Evaluating shared decision making for dialysis initiation: A qualitative study on patient refusal of long-term dialysis in Taiwan.

RATIONALE: Previous studies have explored shared decision making (SDM) implementation to determine the renal replacement therapy modality; however, the SDM approach for dialysis initiation, especially when patients refuse physician suggestions for long-term dialysis, remains unclear. AIMS AND OBJECTIVES: This study aimed to explore physicians' responses towards patients' refusal of long-term dialysis during the SDM process and the thinking processes of both physicians and patients regarding dialysis refusal. METHOD: We conducted in-depth semi-structured interviews with 10 patients diagnosed with end-stage renal disease, each of whom refused long-term dialysis after physicians employed the SDM framework, and nine nephrologists at the Chang Gung Memorial Hospital, Taiwan, from March to May 2020. Interviews were audio-recorded, transcribed, and translated from Mandarin to English. They were then thematically analysed. RESULTS: Three main themes on dialysis initiation SDM implementation and the differences between physician and patient perceptions on patient treatment refusal were yielded. While the SDM approach for dialysis initiation developed by nephrologists in Taiwan respects patient decisions, physicians often actively persuade patients to undergo dialysis in case of treatment refusal. The motivation behind this approach is to promote the patient's best medical interests, particularly post-dialysis life quality, and to ensure a 'rational' medical decision is made. However, patients' perceptions of treatment refusal differ significantly from those of physicians, and their decision-making process is often iterative and based on comprehensive evaluation of immediate concerns beyond biomedical factors. CONCLUSIONS: Findings suggest that the current physician-led SDM approach for dialysis initiation characterises active persuasion with physicians' perspectives predominating the clinical encounter. To improve SDM implementation, we propose that physicians should acknowledge and understand patients' reasoning for dialysis refusal and the distinction between objective health and subjective well-being during the decision-making process.

Combined Effects of Tanshinone IIA and an Autophagy Inhibitor on the Apoptosis of Leukemia Cells via p53, Apoptosis-Related Proteins and Oxidative Stress Pathways.

BACKGROUND: Acute myeloid leukemia (AML) is a kind of hematopoietic malignancy with limited response and acquired resistance to therapy. Inducing apoptosis and inhibiting autophagy in tumor cells is a combinational strategy for the development of anticancer therapeutics. Tanshinone IIA (TAIIA) is one of the major ingredients in Salvia miltiorrhiza, which is the most prescribed herb for the treatment of AML in Taiwan. Therefore, this study aimed to delineate the anticancer effects of TAIIA and its effect when combined with an autophagy inhibitor to treat AML. METHODS: The anticancer effects of a combination of TAIIA and the autophagy inhibitor 3-methladenine (3MA) on the human monocytic leukemia cell line THP-1 were explored. The apoptosis and cell cycle of the leukemia cells were examined by Annexin V and propidium iodide staining and analyzed by flow cytometry. The oxidative stress level was determined by a malondialdehyde (MDA) colorimetric assay, nitric oxide colorimetric assay and glutathione peroxidase (GPx) colorimetric assay. The expression of apoptosis-related proteins was determined by western blotting. RESULTS: TAIIA treatment significantly induced apoptosis via increased p53, Bax/Bcl, PARP, and caspase-3 signals and oxidative stress by enhancing MDA and nitrate/nitrite production and reducing GPx activity in THP-1 cells in a dose-dependent and time-dependent manner. The combination of the autophagy inhibitor 3MA enhanced TAIIA-induced apoptosis via the p53, Bax/Bcl, PARP, caspase-3, and oxidative stress pathways in THP-1 cells. CONCLUSION: The results suggest that TAIIA and autophagy inhibitors have combined effects on the apoptosis of leukemia cells, thus representing a novel and effective combination with the potential for application as a clinical therapy for AML.

Development and validation of the dialysis dementia risk score: A retrospective, population-based, nested case-control study.

BACKGROUND: Dementia is prevalent and underdiagnosed in the dialysis population. We aimed to develop and validate a simple dialysis dementia scoring system to facilitate identification of individuals who are at high risk for dementia. METHODS: We applied a retrospective, nested case-control study design using a national dialysis cohort derived from the National Health Insurance Research Database in Taiwan. Patients aged between 40 and 80 years were included and 2940 patients with incident dementia were matched to 29,248 non-dementia controls. All subjects were randomly divided into the derivation and validation sets with a ratio of 4:1. Conditional logistic regression models were used to identify factors contributing to the risk score. The cutoff value of the risk score was determined by Youden's J statistic and the graphic method. RESULTS: The dialysis dementia risk score (DDRS) finally included age and 10 comorbidities as risk predictors. The C-statistic of the model was 0.71 (95% confidence interval [CI] 0.70-0.72). Calibration revealed a strong linear relationship between predicted and observed dementia risk (R2  = 0.99). At a cutoff value of 50 points, the high-risk patients had an approximately three-fold increased risk of having dementia compared to those with low risk (odds ratio [OR] 3.03, 95% CI 2.78-3.31). The DDRS performance, including discrimination (C-statistic 0.71, 95% CI 0.69-0.73) and calibration (p value of Hosmer-Lemeshow test for goodness of fit = 0.18), was acceptable during validation. The OR value (2.82, 95% CI 2.37-3.35) was similar to those in the derivation set. CONCLUSION: The DDRS system has the potential to serve as an easily accessible screening tool to determine the high-risk groups who deserve subsequent neurological evaluation in daily clinical practice.

Molecular insights into substrate recognition and discrimination by the N-terminal domain of Lon AAA+ protease.

The Lon AAA+ protease (LonA) is a ubiquitous ATP-dependent proteolytic machine, which selectively degrades damaged proteins or native proteins carrying exposed motifs (degrons). Here we characterize the structural basis for substrate recognition and discrimination by the N-terminal domain (NTD) of LonA. The results reveal that the six NTDs are attached to the hexameric LonA chamber by flexible linkers such that the formers tumble independently of the latter. Further spectral analyses show that the NTD selectively interacts with unfolded proteins, protein aggregates, and degron-tagged proteins by two hydrophobic patches of its N-lobe, but not intrinsically disordered substrate, α-casein. Moreover, the NTD selectively binds to protein substrates when they are thermally induced to adopt unfolded conformations. Collectively, our findings demonstrate that NTDs enable LonA to perform protein quality control to selectively degrade proteins in damaged states and suggest that substrate discrimination and selective degradation by LonA are mediated by multiple NTD interactions.

There are many different types of protein which each have different roles in biology. Most proteins are surrounded by water and are folded so that their water-attracting regions are on the outside and more fat-like regions, which repel water, are on the inside. When a protein becomes damaged or is assembled incorrectly, some of the fat-like regions end up on the outside of the protein and become exposed to water. This can prevent the protein from performing its role and harm the cell instead. LonA proteases are responsible for dismantling and recycling these harmful proteins, as well as proteins that have been labelled for destruction. They do this by unfolding the unwanted protein and transporting it into an enclosed chamber made of six LonA molecules. Once inside the chamber, the target protein is broken down into smaller fragments that can be used to build other structures. LonA proteases contain a region called the N-terminal domain, or NTD for short, which is thought to be responsible for identifying which proteins need degrading. Yet it remained unclear how the NTD recognizes and binds to these target proteins. To answer this question, Tzeng et al. studied the detailed structure of a LonA protease that had been purified from bacteria cells. This revealed that the NTD of LonA contains two water-repelling regions which bind to fat-like segments on the surface of proteins that have become unfolded or tagged for destruction. Further experiments showed that the NTD is bound to the main body of LonA via a 'flexible linker'. This led Tzeng et al. to propose that the NTD sways around loosely at the end of LonA searching for proteins with exposed water-repelling regions. Once an NTD identifies and attaches to a target, the NTDs of the other LonA molecules then bind to the protein and help insert it into the chamber. Proteases are a vital component of all biological systems. Controlling protein destruction and recycling is a key factor in how cells divide and respond to a changing environment. This study provides new insights into how LonA operates in bacteria, which may apply to proteases more widely. This contributes to our knowledge of fundamental biology and may also be relevant in a range of diseases where protein recycling is defective or inefficient.

Diterpenoid anthraquinones as chemopreventive agents altered microRNA and transcriptome expressions in cancer cells.

OBJECTIVE: Cryptotanshinone (CPT) and dihydrotanshinone (DHT) are diterpenoid anthraquinone compounds extracted from traditional Chinese herbal medicine (TCM). Recent studies have shown that CPT regulates the signal transduction pathways via microRNA (miRNA) alterations. However, few studies have investigated the role of DHT in miRNA alterations affecting cell-signaling pathways. This study aimed to investigate the miRNA alterations and post-transcriptional regulation activities of DHT in comparison to CPT. METHODS: HepG2 and HT-29 cells were treated with DHT or CPT for 72 h. MiRNA, transcription factor encoding mRNA, and downstream gene expression were determined using real-time quantitative PCR. Protein expression was analyzed using western blotting. RESULTS: The results revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via miR-15a-5p, miR-27a-5p, miR-100-5p, and miR-200a-5p alterations.In silico target predictions showed that downregulation of epidermal growth factor receptor (EGFR) mRNA expression by DHT might also suppress the expression of STAT family proteins and lead to anti-proliferation effects. We also found that, compared to CPT, DHT might possess higher potency in cell growth regulation via multi-miRNA and transcription factor alterations. CONCLUSION: This study revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via alterations in miRNAs and transcription factors. In addition, the findings of this study suggest that DHT is more potent than CPT in cancer chemopreventive activities. Therefore, DHT at a low dose is a TCM compound with less toxic side effects and may contribute to the development of natural medicine as a potential cancer chemopreventive agent.

Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy.

BACKGROUND: Ligamentum flavum hypertrophy (LFH) is among the most crucial factors in degenerative lumbar spinal stenosis, which can cause back pain, lower extremity pain, cauda equina syndrome and neurogenic claudication. The exact pathogenesis of LFH remains elusive despite extensive research. Most in vitro studies investigating LFH have been carried out using conventional two-dimensional (2D) cell cultures, which do not resemble in vivo conditions, as they lack crucial pathophysiological factors found in three-dimensional (3D) LFH tissue, such as enhanced cell proliferation and cell cluster formation. In this study, we generated ligamentum flavum (LF) clusters using spheroid cultures derived from primary LFH tissue. RESULTS: The cultured LF spheroids exhibited good viability and growth on an ultra-low attachment 96-well plate (ULA 96-plate) platform according to live/dead staining. Our results showed that the 100-cell culture continued to grow in size, while the 1000-cell culture maintained its size, and the 5000-cell culture exhibited a decreasing trend in size as the culture time increased; long-term culture was validated for at least 28 days. The LF spheroids also maintained the extracellular matrix (ECM) phenotype, i.e., fibronectin, elastin, and collagen I and III. The 2D culture and 3D culture were further compared by cell cycle and Western blot analyses. Finally, we utilized hematoxylin and eosin (H&E) staining to demonstrate that the 3D spheroids resembled part of the cell arrangement in LF hypertrophic tissue. CONCLUSIONS: The developed LF spheroid model has great potential, as it provides a stable culture platform in a 3D model that can further improve our understanding of the pathogenesis of LFH and has applications in future studies.

A comparison of the risk of acute myocardial infarction in patients receiving hemodialysis and peritoneal dialysis: A population-based, propensity score-matched cohort study.

BACKGROUND AND AIMS: Acute myocardial infarction (AMI) remains the major cause of morbidity and mortality in the dialysis population. Traditional cardiovascular (CV) risk factors are unable to fully account for the high incidence of AMI in the dialysis population. In this study, we investigated whether dialysis modalities could be one of the uremia-specific risk factors for AMI. METHODS: Using the National Health Insurance Research Database, we recruited all incident dialysis patients from the period January 1, 1998 to December 31, 2010. The propensity score matching method was applied to form the matched pairs of hemodialysis (HD) and peritoneal dialysis (PD) patients. Incidence rate (IR), cumulative incidence rate (CIR) and multivariable subdistribution hazards models were employed to compare the risk of AMI in the HD and PD groups. RESULTS: Of the 86,215 incident dialysis patients, 5,513 matched pairs of HD and PD patients were identified. The HD patients had a higher IR of AMI than the PD patients (9.71 vs. 8.35 per 1000 patient-years, respectively, p = 0.01). The CIR was also higher in the HD patients than in the PD patients (0.09 vs. 0.05), especially 4 years after dialysis therapy was initiated (p = 0.04). In the subdistribution hazards models, HD was still significantly associated with a higher risk of developing AMI (adjusted hazard ratio:1.30, 95% confidence interval:1.02-1.65). The results remained unchanged in various stratifications as well as in the analysis of the unmatched cohorts. CONCLUSIONS: Compared to PD, HD was significantly associated with higher risk of developing AMI, especially after 4 years since dialysis was initiated. Prevention and routine surveillance programs for AMI should be individualized according to dialysis modalities and vintage.

A comparison of the risk of congestive heart failure-related hospitalizations in patients receiving hemodialysis and peritoneal dialysis - A retrospective propensity score-matched study.

INTRODUCTION: Congestive heart failure (CHF) is associated with high mortality and a heavy financial and healthcare burden in the dialysis population. Determining which dialysis modality is associated with a higher risk of developing CHF might facilitate clinical decision making and surveillance programs in the dialysis population. METHODS: Using the Taiwan National Health Insurance Database, we recruited all incident dialysis patients during the period from January 1, 1998 to December 31, 2010. The propensity score matching method was applied to establish the matched hemodialysis (HD) and peritoneal dialysis (PD) cohort. Incidence rates and cumulative incidence rates of CHF-related hospitalization were first compared for the HD and PD patients. Multivariable subdistribution hazards models were then constructed to control for potential confounders. RESULTS: Among a total of 65,899 enrolled dialysis patients, 4,754 matched pairs of HD and PD patients were identified. The incidence rates of CHF in the matched HD and PD patients were 25.98 and 19.71 per 1000 patient-years, respectively (P = 0.001). The cumulative incidence rate of CHF was also higher in the matched HD patients (0.16, 95% confidence interval (CI)(0.12-0.21)] than in the corresponding PD patients (0.09, 95% CI [0.08-0.11])(P<0.0001). HD was consistently associated with an increased subdistribution hazard ratio (HR) of CHF compared with PD in the matched cohort (HR: 1.45, 95% CI [1.23-1.7]). Similar phenomenons were observed in either the subgroup analysis stratified by selected confounders or in the HD and PD group without matching. CONCLUSIONS: HD is associated with a higher risk of developing CHF-related hospitalization than PD. The surveillance program for CHF should differ in patients receiving different dialysis modalities.

Risk of dementia in patients with end-stage renal disease under maintenance dialysis-a nationwide population-based study with consideration of competing risk of mortality.

BACKGROUND: Dementia is prevalent in the end-stage renal disease (ESRD) population. However, it is still not clarified whether ESRD is one of the etiology of dementia or its attributable effect on the cumulative risk of dementia. Meanwhile, the effect of competing risk of mortality should be taken into consideration when performing epidemiologic analyses among populations with high risk of mortality. METHODS: By using the National Health Insurance Research Database (1998-2010), we identified 927,142 non-ESRD individuals and 99,158 ESRD patients to investigate the effect of ESRD on the risk of dementia. Age- and sex-specific incidence rates (IRs) and cumulative incidence rates (CIRs) were first compared between these two cohorts. Competing risk analyses including cause-specific and subdistribution proportional hazards models were then constructed with adjustments for potential confounders. RESULTS: The overall IR and CIR of dementia were much higher in the ESRD group than in the non-ESRD group (10.73 vs. 1.40 per 1000 person-years and 0.061 vs. 0.017, respectively, both P < 0.0001). Results from the multivariable cause-specific hazard models suggested that ESRD was one of the etiological factors for dementia (cause-specific hazard ratio [csHR] : 2.06 [95% CI : 1.95-2.17]). However, the subdistribution HR (sdHR) of ESRD was 0.51 (95% Cl : 0.49-0.54), which indicated the lower cumulative incidence risk of dementia in ESRD patients. The inverse relationship between csHR and sdHR could be explained by the high mortality rate in the ESRD population. These findings were also essentially consistent across various subgroup analyses according to selected confounders, as well as in the analyses that limited dementia diagnoses made by neurologists or psychologists. CONCLUSIONS: Although ESRD appears directly associated with the risk of dementia, the high competing mortality means that primary prevention of comorbidity associated with dementia may be more effective in reducing overall dementia in the general population, which may also potentially reduce the incidence of ESRD and prevent death from multimorbidity when affected by ESRD.

Age and sex differences in the relationship between neutrophil-to-lymphocyte ratio and chronic kidney disease among an adult population in Taiwan.

BACKGROUND: This study investigated the association between systemic inflammation and chronic kidney disease (CKD), and whether this association changes with aging in adults, by using neutrophil-to-lymphocyte ratio (NLR) as an inflammation marker. MATERIALS AND METHODS: A total of 2954 adults (1815 men and 1139 women) who attended a health examination at a medical center in central Taiwan were included for the final cross-sectional analysis. RESULTS: Compared with participants aged <60 years, participants aged ≥60 years had a markedly higher prevalence rate of CKD in both men (7.6% vs. 37.8%, p < .001) and women (3.8% vs. 28.0%, p < .001). In men aged <60 years, multivariable logistic regression analysis revealed that, after adjusting for conventional CKD risk factors, higher NLR (per 1 unit increment) was independently associated with higher risk of CKD [adjusted OR = 1.48 (95% C.I.: 1.10 to 1.99, p = .009)]. There was no such association in both men and women aged ≧60 years, and woman aged <60 years. CONCLUSIONS: Our study showed a differential effect that aging has on the relationship between NLR and CKD in men but not in women. Being inexpensive and readily available, NLR may potentially be used for CKD risk assessment in men younger than 60 years of age.

Complementary Chinese Herbal Medicine Therapy Improves Survival of Patients With Pancreatic Cancer in Taiwan: A Nationwide Population-Based Cohort Study.

BACKGROUND: Pancreatic cancer is a difficult-to-treat cancer with a late presentation and poor prognosis. Some patients seek traditional Chinese medicine (TCM) consultation. We aimed to investigate the benefits of complementary Chinese herbal medicine (CHM) among patients with pancreatic cancer in Taiwan. METHODS: We included all patients with pancreatic cancer who were registered in the Taiwanese Registry for Catastrophic Illness Patients Database between 1997 and 2010. We used 1:1 frequency matching by age, sex, the initial diagnostic year of pancreatic cancer, and index year to enroll 386 CHM users and 386 non-CHM users. A Cox regression model was used to compare the hazard ratios (HRs) of the risk of mortality. The Kaplan-Meier curve was used to compare the difference in survival time. RESULTS: According to the Cox hazard ratio model mutually adjusted for CHM use, age, sex, urbanization level, comorbidity, and treatments, we found that CHM users had a lower hazard ratio of mortality risk (adjusted HR = 0.67, 95% CI = 0.56-0.79). Those who received CHM therapy for more than 90 days had significantly lower hazard ratios of mortality risk than non-CHM users (90- to 180-day group: adjusted HR = 0.56, 95% CI = 0.42-0.75; >180-day group: HR = 0.33, 95% CI = 0.24-0.45). The survival probability was higher for patients in the CHM group. Bai-hua-she-she-cao (Herba Oldenlandiae; Hedyotis diffusa Spreng) and Xiang-sha-liu-jun-zi-tang (Costus and Chinese Amomum Combination) were the most commonly used single herb and Chinese herbal formula, respectively. CONCLUSIONS: Complementary Chinese herbal therapy might be associated with reduced mortality among patients with pancreatic cancer. Further prospective clinical trial is warranted.

Use of Complementary Traditional Chinese Medicines by Adult Cancer Patients in Taiwan: A Nationwide Population-Based Study.

BACKGROUND: Many patients with cancer seek complementary and alternative medicine treatments. We investigated the use of traditional Chinese medicine (TCM) by adult cancer patients in Taiwan. METHODS: We reviewed the Registry for Catastrophic Illness Patients Database of Taiwan, and included all adult patients diagnosed cancer, based on the International Classification of Diseases (ninth revision), from 2001 to 2009 and followed until 2011. This database allowed categorization of patients as TCM users (n = 74 620) or non-TCM users (n = 508 179). All demographic and clinical claims data were analyzed. RESULTS: Compared with non-TCM users, TCM users were younger and more likely to be female, white-collar workers, and reside in highly urbanized areas. The average interval between cancer diagnosis and TCM consultation was 15.3 months. The most common cancer type was breast cancer in TCM users (19.4%), and intrahepatic bile duct cancer in non-TCM users (13.6%). The major condition for which TCM users visited clinics were endocrine, nutritional and metabolic diseases, and immunity disorders (23.2%). A total of 33.1% of TCM users visited TCM clinics more than 9 times per year and their time from diagnosis to first TCM consultation was 5.14 months. The most common TCM treatment was Chinese herbal medicine. The common diseases for which cancer patients sought TCM treatment were insomnia, malaise and fatigue, dizziness and headache, gastrointestinal disorders, myalgia and fasciitis, anxiety, and depression. Overall, TCM users had a lower adjusted hazard ratio (aHR) for mortality (aHR = 0.69, 95% CI = 0.68-0.70) after adjustment for age, sex, urbanization of residence, occupation, annual medical center visits, and annual non-medical center visits. CONCLUSIONS: This study provides an overview of TCM usage among adult cancer patients in Taiwan. TCM use varied among patients with different types of cancer. Physicians caring for cancer patients should pay more attention to their patients' use of complementary TCM.

Structural basis of adaptor-mediated protein degradation by the tail-specific PDZ-protease Prc.

Peptidoglycan (PG) is a highly cross-linked, protective mesh-like sacculus that surrounds the bacterial cytoplasmic membrane. Expansion of PG is tightly coupled to growth of a bacterial cell and requires hydrolases to cleave the cross-links for insertion of nascent PG material. In Escherichia coli, a proteolytic system comprising the periplasmic PDZ-protease Prc and the lipoprotein adaptor NlpI contributes to PG enlargement by regulating cellular levels of MepS, a cross-link-specific hydrolase. Here, we demonstrate how NlpI binds Prc to facilitate the degradation of its substrate MepS by structural and mutational analyses. An NlpI homodimer binds two molecules of Prc and forms three-sided MepS-docking cradles using its tetratricopeptide repeats. Prc forms a monomeric bowl-shaped structure with a lid-like PDZ domain connected by a substrate-sensing hinge that recognizes the bound C terminus of the substrate. In summary, our study reveals mechanistic details of protein degradation by the PDZ-protease Prc bound to its cognate adaptor protein.

Complementary Chinese herbal medicine therapy improves survival of patients with gastric cancer in Taiwan: A nationwide retrospective matched-cohort study.

ETHNOPHARMACOLOGICAL RELEVANCE: Many patients with gastric cancer seek traditional medicine consultations in Asian countries. This study aimed to investigate the prescription of Chinese herbal medicine (CHM) and its benefits for the patients with gastric cancer in Taiwan. METHODS: From the Registry for Catastrophic Illness Patients Database, we included all patients with gastric cancer whose age at diagnosis was ≥18 from 1997 to 2010 in Taiwan. We used 1:1 frequency matching by age, sex, Charlson comorbidity score, treatment and index year to compare the CHM users and non-CHM users. We used the Cox regression model to compare the hazard ratios (HR) for the risk of mortality and the Kaplan-Meier curve for the survival time. RESULTS: There was a total of 1333 patients in the CHM-cohort and 44786 patients in the non-CHM cohort. After matching, we compared 962 newly diagnosed CHM users and 962 non-CHM users. Adjusted HRs (aHR) were higher among patients of above 60-year-old group, with a Charlson Comorbidity Index score ≥2 before the index date, and those who need surgery combined with chemotherapy or radiotherapy. CHM users had a lower HR of mortality risk (adjusted HR: 0.55, 95% CI: 0.48-0.62). Compared to the non-CHM users, the aHR among CHM-users is 0.37 (95% CI:0.2-0.67) for those who used CHM more than 180 days annually. The Kaplan-Meier curve revealed that the survival probability was higher for complementary CHM-users. Bai-Hua-She-She-Cao (Herba Hedyotidis Diffusae) was the most commonly used single herb and Xiang-Sha-Liu-Jun-Zi-Tang was the most commonly used herbal formula among CHM prescriptions. CONCLUSIONS: Complementary CHM improves the overall survival among patients with gastric cancer in Taiwan. Further ethnopharmacological investigations and clinical trials are required to validate the efficacy and safety.

The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.

Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

Color Doppler evaluation of blood flow changes in leiomyomas after uterine artery ligation.

OBJECTIVE: To detect possible changes in main blood vessels within leiomyomas after uterine artery ligation using color Doppler sonography. METHOD: Blood flow in main leiomyoma blood vessels was measured before and after the procedure in 14 women who also had abnormal uterine bleeding, pelvic pain or pressure, and/or anemia. RESULTS: Of the 14 patients, 13 reported complete disappearance of preoperative pain or pressure and 1 reported significant relief. Within 1 week to 4 months after uterine artery ligation, major blood flow within leiomyomas had significantly decreased in all patients. Eight months after the procedure, 1 of the women became pregnant. CONCLUSION: Laparoscopic uterine artery ligation via a lateral retroperitoneal technique is a safe and effective treatment for leiomyomas. Color Doppler sonography verified the ability of the procedure to diminish blood flow within leiomyomas in all patients.