Resistance studies of a dithiazol analogue, DBPR110, as a potential hepatitis C virus NS5A inhibitor in replicon systems.

Hepatitis C virus (HCV), a member of the Flaviviridae family, affects approximately 3% of the world's population and is becoming the leading cause of liver disease in the world. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In our previous study, we identified a potential HCV NS5A inhibitor, BP008. After further systemic optimization, we discovered a more potent HCV inhibitor, DBPR110. DBPR110 reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC(50)) and a selective index value of 3.9 ± 0.9 pM and >12,800,000, respectively. DBPR110 reduced HCV2a replicon activity with an EC(50) and a selective index value of 228.8 ± 98.4 pM and >173,130, respectively. Sequencing analyses of several individual clones derived from the DBPR110-resistant RNAs purified from cells harboring genotype 1b and 2a HCV replicons revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. P58L/T and Y93H/N in genotype 1b and T24A, P58L, and Y93H in the genotype 2a replicon were the key substitutions for resistance selection. In the 1b replicon, V153M, M202L, and M265V play a compensatory role in replication and drug resistance. Moreover, DBPR110 displayed synergistic effects with alpha interferon (IFN-α), an NS3 protease inhibitor, and an NS5B polymerase inhibitor. In summary, our results present an effective small-molecule inhibitor, DBPR110, that potentially targets HCV NS5A. DBPR110 could be part of a more effective therapeutic strategy for HCV in the future.

Resistance analysis and characterization of a thiazole analogue, BP008, as a potent hepatitis C virus NS5A inhibitor.

Hepatitis C virus (HCV) is a global health problem, affecting approximately 3% of the world's population. The standard treatment for HCV infection is often poorly tolerated and ineffective. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In this report, BP008, a potent small-molecule inhibitor of HCV replication, was developed from a class of compounds with thiazol core structures by means of utilizing a cell-based HCV replicon system. The compound reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC(50)) and selective index value of 4.1 ± 0.7 nM and >12,195, respectively. Sequencing analyses of several individual clones derived from BP008-resistant RNAs purified from cells harboring HCV1b replicon revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. Q24L, P58S, and Y93H are the key substitutions for resistance selection; F149L and V153M play the compensatory role in the replication and drug resistance processes. Moreover, BP008 displayed synergistic effects with alpha interferon (IFN-α), NS3 protease inhibitor, and NS5B polymerase inhibitor, as well as good oral bioavailability in SD rats and favorable exposure in rat liver. In summary, our results pointed to an effective small-molecule inhibitor, BP008, that potentially targets HCV NS5A. BP008 can be considered a part of a more effective therapeutic strategy for HCV in the future.

Successful propagation of flavivirus infectious cDNAs by a novel method to reduce the cryptic bacterial promoter activity of virus genomes.

Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. Full-length DENV2 and JEV cDNAs were obtained by inserting mutations reducing their ECP activity in bacteria without altering amino acid sequences. A severe cytopathic effect occurred when BHK21 cells were transfected with in vitro-transcribed RNAs from either a DENV2 cDNA clone with multiple silent mutations within the prM-E-NS1 region of dengue genome or a JEV cDNA clone with an A-to-C mutation at nt 90 of the JEV genome. The virions derived from the DENV2 or JEV cDNA clone exhibited infectivities similar to those of their parental viruses in C6/36 and BHK21 cells. A cis-acting element essential for virus replication was revealed by introducing silent mutations into the central portion (nt 160 to 243) of the core gene of DENV2 infectious cDNA or a subgenomic DENV2 replicon clone. This novel strategy of constructing DENV2 and JEV infectious clones could be applied to other flaviviruses or pathogenic RNA viruses to facilitate research in virology, viral pathogenesis, and vaccine development.

Determination of critical power in trained rowers using a three-minute all-out rowing test.

The purpose of this study was to determine whether the hyperbolic relationship between power output and time to exhaustion (work - time and power - [1/time] models) could be estimated from a modified version of a three-minute all-out rowing test (3-min RT), and to investigate the test-retest reliability of the 3-min RT. Eighteen male rowers volunteered to participate in this study and underwent an incremental exercise test (IRT), three constant-work rate tests to establish the critical power (CP) and the curvature constant (W'), and two 3-min RTs against a fixed resistance to estimate the end-test power (EP) and work-done-above-EP (WEP) on a rowing ergometer. Peak VO(2max) and maximal VO(2max) oxygen uptakes were calculated as the highest 30 s average achieved during the 3-min RT and IRT tests. The results showed that EP and WEP determinations, based on the 3-min RT, have moderate reproducibility (P = 0.002). EP (269 ± 39 W) was significantly correlated with CP (work - time, 272 ± 30 W; power - [1/time], 276 ± 32 W) (P = 0.000), with no significant differences observed between the EP and CP values (P = 0.474). However, WEP did not significantly correlate with W' (P = 0.254), and was significantly higher than the W' values. There was a significant correlation between the VO(2max) (60 ± 3 ml kg(-1) min(-1)) and VO(2max) (61 ± 4 ml kg(-1) min(-1)) (P = 0.003). These results indicate that the 3-min RT has moderate reliability, and is able to appropriately estimate the aerobic capacity in rowers, particularly for the CP and VO(2max) parameters.

Analysis of Palliative Care Utilization and Medical Expenses among Patients with Chronic Diseases in Taiwan: A Population-Based Cohort Study.

Palliative care (PC) is an important alternative treatment for patients with chronic diseases, particularly for those in the later stages of disease progression. This is because these diseases are often irreversible, with progressive worsening of symptoms. By encouraging the use of tranquility resources for good death and spiritual relief, PC can reduce the physical and psychological burden on patients at the end of their lives. Currently, most discussions on PC have focused on patients with cancers, and few have further discussed the differences in medical expenses between PC and emergency treatment in patients with chronic diseases at the end of their lives. This study analyzed the top three chronic diseases in patients who used PC resources in the past decade and identified the impact of emergency treatment on mean survival time and medical expenses based on the medical records from the National Health Insurance Research Database. In total, 4061 patients with chronic diseases who were admitted to hospice wards were included in this study; of them, 85 patients still received emergency treatment, including urinary catheterization, nasogastric intubation, and respirator use, at the end of their lives. The mean survival time of patients aged 50-64 years who received emergency treatment was longer than that of the same age group who did not receive emergency treatment. Different comparisons of the mean survival time and medical expenses using real-world data provides important insights regarding PC management that may assist in establishing health policies in the future.

Exploratory Study of Palliative Care Utilization and Medical Expense for Inpatients at the End-of-Life.

BACKGROUND: Previous research mostly analyzed the utilization of palliative care for patients with cancer, and data regarding non-cancer inpatients are limited. OBJECTIVES: This research aimed to investigate the current situation regarding palliative care and the important factors that influence its utilization by inpatients (including inpatients with and without cancer) at the end of their lives. We also explored the feasibility of establishing a prediction model of palliative care utilization for inpatients at the end of their lives. These findings will allow medical staff to monitor and focus on those who may require palliative care, resulting in more end-of-life patients receiving palliative care and thereby reducing medical expense and improving their quality of life. METHODS: This was a retrospective study based on real-world health information system (HIS) data from 5 different branches of Taipei City Hospital between 1 January 2018 and 31 December 2018 that enrolled a total of 1668 deceased inpatients. To explore palliative care utilization at the end of life, we used 5-fold cross-validation in four different statistical models to obtain the performance of predictive accuracy: logistic regression (LGR), classification and regression tree (CART), multivariate adaptive regression spline (MARS), and gradient boosting (GB). The important variables that may affect palliative care utilization by inpatients were also identified. RESULTS: The results were as follows: (1) 497 (29.8%) inpatients received palliative care; (2) the average daily hospitalization cost of patients with cancer who received palliative care (NTD 5789 vs. NTD 12,115; p ≤ 0.001) and all patients who received palliative care (NTD 91,527 vs. NTD 186,981; p = 0.0037) were statistically significantly lower than patients who did not receive palliative care; (3) diagnosis, hospital, and length of stay (LOS) may affect palliative care utilization of inpatient; diagnosis, hospitalization unit, and length of hospitalization were statistically significant by LGR; (4) 51.5% of patients utilized palliative consultation services, and 48.5% utilized palliative care units; and (5) MARS had the most consistent results; its accuracy was 0.751, and the main predictors of palliative care utilization are hospital, medical expense, LOS, diagnosis, and Palliative Care Screening Tool-Taiwan version (TW-PCST) scores. CONCLUSIONS: The results reveal that palliative care utilization by inpatients remains low, and it is necessary to educate patients without cancer of the benefits and advantages of palliative care. Although data were limited, the predictability of the MARS model was 0.751; a better prediction model with more data is necessary for further research. Precisely predicting the need for palliative care may encourage patients and their family members to consider palliative care, which may balance both physical and mental care. Therefore, unnecessary medical care can be avoided and limited medical resources can be allocated to more patients in need.

Investigating Key Factors Related to the Decision of a Do-Not-Resuscitate Consent.

BACKGROUND: The decision to sign a do-not-resuscitate (DNR) consent is critical for patients concerned about their end-of-life medical care. Taiwan's National Health Insurance Administration (NHIA) introduced a family palliative care consultation fee to encourage family palliative care consultations; since its implementation, identifying which families require such consultations has become more important. In this study, the Taiwanese version of the Palliative Care Screening Tool (TW-PCST) was used to determine each patient's degree of need for a family palliative care consultation. OBJECTIVE: This study analyzed factors associated with signing DNR consents. The results may inform family palliative care consultations for families in need, thereby achieving a higher DNR consent rate and promoting the effective use of medical resources, including time, labor, and funding. METHOD: In this retrospective study, logistic regression analysis was conducted to determine which factors affected the DNR decisions of 2144 deceased patients (aged ≥ 20 years), whose records were collected from the Taipei City Hospital health information system from 1 January to 31 December 2018. RESULTS: Among the 1730 patients with a DNR consent, 1298 (75.03%) received family palliative care consultations. The correlation between DNR consent and family palliative care consultations was statistically significant (p < 0.001). Through logistic regression analysis, we determined that participation in family palliative care consultation, TW-PCST score, type of ward, and length of stay were significant variables associated with DNR consent. CONCLUSIONS: This study determined that TW-PCST scores can be used as a measurement standard for the early identification of patients requiring family palliative care consultations. Family palliative care consultations provide opportunities for patients' family members to participate in discussions about end-of-life care and DNR consent and provide patients and their families with accurate medical information regarding the end-of-life care decision-making process. The present results can serve as a reference to increase the proportion of patients willing to sign DNR consents and reduce the provision of ineffective life-prolonging medical treatment.

Rhodiola/Cordyceps-Based Herbal Supplement Promotes Endurance Training-Improved Body Composition But Not Oxidative Stress and Metabolic Biomarkers: A Preliminary Randomized Controlled Study.

Rhodiola crenulata (R) and Cordyceps sinensis (C) are commonly used herbs that promote health in traditional Chinese medicine. These two herbs have also been shown to exhibit anti-inflammation and antioxidant functions. Regular endurance training reveals potent endurance capacity, body composition improvement, and metabolic-related biomarker benefits. However, it is not known whether the combination of Rhodiola crenulata and Cordyceps sinensis (RC) supplementation during endurance training provides additive health benefits. The purpose of this study was to investigate the effects of 8-week endurance training plus RC supplementation on body composition, oxidative stress, and metabolic biomarkers in young sedentary adults. METHODS: Fourteen young sedentary adults (8M/6F) participated in this double-blind randomized controlled study. Participants were assigned to exercise training with placebo groups (PLA, n = 7, 4M/3F; age: 21.4 ± 0.4 years) and exercise training with the RC group (RC, 20 mg/kg/day; n = 7, 4M/3F; age: 21.7 ± 0.4 years). Both groups received identical exercise training for eight weeks. The body composition, circulating oxidative stress, and blood metabolic biomarkers were measured before and after the 8-week intervention. RESULTS: Improvement in body composition profiles were significantly greater in the RC group (body weight: p = 0.044, BMI: p = 0.003, upper extremity fat mass: p = 0.032, lower extremity muscle mass: p = 0.029, trunk fat mass: p = 0.011) compared to the PLA group after training. The blood lipid profile and systemic oxidative stress makers (thiobarbituric reactive substanceand total antioxidant capacity) did not differ between groups. Although endurance training markedly improved endurance capacity and glycemic control ability (i.e., fast blood glucose, insulin, and HOMA index), there were no differences in these variables between treatments. CONCLUSIONS: In this preliminary investigation, we demonstrated that an 8-week RC supplementation (20 mg/kg/day) faintly enhanced endurance training-induced positive adaptations in body composition in young sedentary individuals, whereas the blood lipid profile and systemic oxidative stress states were not altered after such intervention.

A novel flavivirus entry inhibitor, BP34610, discovered through high-throughput screening with dengue reporter viruses.

Dengue virus (DENV) is a global health problem that affects approximately 3.9 billion people worldwide. Since safety concerns were raised for the only licensed vaccine, Dengvaxia, and since the present treatment is only supportive care, the development of more effective therapeutic anti-DENV agents is urgently needed. In this report, we identified a potential small-molecule inhibitor, BP34610, via cell-based high-throughput screening (HTS) of 12,000 compounds using DENV-2 reporter viruses. BP34610 reduced the virus yields of type 2 DENV-infected cells with a 50% effective concentration (EC50) and selectivity index value of 0.48 ±â€¯0.06 µM and 197, respectively. Without detectable cytotoxicity, the compound inhibited not only all four serotypes of DENV but also Japanese encephalitis virus (JEV). Time-of-addition experiments suggested that BP34610 may act at an early stage of DENV virus infection. Sequencing analyses of several individual clones derived from BP34610-resistant viruses revealed a consensus amino acid substitution (S397P) in the N-terminal stem region of the E protein. Introduction of S397P into the DENV reporter viruses conferred an over 14.8-fold EC90 shift for BP34610. Importantly, the combination of BP34610 with a viral replication inhibitor, ribavirin, displayed synergistic enhancement of anti-DENV-2 activity. Our results identify an effective small-molecule inhibitor, BP34610, which likely targets the DENV E protein. BP34610 could be developed as an anti-flavivirus agent in the future.

Hepatoprotective effects of Solanum nigrum Linn extract against CCl(4)-induced oxidative damage in rats.

Solanum nigrum L. (SN) is an herbal plant that has been used as hepatoprotective and anti-inflammation agent in Chinese medicine. In this study, the protective effects of water extract of SN (SNE) against liver damage were evaluated in carbon tetrachloride (CCl4)-induced chronic hepatotoxicity in rats. Sprague-Dawley (SD) rats were orally fed with SNE (0.2, 0.5, and 1.0 g kg(-1) bw) along with administration of CCl4 (20% CCl4/corn oil; 0.5 mL kg(-1) bw) for 6 weeks. The results showed that the treatment of SNE significantly lowered the CCl4-induced serum levels of hepatic enzyme markers (GOT, GPT, ALP, and total bilirubin), superoxide and hydroxyl radical. The hepatic content of GSH, and activities and expressions of SOD, GST Al, and GST Mu that were reduced by CCl4 were brought back to control levels by the supplement of SNE. Liver histopathology showed that SNE reduced the incidence of liver lesions including hepatic cells cloudy swelling, lymphocytes infiltration, hepatic necrosis, and fibrous connective tissue proliferation induced by CCl4 in rats. Therefore, the results of this study suggest that SNE could protect liver against the CCl4-induced oxidative damage in rats, and this hepatoprotective effect might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects.

Development of robust genotype 1a hepatitis C replicons harboring adaptive mutations for facilitating the antiviral drug discovery and study of virus replication.

The hepatitis C virus (HCV) subgenomic replicon is a valuable tool for studying virus replication and HCV drug development. Despite the fact that HCV genotype 1a (HCV1a) is the most prevalent genotype in the United States, few HCV1a reporter replicon constructs have been reported, and their replication capacities are not as efficient as those of HCV1b or 2a, especially in transient expression. In this study, we selected efficient HCV1a replicons and characterized the novel adaptive mutations derived from stable HCV1a (strain H77) replicon cells after G418 selection. These novel adaptive mutations were scored in NS3 (A1065V, C1073S, N1227D, D1431Y, and E1556G), NS4A (I1694T and E1709V), and NS4B (G1871C). The D1431Y mutation alone or combinations of other adaptive mutations introduced into the parental HCV1a replicon construct was observed to differentially enhance either transient or stable expression of replicon. In particular, two replicon mutants VDYG (A1065V, N1227D, D1431Y, and E1556G within NS3) and VDYGRG, VDYG with two additional adaptive mutations (NS4A-K1691R and NS4B-E1726G), displayed robust replication and exhibited no impairment in the susceptibility of replicon activity to various known HCV inhibitors.

Effect of Hibiscus anthocyanins-rich extract induces apoptosis of proliferating smooth muscle cell via activation of P38 MAPK and p53 pathway.

Hibiscus sabdariffa L. (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in Sudan and in eastern Taiwan. It has been reported to contain a number of protocatechuic acid and anthocyanins. In vitro experimental studies have shown that anthocyanins administration of the extract produces anti-inflammation and chemoprevention effects. In spite of the wide use of Hibiscus sabdariffa L. in folk medicine for treating various diseases, our previous study indicated a potency of Hibiscus sabdariffa extract (HSE) in anti-atherosclerosis. The mechanisms of anthocyanins administration of the extract produce from Hibiscus sabdariffa L. to attenuate atherosclerosis were not clarified. In this study, we found that Hibiscus anthocyanins (HAs) could inhibit the serum-stimulated proliferation of smooth muscle cell (SMC) and result in cell apoptosis. The HAs inducing cell apoptosis was dose dependent. We further used SB203580 (p38 inhibitor) to block cellular apoptosis and evaluate its effect on the HAs-inducing SMC death via some apoptosis criteria including DNA fragmentation and flow cytometry. We suggested that the mechanisms of the inhibitory effect of HAs on atherosclerosis could be via inhibiting the proliferation of SMC. HAs induces apoptosis via (i) activating p38 MAP kinase that subsequently phosphorylates target protein c-Jun and transduces the signal to further activate the apoptotic protein cascades that contain Fas-mediated signaling (Fas/caspase-8 signaling module) and (ii) activating p53 and inducing bax expression. As an outcome of the events, cytochrome c releases from the mitochondria, leading to cell apoptosis. In these experiments, HAs showed strong potential to induce SMC cell apoptosis via p38 and p53 pathway. In consequence, the rate of atherosclerotic formation is slowed down, and the progress is suppressed.

Induction of autophagy and apoptosis by the extract of Solanum nigrum Linn in HepG2 cells.

Solanum nigrum L. (SN) has been used in traditional folk medicine to treat different cancers. It is also used as a hepatoprotective and anti-inflammatory agent. In this study, we demonstrated that the extract of SN (SNE) induced a strong cytotoxic effect toward HepG2 cells but much less to Chang liver and WRL-68 cells. The mechanisms of the cytotoxic effect were concentration-dependent. High doses of SNE (2 and 5 mg/mL) induced apoptotic cell death in HepG2 cells, as evidenced by increases in the expressions of p-JNK and Bax, mitochodrial release of cytochrome c, and caspase activation. On the other hand, cells treated with low concentrations of SNE (50-1000 microg/mL) revealed morphological and ultrastructural changes of autophagocytic death under electron microscopic observation. Furthermore, these cells showed increased levels of autophagic vacuoles and LC3-I and LC3-II proteins, specific markers of autophagy. The levels of Bcl-2 and Akt that have been implicated in the down-regulation of autophagy were decreased upon SNE treatment. Taken together, these findings indicate that SNE induced cell death in hepatoma cells via two distinct antineoplastic activities of SNE, the ability to induce apoptosis and autophagocytosis, therefore suggesting that it may provide leverage to treat liver cancer.

Construction of a hydrazone-linked chiral covalent organic framework-silica composite as the stationary phase for high performance liquid chromatography.

Covalent organic frameworks (COFs), as an emerging class of crystalline porous organic polymers, have great potential for applications in chromatographic separation owning to their fascinating crystalline structures and outstanding properties. However, development of COF materials as novel stationary phases in high performance liquid chromatography (HPLC) is just in its infancy. Herein, we report the design and construction of a new hydrazone-linked chiral COF, termed BtaMth COF, from a chiral hydrazide building block (Mth) and present a one-pot synthetic method for the fabrication of BtaMth@SiO2 composite for HPLC separation of isomers. The as-synthesized BtaMth chiral COF displays good crystallinity, high porosity, as well as excellent chemical stability. Meanwhile, the fabricated HPLC column by using BtaMth@SiO2 composite as the new stationary phase exhibits high resolution performances for the separation of positional isomers including nitrotoluene and nitrochlorobenzene, as well as cis-trans isomers including beta-cypermethrin and metconazole. Additionally, some effects such as the composition of the mobile phase and column temperature for HPLC separations on the BtaMth@SiO2 packed column also have been studied in detail. The successful applications indicate the great potentials of hydrazone-linked chiral COF-silica composite as novel stationary phase for the efficient HPLC separation.

Characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus.

Dengue virus (DENV) is a public health threat to approximately 40% of the global population. At present, neither licensed vaccines nor effective therapies exist, and the mechanism of viral RNA replication is not well understood. Here, we report the development of efficient Renilla luciferase reporter-based DENV replicons that contain the full-length capsid sequence for transient and stable DENV RNA replication. A comparison of the transient and stable expression of this RNA-launched replicon to replicons containing various deletions revealed dengue replicon containing entire mature capsid RNA element has higher replicon activity. An efficient DNA-launched DENV replicon, pCMV-DV2Rep, containing a full-length capsid sequence, was created and successfully applied to evaluate the potency of known DENV inhibitors. Stable cell lines harboring the DENV replicon were easily established by transfecting pCMV-DV2Rep into BHK21 cells. Steady and high replicon reporter signals were observed in the stable DENV replicon cells, even after 30 passages. The stable DENV replicon cells were successfully used to determine the potency of known DENV inhibitors. A high-throughput screening assay based on stable DENV replicon cells was evaluated and shown to have an excellent Z' factor of 0.74. Altogether, the development of our efficient DENV replicon system will facilitate the study of virus replication and the discovery of antiviral compounds.

Low copy number and high 4977 deletion of mitochondrial DNA in uterosacral ligaments are associated with pelvic organ prolapse progression.

INTRODUCTION AND HYPOTHESIS: The pathophysiology of pelvic organ prolapse (POP) is related to aging in the pelvic organ support, and mitochondrial dysfunction is one of the major contributors to aging. Therefore, the objective of this study was to investigate the correlation between alternations of mitochondrial DNA and progression of POP. METHODS: Polymerase chain reaction (PCR) was applied in the present study. Uterosacral ligaments (UL) were obtained from 45 POP patients and 38 myoma patients without POP. Chi-square test, Student's t-test, Mann-Whitney U test, and Spearman correlation analysis were applied in the comparison between POP and non-POP patients. RESULTS: The results revealed that significant depletion of mitochondrial DNA (mtDNA) and an increase in the incidence of 4977 deletion of mtDNA (mtDNA(4977)) in the UL tissue of POP patients. CONCLUSIONS: The alternations of mtDNA may play an important role in the molecular pathogenesis and process of POP formation.