Risk of Hepatocellular Carcinoma Remains High in Patients with HBV-Related Decompensated Cirrhosis and Long-Term Antiviral Therapy.

Background: This study aimed to evaluate the risk factors of HCC development in patients with hepatitis B virus (HBV)-related DC and who underwent long-term antiviral therapy. Methods: Data from 308 patients with HBV-related DC and long-term antiviral therapy were collected and retrospectively reviewed. Cox regression analysis was used to analyze independent risk factors of HCC development. Results: Data from 129 patients with definite records were analyzed. The median follow-up time was 5 years (range, 1 to 8 years). At the end of the follow-up, 41 (31.8%) patients developed HCC, and the time from DC diagnosis to HCC incidence who received antiviral therapy was 4.4 years (range, 1-7 years). The incidence of HCC was higher in males (30/78, 38.5%) than in females (11/51, 21.6%) (P = 0.04). Patients who developed HCC were significantly older than those who did not develop HCC (P < 0.01). The incidence of HCC in patients receiving nucleoside analogues, nucleotide analogues, and combination therapy was 34.7%, 38.1%, and 33.3%, respectively, and the difference showed no significant differences (P = 0.95). Multivariate Cox regression analysis demonstrated that male gender and age ≥50 years are independent risk factors of HCC development (OR = 2.987 and 2.408; 95% CI (1.301-6.858) and (1.126-5.149); P = 0.01 and 0.02, respectively). Conclusion: The risk of HCC remains to be high in patients with HBV-related DC, especially in males aged ≥50 years.

Iron-induced energy supply deficiency and mitochondrial fragmentation in neurons.

Iron dyshomeostasis and mitochondrial impairments are both vitally important for the progression of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Nevertheless, how these two pathological phenomena are linked with one another remains unclear, especially in neurons. To address the question, a model of iron overload was established with exposure of rat primary cortical neurons to excessive iron. We first verified that iron overload resulted in a decrease in adenosine triphosphate (ATP) production in neurons. Meanwhile, the release of mitochondrial cytochrome c was significantly increased after iron overload and consequently triggered an apoptosis signal, as revealed by Caspase 3 cleavage. To explore the potential underlying molecular mechanisms, an unlabeled quantitative proteomics approach was applied to primary neurons. Gene Ontology enrichment analysis revealed that 58 mitochondria-associated proteins were significantly altered, including three subunits of mitochondrial complex I and optic atrophy 1(OPA1). Increased NADH-ubiquinone oxidoreductase 75 kDa subunit and decreased NADH-ubiquinone oxidoreductase subunit A10 levels were further validated by a western blot, and more importantly, complex I activity markedly declined. Iron-induced down-regulation on the OPA1 level was also validated by a western blot, which was not reversed by the anti-oxidant but was reversed by the iron chelator. Moreover, an OPA1-associated key downstream effect, mitochondrial fragmentation, was found to be aggravated in neurons exposed to excessive iron, which is consistent with the down-regulation of OPA1. Furthermore, the protein level of PTEN-induced putative kinase 1, an important protein closely related to complex I activity and mitochondrial fragmentation, also significantly declined in neurons by iron overload. Thus, our findings may shed new light on the linkage between iron toxicity and mitochondrial impairments, such as energy supply deficiency and mitochondrial fragmentation, and further expand the toxic repertoire of iron in the central nerve system. Cover Image for this issue: doi: 10.1111/jnc.14205.

Evolution of drug-resistant mutations in HBV genomes in patients with treatment failure during the past seven years (2010-2016).

The objective of this study was to analyze the prevalence of drug-resistant HBV mutants in patients with treatment failure during the past seven years (2010-2016). 4055 HBV-infected patients who underwent HBV polymerase gene mutation test from 2010 to 2016 were enrolled. The nucleos(t)ide analogues (NAs) resistance mutation positions, including rtL180, rtA181, rtT184, rtS202, rtM204, rtI233, rtN236, rtI169, rtV173, and rtM250 were analyzed. Genotypic resistance mutations were detected in 30.8% (1248/4055) of the patients with treatment failure. Rates of drug-resistant mutations associated with LAM, ADV, ETV, and multidrug were 27.23% (1104/4055), 9.67% (392/4055), 3.69% (150/4055), and 0.79% (32/4055). Among the primary NA-resistant mutations, rtM204I (13.44%, 545/4055) occurred more frequently, followed by rtM204V, rtN236T, rtA181T, and rtA181V. For single-base mutations, rtL180M and rtA181V increased gradually during the past seven years, while rtM204I/V and rtN236T decreased after 2015. The development of drug-resistant mutations positively correlated with the consumption of ETV (r = 0.964, P = 0.002), and weakly correlated with that of LAM (r = 0.679, P = 0.109) and ADV (r = 0.429, P = 0.354). Moreover, single-base mutation rtA181V and multi-base mutations (rtL180M + M204I and rtL180M + M204V + M204I) were more common in HBV genotype C than those in genotype B (1.94% vs. 0.66%, 1.84% vs. 0.16%, 1.02% vs. 0.16%, respectively). NA-related mutations in HBV RT region increased in the past seven years, especially for LAM. Frequencies of rtL180M and rtA181T/V increased gradually in the past seven years, to which we should pay more attention.

Involvement of Intracellular and Mitochondrial Aß in the Ameliorative Effects of Huperzine A against Oligomeric Aß42-Induced Injury in Primary Rat Neurons.

Considerable studies indicate huperzine A is a promising natural product to suppress neuronal damages induced by ß-amyloid (Aß), a key pathogenic event in the Alzheimer's disease (AD). As an extension, the present study for the first time explored whether the beneficial profiles of huperzine A against oligomeric Aß(42) induced neurotoxicity are associated with the accumulation and detrimental function of intraneuronal/mitochondrial Aß, on the basis of the emerging evidence that intracellular Aß is more relevant to AD progression as compared with extracellular Aß. Huperzine A treatment was shown to significantly attenuate the neurotoxicity of oligomeric Aß(42), as demonstrated by increased neuronal viability. Interestingly, our results proved that exogenous Aß(42) could accumulate intraneuronally in a dose- and time-dependent manner, while huperzine A treatment markedly reduced the level of intracellular Aß(42). Moreover, huperzine A treatment rescued mitochondrial dysfunction induced by oligomeric Aß(42), including adenosine triphosphate (ATP) reduction, reactive oxygen species (ROS) overproduction and membrane potential depolarization. Further study demonstrated that huperzine A also significantly reduced the level of Aß(42) in the mitochondria-enriched subcellular fractions, as well as the Aß(42) fluorescent signals colocalized with mitochondrial marker. This study indicates that interfering intracellular Aß especially mitochondrial Aß accumulation, together with ameliorating Aß-associated mitochondrial dysfunction, may contribute to the protective effects of huperzine A against Aß neurotoxicity. Above results may shed more light on the pharmacological mechanisms of huperzine A and provide important clues for discovering novel therapeutic strategies for AD.

Donepezil attenuates Aß-associated mitochondrial dysfunction and reduces mitochondrial Aß accumulation in vivo and in vitro.

The main purpose of the present study is to investigate the influence of donepezil, a well-known acetylcholinesterase (AChE) inhibitor, on amyloid-ß (Aß)-associated mitochondrial dysfunction, in order to gain a better understanding of the neuroprotective effects of this clinically used anti-Alzheimer's disease (AD) drug. First, our study verifies the ameliorative effects of donepezil on behavioral deficits in both working memory and anxiety in APP/PS1 double transgenic mice, at a time point that AChE is not inhibited. Meanwhile, we demonstrate that donepezil enhances the resistance of brain mitochondria of APP/PS1 mice to the induction of mitochondrial permeability transition (MPT) by calcium ions. Moreover, the level of mitochondrial Aß in the brain of donepezil-treated APP/PS1 transgenic mice is significantly lower than that of vehicle-treated APP/PS1 mice. Our in vitro study using isolated mitochondria from rat brains, which is expected as an AChE-free subcellular system, further confirms the ameliorative effects of donepezil on oligomeric Aß1-42 induced mitochondrial swelling and ATP reduction. In addition, donepezil treatment also significantly blocks the Aß accumulation in the isolated mitochondria. Our study reported for the first time that the protective effects of donepezil against Aß-associated mitochondrial dysfunction are closely associated with the reduction of Aß accumulation in the mitochondria. Above observation led us to assume that, besides potent AChE inhibitory effect, other non-cholinergic mechanisms may be involved in the neuroprotective profiles of donepezil.

A novel caffeoyl triterpene attenuates cerebral ischemic injury with potent anti-inflammatory and hypothermic effects.

Despite the intense efforts in searching for stroke therapies, an urgent need still exists to explore novel neuroprotective agents for ischemic stroke that have high efficacy and wide therapeutic time-window. Here, we provide the first demonstration that 28-O-caffeoyl betulin (B-CA), a novel derivative of naturally occurring caffeoyl triterpene, could significantly alleviate brain infarction and neurological deficit when given as late as 6 h after transient middle cerebral artery occlusion in the rat. Moreover, post-ischemia B-CA administration exhibited long-term (14 days post stroke) protective effects on both brain infarction and functional (i.e., motor and sensory) deficits. Protective B-CA effects correlated with decreased inflammatory responses as indicated by inhibition of microglia and astrocyte activation [stained with ionized calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) antibody, respectively], as well as suppression of tumor necrosis factor-α, interleukin-1ß, and cyclooxygenase-2 overproduction in the ipsilateral cortex of ischemic rat. B-CA administration caused significant hypothermia in the focal cerebral ischemic rat, which may contribute to its ameliorative effects on brain damage and inflammation. In view of its potency in wide therapeutic time-window, robust anti-inflammatory and hypothermic effects, this novel caffeoyl triterpene derivative may lead toward the development of effective therapeutic strategies for the treatment of ischemic stroke.

Decreased accumulation of subcellular amyloid-ß with improved mitochondrial function mediates the neuroprotective effect of huperzine A.

A number of recent discoveries indicate that huperzine A, an active herbal medicine employed for the treatment of Alzheimer's disease (AD) in China, can afford neuroprotection on in vitro and in vivo models related to mitochondrial dysfunction. However, it is an intricate and highly debated research topic about whether another pharmacological mechanism is involved in the beneficial profiles of huperzine A, independent of its well-recognized potent acetycholinesterase (AChE) inhibitory effect. As an extension, this study for the first time verified the co-occurrence of the beneficial effects of huperzine A on mitochondrial dysfunction and memory deficits in AßPP/PS1 double transgenic mice, at a time point that AChE was not inhibited. Moreover, using isolated brain cortical mitochondria, we confirmed the ameliorating effect of huperzine A on oligomeric Aß1-42-induced ATP reduction and mitochondrial swelling, as well as a decrease in the enzymatic activities of respiratory chain complexes, especially complex II-III and complex IV, which may be attributed to the blockage of oligomeric Aß1-42 from penetrating into mitochondria. These results shed more light on a potential direct target of huperzine A on isolated mitochondria, which may be largely different from its specific inhibition on AChE. This work describes a novel mechanism of neuroprotection by huperzine A and provides important clues for discovering novel therapeutic strategy for AD.

Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction

OBJECTIVES: Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1 percent) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100 percent) at codon 180 and codon 204. Concordant results were observed for 99.4 percent of the 158 samples at codon 181 and 98.7 percent at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1 percent mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.

Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction.

OBJECTIVES: Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1%) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100%) at codon 180 and codon 204. Concordant results were observed for 99.4% of the 158 samples at codon 181 and 98.7% at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1% mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.