Chronic hepatitis B (CHB) is caused by the Hepatitis B virus (HBV) and affects millions of people worldwide. Developing an effective CHB therapy requires using in vivo screening methods, such as mouse models reflecting CHB based on hydrodynamic delivery of plasmid vectors containing a replication-competent HBV genome. However, long-term expression of HBV proteins is accompanied by production of progeny virions, thereby requiring a Biosafety Level (BSL) 3 animal facility. In the present study, we introduced a point mutation in the START codon of the HBV polymerase to develop a mouse model reflecting chronic hepatitis B infection without formation of viral progeny. We induced the mouse model by hydrodynamic injection of adeno-associated virus plasmid vector (pAAV) and minicircle plasmid (pMC) constructs into C57Bl/6 and C3H/HeN mouse strains, monitoring HBV antigens and antibodies in blood by enzyme-linked immunosorbent assay and analyzing liver expression of HBV core antigen by immunohistology. Persisting expression of viral antigens over 140 days (study endpoint) was observed only in the C3H/HeN mouse strain when using pAAV/1.2HBV-A and pMC/1.0HBV-D with pre-C and pre-S recombination sites. In addition, pAAV/1.2HBV-A in C3H/HeN sustained HBV core antigen positivity up to the study endpoint in C3H/HeN mice. Moreover, introducing the point mutation in the START codon of polymerase effectively prevented the formation of viral progeny. Our study establishes an accessible and affordable experimental paradigm for developing a robust mouse model reflecting CHB suitable for preclinical testing of anti-HBV therapeutics in a BSL2 animal facility.

• MeSH
• chronická hepatitida B * genetika   MeSH
• kodon iniciační MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myši inbrední C3H MeSH
• myši MeSH
• virus hepatitidy B genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Biosensors based on graphene field-effect transistors have become a promising tool for detecting a broad range of analytes. However, their performance is substantially affected by the functionalization protocol. In this work, we use a controlled in-vacuum physical method for the covalent functionalization of graphene to construct ultrasensitive aptamer-based biosensors (aptasensors) able to detect hepatitis C virus core protein. These devices are highly specific and robust, achieving attomolar detection of the viral protein in human blood plasma. Such an improved sensitivity is rationalized by theoretical calculations showing that induced polarization at the graphene interface, caused by the proximity of covalently bound molecular probe, modulates the charge balance at the graphene/aptamer interface. This charge balance causes a net shift of the Dirac cone providing enhanced sensitivity for the attomolar detection of the target proteins. Such an unexpected effect paves the way for using this kind of graphene-based functionalized platforms for ultrasensitive and real-time diagnostics of different diseases.

• MeSH
• aptamery nukleotidové * MeSH
• biosenzitivní techniky * MeSH
• grafit * MeSH
• hepatitida C * diagnóza   MeSH
• lidé MeSH
• proteiny virového jádra MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND & AIMS: Severity of portal hypertension is usually quantified by measuring the hepatic venous pressure gradient (HVPG). However, due to its invasiveness, alternative markers are being sought. Bile acids (BA), being synthesized, metabolized, and transported by the liver, seem to have the potential to serve as endogenous markers. The aim of the present study was to determine whether serum BA reflect the severity of portal hypertension. METHODS: We correlated serum concentrations of individual BA with portal pressure (as HVPG) in an exploratory cohort of 21 cirrhotic patients with portal hypertension. The predictive potential of selected candidates was then confirmed in an independent validation cohort (n = 214). Additionally, nine previously published noninvasive markers were added to the stepwise logistic regression model to identify the most relevant ones, which were eventually used to create a prognostic index of portal hypertension. RESULTS: Serum levels of taurochenodeoxycholic acid (TCDCA) significantly correlated with HVPG and showed a high potential to predict clinically significant portal hypertension (HVPG ≥ 10 mm Hg: AUROC = 0.97 ± 0.06). This was confirmed in the validation cohort (AUROC = 0.96 ± 0.01). The predictive index (constructed based on AST/ALT, spleen diameter, and TCDCA concentration) was able to distinguish clinically significant portal hypertension with 95% sensitivity and 76% specificity. CONCLUSIONS: TCDCA seems to be a promising noninvasive marker of clinically significant portal hypertension. Its predictive potential may be further enhanced when it is combined with both the AST/ALT ratio and spleen diameter.

• MeSH
• jaterní cirhóza komplikace   diagnóza   MeSH
• játra MeSH
• kyselina taurochenodeoxycholová * MeSH
• lidé MeSH
• portální hypertenze * diagnóza   MeSH
• portální tlak MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

U zdravých jedinců jsou kosterní svaly největší tkání v těle (30–40 % celkové tělesné hmotnosti) a jsou v nich uloženy tři čtvrtiny všech tělesných proteinů. Změny svalové soustavy, zejména ve smyslu snížení svalové hmoty a funkce, významně ovlivňují prognózu a průběh řady chronických onemocnění, včetně chronických onemocnění jater. Jejich přítomnost je asociována s vyšší morbiditou, mortalitou a horší kvalitou života pacientů. K popisu změn svalové tkáně jsou používány pojmy sarkopenie, myosteatóza a s poruchou svalstva úzce souvisí i komplexnější pojem „frailty“, syndrom křehkosti. Sarkopenie je definována jako snížení množství svalové hmoty a/nebo svalové funkce, k diagnostice se nejčastěji používá stanovení tzv. skeletal muscle index (SMI) na úrovni třetího lumbálního obratle (L3) dle CT. Myosteatóza je definována jako přítomnost tuku ve svalech jak intramyocelulárně, tak intermyocelulárně a je diagnostikována na základě stanovení denzity svalů na CT v úrovni L3 (skeletal muscle radiation attenuation – SM-RA). Syndrom křehkosti (frailty) zahrnuje kromě změn svalové tkáně a poklesu schopnosti pohybu i změny dalších orgánových systémů, jejichž důsledkem je snížení rezerv organizmu a vyšší riziko nepříznivého průběhu onemocnění. K diagnostice se využívají různé skórovací systémy, např. Fried Frailty Index nebo modifikovaný Liver Frailty Index (LFI) u pacientů s jaterní cirhózou. Základem léčby pacientů s poruchami svalové tkáně je léčba základního onemocnění (včetně transplantace jater u pokročilé cirhózy), zlepšení výživy, dostatečná fyzická aktivita a léčba eventuálního deficitu vitaminu D. V tomto přehledovém článku shrnujeme aktuální pohled na patofyziologii, diagnostiku a léčbu sarkopenie, myosteatózy a syndromu křehkosti u pacientů s jaterní cirhózou.

Skeletal muscles are the biggest tissue in healthy people (30–40% of total body mass) and they comprise three quarters of total body proteins. Muscle alterations, especially muscle wasting and loss of muscle function, have an indisputable prognostic value in the outcome of chronic diseases, including chronic liver diseases. Muscle wasting is associated with higher morbidity, mortality and poor quality of life. The terms sarcopenia and myosteatosis are used to describe specific muscle alterations, both forming substantial components of multidimensional construct “frailty syndrome”. Sarcopenia is defined as loss of muscle mass and/or loss of muscle function. It is usually diagnosed using the skeletal muscle index from computed tomography (CT) image analysis at the L3 vertebra. Myosteatosis is defined as an excess intramyocelullar and intermyocellular fat deposition. Diagnosis of myosteatosis is based on a measurement of skeletal muscle density by CT imaging at the L3 vertebra (skeletal muscle radiation attenuation – SM-RA). Besides muscle alterations and decreased physical performance, frailty syndrome also comprises changes of other organ systems, leading to the loss of functional reserves and higher vulnerability. Different scoring systems, such as Fried Frailty Index (FFI) or modified Liver Frailty Index (LFI) for patients with liver cirrhosis, are used to diagnose patients with frail phenotype. The principle of the treatment of patients with muscle alterations is therapy of liver disease (including liver transplant in advanced cirrhosis), improvement of the nutritional status, adequate physical activity and supplementation of vitamin D deficiency, if necessary. In this review, we summarize up-to-date knowledge about pathophysiology, diagnostic tools and treatment options of sarcopenia, myosteatosis and frailty syndrome in patients with liver cirrhosis

• Klíčová slova
• myosteatóza, syndrom křehkosti,
• MeSH
• jaterní cirhóza * komplikace   MeSH
• lidé MeSH
• sarkopenie * etiologie   patofyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

BACKGROUND: Following the introduction of direct-acting antiviral therapy in 2013, WHO launched the first Global Health Sector Strategy on Viral Hepatitis. We describe a hepatitis C virus (HCV) cascade of care in people with HIV (PWH) across Europe in terms of reaching the WHO elimination targets of diagnosing 90% and treating 80% of HCV-infected individuals. METHODS: HIV/HCV-coinfected participants in the EuroSIDA cohort under prospective follow-up at October 1, 2019, were described using a nine-stage cascade of care. Care cascades were constructed across Europe, on a regional (n = 5) and country (n = 21) level. RESULTS: Of 4773 anti-HCV positive PWH, 4446 [93.1%, 95% confidence interval (CI) 92.4-93.9)] were ever tested for HCV RNA, and 19.0% (95% CI 16.4-21.6) were currently HCV RNA positive, with the highest prevalence in Eastern and Central-Eastern Europe (33.7 and 29.6%, respectively). In Eastern Europe, 78.1% of the estimated number of chronic infections have been diagnosed, whereas this proportion was above 95% in the other four regions. Overall, 3116 persons have ever started treatment (72.5% of the ever chronically infected, 95% CI 70.9-74.0) and 2404 individuals (55.9% of the ever chronically infected, 95% CI 53.9-57.9) were cured. Cure proportion ranged from 11.2% in Belarus to 87.2% in Austria. CONCLUSION: In all regions except Eastern Europe, more than 90% of the study participants have been tested for HCV-RNA. In Southern and Central-Western regions, more than 80% ever chronically HCV-infected PWH received treatment. The proportion with cured HCV infection did not exceed 80% in any region, with significant heterogeneity between countries. SUMMARY: In a pan-European cohort of PWH, all regions except Eastern Europe achieved the WHO target of diagnosing 90% of chronic HCV infections, while the target of treating 80% of eligible persons was achieved in none of the five regions.

• MeSH
• antivirové látky terapeutické užití   MeSH
• chronická hepatitida C * komplikace   farmakoterapie   epidemiologie   MeSH
• Hepacivirus genetika   MeSH
• hepatitida C * komplikace   farmakoterapie   epidemiologie   MeSH
• HIV infekce * komplikace   farmakoterapie   epidemiologie   MeSH
• koinfekce * farmakoterapie   MeSH
• lidé MeSH
• prospektivní studie MeSH
• RNA terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

Liver cell types derived from induced pluripotent stem cells (iPSCs) share the potential to investigate development, toxicity, as well as genetic and infectious disease in ways currently limited by the availability of primary tissue. With the added advantage of patient specificity, which can play a role in all of these areas. Many iPSC differentiation protocols focus on 3 dimensional (3D) or organotypic differentiation, as these offer the advantage of more closely mimicking in vivo systems including; the formation of tissue like architecture and interactions/crosstalk between different cell types. Ultimately such models have the potential to be used clinically and either with or more aptly, in place of animal models. Along with the development of organotypic and micro-tissue models, there will be a need to co-develop imaging technologies to enable their visualization. A variety of liver models termed "organoids" have been reported in the literature ranging from simple spheres or cysts of a single cell type, usually hepatocytes, to those containing multiple cell types combined during the differentiation process such as hepatic stellate cells, endothelial cells, and mesenchymal cells, often leading to an improved hepatic phenotype. These allow specific functions or readouts to be examined such as drug metabolism, protein secretion or an improved phenotype, but because of their relative simplicity they lack the flexibility and general applicability of ex vivo tissue culture. In the liver field these are more often constructed rather than developed together organotypically as seen in other organoid models such as brain, kidney, lung and intestine. Having access to organotypic liver like surrogates containing multiple cell types with in vivo like interactions/architecture, would provide vastly improved models for disease, toxicity and drug development, combining disciplines such as microfluidic chip technology with organoids and ultimately paving the way to new therapies.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.

• MeSH
• arginin genetika   MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• HEK293 buňky MeSH
• hepatitida B genetika   MeSH
• hepatocelulární karcinom genetika   MeSH
• lidé MeSH
• lysin genetika   MeSH
• nádorové buněčné linie MeSH
• posttranslační úpravy proteinů genetika   MeSH
• ubikvitin genetika   MeSH
• ubikvitinace genetika   MeSH
• ubikvitinligasy genetika   MeSH
• virové proteiny genetika   MeSH
• virus hepatitidy B genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

• MeSH
• buněčné linie MeSH
• diazepam farmakologie   MeSH
• dospělí MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• lidé středního věku MeSH
• lidé MeSH
• ligandy MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• proteinové domény účinky léků   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• reportérové geny účinky léků   genetika   MeSH
• transport proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A thermoresponsive Pluronic/alginate semisynthetic hydrogel is used to bioprint 3D hepatic constructs, with the aim to investigate liver-specific metabolic activity of the 3D constructs compared to traditional 2D adherent cultures. The bioprinting method relies on a bioinert hydrogel and is characterized by high-shape fidelity, mild depositing conditions and easily controllable gelation mechanism. Furthermore, the dissolution of the sacrificial Pluronic templating agent significantly ameliorates the diffusive properties of the printed hydrogel. The present findings demonstrate high viability and liver-specific metabolic activity, as assessed by synthesis of urea, albumin, and expression levels of the detoxifying CYP1A2 enzyme of cells embedded in the 3D hydrogel system. A markedly increased sensitivity to a well-known hepatotoxic drug (acetaminophen) is observed for cells in 3D constructs compared to 2D cultures. Therefore, the 3D model developed herein may represent an in vitro alternative to animal models for investigating drug-induced hepatotoxicity.

• MeSH
• 3D tisk MeSH
• bioprinting * MeSH
• hydrogely MeSH
• lékové postižení jater * MeSH
• tkáňové inženýrství MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Our recent experience of the COVID-19 pandemic has highlighted the importance of easy-to-use, quick, cheap, sensitive and selective detection of virus pathogens for the efficient monitoring and treatment of virus diseases. Early detection of viruses provides essential information about possible efficient and targeted treatments, prolongs the therapeutic window and hence reduces morbidity. Graphene is a lightweight, chemically stable and conductive material that can be successfully utilized for the detection of various virus strains. The sensitivity and selectivity of graphene can be enhanced by its functionalization or combination with other materials. Introducing suitable functional groups and/or counterparts in the hybrid structure enables tuning of the optical and electrical properties, which is particularly attractive for rapid and easy-to-use virus detection. In this review, we cover all the different types of graphene-based sensors available for virus detection, including, e.g., photoluminescence and colorimetric sensors, and surface plasmon resonance biosensors. Various strategies of electrochemical detection of viruses based on, e.g., DNA hybridization or antigen-antibody interactions, are also discussed. We summarize the current state-of-the-art applications of graphene-based systems for sensing a variety of viruses, e.g., SARS-CoV-2, influenza, dengue fever, hepatitis C virus, HIV, rotavirus and Zika virus. General principles, mechanisms of action, advantages and drawbacks are presented to provide useful information for the further development and construction of advanced virus biosensors. We highlight that the unique and tunable physicochemical properties of graphene-based nanomaterials make them ideal candidates for engineering and miniaturization of biosensors.

• MeSH
• Betacoronavirus genetika   izolace a purifikace   patogenita   MeSH
• biosenzitivní techniky * přístrojové vybavení   metody   trendy   MeSH
• design vybavení MeSH
• DNA virů analýza   genetika   MeSH
• elektrochemické techniky MeSH
• grafit * chemie   MeSH
• hybridizace nukleových kyselin MeSH
• klinické laboratorní techniky * přístrojové vybavení   metody   statistika a číselné údaje   MeSH
• kolorimetrie MeSH
• koronavirové infekce diagnóza   epidemiologie   virologie   MeSH
• kvantové tečky chemie   MeSH
• lidé MeSH
• luminiscence MeSH
• nanostruktury chemie   MeSH
• pandemie MeSH
• povrchová plasmonová rezonance MeSH
• Ramanova spektroskopie MeSH
• reakce antigenu s protilátkou MeSH
• virologie metody   MeSH
• virová pneumonie diagnóza   epidemiologie   virologie   MeSH
• viry genetika   izolace a purifikace   patogenita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH