Репозиторий Университета

A Scientific Methodology for Expansion of Anti-Parkinson Drug Product Range Nat a lia Valeryevna Pyatigorskaya Galina Eduardovna Brkich Alexey Nikitich Pavlov Valery Vasilyevich Beregovykh Olga Vladimirovna Evdokimova Sechenov First Moscow State Medical University, Russian Fedetation, 119991, Moscow, Trubetskaya Street, 8-2 Abstract Parkinson's disease (PD) is a chronic and progressive brain disease associat ed primarily with dopamine neurons degeneration of substantia nigra. More than 10 millions of people worldwide are affected by this disease manifested by combination of hypokinesia and rigidity, shaking, and postural instability. The high prevalence of disease has determined the aim of the study: to deve lop a methodology for expansion of anti-Parkinson drug product range. The information analysis methods were used for unbiased evaluation of novel anti-Parkinson drugs creation prospects. A systemic analysis of active pharmaceu tical ingredients (AFIs) used in anti- Parkinson drug products allowed for discovery of most widely used, levodopa being the top one. A comparative assessment of dosage forms used in Parkinson’s disease treatment showed th at they are represented primarily by intestinal gels, tablets, dispersible tablets, capsules, and modified release capsules. The authors conclude, that the novel anti-Parkinson drug pr oduct should contain levodopa in form of an endonasal spray which provides optimal bioavailability due to neces sary excipients (triglycerides, sodium monohydrogen phosphate, volatile and fatty oils, herbal extracts, stabilizers, and flavouring agents).

Современная терапия, направленная на эрадикацию H.pylori, включает комплекс антисекреторных и антибактериальных препаратов, иногда препаратов висмута. Особенностью современных эрадикационных схем является четырнадцатидневное применение антибактериальных средств, назначаемых в высоких суточных дозах и избираемых преимущественно с учётом резистентности микроорганизма к кларитромицину и метронидазолу в соответствующем регионе. Однако каждый компонент эрадикационной схемы может иметь достаточно серьёзные неблагоприятные побочные реакции, а также влиять на биодоступность, биотрансформацию, выведение и потенцирование эффектов лекарственных препаратов, которые больной может принимать одновременно с антихеликобактерной терапией. В статье перечислены наиболее серьёзные и распространённые варианты лекарственных взаимодействий компонентов эрадикационных схем, дано описание механизма их развития, если таковой выяснен. До появления практических рекомендаций относительно профилактики лекарственных взаимодействий препаратов, включаемых в эрадикационные схемы, следует использовать общедоступные базы данных, содержащие сведения о таких взаимодействиях.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC–MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that “spacer peptides” can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC–tandem MS methods.

Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N6-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N6 by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.

Over the last decade, a dual character of cell response to oxidative stress, eustress versus distress, has become increasingly recognized. A growing body of evidence indicates that under physiological conditions, low concentrations of reactive oxygen and nitrogen species (RONS) maintained by the activity of endogenous antioxidant system (AOS) allow reversible oxidative/nitrosative modifications of key redox-sensitive residues in regulatory proteins. The reversibility of redox modifications such as Cys S-sulphenylation/S-glutathionylation/S-nitrosylation/S-persulphidation and disulphide bond formation, or Tyr nitration, which occur through electrophilic attack of RONS to nucleophilic groups in amino acid residues provides redox switches in the activities of signalling proteins. Key requirement for the involvement of the redox modifications in RONS signalling including ROS-MAPK, ROS-PI3K/Akt, and RNS-TNF-α/NF-kB signalling is their specificity provided by a residue microenvironment and reaction kinetics. Glutathione, glutathione peroxidases, peroxiredoxins, thioredoxin, glutathione reductases, and glutaredoxins modulate RONS level and cell signalling, while some of the modulators (glutathione, glutathione peroxidases and peroxiredoxins) are themselves targets for redox modifications. Additionally, gene expression, activities of transcription factors, and epigenetic pathways are also under redox regulation. The present review focuses on RONS sources (NADPH-oxidases, mitochondrial electron-transportation chain (ETC), nitric oxide synthase (NOS), etc.), and their cross-talks, which influence reversible redox modifications of proteins as physiological phenomenon attained by living cells during the evolution to control cell signalling in the oxygen-enriched environment. We discussed recent advances in investigation of mechanisms of protein redox modifications and adaptive redox switches such as MAPK/PI3K/PTEN, Nrf2/Keap1, and NF-κB/IκB, powerful regulators of numerous physiological processes, also implicated in various diseases.

The LysM receptor-like kinase K1 is involved in regulation of pea-rhizobial symbiosis development.

The ability of the crop legume Pisum sativum L. to perceive the Nod factor rhizobial signals may depend on several receptors that differ in ligand structure specificity. Identification of pea mutants defective in two types of LysM receptor-like kinases (LysM-RLKs), SYM10 and SYM37, featuring different phenotypic manifestations and impaired at various stages of symbiosis development, corresponds well to this assumption. There is evidence that one of the receptor proteins involved in symbiosis initiation, SYM10, has an inactive kinase domain. This implies the presence of an additional component in the receptor complex, together with SYM10, that remains unknown. Here, we describe a new LysM-RLK, K1, which may serve as an additional component of the receptor complex in pea. To verify the function of K1 in symbiosis, several P. sativum non-nodulating mutants in the k1 gene were identified using the TILLING approach. Phenotyping revealed the blocking of symbiosis development at an appropriately early stage, strongly suggesting the importance of LysM-RLK K1 for symbiosis initiation. Moreover, the analysis of pea mutants with weaker phenotypes provides evidence for the additional role of K1 in infection thread distribution in the cortex and rhizobia penetration. The interaction between K1 and SYM10 was detected using transient leaf expression in Nicotiana benthamiana and in the yeast two-hybrid system. Since the possibility of SYM10/SYM37 complex formation was also shown, we tested whether the SYM37 and K1 receptors are functionally interchangeable using a complementation test. The interaction between K1 and other receptors is discussed.

Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete with other organisms (fungi, bacteria, nematodes, and insects) and help them occupy more preferential areas at the expense of effective antagonism. These compounds possess antibiotic activity towards a wide range of other microbes, particularly fungi that belong to different taxonomical units. These compounds include peptaibols, which are non-ribosomal synthesized polypeptides containing non-standard amino acid residues (alpha-aminoisobutyric acid mandatory) and some posttranslational modifications. We isolated a novel antibiotic peptide from the culture medium of Emericellopsis alkalina, an alkalophilic strain. This peptide, called emericellipsin A, exhibited a strong antifungal effect against the yeast Candida albicans, the mold fungus Aspergillus niger, and human pathogen clinical isolates. It also exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. Additionally, emericellipsin A showed a significant cytotoxic effect and was highly active against Hep G2 and HeLa tumor cell lines. We used NMR spectroscopy to reveal that this peptaibol is nine amino acid residues long and contains non-standard amino acids. The mode of molecular action of emericellipsin A is most likely associated with its effects on the membranes of cells. Emericellipsin A is rather short peptaibol and could be useful for the development of antifungal, antibacterial, or anti-tumor remedies. View Full-Text

Blue biotechnologies implement marine bio-resources for addressing practical concerns. The isolation of biologically active molecules from marine animals is one of the main ways this field develops. Strikingly, cnidaria are considered as sustainable resources for this purpose, as they possess unique cells for attack and protection, producing an articulated cocktail of bioactive substances. The Mediterranean sea anemone Anemonia viridis has been studied extensively for years. In this short review, we summarize advances in bioprospecting of the A. viridis toxin arsenal. A. viridis RNA datasets and toxin data mining approaches are briefly described. Analysis reveals the major pool of neurotoxins of A. viridis, which are particularly active on sodium and potassium channels. This review therefore integrates progress in both RNA-Seq based and biochemical-based bioprospecting of A. viridis toxins for biotechnological exploitation. View Full-Text

Chronic hepatitis B virus (HBV) infection has long remained a critical global health issue. Covalently closed circular DNA (cccDNA) is a persistent form of the HBV genome that maintains HBV chronicity. Decades of extensive research resulted in the two therapeutic options currently available: nucleot(s)ide analogs and interferon (IFN) therapy. A plethora of reliable markers to monitor HBV patients has been established, including the recently discovered encapsidated pregenomic RNA in serum, which can be used to determine treatment end-points and to predict the susceptibility of patients to IFN. Additionally, HBV RNA splice variants and cccDNA and its epigenetic modifications are associated with the clinical course and risks of hepatocellular carcinoma (HCC) and liver fibrosis. However, new antivirals, including CRISPR/Cas9, APOBEC-mediated degradation of cccDNA, and T-cell therapies aim at completely eliminating HBV, and it is clear that the diagnostic arsenal for defining the long-awaited sterilizing cure is missing. In this review, we discuss the currently available tools for detecting and measuring HBV RNAs and cccDNA, as well as the state-of-the-art in clinical implications of these markers, and debate needs and goals within the context of the sterilizing cure that is soon to come. View Full-Text

The heme in the active center of peroxidases reacts with hydrogen peroxide to form highly reactive intermediates, which then oxidize simple substances called peroxidase substrates. Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body, but under the influence of external conditions they can display peroxidase-like activity. As oxidative intermediates, these peroxidases produce not only active heme compounds, but also protein-based tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are considered as pseudo-peroxidases. Рeroxidases play an important role in innate immunity and in a number of physiologically important processes like apoptosis and cell signaling. Unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance. Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase activity and the biological effects of peroxidase products are specific for each hemoprotein. This review summarizes the knowledge about the properties, activities, regulations and biological effects of true and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and adverse effects of this class of enzymes. View Full-Text

Purpose

The interaction between malignant cells and surrounding healthy tissues is a critical factor in the metastatic progression of breast cancer (BC). Extracellular vesicles, especially exosomes, are known to be involved in inter-cellular communication during cancer progression. In the study presented herein, we aimed to evaluate the role of circulating plasma exosomes in the metastatic dissemination of BC and to investigate the underlying molecular mechanisms of this phenomenon.

Methods

Exosomes isolated from plasma of healthy female donors were applied in various concentrations into the medium of MDA-MB-231 and MCF-7 cell lines. Motility and invasive properties of BC cells were examined by random migration and Transwell invasion assays, and the effect of plasma exosomes on the metastatic dissemination of BC cells was demonstrated in an in vivo zebrafish model. To reveal the molecular mechanism of interaction between plasma exosomes and BC cells, a comparison between un-treated and enzymatically modified exosomes was performed, followed by mass spectrometry, gene ontology, and pathway analysis.

A Scientific Methodology for Expansion of Anti-Parkinson Drug Product Range Nat a lia Valeryevna Pyatigorskaya Galina Eduardovna Brkich Alexey Nikitich Pavlov Valery Vasilyevich Beregovykh Olga Vladimirovna Evdokimova Sechenov First Moscow State Medical University, Russian Fedetation, 119991, Moscow, Trubetskaya Street, 8-2 Abstract Parkinson's disease (PD) is a chronic and progressive brain disease associat ed primarily with dopamine neurons degeneration of substantia nigra. More than 10 millions of people worldwide are affected by this disease manifested by combination of hypokinesia and rigidity, shaking, and postural instability. The high prevalence of disease has determined the aim of the study: to deve lop a methodology for expansion of anti-Parkinson drug product range. The information analysis methods were used for unbiased evaluation of novel anti-Parkinson drugs creation prospects. A systemic analysis of active pharmaceu tical ingredients (AFIs) used in anti- Parkinson drug products allowed for discovery of most widely used, levodopa being the top one. A comparative assessment of dosage forms used in Parkinson’s disease treatment showed th at they are represented primarily by intestinal gels, tablets, dispersible tablets, capsules, and modified release capsules. The authors conclude, that the novel anti-Parkinson drug pr oduct should contain levodopa in form of an endonasal spray which provides optimal bioavailability due to neces sary excipients (triglycerides, sodium monohydrogen phosphate, volatile and fatty oils, herbal extracts, stabilizers, and flavouring agents).

Современная терапия, направленная на эрадикацию H.pylori, включает комплекс антисекреторных и антибактериальных препаратов, иногда препаратов висмута. Особенностью современных эрадикационных схем является четырнадцатидневное применение антибактериальных средств, назначаемых в высоких суточных дозах и избираемых преимущественно с учётом резистентности микроорганизма к кларитромицину и метронидазолу в соответствующем регионе. Однако каждый компонент эрадикационной схемы может иметь достаточно серьёзные неблагоприятные побочные реакции, а также влиять на биодоступность, биотрансформацию, выведение и потенцирование эффектов лекарственных препаратов, которые больной может принимать одновременно с антихеликобактерной терапией. В статье перечислены наиболее серьёзные и распространённые варианты лекарственных взаимодействий компонентов эрадикационных схем, дано описание механизма их развития, если таковой выяснен. До появления практических рекомендаций относительно профилактики лекарственных взаимодействий препаратов, включаемых в эрадикационные схемы, следует использовать общедоступные базы данных, содержащие сведения о таких взаимодействиях.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC–MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that “spacer peptides” can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC–tandem MS methods.

Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N6-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N6 by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.

Over the last decade, a dual character of cell response to oxidative stress, eustress versus distress, has become increasingly recognized. A growing body of evidence indicates that under physiological conditions, low concentrations of reactive oxygen and nitrogen species (RONS) maintained by the activity of endogenous antioxidant system (AOS) allow reversible oxidative/nitrosative modifications of key redox-sensitive residues in regulatory proteins. The reversibility of redox modifications such as Cys S-sulphenylation/S-glutathionylation/S-nitrosylation/S-persulphidation and disulphide bond formation, or Tyr nitration, which occur through electrophilic attack of RONS to nucleophilic groups in amino acid residues provides redox switches in the activities of signalling proteins. Key requirement for the involvement of the redox modifications in RONS signalling including ROS-MAPK, ROS-PI3K/Akt, and RNS-TNF-α/NF-kB signalling is their specificity provided by a residue microenvironment and reaction kinetics. Glutathione, glutathione peroxidases, peroxiredoxins, thioredoxin, glutathione reductases, and glutaredoxins modulate RONS level and cell signalling, while some of the modulators (glutathione, glutathione peroxidases and peroxiredoxins) are themselves targets for redox modifications. Additionally, gene expression, activities of transcription factors, and epigenetic pathways are also under redox regulation. The present review focuses on RONS sources (NADPH-oxidases, mitochondrial electron-transportation chain (ETC), nitric oxide synthase (NOS), etc.), and their cross-talks, which influence reversible redox modifications of proteins as physiological phenomenon attained by living cells during the evolution to control cell signalling in the oxygen-enriched environment. We discussed recent advances in investigation of mechanisms of protein redox modifications and adaptive redox switches such as MAPK/PI3K/PTEN, Nrf2/Keap1, and NF-κB/IκB, powerful regulators of numerous physiological processes, also implicated in various diseases.

The LysM receptor-like kinase K1 is involved in regulation of pea-rhizobial symbiosis development.

The ability of the crop legume Pisum sativum L. to perceive the Nod factor rhizobial signals may depend on several receptors that differ in ligand structure specificity. Identification of pea mutants defective in two types of LysM receptor-like kinases (LysM-RLKs), SYM10 and SYM37, featuring different phenotypic manifestations and impaired at various stages of symbiosis development, corresponds well to this assumption. There is evidence that one of the receptor proteins involved in symbiosis initiation, SYM10, has an inactive kinase domain. This implies the presence of an additional component in the receptor complex, together with SYM10, that remains unknown. Here, we describe a new LysM-RLK, K1, which may serve as an additional component of the receptor complex in pea. To verify the function of K1 in symbiosis, several P. sativum non-nodulating mutants in the k1 gene were identified using the TILLING approach. Phenotyping revealed the blocking of symbiosis development at an appropriately early stage, strongly suggesting the importance of LysM-RLK K1 for symbiosis initiation. Moreover, the analysis of pea mutants with weaker phenotypes provides evidence for the additional role of K1 in infection thread distribution in the cortex and rhizobia penetration. The interaction between K1 and SYM10 was detected using transient leaf expression in Nicotiana benthamiana and in the yeast two-hybrid system. Since the possibility of SYM10/SYM37 complex formation was also shown, we tested whether the SYM37 and K1 receptors are functionally interchangeable using a complementation test. The interaction between K1 and other receptors is discussed.

Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete with other organisms (fungi, bacteria, nematodes, and insects) and help them occupy more preferential areas at the expense of effective antagonism. These compounds possess antibiotic activity towards a wide range of other microbes, particularly fungi that belong to different taxonomical units. These compounds include peptaibols, which are non-ribosomal synthesized polypeptides containing non-standard amino acid residues (alpha-aminoisobutyric acid mandatory) and some posttranslational modifications. We isolated a novel antibiotic peptide from the culture medium of Emericellopsis alkalina, an alkalophilic strain. This peptide, called emericellipsin A, exhibited a strong antifungal effect against the yeast Candida albicans, the mold fungus Aspergillus niger, and human pathogen clinical isolates. It also exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. Additionally, emericellipsin A showed a significant cytotoxic effect and was highly active against Hep G2 and HeLa tumor cell lines. We used NMR spectroscopy to reveal that this peptaibol is nine amino acid residues long and contains non-standard amino acids. The mode of molecular action of emericellipsin A is most likely associated with its effects on the membranes of cells. Emericellipsin A is rather short peptaibol and could be useful for the development of antifungal, antibacterial, or anti-tumor remedies. View Full-Text

Blue biotechnologies implement marine bio-resources for addressing practical concerns. The isolation of biologically active molecules from marine animals is one of the main ways this field develops. Strikingly, cnidaria are considered as sustainable resources for this purpose, as they possess unique cells for attack and protection, producing an articulated cocktail of bioactive substances. The Mediterranean sea anemone Anemonia viridis has been studied extensively for years. In this short review, we summarize advances in bioprospecting of the A. viridis toxin arsenal. A. viridis RNA datasets and toxin data mining approaches are briefly described. Analysis reveals the major pool of neurotoxins of A. viridis, which are particularly active on sodium and potassium channels. This review therefore integrates progress in both RNA-Seq based and biochemical-based bioprospecting of A. viridis toxins for biotechnological exploitation. View Full-Text

Chronic hepatitis B virus (HBV) infection has long remained a critical global health issue. Covalently closed circular DNA (cccDNA) is a persistent form of the HBV genome that maintains HBV chronicity. Decades of extensive research resulted in the two therapeutic options currently available: nucleot(s)ide analogs and interferon (IFN) therapy. A plethora of reliable markers to monitor HBV patients has been established, including the recently discovered encapsidated pregenomic RNA in serum, which can be used to determine treatment end-points and to predict the susceptibility of patients to IFN. Additionally, HBV RNA splice variants and cccDNA and its epigenetic modifications are associated with the clinical course and risks of hepatocellular carcinoma (HCC) and liver fibrosis. However, new antivirals, including CRISPR/Cas9, APOBEC-mediated degradation of cccDNA, and T-cell therapies aim at completely eliminating HBV, and it is clear that the diagnostic arsenal for defining the long-awaited sterilizing cure is missing. In this review, we discuss the currently available tools for detecting and measuring HBV RNAs and cccDNA, as well as the state-of-the-art in clinical implications of these markers, and debate needs and goals within the context of the sterilizing cure that is soon to come. View Full-Text