RESUMO
Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.
Assuntos
Vírus da Hepatite B , Hepatite B , Animais , Humanos , Camundongos , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Fucosiltransferases/genética , Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hipóxia , Oxigênio , RNA , TranscriptomaRESUMO
Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.
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Vírus da Hepatite B , Histona Desmetilases com o Domínio Jumonji , Oxigênio , Replicação Viral , Humanos , DNA Viral/genética , Genoma Viral/genética , Hepatite B/enzimologia , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/virologia , Oxigênio/metabolismo , Plasmídeos/genética , Transcriptoma , Replicação Viral/genéticaRESUMO
Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.
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Vírus da Hepatite B , Hepatite B , Vírus Delta da Hepatite , Replicação Viral , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Vírus da Hepatite B/imunologia , Humanos , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/fisiologia , Hepatite B/virologia , Hepatite B/imunologia , Biologia Molecular , Japão , Hepatite D/virologia , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/genéticaRESUMO
Understanding the host pathways that define susceptibility to Severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in the Syrian hamster model. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.
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COVID-19 , Inibidores de Prolil-Hidrolase , Animais , Antivirais , Cricetinae , Hipóxia , Pulmão/patologia , Mesocricetus , Oxigênio , RNA Viral , SARS-CoV-2RESUMO
Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25â% of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from de novo HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5' truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome.
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Vírus da Hepatite B , Transcriptoma , Humanos , Vírus da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA Viral/genéticaRESUMO
Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin-organising transcriptional insulator protein, CCCTC-binding factor (CTCF), has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies of the viral genome. siRNA knock-down of CTCF results in a significant increase in pre-genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription.
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Fator de Ligação a CCCTC/fisiologia , Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/fisiologia , Transcrição Viral , Sítios de Ligação , Linhagem Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina , DNA Viral/metabolismo , Epigenômica , Células Hep G2 , Hepatite B/virologia , Humanos , Mutação , RNA Viral , Replicação ViralRESUMO
BACKGROUND & AIMS: Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication. METHODS: Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle. RESULTS: Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family. CONCLUSIONS: Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection. LAY SUMMARY: Viral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets.
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Vírus da Hepatite B , Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Fator 6 Semelhante a Kruppel/metabolismo , Oxigênio/metabolismo , Replicação Viral/fisiologia , Animais , Microambiente Celular , Hepadnaviridae/fisiologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Fígado/metabolismo , Transdução de Sinais , Ativação TranscricionalRESUMO
Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae and replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome of approximately 3.2 kb. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate HBV transcription. However, the host pathways that regulate HBV transcription and the temporal nature of promoter usage in infected cells are not well understood, in part due to the compact genome structure and overlapping open reading frames. To address this we developed a simple and cost-effective PCR assay to quantify the major viral RNAs and validated this technique using current state-of-art de novo HBV infection model systems. Our PCR method is three orders of magnitude more sensitive than Northern blot and requires relatively small amounts of starting material, making this an attractive tool for assessing HBV transcription.
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Vírus da Hepatite B/genética , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Transcrição Gênica , Células Hep G2 , Humanos , RNA Viral/genética , Sensibilidade e Especificidade , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.
Assuntos
Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Estágios do Ciclo de Vida/fisiologia , Replicação Viral , DNA Viral/genética , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Humanos , Técnicas In Vitro , Cinética , RNA Viral/metabolismo , Simportadores/genética , Simportadores/metabolismo , Internalização do VírusRESUMO
INTRODUCTION: Smoking in pregnancy in the United Kingdom remains prevalent (11%). To encourage and support pregnant smokers to quit, midwives must be adequately trained to do so. Substantial curricular gaps have been identified in the smoking cessation training of medical, nursing, and optometry schools. This study aimed to identify the extent of smoking cessation training and assessment in UK midwifery schools. METHODS: All UK undergraduate midwifery schools (n = 53) were invited to complete a web-based survey of their curricular coverage and assessment related to smoking cessation, and perceived barriers to delivering smoking cessation training. RESULTS: Twenty-nine (55%) midwifery schools responded. Most teaching was completed in the initial year of study. All reported teaching the harmful effects of tobacco use. The majority of respondents (83%) reported training students in brief intervention delivery and ways to assist quit attempts. Only 24% of schools in this study included relapse prevention in their curriculum. The most frequently reported barriers to teaching smoking cessation were "lack of knowledge amongst staff" (17%), "no space in a crowded curriculum" (17%), and "administrative problems" (13%). CONCLUSIONS: Midwifery schools are teaching the harmful effects of smoking and providing training on brief interventions. However, in some schools student midwives are not being sufficiently trained on relapse prevention or assessed in the practical skills necessary for delivering evidence-based interventions. IMPLICATIONS: Midwifery schools should revise the content and delivery of smoking cessation training to ensure midwives are equipped with the necessary knowledge and skills to contribute to the challenge of smoking cessation in pregnancy.
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Currículo , Tocologia/educação , Abandono do Hábito de Fumar , Fumar , Tabagismo , Estudos Transversais , Feminino , Humanos , Gravidez , Escolas de Enfermagem , Inquéritos e Questionários , Reino UnidoRESUMO
Many pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrial-derived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1α (hif1α). Epistasis assays demonstrated that hif1α regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Indução Embrionária , Glucose/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Animais , Animais Geneticamente Modificados , Metabolismo dos Carboidratos/genética , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero , Indução Embrionária/efeitos dos fármacos , Indução Embrionária/genética , Regulação da Expressão Gênica no Desenvolvimento , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/genética , Glicólise/fisiologia , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Fosforilação Oxidativa , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Background and aims: The intrahepatic processes associated with chronic hepatitis B (CHB), especially in the context of hepatitis delta virus (HDV) and HIV co-infection, require a better understanding. Spatial transcriptomics can provide new insights into the complex intrahepatic biological processes, guiding new personalised treatments. Our aim is to evaluate this method characterising the intrahepatic transcriptional landscape, cellular composition and biological pathways in liver biopsy samples from patients with hepatitis B virus (HBV) and HDV or HIV co-infection. Method: The NanoString GeoMx digital spatial profiling platform was employed to assess expression of HBV surface antigen and CD45 in formalin-fixed paraffin-embedded (FFPE) biopsies from three treatment-naïve patients with chronic HBV and HDV or HIV co-infection. The GeoMx Human Whole Transcriptome Atlas assay quantified the expression of genes enriched in specific regions of interest (ROIs). Cell type proportions within ROIs were deconvoluted using a training matrix from the human liver cell atlas. A weighted gene correlation network analysis evaluated transcriptomic signatures across sampled regions. Results: Spatially discrete transcriptomic signatures and distinct biological pathways were associated with HBV infection/disease status and immune responses. Shared features including 'cytotoxicity' and 'B cell receptor signalling' were consistent across patients, suggesting common elements alongside individual traits. HDV/HBV co-infection exhibited upregulated genes linked to apoptosis and immune cell recruitment, whereas HIV/HBV co-infection featured genes related to interferon response regulation. Varied cellular characteristics and immune cell populations, with an abundance of γδT cells in the HDV/HBV sample, were observed within analysed regions. Transcriptional differences in hepatocyte function suggest disrupted metabolic processes in HDV/HBV co-infection potentially impacting disease progression. Conclusion: This proof-of-principle study shows the value of this platform in investigating the complex immune landscape, highlighting relevant host pathways to disease pathogenesis.
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BACKGROUND: Most patients with signs or symptoms (s/s) of suspected preeclampsia are not diagnosed with preeclampsia. We sought to determine and compare the prevalence of s/s, pregnancy outcomes, and costs between patients with and without diagnosed preeclampsia. METHODS: This retrospective cohort study analyzed a large insurance research database. Pregnancies with s/s of preeclampsia versus a confirmed preeclampsia diagnosis were identified using International Classification of Diseases codes. S/s include hypertension, proteinuria, headache, visual symptoms, edema, abdominal pain, and nausea/vomiting. Pregnancies were classed as 1) s/s of preeclampsia without a confirmed preeclampsia diagnosis (suspicion only), 2) s/s with a confirmed diagnosis (preeclampsia with suspicion), 3) diagnosed preeclampsia without s/s recorded (preeclampsia only), and 4) no s/s, nor preeclampsia diagnosis (control). RESULTS: Of 1,324,424 pregnancies, 29.2 % had ≥1 documented s/s of suspected preeclampsia, and 14.2 % received a preeclampsia diagnosis. Hypertension and headache were the most common s/s, leading 20.2 % and 9.2 % pregnancies developed to preeclampsia diagnosis, respectively. Preeclampsia, with or without suspicion, had the highest rates of hypertension-related severe maternal morbidity (HR [95 % CI]: 3.0 [2.7, 3.2] and 3.6 [3.3, 4.0], respectively) versus controls. A similar trend was seen in neonatal outcomes such as preterm delivery and low birth weight. Cases in which preeclampsia was suspected but not confirmed had the highest average total maternal care costs ($6096 [95 % CI: 602, 6170] over control). CONCLUSION: There is a high prevalence but poor selectivity of traditional s/s of preeclampsia, highlighting a clinical need for improved screening method and cost-effectiveness disease management.
Assuntos
Bases de Dados Factuais , Pré-Eclâmpsia , Resultado da Gravidez , Humanos , Feminino , Gravidez , Pré-Eclâmpsia/epidemiologia , Pré-Eclâmpsia/economia , Pré-Eclâmpsia/diagnóstico , Estudos Retrospectivos , Adulto , Prevalência , Resultado da Gravidez/epidemiologia , Adulto Jovem , Estados Unidos/epidemiologia , Custos de Cuidados de Saúde/estatística & dados numéricosRESUMO
Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. Our knowledge of the host pathways that define susceptibility to infection and disease severity are limited. Hypoxia inducible factors (HIFs) define metabolic responses to low oxygen and regulate inflammatory responses in the lower respiratory tract. We demonstrate a role for HIFs to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors reduce the expression of the RSV entry receptor nucleolin and inhibit viral cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for this respiratory pathogen.
RESUMO
Developmental signals determine organ morphology and position during embryogenesis. To discover novel modifiers of liver development, we performed a chemical genetic screen in zebrafish and identified retinoic acid as a positive regulator of hepatogenesis. Knockdown of the four RA receptors revealed that all receptors affect liver formation, however specific receptors exert differential effects. Rargb knockdown results in bilateral livers but does not impact organ size, revealing a unique role for Rargb in conferring left-right positional information. Bilateral populations of hepatoblasts are detectable in rargb morphants, indicating Rargb acts during hepatic specification to position the liver, and primitive endoderm is competent to form liver on both sides. Hearts remain at the midline and gut looping is perturbed in rargb morphants, suggesting Rargb affects lateral plate mesoderm migration. Overexpression of Bmp during somitogenesis similarly results in bilateral livers and midline hearts, and inhibition of Bmp signaling rescues the rargb morphant phenotype, indicating Rargb functions upstream of Bmp to regulate organ sidedness. Loss of rargb causes biliary and organ laterality defects as well as asplenia, paralleling symptoms of the human condition right atrial isomerism. Our findings uncover a novel role for RA in regulating organ laterality and provide an animal model of one form of human heterotaxia.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal , Embrião não Mamífero/metabolismo , Fígado/embriologia , Fígado/metabolismo , Modelos Animais , Proteína Nodal/metabolismo , Fenótipo , Receptores do Ácido Retinoico/genética , Transdução de Sinais , Tretinoína/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Receptor gama de Ácido RetinoicoRESUMO
We evaluated the effect of storage at 2 to 8°C on the stability of human genomic and human papillomavirus (HPV) DNA stored in BD SurePath and Hologic PreservCyt liquid-based cytology media. DNA retained the ability to be extracted and PCR amplified for more than 2.5 years in both medium types. Prior inability to detect DNA in archived specimens may have been due to failure of the extraction method to isolate DNA from fixed cells.
Assuntos
Técnicas Citológicas/métodos , DNA Viral/isolamento & purificação , Papillomaviridae/isolamento & purificação , Manejo de Espécimes/métodos , Virologia/métodos , Meios de Cultura/química , DNA Viral/genética , Humanos , Papillomaviridae/genética , Refrigeração , Fatores de TempoRESUMO
OBJECTIVES: To evaluate how women access and engage with different models of maternity care, whether specialist models improve access and engagement for women with social risk factors, and if so, how? DESIGN: Realist evaluation. SETTING: Two UK maternity service providers. PARTICIPANTS: Women accessing maternity services in 2019 (n=1020). METHODS: Prospective observational cohort with multinomial regression analysis to compare measures of access and engagement between models and place of antenatal care. Realist informed, longitudinal interviews with women accessing specialist models of care were analysed to identify mechanisms. MAIN OUTCOME MEASURES: Measures of access and engagement, healthcare-seeking experiences. RESULTS: The number of social risk factors women were experiencing increased with deprivation score, with the most deprived more likely to receive a specialist model that provided continuity of care. Women attending hospital-based antenatal care were more likely to access maternity care late (risk ratio (RR) 2.51, 95% CI 1.33 to 4.70), less likely to have the recommended number of antenatal appointments (RR 0.61, 95% CI 0.38 to 0.99) and more likely to have over 15 appointments (RR 4.90, 95% CI 2.50 to 9.61) compared with community-based care. Women accessing standard care (RR 0.02, 95% CI 0.00 to 0.11) and black women (RR 0.02, 95% CI 0.00 to 0.11) were less likely to have appointments with a known healthcare professional compared with the specialist model. Qualitative data revealed mechanisms for improved access and engagement including self-referral, relational continuity with a small team of midwives, flexibility and situating services within deprived community settings. CONCLUSION: Inequalities in access and engagement with maternity care appears to have been mitigated by the community-based specialist model that provided continuity of care. The findings enabled the refinement of a realist programme theory to inform those developing maternity services in line with current policy.
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Serviços de Saúde Materna , Obstetrícia , Feminino , Gravidez , Humanos , Cuidado Pré-Natal , Fatores de Risco , Reino UnidoRESUMO
Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5' stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.