Comparing the risk of hepatitis B virus reactivation between direct-acting antiviral therapies and interferon-based therapies for hepatitis C.

Hepatitis B virus (HBV) reactivation has been reported during antihepatitis C treatment in patients with hepatitis C virus (HCV) and HBV co-infection. We aimed to evaluate the frequency and risk factors of HBV reactivation during anti-HCV therapy and compared those between interferon (IFN)-free direct-acting antiviral (DAA) therapies and IFN-based therapies. Three hundred and twenty-two patients with HCV infection receiving anti-HCV therapy were retrospectively screened. The baseline HBV infection statuses of all eligible patients and the HBV-DNA level of all patients with current or previous HBV infection were examined at the end of treatment. In patients with baseline anti-HBs positivity, changes in anti-HBs titre were evaluated. Of 287 patients who met the inclusion criteria, 157 had current (n=4) or previous (n=153) HBV infection; 85 were treated with IFN-free DAA therapies and 72 were treated with IFN-based therapies. Six patients experienced HBV reactivation (n=2) or HBV reappearance (n=4) after IFN-free DAA therapies, while no patient developed HBV reactivation after IFN-based therapies. The risk factors of HBV reactivation or reappearance were DAA therapies and a reduction in anti-HBs titre to <12 mIU mL-1 by the end of treatment. The decline changes of anti-HBs titre were significantly higher in patients treated with DAA therapies. Although HBV reactivation hepatitis was not observed, three of four patients with HBV reactivation or reappearance after achieving HCV eradication had viremia 8 weeks after completion of therapy. A significant proportion of patients develop HBV reactivation or reappearance without hepatitis after IFN-free DAA therapies. Low levels of anti-HBs and their decrease to <12 mIU mL-1 after treatment are significant risk factors for HBV reactivation or reappearance.

Micro/extended-nano sampling interface from a living single cell.

Single-cell analysis is of increasing importance in many fields, but is challenging due to the ultra-small volumes (picoliters) of single cells. Indeed, analysis of a specific analyte might require the analysis of a single molecule or several molecules. Analytical processes usually include sampling, chemical processing, and detection. Although several papers have reported chemical processing and detection methods for single cells, a sampling method compatible with maintaining the viability of a single cell during sampling has yet to be developed. Here, we propose a femtoliter sampling method from a living single cell using micro/nanofluidic device technology. The sampling of 39 fL of cytoplasm from a single human aortic endothelial cell was demonstrated and its viability after sampling was confirmed.

Lymphotropic hepatitis C virus has an interferon-resistant phenotype.

Hepatitis C virus (HCV) infects and associates with B cells, leading to abnormal B-cell activation and development of lymphoproliferative and autoimmune disorders. This immune perturbation may in turn be associated with the resistance of HCV against the host immune system. The objective of this study was to analyse the effects of HCV infection of B cells on the efficacy of interferon (IFN)-based therapy. The study enrolled 102 patients with chronic hepatitis C who were treated with pegylated IFN plus ribavirin. HCV RNA titres in B cells were compared in patients with rapid viral responder (RVR) vs non-RVR, sustained viral responder (SVR) vs non-SVR and null viral responder (NVR) vs VR. The levels of HCV RNA in B cells were significantly higher in non-RVR, non-SVR and NVR groups. Association between the therapy outcome and the positive B-cell HCV RNA was also investigated in relation to other known viral and host factors. Multivariable analyses showed that the positive B-cell HCV RNA and the minor single-nucleotide polymorphism near the IL28B gene (rs8099917) were independent factors associated with NVR in patients infected with HCV genotype 1. When these two factors were combined, the sensitivity, specificity, positive and negative predictive values for NVR were 92.3%, 98.2%, 92.3% and 98.2%, respectively. Genotype 1 and the presence of one or no mutations in the IFN-sensitivity determining region were associated with higher levels of B-cell HCV RNA. B-cell-tropic HCV appears to have an IFN-resistant phenotype. B-cell HCV RNA positivity is a predictive factor for resistance to IFN-based therapy.

The clinical utility of comprehensive measurement of autoimmune disease-related antibodies in patients with advanced solid tumors receiving immune checkpoint inhibitors: a retrospective study.

BACKGROUND: The comprehensive measurement of autoimmune disease-related antibodies (Abs) before immune checkpoint inhibitor (ICI) treatment may be useful for predicting the development of immune-related adverse events (irAEs); however, the clinical utility is not well known. MATERIALS AND METHODS: We retrospectively analyzed patients with advanced solid tumors treated with ICI monotherapy or doublet combination therapy between July 2014 and December 2020 at single institute. Anti-nuclear antibody (ANA), anti-thyroglobulin (Tg) Ab, anti-thyroid peroxidase (TPO) Ab, anti-glutamic acid decarboxylase (GAD) Ab, anti-acetylcholine esterase receptor (AchR) Ab, and platelet-associated immunoglobulin G (PA-IgG) Ab were comprehensively measured for the screening before ICI therapy. RESULTS: Of 275 registered patients (median age, 70 years; male, 64.4%; Eastern Cooperative Oncology Group performance status of 0 or 1, 88.7%; and prior regimen of 0-1/≥2, 88.7%/11.3%), 128 non-small-cell lung cancer, 35 gastric cancer, 33 head and neck cancer, 24 melanoma, 19 renal cell carcinoma, 13 urothelial carcinoma, 12 esophageal cancer, 5 malignant mesothelioma of pleura, 2 endometrial cancer, and 4 other cancer were included. The number of patients with positive ANA, Tg, TPO, PA-IgG, GAD, and AchR Abs was 52 (24.9%), 38 (14.5%), 11 (10.1%), 6 (3.5%), 5 (2.0%), and 1 (0.5%), respectively. There was no association between the development of any irAEs and Abs positivity, while thyroid dysfunction developed more frequently among patients with than without Tg Ab or TPO Ab (39.5% versus 12.5%, P < 0.01; 45.5% versus 14.3%, P = 0.02). CONCLUSIONS: The clinical utility of comprehensive measurement of autoimmune disease-related Abs before introduction of ICI therapy was limited for predicting irAE. However, Tg and TPO Abs were risk factors as regards the development of ICI-induced thyroid dysfunction.

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.

Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.

Restricted antibody formation to sheep erythrocytes of allogeneic bone marrow chimeras histoincompatible at the K end of the H-2 complex.

Employing a new method for allogeneic bone marrow transplantation, irradiation chimeras constructed from various combinations of marrow cells from B10 H-2 recombinant mice and AKR recipients were prepared. Though these chimeras had well-developed populations of T and B cells, they showed strikingly different patterns of responses in the primary antibody formation to sheep erythrocytes (SRBC), a T dependent antigen. These are (a) AKR mice treated with C57BL/10 cells, [B10 leads to AKR] fully H-2 incompatible, and AKR mice treated with B10.A (5R) cells, [5R leads to AKR] I-J,E compatible chimeras that were almost completely unresponsive to SRBC; (b) AKR mice treated with B10.BR cells [BR leads to AKR] fully H-2 compatible, and AKR mice treated with B10 AKM cells, [AKM leads to AKR] chimeras where donor and recipient differed only at H-2D, showed the same number of plaque-forming cells (PFC) as B10 control mice; (c) AKR mice treated with B10.A cells, [B10 leads to AKR] chimeras, where donor and recipient were matched at H-2K-I-E region, showed about one-half the number of PFC as the control mice. From these results we conclude that in allogeneic bone marrow chimeras primary antibody response to T-dependent antigen, such as SRBC, is generated when at least the K end of the H-2 complex is compatible between donor and recipient.

Gallamine (Flaxedil) and synaptic transmission in the spinal cord.

A paralyzing dose of gallamine (Flaxedil) (1 to 2 milligrams per kilogram of body weight) has no effect on synaptic transmission in the cat's spinal cord. In spinal cats ventilated with oxygen, we stimulated a dorsal spinal root and recorded the compound ventral root potential. The reflex potential was not affected by 6.25 milligrams of gallamine per kilogram. Giving 12.5 milligrams of gallamine per kilogram had no significant effect on the monosynaptic spike height, but the polysynaptic response rose briefly to 12 percent above control. Increased magnitude of the polysynaptic response appeared related to a concomitant rise in blood pressure.

Architectures of class-defining and specific domains of glutamyl-tRNA synthetase.

The crystal structure of a class I aminoacyl-transfer RNA synthetase, glutamyl-tRNA synthetase (GluRS) from Thermus thermophilus, was solved and refined at 2.5 A resolution. The amino-terminal half of GluRS shows a geometrical similarity with that of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of the same subclass in class I, comprising the class I-specific Rossmann fold domain and the intervening subclass-specific alpha/beta domain. These domains were found to have two GluRS-specific, secondary-structure insertions, which then participated in the specific recognition of the D and acceptor stems of tRNA(Glu) as indicated by mutagenesis analyses based on the docking properties of GluRS and tRNA. In striking contrast to the beta-barrel structure of the GlnRS carboxyl-terminal half, the GluRS carboxyl-terminal half displayed an all-alpha-helix architecture, an alpha-helix cage, and mutagenesis analyses indicated that it had a role in the anticodon recognition.

X-ray structure of T4 endonuclease V: an excision repair enzyme specific for a pyrimidine dimer.

The x-ray structure of T4 endonuclease V, an enzyme responsible for the first step of a pyrimidine-dimer-specific excision-repair pathway, was determined at a 1.6-angstrom resolution. The enzyme consists of a single compact domain classified into an all-alpha structure. This single domain has two distinct catalytic activities; it functions as a pyrimidine dimer glycosylase and as an apurinic-apyrimidinic endonuclease. The amino-terminal segment penetrates between two major helices and prevents their direct contact. The refined structure suggests the residues involved in the substrate binding and the catalysis of the glycosylation reaction.

The beta-prism: a new folding motif.

A new protein fold with internal symmetry has been observed in two proteins: vitelline membrane outer layer protein I (VMO-I) and delta-endotoxin. Despite lacking any discernible sequence similarity, both proteins have similar three-dimensional structures as well as a carbohydrate-binding site in the top region of the common fold.

DNA-repair enzymes.

Recent crystallographic studies of DNA-repair enzymes have provided the structural basis for the recognition of damaged DNA. The results imply that flipping out of the base is a common and crucial event in DNA repair. Two classes of repair enzymes that recognize distinct types of damage may exist. DNA-repair enzymes that share similar folds and DNA binding motifs have been proposed to belong to a superfamily.

Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.

Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions.

The structural basis of damaged DNA recognition and endonucleolytic cleavage for very short patch repair endonuclease.

Endonucleases in DNA repair must be able to recognize damaged DNA as well as cleave the phosphodiester backbone. These functional prerequisites are manifested in very short patch repair (Vsr) endonuclease through a common endonuclease topology that has been tailored for recognition of TG mismatches. Structural and biochemical comparison with type II restriction enzymes illustrates how Vsr resembles these endonucleases in overall topology but also how Vsr diverges in terms of the detailed catalytic mechanism. A histidine and two metal-water clusters catalyze the phosphodiester cleavage. The mode of DNA damage recognition is also unique to Vsr. All other structurally characterized DNA damage-binding enzymes employ a nucleotide flipping mechanism for substrate recognition and for catalysis. Vsr, on the other hand, recognizes the TG mismatch as a wobble base pair and penetrates the DNA with three aromatic residues on one side of the mismatch. Thus, Vsr endonuclease provides important counterpoints in our understanding of endonucleolytic mechanisms and of damaged DNA recognition.

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.

PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing endonucleases and provides some of the catalytic residues necessary for DNA cleavage activity. Site-specific mutagenesis studies showed that two acidic residues in the motifs, Asp149 and Glu250 in PI- Pfu I, and Asp156 and Asp249 in PI- Pfu II, were critical for catalysis. The third residues of the active site triads, as predicted from the structure of PI- Sce I, were Asn225 in PI- Pfu I and Lys224 in PI- Pfu II. Substitution of Asn225 in PI- Pfu I by Ala did not affect catalysis. The cleavage activity of PI- Pfu II was 50-fold decreased by the substitution of Ala for Lys224. The binding affinity of the mutant protein for the substrate DNA also decreased 6-fold. The Lys in PI- Pfu II may play a direct or indirect role in catalysis of the endonuclease activity.

Biochemical characterization of the hjc holliday junction resolvase of Pyrococcus furiosus.

The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination. The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases. In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K:(d)) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic Holliday junction as substrate. Hjc required a divalent cation for cleavage activity and Mg(2+) at 5-10 mM was optimal. Mn(2+) could substitute for Mg(2+), but it was much less efficient than Mg(2+) as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCl at approximately 200 mM. In addition to the high specific activity, Hjc was found to be extremely heat stable. In contrast to the case of SULFOLOBUS:, the Holliday junction resolving activity detected in P. furiosus cell extract thus far is only derived from Hjc.

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities.

We screened for proteins with specific binding activity to Holliday junction DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a protein that has specific affinity for DNA with a branched structure, like a three-way or four-way junction. The protein was identified as one of the two inteins encoded in the gene for ribonucleotide reductase (RNR) by gene cloning. These two inteins were spliced out from the precursor protein as polypeptides with molecular weights of 53.078 and 43.976 kDa, respectively. The amino acid sequences of these inteins have two copies of the LAGLIDADG motif, which is found in the site-specific DNA endonucleases. The purified proteins actually cleaved double-stranded DNA with the sequence of the intein(-)allele, and, therefore, they were designated PI- Pfu I and PI- Pfu II. They generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homing endonucleases originating from inteins. The optimal conditions of the DNA cleavage reaction, including temperature, pH, and concentrations of KCl and MgCl(2), have been determined. The high affinity for junction DNA of PI- Pfu I was confirmed using the purified protein.

Precluding uracil from DNA.

Two enzymes, dUTP pyrophosphatase and uracil-DNA glycosylase, prevent the misincorporation of uracil into the genome in distinct manners. The atomic structures of these proteins complexed with substrate analogs reveal the structural basis for uracil recognition and suggest a novel mechanism of DNA repair.

Functional analyses of the domain structure in the Holliday junction binding protein RuvA.

BACKGROUND: Homologous recombination is crucial for genetic diversity and repairing damaged chromosomes. In Escherichia coli cells, the RuvA, RuvB and RuvC proteins participate in the processing of an important intermediate, the Holliday junction. The RuvA-RuvB protein complex facilitates branch migration of the junction, depending on ATP hydrolysis. The atomic structure of RuvA should enable critical questions to be addressed about its specific interactions with the Holliday junction and the RuvB protein. RESULTS: The crystal structure of RuvA shows the tetrameric molecules with a fourfold axis at the center. Each subunit consists of three distinct domains, some of which contain important secondary structure elements for DNA binding. Together with the detailed structural information, the biochemical assays of various mutant RuvA proteins and domains, isolated by partial proteolysis, allowed us to define the functional roles of these domains in Holliday junction binding and the RuvB interaction. CONCLUSIONS: The RuvA molecule is formed by four identical subunits, each with three domains, I, II and III. The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction. Domain III is weakly attached to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III. Functional analyses of the mutant proteins and the partial digestion products, including Holliday junction binding and branch-migration assays, revealed that domain III and the preceding loop are crucial for RuvB binding and branch migration, although this region is not required for the junction-DNA binding.

Crystal structure of the archaeal holliday junction resolvase Hjc and implications for DNA recognition.

BACKGROUND: Homologous recombination is a crucial mechanism in determining genetic diversity and repairing damaged chromosomes. Holliday junction is the universal DNA intermediate whose interaction with proteins is one of the major events in the recombinational process. Hjc is an archaeal endonuclease, which specifically resolves the junction DNA to produce two separate recombinant DNA duplexes. The atomic structure of Hjc should clarify the mechanisms of the specific recognition with Holliday junction and the catalytic reaction. RESULTS: The crystal structure of Hjc from the hyperthermophilic archaeon Pyrococcus furiosus has been determined at 2.0 A resolution. The active Hjc molecule forms a homodimer, where an extensive hydrophobic interface tightly assembles two subunits of a single compact domain. The folding of the Hjc subunit is clearly different from any other Holliday junction resolvases thus far known. Instead, it resembles those of type II restriction endonucleases, including the configurations of the active site residues, which constitute the canonical catalytic motifs. The dimeric Hjc molecule displays an extensive basic surface on one side, which contains many conserved amino acids, including those in the active site. CONCLUSIONS: The architectural similarity of Hjc to restriction endonucleases allowed us to construct a putative model of the complex with Holliday junction. This model accounts for how Hjc recognizes and resolves the junction DNA in a specific manner. Mutational and biochemical analyses highlight the importance of some loops and the amino terminal region in interaction with DNA.

The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin.

BACKGROUND: Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. RESULTS: Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. CONCLUSIONS: The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range. The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.