A mutual regulatory loop between transcription factor Yin Yang 1 and hepatitis B virus replication influences chronic hepatitis B.

Hepatitis B virus (HBV) infections pose a major threat to human health. HBV can upregulate the expression of the transcription factor Yin Yang 1 (YY1) in in vitro cytological experiments, suggesting an association between YY1 and HBV infection. However, data on YY1 expression in chronic hepatitis B (CHB) patients are lacking. In this study, we aimed to assess the correlation between YY1 expression and HBV infection. We detected serum YY1 levels in 420 patients with chronic HBV infection, 30 patients with chronic hepatitis C virus infection, and 32 healthy controls using an enzyme-linked immunosorbent assay. The correlation between YY1 levels and clinical parameters was analyzed. Meanwhile, the changes of YY1 before and after interferon or entecavir treatment were analyzed. YY1 levels in the liver tissues were detected using immunofluorescence staining. The expression of YY1 in HBV-expressing cells was detected through western blotting. Meanwhile, we explored the effects of YY1 on HBV replication and gene expression. We found that YY1 was highly expressed in the serum and liver tissues of CHB patients. Serum YY1 levels positively correlated with HBV DNA and hepatitis B surface antigen (HBsAg). Additionally, HBV DNA levels increased but HBsAg levels decreased after HBV-expressing cells overexpress YY1. In conclusion, our study demonstrates that YY1 plays an important role in HBV replication and gene expression, providing a potential target for the treatment of CHB.

Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

Hydrophilic interaction liquid chromatography based method for simultaneous determination of purines and their derivatives in food spices.

This work presents method for separation and quantification of adenine, guanine, xanthine, hypoxanthine, uric acid, and creatinine in food spices using hydrophilic interaction liquid chromatography with UV detection. Optimized conditions allowed separation with mobile phases containing acetonitrile and additives ammonium acetate (90:10, v/v, pH 6.1) or formate (90:10, v/v, pH 3.2). In food spices no uric acid was detected, creatinine (16 ± 2 µg g-1) was found only in instant dried yeast. The highest content of purines was determined in dried yeast (xanthine 110 ± 8 µg g-1, hypoxanthine 441 ± 24 µg g-1, adenine 84 ± 16 µg g-1, guanine 163 ± 12 µg g-1), high in curry, herbal pepper, and chicken seasoning, the lowest concentration was in black pepper (hypoxanthine 12 ± 2 µg g-1, adenine 27 ± 3 µg g-1). To best of our knowledge, no such complementary method and obtained data have been reported so far.

Repair-driven DNA tetrahedral nanomachine combined with DNAzyme for 8-oxo guanine DNA glycosylase activity assay, drug screening and intracellular imaging.

8-oxo guanine DNA glycosylase (8-oxoG DNA glycosylase), a crucial DNA repair enzyme, is essential for maintaining genome integrity and preventing diseases caused by DNA oxidative damage. Imaging 8-oxoG DNA glycosylase in living cells requires a dependable technique. In this study, we designed a DNAzyme-modified DNA tetrahedral nanomachine (DTDN) powered by 8-oxoG restoration. Incorporating a molecular beacon probe (MB), the constructed platform was used for amplified in situ monitoring of 8-oxoG DNA glycosylase. Under normal conditions, duplexing with a complementary strand modified with two 8-oxoG sites inhibited the activity of DNAzyme. The restoration of DNAzyme activity by the repair of intracellular 8-oxoG DNA glycosylase on 8-oxoG bases can initiate a signal amplification reaction. This detection system can detect 8-oxoG DNA glycosylase activity linearly between 0 and 20 U mL-1, with a detection limit as low as 0.52 U mL-1. Using this method, we were able to screen 14 natural compounds and identify 6 of them as 8-oxoG DNA glycosylase inhibitors. In addition, a novel approach was utilized to assess the activity of 8-oxoG DNA glycosylase in living cells. In conclusion, this method provides a universal tool for monitoring the activity of 8-oxoG DNA glycosylase in vitro and in living cells, which holds great promise for elucidating the enzyme's functionality and facilitating drug screening endeavors.

Pilot Study of High-Dose Pemetrexed in Patients with Progressive Chordoma.

PURPOSE: Chordomas are ultrarare tumors of the axial spine and skull-base without approved systemic therapy. Most chordomas have negative expression of thymidylate synthase (TS), suggesting a potential for responding to the antifolate agent pemetrexed, which inhibits TS and other enzymes involved in nucleotide biosynthesis. We evaluated the therapeutic activity and safety of high-dose pemetrexed in progressive chordoma. PATIENTS AND METHODS: Adult patients with previously treated, progressive chordoma participated in an open-label, single-institution, single-arm, pilot clinical trial of intravenous pemetrexed 900 mg/m2 every 3 weeks and supportive medications of folic acid, vitamin B12, and dexamethasone. The primary endpoint was objective response rate according to RECIST v1.1. Secondary endpoints included adverse events, progression-free survival (PFS), tumor molecular profiles, and alterations in tissue and blood-based biomarkers. RESULTS: Fifteen patients were enrolled and the median number of doses administered was 15 (range, 4-31). One patient discontinued treatment due to psychosocial issues after four cycles and one contracted COVID-19 after 13 cycles. Of the 14 response-evaluable patients, 2 (14%) achieved a partial response and 10 (71%) demonstrated stable disease. Median PFS was 10.5 months (95% confidence interval: 9 months-undetermined) and 6-month PFS was 67%. Adverse events were expected and relatively mild, with one grade 3 creatinine increased, and one each of grade 3 and 4 lymphopenia. No grade 5 adverse events, unexpected toxicities, or dose-limiting toxicities were observed. Several patients reported clinical improvement in disease-related symptoms. CONCLUSIONS: High-dose pemetrexed appears tolerable and shows objective antitumor activity in patients with chordoma. Phase II studies of high-dose pemetrexed are warranted.

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e.

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-L-Lysine Induce Antibody Production as Vaccine Adjuvants.

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-ß. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.

Structure of the major oxidative damage 7,8-dihydro-8-oxoguanine presented into a catalytically competent DNA glycosylase.

If left unrepaired, the major oxidative DNA lesion 7,8-dihydro-8-oxoguanine (oxoG) promotes G-to-T transversions by favorably adopting a syn conformation and base pairing with dATP during replication. The human oxoG DNA glycosylase hOGG1 senses and removes oxoG amid millions-fold excess of guanine, thereby counteracting the genotoxic effects of the major oxidative damage. Crystal structures of hOGG1 in complex with oxoG-containing DNA have provided key insights into the lesion recognition and catalysis mechanisms of the enzyme. These lesion-recognition complex (LRC) structures typically involve a catalytically inactive hOGG1 mutant, where one of the catalytic-site amino acid residues is mutated to prevent the cleavage of oxoG. The use of a catalytically incompetent hOGG1 mutant has thus precluded understanding of unscathed interactions between oxoG and hOGG1 catalytic site as well as interactions among catalytic-site amino acid residues. As an orthogonal approach to visualize such interactions, we have co-crystallized a catalytically competent hOGG1 bound to 2'-fluoro-oxodG-containing DNA, a transition state destabilizing inhibitor that binds hOGG1 but is not processed by the enzyme. In this fluorinated lesion-recognition complex (FLRC), the 8-oxo moiety of oxoG is recognized by Gly42 and the Watson-Crick edge of oxoG is contacted by Gln315 and Pro266. The previously observed salt bridge between Lys249 and Cys253 is lacking in the FLRC, suggesting Lys249 is primed by Cys253 and poised for nucleophilic attack on C1' of oxodG. Overall, hOGG1 FLRC marks the first structure of oxoG presented into an intact catalytic site of hOGG1 and provides complementary insights into the glycosylase mechanisms of the enzyme.

A generally minimalist strategy of constructing biomineralized high-efficiency personalized nanovaccine combined with immune checkpoint blockade for cancer immunotherapy.

As a representative of tumor immunotherapy, tumor vaccine can inhibit tumor growth by activating tumor-specific immune response, which has the advantages of relatively low toxicity and high efficiency, and has attracted much attention in recent years. However, there are still difficulties in how to effectively deliver tumor vaccines in vivo and make them work efficiently. It is a relatively mature method to load tumor specific antigens with suitable carriers to produce tumor vaccines. Here, a generally minimalist construction method of tumor nanovaccine was developed. A high-efficiency tumor nanovaccine (NV) was prepared in one step by a biomineralization-like method, which contained ovalbumin (OVA, model antigen), unmethylated cytosine-phosphate-guanine (CpG, adjuvant) and Mn-NP (carrier and adjuvant). NV not only showed good tumor preventive effect, but also could successfully inhibited tumor development and metastasis when combined with anti-PD-L1, and induced long-term immune memory effect. However, the method of screening tumor specific antigen to construct nanovaccine is cumbersome and tumors are heterogeneous. Therefore, surgically resected tumor tissue is the best source of antigens for preparing tumor vaccines. Next, based on the strong loading ability of the carrier, we designed a personalized tumor nanovaccine (PNV) using the supernatant of tumor abrasive fluid (STAF) as antigen based on the generally minimalist tumor nanovaccine construction strategy. PNV combined with anti-PD-L1 could successfully inhibit post-surgical tumor recurrence and induce strong and durable immune memory effects. This study presents a novel, general, and minimalist strategy to construct high-efficiency personalized nanovaccine, which has a wide range of potential applications in the field of tumor treatment.

Efficacy and safety of Fuzheng Huayu tablet on persistent advanced liver fibrosis following 2 years entecavir treatment: A single arm clinical objective performance criteria trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Antiviral therapy can alleviate liver fibrosis in chronic hepatitis B, but it has a limited effect on advanced liver fibrosis/cirrhosis. Traditional Chinese medicine (TCM), particularly FuZheng HuaYu (FZHY) tablet, appears to have an antifibrotic effect, but its improving resolution of hepatitis b virus (HBV) -associated advanced fibrosis and experienced anti-viral treatment has not been investigated. AIM OF THE STUDY: To observe the safety and efficacy of adjunctive FZHY on the HBV-associated cirrhosis patients who received 2 years of entecavir but still with advanced fibrosis. METHODS: An open-label, multicentre, single arm trial. 251 patients were included and treated with TCM consisted of FZHY tablets 1.6 g and granules, three times a day in addition to entecavir 0.5 mg daily for an additional 48 weeks. Primary outcome was regression of fibrosis (the proportion of patients with a 1-point decrease in the Ishak liver fibrosis score from baseline to week 48). RESULTS: Fibrosis regression occurred in 94 of 184 patients with paired liver biopsy (51.09%, 95% CI: 43.9～58.0). In 132 compensated cirrhosis patients (Ishak score ≥5), 56.06% (74/132, 95% CI: 47.5～64.2) showed fibrosis regression and reached the goal of 54% (15% more than entecavir mono-therapy). 10 patients occurred adverse reaction, most of them were mild, and all recovered or achieved remission. CONCLUSIONS: The combination therapy of FZHY, TCM granules and ETV could regress the liver fibrosis in the patients with HBV cirrhosis, who experienced 2 years of ETV treatment, and it is safe and well tolerated.

A Pilot Study on the Association of Lead, 8-Hydroxyguanine, and Malondialdehyde Levels in Opium Addicts' Blood Serum with Illicit Drug Use and Non-Addict Persons.

While a large body of literature has shown the health problems of illicit drug use, research is needed on how substance abuse impacts DNA damage and contaminants in blood, especially given Pb-contaminated opium. This pilot study aimed to evaluate the levels of lead (Pb), 8-hydroxy di-guanine (8-oxo-Gua), and malondialdehyde (MDA) in the blood serum of opium addicts and non-addict people. The current study is a case-control study with a cross-sectional design. A sample of 50 opium-addicted and non-addict adults were chosen for this study using convenience and random sampling methods. Participants were divided into two groups: addicts and non-addicts. The atomic absorption spectroscopy method was used to measure the quantity of Pb, and the Enzyme-Linked Immunosorbent Assay (ELISA) method was used to measure the amount of 8-oxo-Gua and MDA. The data were analyzed using an independent t-test. The results show that the amount of Pb in the blood serum of addicted women and men was higher than levels in non-addict men and women, for the study participants (p-value = 0.001). Blood levels were not significantly different between addicts and non-addicts for men or women for 8-oxo-Gua (p-value = 0.647 for women and p-value = 0.785 for men) and MDA (p-value = 0.867 for women and p-value = 0.995 for men). In general, addicts' blood Pb levels were found to be substantially higher than those of normal non-addict persons in this pilot study. As a result, testing for blood Pb levels in addicts may be informative in instances when symptoms are inconclusive.

N/O→B dative bonds supplemented by N-HN/HC hydrogen bonds make BN-cages an attractive candidate for DNA-nucleobase adsorption - an MP2 prediction.

The response of B12N12-nanocages towards DNA-nucleobases (adenine, guanine, cytosine, and thymine) is investigated using MP2 and DFT (M06-2X) levels of theory with the 6-311+G** basis set. Multiple BN-cage-nucleobase structures for each nucleobase emerged depending on the number of Lewis base centers of nucleobases. The main source of stability of these complexes is the N/O→B dative bond, where the N or O atom of nucleobases donates the lone-pair electron to one of the boron atoms of the nanocage. Nitrogen atoms of the BN-cage, adjacent to the B-site forming dative bond, act as a proton acceptor to form multiple (N-HN and N-HC) hydrogen bonds, where proton-donors NH and CH are part of nucleobases. MP2/6-311+G** adsorption energies are -43.1, -43.4 and -45.3 kcal mol-1 (B12N12-adenine), -37.1, -41.9 and -43.3 kcal mol-1 (B12N12-guanine), -41.3 and -43.4 (B12N12-cytosine), and -29.3 and -31.3 (B12N12-thymine). Similar adsorption energies were recorded for larger BN-fullerenes-nucleobases, namely B16N16 and B24N24. Changes in adsorption energies and structures of these nano-bio-hybrid materials in aqueous media are also discussed. Computationally cost-effective MP2 single point calculations at the M06-2X optimized geometries were found to be reliable in predicting adsorption energies. The effect of the BN-network and H-bonds on the adsorption process is assessed by comparing the results with simple BH3-nucleobase models. BSSE correction to the adsorption energy is not recommended.

CpG ODN (K3)-toll-like receptor 9 agonist-induces Th1-type immune response and enhances cytotoxic activity in advanced lung cancer patients: a phase I study.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients' immune responses. METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29. RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days. CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined. TRIAL REGISTRATION: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

Stoichiometry versus ecology: the relationships between genome size and guanine-cytosine content, and tissue nitrogen and phosphorus in grassland herbs.

BACKGROUND AND AIMS: Plant tissue nitrogen (N) and phosphorus (P) and genome traits, such as genome size and guanine-cytosine (GC) content, scale with growth or metabolic rates and are linked to plant ecological strategy spectra. Tissue NP stoichiometry and genome traits are reported to affect plant growth, metabolic rates or ecological strategies in contrasting ways, although the elemental costs for building and maintaining DNA are typically overlooked. METHODS: We formulated stoichiometry- and ecology-based predictions on the relationship between genome size and GC content to tissue N, P and N : P and tested them on a set of 130 herbaceous species from a temperate grassland using ordinary, phylogenetic and quantile regression. KEY RESULTS: Genome size was only negatively linked to plant N and N : P in species with very small genomes. We found no link between genome size and plant P. GC content was negatively linked to plant N and P but we found these significant links consistently in both GC-rich and GC-poor species. Finally, GC content correlated positively with plant N : P but only in species with GC-rich genomes. CONCLUSIONS: Our results provide stronger support for the ecology-based predictions than the stoichiometry-based predictions, and for the links between GC content and plant N and P stoichiometry than for genome size. We argue that the theories of plant metabolic rates and ecological strategies (resource-acquisitive vs. conservative or ruderal vs. stress-tolerator spectra) better explain interspecific genome-NP stoichiometry relationships at the tissue level (although relatively weakly) than the stoichiometric theory based on the elemental costs for building and maintaining DNA.

Impact of Fuzheng Huayu tablet on antiviral effect of entecavir in patients with hepatitis B cirrhosis.

BACKGROUND: Fuzheng Huayu tablet is a traditional Chinese medicine (TCM) used for the treatment of liver fibrosis and cirrhosis. However, whether the combination with Fuzheng Huayu tablet could affect the antiviral efficacy of nucleos(t)ide remains a concern. The objective of this trial was to explore the impact of Fuzheng Huayu tablet on antiviral effect of entecavir in patients with hepatitis B cirrhosis. METHODS: A prospective, randomized control trial was conducted. Patients with compensated hepatitis B cirrhosis were randomly divided into the treatment group (entecavir capsule plus Fuzheng Huayu tablet) and the control group (entecavir capsule plus simulant of Fuzheng Huayu), and followed up for 48 weeks. The dynamic changes of HBV DNA load, the rate of serological conversion of HBeAg, liver function, renal function and liver stiffness measurement (LSM) were monitored. The general clinical data and adverse events were also recorded. RESULTS: There was no significant difference in the rate of virological response and cumulative virological response between the treatment group and the control group (P > 0.05). After 48 weeks of treatment, the HBeAg seroconversion rate, biochemical response rate and LSM value were 21.05% and 4.76% (P = 0.164), 86.96% and 65.96% (P = 0.017), 9.5 kpa and 10.6 kpa (P = 0.827) in the treatment group and the control group, respectively. No serious adverse events related to the study therapy occurred during the trial. CONCLUSIONS: The antiviral entecavir combined with Fuzheng Huayu tablet did not affect the antiviral efficacy of entecavir, but could improve the rate of biochemical response, and had a tendency to improve the rate of serological conversion of HBeAg and liver fibrosis in patients with hepatitis B cirrhosis. Fuzheng Huayu tablet is clinically safe for patients with hepatitis B cirrhosis.

Predictive Performance of CAGE-B and SAGE-B Models in Asian Treatment-Naive Patients Who Started Entecavir for Chronic Hepatitis B.

BACKGROUND & AIMS: Cirrhosis and age (CAGE-B) and stiffness and age (SAGE-B) models assess the risk of hepatocellular carcinoma (HCC) development in white patients with chronic hepatitis B (CHB) undergoing sustained antiviral therapy (AVT). Herein, we checked the predictive performance of these models in Asian patients with CHB. METHODS: We reviewed 734 treatment-naive patients with CHB who started entecavir between 2006 and 2011 and were followed up for more than 5 years without HCC development during AVT. The predictive performance of CAGE-B and SAGE-B models was calculated using area under the receiver operating characteristic curves (AUROCs). RESULTS: Median liver stiffness assessed using transient elastography after 5 years of AVT was 6.8 kPa. Median CAGE-B and SAGE-B models after 5 years of AVT were 7.0 and 6.0, respectively. More than 5 years after AVT initiation, 66 patients (9.0%) developed HCC. The AUROCs of the CAGE-B and SAGE-B models were 0.764 and 0.785 after 7 years and 0.799 and 0.802 after 10 years of AVT, respectively. The cumulative incidence of HCC was significantly higher in the high-risk groups according to CAGE-B and SAGE-B risk stratification than in the medium- and low-risk groups (P < .05 in all cases). The SAGE-B model showed a higher likelihood ratio (χ2) (76.2 vs 71.4) and linear trend (χ2) (74.1 vs 58.6) than the CAGE-B model, whereas the CAGE-B model showed higher Akaike information criteria (64.3 vs 50.3). CONCLUSIONS: Both SAGE-B and CAGE-B showed acceptable performance in predicting HCC after 5 years of AVT in Asian patients with CHB.

Development of Hydrophobic Interaction-based DNA Supramolecules as Efficient Delivery Carriers of CpG DNA to Immune cells.

Unmethylated cytosine-phosphate-guanine (CpG) DNA stimulates mammalian immune cells through recognition by Toll-like receptor 9 (TLR9). Therefore, CpG DNA is expected to be an effective adjuvant for the treatment of immune and allergic diseases. However, challenges, such as low stability against DNase and low delivery efficiency for immune cells, still need to be resolved for the application of CpG DNA. To overcome these challenges, we developed DNA supramolecules consisting of long single-stranded DNA (lss-DNA) synthesized using rolling circle amplification (RCA) and cholesterol-modified DNA (chol-DNA). Lss-DNAs containing multiple CpG motifs were annealed with complementary chol-DNAs to form DNA supramolecules through hydrophobic interactions. Transmission electron microscopy revealed that lss-DNA mixed with chol-DNA formed micrometer-sized DNA supramolecules. The formation of DNA supramolecules increased their stability against DNase compared to lss DNA, which was evaluated using FBS. Furthermore, DNA supramolecules induced three-times higher TNF-α release from RAW264.7 cells than lss-DNA alone. These results demonstrate that DNA supramolecules are efficient delivery carriers of CpG DNA to immune cells.

Mechanism of DNA methylation-mediated downregulation of O6-methylguanine-DNA methyltransferase in cartilage injury of Kashin-Beck disease.

OBJECTIVE: Kashin-Beck disease (KBD) is an endemic osteoarthropathy, in which excessive apoptosis of chondrocytes occurs. O6-methylguanine-DNA methyltransferase (MGMT), a DNA damage repair gene, plays an important role in apoptosis, but the mechanism is unclear in KBD cartilage injury. This study was to investigate the expression and promoter methylation of MGMT in KBD patients and its role in DNA damage and apoptosis of chondrocytes. METHODS: MGMT mRNA and protein level were detected by quantitative real-time PCR and immunohistochemistry. Demethylation of MGMT was carried out using 5-Aza-2'-deoxycytidine, and the methylation level of MGMT promoter was measured by quantitative methylation specific PCR. Next, small hairpin RNA was used to knockdown the expression of MGMT. Cell viability, apoptosis and DNA damage were determined by MTT assay, flow cytometry, Hoechst 33342 staining and alkaline comet assay following T-2 toxin and selenium treatment. RESULTS: MGMT protein expression and mRNA levels were decreased (P = 0.02, P = 0.007) and promoter methylation was increased (P = 0.008) in KBD patients. Meanwhile, MGMT level was upregulated by 5-Aza-2'-deoxycytidine in chondrocytes (P = 0.0002). DNA damage and apoptosis rates were increased in MGMT-silenced chondrocytes (all P < 0.0001). Furthermore, DNA damage and apoptosis were increased in chondrocytes treated with T-2 toxin (all P < 0.0001), but were decreased after selenium treatment (P < 0.0001, P = 0.01). Decreased mRNA level and increased methylation of MGMT were found in the T-2 toxin group (P = 0.005, P = 0.002), while selenium reversed it (P = 0.02, P = 0.004). CONCLUSIONS: MGMT might play a crucial part in the pathogenesis of KBD cartilage injury, which could provide a therapeutic target for KBD.

Synergistic Effect of Biejia-Ruangan on Fibrosis Regression in Patients With Chronic Hepatitis B Treated With Entecavir: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Long-term nucleos(t)ide analogue (NA) treatment can reverse liver fibrosis in chronic hepatitis B (CHB), but its effect on fibrosis regression remains limited. Biejia-Ruangan (BR) has been approved in China as an antifibrotic traditional Chinese medicine drug in patients with chronic liver diseases. A multicenter randomized controlled trial aims to evaluate the effect of BR on fibrosis regression in CHB patients treated with NAs. METHODS: CHB patients with histologically confirmed advanced fibrosis or cirrhosis were randomly assigned to receive entecavir (ETV) (0.5 mg per day) plus BR (2 g 3 times a day) or placebo for 72 weeks. Liver fibrosis regression was defined as a reduction of ≥ 1 point by the Ishak fibrosis stage (IFS). RESULTS: Overall, 500 patients were enrolled in each group as the intention-to-treat population. The rate of fibrosis regression after 72 weeks of treatment was significantly higher in the ETV + BR group (40% vs 31.8%; P = .0069). Among 388 patients with cirrhosis (ie, IFS ≥ 5) at baseline, the rate of cirrhosis reversal (ie, IFS ≤ 4) was significantly higher in the ETV + BR group (41.5% vs 30.7%; P = .0103). CONCLUSIONS: Addition of BR to the current standard treatment with NAs in CHB patients with advanced fibrosis or cirrhosis can improve liver fibrosis regression. CLINICAL TRIALS REGISTRATION: NCT01965418.

Mechanisms, Management and Prevention of Pemetrexed-Related Toxicity.

Pemetrexed is a cytostatic antifolate drug and a cornerstone in the treatment of lung cancer. Although generally well tolerated, a substantial part of the patient population experiences dose-limiting or even treatment-limiting toxicities. These include mucositis, skin problems, fatigue, renal toxicity, and neutropenia. Several studies confirmed that pemetrexed pharmacokinetics can serve as a prognostic factor for the development of toxicity, especially for neutropenia. Preventing and managing toxicity of pemetrexed can help to ensure durable treatment. Several evidence-based strategies are already implemented in clinical care. With the introduction of standard vitamin supplementation and dexamethasone, the incidence of hematological toxicity and skin reactions substantially decreased. In the case of high risk for toxicity, granulocyte colony-stimulating factor can be used to prevent severe hematological toxicity. Moreover, high-dose folinic acid can resolve severe pemetrexed-induced toxicity. There are several experimental options to prevent or manage pemetrexed-related toxicity, such as the use of standard folinic acid, hemodialysis, antidotes such as thymidine, hypoxanthine, and glucarpidase, and the use of therapeutic drug monitoring. These strategies still need clinical evaluation before implementation, but could enable treatment with pemetrexed for patients who are at risk for toxicity, such as in renal impairment.