Opioids are often prescribed for pain in cirrhosis and may increase the risk of hepatic encephalopathy (HE). AIMS:To assess the association between opioids and HE in patients with well-compensated cirrhosis. METHODS:We used the IQVIA PharMetrics (Durham, NC) database to identify patients aged 18-64 years with cirrhosis. We excluded patients with any decompensation event from 1 year before cirrhosis diagnosis to 6 months after cirrhosis diagnosis. Over the 6 months after cirrhosis diagnosis, we determined the duration of continuous opioid use and classified use into short term (1-89 days) and chronic (90-180 days). We assessed whether patients developed HE over the subsequent year (ie 6-18 months after cirrhosis diagnosis). We used a landmark analysis and performed multivariable Cox proportional hazards regression to assess associations between opioid use and HE, adjusting for relevant confounders. RESULTS:The cohort included 6451 patients with compensated cirrhosis, of whom 23.3% and 4.7% had short-term and chronic opioid prescriptions respectively. Over the subsequent year, HE occurred in 6.3% patients with chronic opioid prescriptions, 5.0% with short-term opioid prescriptions and 3.3% with no opioid prescriptions. In the multivariable model, an increased risk of HE was observed with short-term (adjusted hazard ratio, HR 1.44, 95% CI 1.07-1.94) and chronic opioid prescriptions (adjusted HR 1.83, 95% CI 1.07-3.12) compared to no opioid prescriptions. CONCLUSION:In this national cohort of privately insured patients with cirrhosis, opioid prescriptions were associated with the risk of incident HE. Opioid use should be minimised in those with cirrhosis and, when required, limited to short duration.
METHODS:BACKGROUND AND AIMS:Cirrhosis is characterized by extensive fibrosis of the liver and is a major cause of liver-related mortality. Cirrhosis is partially heritable but genetic contributions to cirrhosis have not been systemically explored. Here, we carry out association analyses with cirrhosis in two large biobanks and determine the effects of cirrhosis associated variants on multiple human disease/traits. METHODS:We carried out a genome-wide association analysis of cirrhosis as a diagnosis in UK BioBank (UKBB; 1088 cases vs. 407 873 controls) and then tested top-associating loci for replication with cirrhosis in a hospital-based cohort from the Michigan Genomics Initiative (MGI; 875 cases of cirrhosis vs. 30 346 controls). For replicating variants or variants previously associated with cirrhosis that also affected cirrhosis in UKBB or MGI, we determined single nucleotide polymorphism effects on all other diagnoses in UKBB (PheWAS), common metabolic traits/diseases and serum/plasma metabolites. RESULTS:Unbiased genome-wide association study identified variants in/near PNPLA3 and HFE, and candidate variant analysis identified variants in/near TM6SF2, MBOAT7, SERPINA1, HSD17B13, STAT4 and IFNL4 that reproducibly affected cirrhosis. Most affected liver enzyme concentrations and/or aspartate transaminase-to-platelet ratio index. PheWAS, metabolic trait and serum/plasma metabolite association analyses revealed effects of these variants on lipid, inflammatory and other processes including new effects on many human diseases and traits. CONCLUSIONS:We identified eight loci that reproducibly associate with population-based cirrhosis and define their diverse effects on human diseases and traits.
METHODS:BACKGROUND:Spontaneous bacterial peritonitis (SBP) is a serious complication in patients with liver cirrhosis. In recent years, it has been postulated that the rate of multidrug-resistant organisms (MDROs) is increasing, especially in nosocomial SBP patients. Aim of the present work was to investigate this hypothesis and its possible clinical consequences. MATERIALS AND METHODS:One hundred and three culture-positive patients between 2007 and 2014 were compared with 81 patients between 2015 and 2017, to study the change of microbiological profiles and their clinical consequences. The cirrhosis patients with bacterascites requiring treatment were included as well. RESULTS:The most prevalent Gram-negative bacteria isolated from ascites were Enterobacterales (31.6%) and in Gram-positive pathogens Staphylococci (22.8%). There was a significant increase in MDROs (22.3% ICU 40.7%, P = .048), accompanied by an increased incidence of sepsis (from 21.4% to 37.0%, P = .021), hepatorenal syndrome (from 40.8% to 58.0%, P = .007) and the need of catecholamine therapy (from 21.4% to 38.8%, P = .036). Nosocomial origin correlated with higher MDRO proportion, more complications and lower antimicrobial susceptibility rates in 12 commonly used antibiotics. MDROs were confirmed as an isolated predictor for inpatient mortality and complications in multivariable logistic regression. CONCLUSIONS:The feeling in clinical practice that MDROs have increased in the last 11 years could be confirmed in our study in Munich, Germany. Nosocomial SBP correlated with significantly higher MDRO rates (nearly 50%) and complication rates. In our opinion, an antibiotic combination with comprehensive effect should be taken into account in nosocomial SBP patients in this region.