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In this study, we demonstrated that the choice of precursor has a strong effect on the reduction of nitrate (NO 3 - ) using zero-valent copper (Cu 0 ) synthesized by sodium borohydride (NaBH 4 ). Different precursors: CuSO 4 , CuO, Cu 2 O, Cu powder, and Cu mesh were used to reduce NO 3 - at 677 mg-N/L under the reducing conditions of NaBH 4 . Compared with the prehydrolyzed samples, those prepared without prehydrolysis exhibited lower reduction rates, longer times and higher concentrations of nitrite (NO 2 - ) intermediate. It was found that one-time addition of NaBH 4 resulted in higher reduction rate and less NO 2 - intermediate than two-step addition. Results showed that Cu 0 from CuSO 4 possessed the smallest particle size (890.9 nm), highest surface area (26.0 m 2 /g), and highest reaction rate (0.166 min -1 ). Values of pseudo-first-order constant (k obs ) were in the order: CuSO 4 > CuO > Cu 2 O > Cu powder >Cu mesh. However, values of surface area-normalized reaction rate (k SA ) were approximately equal. It was proposed that NO 3 - was reduced to NO 2 - on Cu 0 , and then converted to NH 4 + and N 2 , respectively; H 2 generated from both NaBH 4 hydration and Cu (II) reduction contributed to NO 3 - reduction as well.

Purpose/objectives: To describe the presence, frequency, severity, and distress of symptoms in outpatients with advanced hepatocellular carcinoma toward the end of life, and the variability in psychological and physical symptom distress between and within patients over time. 
., Design: A prospective, longitudinal, descriptive design. 
., Setting: Outpatient clinics at two healthcare institutions. 
., Sample: 18 patients (15 men and 3 women) with hepatocellular carcinoma and a mean age of 63.3 years (range = 54-81 years).
., Methods: Data were collected monthly for six months. Patients completed the Memorial Symptom Assessment Scale, which reports a total score, and three subscales that provide global distress, psychological distress, and physical distress scores.
., Main Research Variables: Global, psychological, and physical distress.
., Findings: Patients reported lack of energy and pain as the most frequent and distressing symptoms. Problems with sexual interest or activity was the fourth most present symptom after drowsiness. Global Distress Index mean scores had notable variability between and within patients over time. During data collection, six patients died. None were referred to palliative care.
., Conclusions: Gaining knowledge about symptom distress and prevalent symptoms experienced by patients with advanced hepatocellular carcinoma is critical for designing symptom management strategies that are comprehensive and tailored to patients to optimize their quality of life as they approach death.
., Implications for Nursing: Nurses play a vital role in advocating for, initiating, and providing comprehensive holistic care based on individual patient needs by facilitating discussions about apparent and less apparent distressing symptoms, including those related to sexuality.

Over half a million Americans are affected by cirrhosis, the cause of end-stage liver disease (ESLD). Little is known about how symptom burden changes over time in adults with ESLD and their informal caregivers, which limits our ability to develop palliative care interventions that can optimize symptom management and quality of life in different patient-caregiver dyads. The purpose of this article is to describe the background and design of a prospective, longitudinal descriptive study, "Symptom Burden in End-Stage Liver Disease Patient-Caregiver Dyads," which is currently in progress. The study is designed to (i) identify trajectories of change in physical and psychological symptom burden in adults with ESLD; (ii) identify trajectories of change in physical and psychological symptom burden in caregivers of adults with ESLD; and (iii) determine predictors of types of patient-caregiver dyads that would benefit from tailored palliative care interventions. We aim for a final sample of 200 patients and 200 caregivers who will be followed over 12 months. Integrated multilevel and latent growth mixture modeling will be used to identify trajectories of change in symptom burden, linking those changes to clinical events, and quality of life outcomes and characterizing types of patient-caregiver dyads based on patient-, caregiver-, and dyad-level factors. Challenges we have encountered include unexpected attrition of study participants, participants not returning their baseline questionnaires, and hiring and training of research staff. The study will lay the foundation for future research and innovation in ESLD, end-of-life and palliative care, and caregiving., (© 2017 Wiley Periodicals, Inc.)

Background: Population-based data for diabetic hand syndrome (DHS) are limited. The aim of the present epidemiological study was to estimate the overall and cause-specific prevalence and rate ratio (RR) of DHS in patients with diabetes., Methods: The present study was a cross-sectional study based on a random sample of 57 093 diabetics and matched controls, both identified from Taiwan National Health Insurance claims in 2010. The DHS analyzed in the present study included carpal tunnel syndrome (CTS), stenosing flexor tenosynovitis (SFT), limited joint mobility (LJM), and Dupuytren's disease (DD)., Results: The prevalence of overall DHS was estimated at 2472 per 10 5 for the diabetics, compared with 1641 per 10 5 for the controls, representing a prevalence RR of 1.51 (95 % confidence interval [CI] 1.39-1.64). Stratified analyses further revealed that the significantly increased prevalence of overall DHS was more evident in females than males (1.59 vs 1.36) and was only noted in diabetics aged ≥35 years. Cause-specific analysis suggested that patients with diabetes had the highest prevalence of CTS (1244 per 10 5 ), followed by SFT (1209 per 10 5 ), LJM (39 per 10 5 ), and DD (6 per 10 5 ). In addition, diabetes was only significantly associated with CTS (RR 1.34; 95 % CI 1.20-1.51) and SFT (1.74; 95 % CI 1.54-1.97)., Conclusion: The prevalence of overall and certain cause-specific DHS was significantly elevated in patients with diabetes in Taiwan., (© 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

Background: The Department of Veterans Affairs is the largest US provider of hepatitis C treatment. Although antiviral regimens are becoming simpler, hepatitis C antivirals are not typically prescribed by primary care providers. The Veterans Affairs Extension for Community Health Outcomes (VA-ECHO) program was launched to promote primary care-based hepatitis C treatment using videoconferencing-based specialist support. We aimed to assess whether primary care provider participation in VA-ECHO was associated with hepatitis C treatment and sustained virologic response., Methods: We identified 4173 primary care providers (n = 152 sites) responsible for 38,753 patients with chronic hepatitis C infection. A total of 6431 patients had a primary care provider participating in VA-ECHO; 32,322 patients had an unexposed primary care provider. Exposure was modeled as a patient-level time-varying covariate. Patients became exposed after primary care provider participation in ≥1 VA-ECHO session. Multivariable Cox proportional hazards frailty modeling assessed the association between VA-ECHO exposure and hepatitis C treatment. Among treated patients, modified Poisson regression assessed the relationship between exposure and sustained virologic response., Results: After adjustment, exposed patients received significantly higher rates of antiviral treatment compared with unexposed patients (adjusted hazard ratio, 1.20; 95% confidence interval, 1.10-1.32; P <.01). The rate of primary care provider-initiated antiviral medication was 21.4% among treated patients reviewed on VA-ECHO teleconferences compared with 2.5% among unexposed patients (P <.01). No difference in adjusted rates of sustained virologic response was observed for patients with exposed primary care providers (P = .32), with similar crude rates for primary care providers versus specialists., Conclusions: National implementation of VA-ECHO was positively associated with hepatitis C treatment initiation by primary care providers, without differences in sustained virologic response., (Published by Elsevier Inc.)

Rationale: Primary care providers (PCPs) frequently encounter sleep complaints, especially in regions with limited specialty care access., Objectives: The U.S. Department of Veterans Affairs Extension for Community Healthcare Outcomes (VA-ECHO) program (based on Project ECHO) has successfully provided rural PCP education in subspecialty areas, including hepatitis C. We describe the feasibility of an ECHO program for sleep medicine., Methods: ECHO creates a virtual learning community through video-teleconferencing, combining didactics with individualized clinical case review. We invited multidisciplinary providers to attend up to 10 stand-alone, 1-hour sessions. Invitees completed a needs assessment, which guided curriculum development. After program completion, we examined participant characteristics and self-reported changes in practice and comfort with managing sleep complaints. We surveyed participation barriers among invitees with low/no attendance., Measurements and Main Results: Of the 39 program participants, 38% worked in rural healthcare. Participants included nurse practitioners (26%), registered nurses (21%), and physicians (15%). Seventeen (44%) completed the summative program evaluation. Respondents anticipated practice change from the program, especially in patient education about sleep disorders (93% of respondents). Respondents reported improved comfort managing sleep complaints, especially sleep-disordered breathing, insomnia, and sleep in post-traumatic stress disorder (80% of respondents each). A follow-up survey of program invitees who attended zero to two sessions reported scheduling conflicts (62%) and lack of protected time (52%) as major participation barriers., Conclusions: Participants in a pilot sleep medicine VA-ECHO program report practice change and increased comfort managing common sleep complaints. Future work is needed to identify objective measures of return on investment and address participation barriers.

Considering the important role of metal ions including copper ions are playing in human body, a novel single-Trp peptide WDAHSS was designed and synthesized in this study to achieve sensitive detection of copper ions via fluorescence spectroscopy. The intrinsic fluorescence of a tryptophan residue in WDAHSS, which was the only source of the molecular fluorescence, could be easily quenched with copper ions. By comparing fluorescence spectra of WDAHSS with those of tryptophan molecules at different pH values, the quenching mechanism of WDAHSS was explored in detail. Research showed that the histidine in WDAHSS bound copper ions with metal coordination. With participation of peptide bond, a square planar structure was formed. It was a consequent chelation of copper ions that caused the quenching of tryptophan residue. At the same time, this study discussed how pH conditions affected the fluorescence spectra of WDAHSS. Furthermore, association constants of copper ions towards WDAHSS were calculated through fluorescence measurements and fitting analyses. To enhance the anti-jamming ability to pH variation, the amino terminal of WDAHSS was intentionally acetylized, leading to a stable fluorescence emission under physiological pH conditions. Besides, WDAHSS was designed as a special structure to enhance the selectivity and biocompatibility of its sensitive detection of copper ions. Further studies on WDAHSS may help to improve the fluorescence imaging detection in vivo.

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro 205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: The purpose of this study was to understand the situation of diabetes patients receiving examinations for diabetes complications and to explore the factors influencing their intention to receive examinations for diabetes complications., Methods: A cross-sectional study was performed that included 251 diabetes patients who visited outpatient clinics in Southern Taiwan. A survey using a self-administered questionnaire was conducted from October 2015 to January 2016. The questionnaire included items on demographic characteristics, perceived susceptibility to diabetes complications, perceived seriousness of diabetes complications, perceived benefits of taking action to receive diabetes complication examinations, perceived barriers to taking action to receive diabetes complication examinations, and the intention to receive diabetes complication examinations. The data were analyzed using regression analysis., Results: The percentage of participants who received fundus, foot, and kidney examinations was 67.7%, 61.4%, and 73.3%, respectively. Every point increase on the perceived barriers to taking action to receive diabetes complication examinations scale increased the intention to receive a foot examination in the following year by 0.91 times (p=.002), and every point increase on the perceived susceptibility to diabetes complications scale increased the intention to receive a kidney examination in the following year by 1.19 times (p=.045)., Conclusions: Nurses should shoulder the responsibility to increase patients' intention to receive examination of diabetes complications. The results of this study can be used to promote nurses' care efficacy in preventing diabetes complications. They can also provide medical institutions with information to establish prevention and control policies for diabetes complications., (Copyright © 2016. Published by Elsevier B.V.)

Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection.

Background & Aims: We investigated the real-world effectiveness of sofosbuvir, ledipasvir/sofosbuvir, and paritaprevir/ritonavir/ombitasvir and dasabuvir (PrOD) in treatment of different subgroups of patients infected with hepatitis C virus (HCV) genotypes 1, 2, 3, or 4., Methods: We performed a retrospective analysis of data from 17,487 patients with HCV infection (13,974 with HCV genotype 1; 2131 with genotype 2; 1237 with genotype 3; and 135 with genotype 4) who began treatment with sofosbuvir (n = 2986), ledipasvir/sofosbuvir (n = 11,327), or PrOD (n = 3174), with or without ribavirin, from January 1, 2014 through June 20, 2015 in the Veterans Affairs health care system. Data through April 15, 2016 were analyzed to assess completion of treatments and sustained virologic response 12 weeks after treatment (SVR12). Mean age of patients was 61 ± 7 years, 97% were male, 52% were non-Hispanic white, 29% were non-Hispanic black, 32% had a diagnosis of cirrhosis (9.9% with decompensated cirrhosis), 36% had a Fibrosis-4 index score >3.25 (indicator of cirrhosis), and 29% had received prior antiviral treatment., Results: An SVR12 was achieved by 92.8% (95% confidence interval [CI], 92.3%-93.2%) of subjects with HCV genotype 1 infection (no significant difference between ledipasvir/sofosbuvir and PrOD regimens), 86.2% (95% CI, 84.6%-87.7%) of those with genotype 2 infection (treated with sofosbuvir and ribavirin), 74.8% (95% CI, 72.2%-77.3%) of those with genotype 3 infection (77.9% in patients given ledipasvir/sofosbuvir plus ribavirin, 87.0% in patients given sofosbuvir and pegylated-interferon plus ribavirin, and 70.6% of patients given sofosbuvir plus ribavirin), and 89.6% (95% CI 82.8%-93.9%) of those with genotype 4 infection. Among patients with cirrhosis, 90.6% of patients with HCV genotype 1, 77.3% with HCV genotype 2, 65.7% with HCV genotype 3, and 83.9% with HCV genotype 4 achieved an SVR12. Among previously treated patients, 92.6% with genotype 1; 80.2% with genotype 2; 69.2% with genotype 3; and 93.5% with genotype 4 achieved SVR12. Among treatment-naive patients, 92.8% with genotype 1; 88.0% with genotype 2; 77.5% with genotype 3; and 88.3% with genotype 4 achieved SVR12. Eight-week regimens of ledipasvir/sofosbuvir produced an SVR12 in 94.3% of eligible patients with HCV genotype 1 infection; this regimen was underused., Conclusions: High proportions of patients with HCV infections genotypes 1-4 (ranging from 75% to 93%) in the Veterans Affairs national health care system achieved SVR12, approaching the results reported in clinical trials, especially in patients with genotype 1 infection. An 8-week regimen of ledipasvir/sofosbuvir is effective for eligible patients with HCV genotype 1 infection and could reduce costs. There is substantial room for improvement in SVRs among persons with cirrhosis and genotype 2 or 3 infections., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

In an intensity-modulation direct-detection (IMDD) orthogonal frequency division multiplexing (OFDM) signal format, radio frequency (RF) power fading produces non-uniform receiving performance among the subcarriers due to chromatic dispersion. Hence each subscriber experiences distinct quality of service (QoS) in conventional frequency division multiple access (FDMA). In this paper, a multicarrier code division multiple access (MC-CDMA) signal along with multicode interference (MCI) cancellation process is proposed and experimentally demonstrated to enhance the receiving performance in an IMDD long reach passive optical network (LR-PON). With the proposed scheme, the receiving performance of each subcarrier can be equalized so that a universal transmitter design at central office (CO) can support all the subscribers with their locations ranging from back-to-back to 100 km. Our experimental results also reveal that, even under 20 dB RF power fading, the proposed scheme can still provide over 21.7 dB power budget and only approximately 2 dB sensitivity deviation is observed in an IMDD LR-PON system.

Background: Primary care providers who participate in structured specialty telemedicine mentorship report improvements in clinical content mastery, professional satisfaction, and specialist communication., Introduction: Although these programs require investments of infrastructure resources and time, the duration of participation required to accrue optimal benefits is not known. We aimed to assess whether duration of participation is related to improved benefits of a longitudinal telemedicine-based mentorship program, specifically regarding perceived specialty care access, acquisition of new knowledge and skills, team integration, and overall job satisfaction., Materials and Methods: We conducted an e-mail survey of Veterans Affairs-based primary care team members in the United States' Pacific Northwest region who engaged in a longitudinal telemedicine mentorship program (n = 78)., Results: After adjustment for potential confounding factors, respondents who engaged in telemedicine mentorship for ≥1 year were significantly more likely to strongly agree that telemedicine mentorship improved patient access to specialty care (adjusted odds ratio [AOR] = 9.3, p < 0.005) and was useful in treating other patients on their panels (AOR = 3.7, p = 0.04). Participation ≥1 year was also associated with higher self-reported knowledge and competencies (AOR = 4.0, p = 0.03) and with perception of integration into a clinical team (AOR = 5.6, p = 0.01), but not with overall job satisfaction., Conclusion: Telemedicine-based specialty mentorship programs are highly valued by primary care-based participants, and self-reported benefits accumulate beyond 1 year of participation.

Background: Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex., Results: A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein., Conclusions: A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.

A simple polarization division multiplexed (PDM) multiband direct-detection optical orthogonal frequency division multiplexing (DDO-OFDM) long reach passive optical network (LR-PON) with net data rate over 210 Gb/s on single wavelength channel is proposed and experimentally demonstrated with self-polarization diversity technique. The proposed self-polarization diversity function is realized at a powered remote node with all passive components to achieve cost-effectiveness and simultaneously double both the channel capacity and subscriber numbers. Meanwhile, this architecture retains the simplicity of direct-detection single receiver-end without any hardware or software modification at the optical network units. The measured power penalty of the proposed PDM multiband DDO-OFDM LR-PON is 0.8 dB over 100 km transmission with respect to that of the ordinary single polarization scheme at a specified forward error correction threshold.

Background: In the United States, the incidence of hepatocellular carcinoma (HCC) is rising. For those diagnosed with terminal HCC, there is no curative treatment and duration of survival is typically 1 to 2 years. Research on illness and treatment experiences toward the end of life for patients with terminal HCC is limited., Objective: The aim of this study was to explore the illness experiences of patients with terminal HCC as they approached the end of life., Methods: This study used a prospective, longitudinal descriptive design. Interview data were collected from 14 patients once a month for up to 6 months, for a total of 45 interviews. Data were analyzed using conventional content analysis., Results: Three major themes (illness perceptions, decision to start treatment, and navigating treatment over time) and 10 subthemes were identified that were reflected across time in all patient experiences. Patients faced challenges with symptom experiences, treatment decisions, and unmet information needs affecting their quality of life., Conclusions: Gaining knowledge about the challenges facing patients with HCC is crucial for designing interventions that optimize their quality of life., Implications for Practice: Healthcare professionals may improve the quality of life of patients with terminal HCC by eliciting patients' perceptions of their illness and treatment decisions, symptom experiences, and information needs as the disease progresses and providing symptom management and offering information tailored to their needs. Care for patients with HCC who are approaching the end of life should be multidisciplinary and include timely referral to palliative care.

Research on symptom distress experienced by patients with end-stage liver disease at the end of life is limited. The aims of the study were to describe presence, frequency, severity, and distress of symptoms in patients with end-stage liver disease toward the end of life and to describe the variability in psychological and physical symptom distress between and within patients over time. This study used a prospective, longitudinal descriptive design. Data were collected from 20 patients once a month for up to 6 months. Participants completed the Memorial Symptom Assessment Scale, which reports a total score, a Global Distress Index score, and a psychological and a physical distress score. Patients reported lack of energy, pain, difficulty sleeping, and feeling drowsy as the most frequent, severe, and distressing symptoms. Global Distress Index mean scores (measured on a 1-4 scale) ranged from 2.6 to 2.9 across time. There was notable variability in psychological and physical distress scores between and within patients across time. Gaining knowledge about the prevalent symptoms experienced by patients with end-stage liver disease and the trajectory of these symptoms is crucial for designing interventions that optimize well-being in patients with end-stage liver disease as they are approaching death.

Background & Aims: Surveillance of patients with cirrhosis for hepatocellular carcinoma (HCC) with liver ultrasound every 6 months has been linked to longer survival and greater use of definitive treatment. However, less than 20% of patients typically undergo routine surveillance., Methods: We conducted a quasi-experimental study to assess whether a primary care-oriented, point-of-care clinical reminder improves HCC surveillance. Our study included patients with cirrhosis who made 1 or more primary care visits to 8 Veterans Affairs (VA) facilities over 18 months. Clinicians at 1 facility were sent a reminder to perform liver ultrasound assessments for patients with cirrhosis who had not received surveillance in the preceding 6 months. Outcomes included the proportion of patients receiving adequate HCC surveillance (defined as >2 instances of liver imaging >6 months apart) and HCC diagnosis and stage. Because it was a quality improvement project, this study did not require approval by an institutional review board under Federal law and VA policy., Results: Baseline rates of adequate HCC surveillance were similar at all facilities (18.2% at the intervention site vs 16.1% elsewhere; P = .23). After the reminder was implemented, adequate surveillance at the intervention site (for 790 patients) increased by 51%, but was unchanged at the other facilities (for 2094 patients) (27.6% vs 17.5%; P < .001). Adequate surveillance occurred more often at the intervention site (adjusted odds ratio, 1.29; 95% confidence interval, 1.03-1.61; P = .02). A higher crude percentage of patients was diagnosed with HCC at the intervention site than elsewhere (3.2% vs 1.9%; P = .03). We detected no difference in tumor stage at diagnosis., Conclusions: In a VA population, a clinical reminder system increased HCC surveillance in patients with cirrhosis., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Although zero-valent iron represents a promising approach for reduction of nitrate (NO(3)(-)) in water, its application in concentrated nitrate is limited by surface passivation. In this study, an alternative approach using in situ synthesized zero-valent copper (Cu(0)) produced by borohydride (NaBH(4)) was investigated. Complete reduction was observed within 55 min by reacting 677 mg-N/L of NO(3)(-) with CuO (0.312 g/L) and NaBH(4) (4.16 g/L) at 60 °C. The pseudo-first-order rate constant was 0.059 min(-1), and it increased threefold when the CuO dose was increased to 1.24 g/L. Increasing the NaBH(4) dose produced less nitrite (NO(2)(-)) throughout the experiments, indicating that it is the primary agent for reducing NO(2)(-). The initial pH exerted a significant effect on the reaction rate, and NO(3)(-) was rapidly reduced when the initial pH was less than 4. Based on the research findings, possible reaction pathways for NO(3)(-) reduction by Cu(0) are proposed in this work.

Ventilator-associated pneumonia (VAP) still lacks a rapid diagnostic strategy. This study proposes installing a nose-on-a-chip at the proximal end of an expiratory circuit of a ventilator to monitor and to detect metabolite of pneumonia in the early stage. The nose-on-a-chip was designed and fabricated in a 90-nm 1P9M CMOS technology in order to downsize the gas detection system. The chip has eight on-chip sensors, an adaptive interface, a successive approximation analog-to-digital converter (SAR ADC), a learning kernel of continuous restricted Boltzmann machine (CRBM), and a RISC-core with low-voltage SRAM. The functionality of VAP identification was verified using clinical data. In total, 76 samples infected with pneumonia (19 Klebsiella, 25 Pseudomonas aeruginosa, 16 Staphylococcus aureus, and 16 Candida) and 41 uninfected samples were collected as the experimental group and the control group, respectively. The results revealed a very high VAP identification rate at 94.06% for identifying healthy and infected patients. A 100% accuracy to identify the microorganisms of Klebsiella, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida from VAP infected patients was achieved. This chip only consumes 1.27 mW at a 0.5 V supply voltage. This work provides a promising solution for the long-term unresolved rapid VAP diagnostic problem.

Background & Aims: Hepatitis C virus (HCV) infection is highly associated with the type 2 diabetes mellitus, but the detailed mechanisms by which the viral proteins are involved in the clinical outcome remain unclear., Methods: A cDNA microarray analysis was performed following introducing an NS5A-encoding plasmid or a control vector into a mouse system by hydrodynamics- based transfection. Differentially expressed genes that are associated with gluconeogenesis were selected and their expression levels in HCV patients, in NS5A-expressing systems, and in the viral subgenomic replicon system were further examined by real-time quantitative polymerase chain reaction and Western blot analysis., Results: Differential gene expression including an upregulation of the gluconeogenic rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK) compared with controls was detected in mouse hepatocytes expressing HCV NS5A and in HCV patients with diabetes. In addition, an NS5A-dependent increase in glucose production was demonstrated in human primary hepatocytes. The upregulation of PEPCK and peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a) were also detected in NS5A-expressing cells and in the viral genotype 1b subgenomic replicon system. Further studies demonstrated that the NS5A-mediated upregulation of PEPCK and PGC-1a genes were resulted from the activation of PI3K-Akt and JNK signalling pathways. In addition, the expression levels of the forkhead transcription factor FoxO1 and the liver-enriched transcription factor HNF-4a were increased in HCV NS5A expressing cells., Conclusions: By upregulating the expression of PEPCK gene via its transactivators FoxO1 and HNF-4a, and the coactivator PGC-1a, the NS5A promotes the production of hepatic glucose which may contribute to the development of HCV-associated type 2 diabetes mellitus.

Photodynamic therapy (PDT) is a novel cancer treatment based on the tumor-specific accumulation of a photosensitizer followed by irradiation with visible light, which induces selective tumor cell death via production of reactive oxygen species. To elucidate the underlying mechanisms, microarray analysis was used to analyze the changes in gene expression patterns during PDT induced by various photosensitizers. Cancer cells were subjected to four different photosensitizer-mediated PDT and the resulting gene expression profiles were compared. We identified many differentially expressed genes reported previously as well as new genes for which the functionfunctions in PDT are still unclear. Our current results not only advance the general understanding of PDT but also suggest that distinct molecular mechanisms are involved in different photosensitizer-mediated PDT. Elucidating the signaling mechanisms in PDT will provide information to modulate the antitumor effectiveness of PDT using various photosensitizers., (© The Author(s) 2014.)

This prospective descriptive study investigated pain characteristics in 20 outpatients with endstage liver disease (ESLD) who were approaching the end of life, described variability in pain between and within patients, and described the pharmacological and nonpharmacological pain management strategies used. The instruments we utilized were the Brief Pain Inventory (BPI) and the self-care behaviour (SCB) log for pain. Data were collected once a month over a six-month period. BPI severity of, and interference from pain mean scores ranged from 5.52 to 6.03 and 5.36 to 6.64, respectively. The top three pain-relieving behaviours reported by patients were "taking pain medication," "taking a nap," and "asking for help." Pain medication intake-differed between patients who were pursuing a liver transplant and those who were not eligible for one. If we are to effectively improve care for ESLD patients, it is essential that we understand the ways in which these patients experience pain and the pain management strategies they employ.

Energy homeostasis is crucial for maintaining normally functioning cells; disturbances in this balance often cause various diseases. The limb region 1 (LMBR1) domain containing 1 gene (lmbrd1) encodes the LMBD1 protein that possesses 9 putative transmembrane domains. LMBD1 has been suggested to be involved in the lysosome in aiding the export of cobalamin. In this study, we determined that LMBD1 plays a regulatory role in the plasma membrane. A micro-positron emission tomography analysis showed that a single-allele knock-out of lmbrd1 increased the (18)F-fluorodeoxyglucose uptake in murine hearts. In addition, the knockdown of lmbrd1 resulted in an up-regulated signaling of the insulin receptor (IR) and its downstream signaling molecule, Akt. Confocal and live total internal reflection fluorescence microscopy showed that LMBD1 co-localized and co-internalized with clathrin and the IR, but not with the transferrin receptor. The results of the mutation analysis and phenotypic rescue experiments indicate that LMBD1 interacts with adaptor protein-2 and is involved in the unique clathrin-mediated endocytosis of the IR. LMBD1 selectively interacts with the IR. The knockdown of lmbrd1 attenuated IR endocytosis, resulting in the perturbation of the IR recycling pathway and consequential enhancement of the IR signaling cascade. In summary, LMBD1 plays an imperative role in mediating and regulating the endocytosis of the IR.

Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

Background: Few published studies have explored the clinical manifestations of nontyphoid salmonellosis in children <2 years of age. The aim of this study was to investigate the clinical manifestations, microbiological features, complications, fecal excretion time, and responses to treatment in children <2 years of age with nontyphoid salmonellosis., Methods: Between January 2005 and December 2009, pediatric patients who were admitted to Kaohsiung Veterans General Hospital with positive cultures for nontyphoid Salmonella were enrolled. The following data were recorded: demographic, clinical, and microbiological features, underlying diseases, treatment regimen, complications, responses to treatment, and fecal excretion time. The clinical manifestations were compared between patients <2 years of age and patients >2 years of age., Results: Of a total 279 enrolled patients, 179 were >2 years of age. Compared with the patients who were ≥2 years of age, patients <2 years of age demonstrated a significantly higher incidence of bloody stool, mixed infection, extraintestinal infection, longer course of antibiotics, longer course of diarrhea after admission, and more days spent in the hospital. The rates of insusceptibility of nontyphoid Salmonella to ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole, ceftriaxone, and ciprofloxacin in patients <2 years of age were 37.87%, 29.09%, 23.73%, 3.26%, and 2.25%, respectively. Younger patients were generally more susceptible to antibiotics than patients ≥2 years of age, although this result was not statistically significant., Conclusion: The clinical manifestations of nontyphoid salmonellosis are more severe in younger children <2 years of age than older children. Local susceptibility patterns could serve as a guide for the prescription of antibiotics by clinicians., (Copyright © 2012. Published by Elsevier B.V.)

Chronic hepatitis C virus (HCV) infection affects approximately 1.3 % of the United States population and 4 % of veterans who use Department of Veterans Affairs medical services. Chronic HCV is the primary cause of cirrhosis, hepatocellular carcinoma (HCC), and end-stage liver disease requiring liver transplantation in the United States. Management of chronic HCV is aimed at halting disease progression, preventing cirrhosis decompensation, reducing the risk of HCC, and treating extrahepatic complications of the infection. As part of a comprehensive HCV management strategy, peginterferon alfa and ribavirin, along with the addition of a hepatitis C protease inhibitor therapy for many genotype 1-infected patients, are the current standard of care. Antiviral therapy should be provided to those individuals who are clinically stable, have moderate liver disease or compensated cirrhosis, and are motivated to pursue therapy. Many patients have comorbid medical and psychiatric conditions, which may affect their adherence to antiviral therapy or worsen while on antiviral therapy. To optimally manage hepatitis C and associated comorbidities, patients benefit from multidisciplinary teams that can provide HCV-specific care and treatment. Sustained virologic response is associated with "cure" of chronic HCV, and results in improved liver disease outcomes and prolonged survival.

Objective: To compare occupational exposure to extremely-low-frequency magnetic field (ELF-MF) between dentists practicing in dental clinics and those employed in hospitals., Methods: Thirty-two dentists who worked at clinics (n=15) and 33 dentists employed at hospital dental departments (n=7) voluntarily provided their informed consent to participate in this measurement study. The study dentists were requested to wear an ELF-MF dosimeter for some 3 h at work to determine their personal exposure. Spot measurements taken at a number of locations in each dental office were used to indicate the work environment exposure level. Additionally, ELF-MF emitted from common dental equipment was also measured. All measurements were performed with EMDEX Lite meters., Results: The average environmental exposure to ELF-MF is higher in clinic dental offices than in hospital dental departments (0.55 vs. 0.15 µT, p=0.008). Personal dosimetry showed that on average, clinic dentists spent 35.71 and 19.39% of their time at exposures above 0.3 and 0.4 µT at work, respectively. The corresponding figures for hospital dentists were 19.61 and 13.92%. Additionally, ELF-MF was greater than 0.4 µT at 30 cm from all selected equipment, but the ELF-MF generally diminished as the distance from dental equipment increased. Uultraviolet air sterilization system produced 3 times as much ELF-MF as other dental equipment., Conclusions: This study suggests the possibility of over-exposure of dentists to power frequency ELF-MF. Additionally, certain dental equipment may produce ELF-MF levels greater than 0.4 µT in areas where dentists usually work when treating patients.

Severe acute respiratory syndrome coronavirus (SARS-CoV) was identified to be the causative agent of SARS with atypical pneumonia. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV. It is not clear whether ACE2 conveys signals from the cell surface to the nucleus and regulates expression of cellular genes upon SARS-CoV infection. To understand the pathogenesis of SARS-CoV, human type II pneumocyte (A549) cells were incubated with the viral spike protein or with SARS-CoV virus-like particles containing the viral spike protein to examine cytokine modulation in lung cells. Results from oligonucleotide-based microarray, real-time PCR, and enzyme-linked immunosorbent assays indicated an upregulation of the fibrosis-associated chemokine (C-C motif) ligand 2 (CCL2) by the viral spike protein and the virus-like particles. The upregulation of CCL2 by SARS-CoV spike protein was mainly mediated by extracellular signal-regulated kinase 1 and 2 (ERK1/2) and AP-1 but not the IkappaBalpha-NF-kappaB signaling pathway. In addition, Ras and Raf upstream of the ERK1/2 signaling pathway were involved in the upregulation of CCL2. Furthermore, ACE2 receptor was activated by casein kinase II-mediated phosphorylation in cells pretreated with the virus-like particles containing spike protein. These results indicate that SARS-CoV spike protein triggers ACE2 signaling and activates fibrosis-associated CCL2 expression through the Ras-ERK-AP-1 pathway.

Hepatitis C virus (HCV) internal non-structural protein 3 (NS3) cleavage can occur in trans in the presence of NS4A. In this study, we have further demonstrated a critical role of the helicase domain in the internal NS3 cleavage, different from HCV polyprotein processing which requires only the serine protease domain. The NTPase domain of NS3 helicase interacts with the RNA binding domain to facilitate internal NS3 cleavage. In addition, NS3 protease activity contributes to the transforming ability of the major internal cleavage product NS3(1-402). These findings imply important roles of the internal cleavage and protease activity of the NS3 protein in the pathogenesis of HCV., (2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Oval cells and hepatocytes rarely proliferate simultaneously. This study aimed to determine the impacts of hepatocyte transplantation on the response and fate of oval cells that are activated to proliferate in acute severe hepatic injury. Retrorsine + D-galactosamine (R+D-gal) treatment was used to induce acute hepatic injury and to elicit extensive activation of oval cells in male dipeptidyl peptidase IV-deficient F344 rats. These rats were then randomized to receive wild-type hepatocyte transplantation or vehicle intraportally. The kinetics of oval cell response and their differentiation fate were analyzed. Results showed that oval cells were activated early and differentiated into hepatocytes in R+D-gal-treated rats without hepatocyte transplantation. With hepatocyte transplantation, the oval cells were recruited later and continued to proliferate in parallel with the massive proliferation of transplanted hepatocytes. They formed ductules and differentiated into biliary cells. When hepatocytes were transplanted at the day when oval cells were at their peak response, the numerous activated oval cells ceased to differentiate into hepatocytes and remained in ductular form. The ductular oval cells were capable of differentiating into hepatocytes again when the donor hepatocytes were inhibited to proliferate. We conclude that hepatocyte transplantation changes the mechanism of liver reconstitution and affects the differentiation fate of host oval cells in acute severe hepatic injury.

We assessed the effects of treatment with 4-methylcatechol (4MC), a known inducer of nerve growth factor, on peripheral nerve regeneration by analyzing cutaneous and muscular reinnervation in mice after sciatic nerve crush injury. At 3 months postinjury, the skin innervation index was significantly higher in the 4MC group than the control group (p=0.0002); there was also increased unmyelinated fiber density (p=0.0042) and unmyelinated fibers/Remak bundle (p = 0.001) in sural nerves, indicating unmyelinated nerve fiber regeneration. These changes were accompanied by increases of transcripts for nerve growth factor (p = 0.0026) and glial cell line-derived neurotrophic factor (p=0.03) in the 4MC group. In contrast, muscle innervation indices were similar in both groups and were higher than the skin innervation index (p < 0.0001). The regeneration of myelinated nerve fibers, as assessed by fiber density, diameter and g ratio analyses in sural nerves, and amplitudes of muscle action potential in sciatic nerves, was similar in both groups. Taken together, these data suggest that 4MC specifically promoted the regeneration of unmyelinated nerve fibers and reinnervation of the skin by increasing the expression of nerve growth factor and glial cell line-derived neurotrophic factor.

Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.

Unlabelled: Liver cirrhosis is characterized by progressive accumulation of extracellular matrix following chronic liver injuries. In the extracellular space, the constant turnover of liver matrix is regulated by the matrix metalloproteinase (MMP) class of enzyme. To assess whether genetic variations in MMP would result in diversity of liver cirrhosis, a case-control study of 320 patients with hepatocellular carcinoma, with or without cirrhosis, was conducted. Ten single-nucleotide polymorphism markers from four potential fibrosis-associated genes were selected for genotyping. Among these genes, a nonsynonymous single-nucleotide polymorphism which generates the variation of Gly-137 and Asp-137 in the MMP-7 gene was found to be strongly associated with the development of liver cirrhosis. In contrast to MMP-7(Gly-137) that predominantly secretes out into the cell culture medium, the cirrhosis-associated MMP-7(Asp-137) variant is preferentially localized on the extracellular membranes where it exerts its proteolytic activity on pericellular substrates. Functional analysis demonstrated an increased ability of the MMP-7(Asp-137) variant to associate with the cell surface CD151 molecule. In wound-healing and Boyden chamber assays, cell motility was specifically enhanced with the expression of MMP-7(Asp-137) as compared to the cells expressing MMP-7(Gly-137). These results demonstrate that the MMP-7(Asp-137) variant confers a gain-of-function phenotype for MMP-7., Conclusion: We have identified a novel genetic association of MMP-7(Asp-137) variant with liver cirrhosis in patients with hepatocellular carcinoma. Whether the MMP-7 variant can be a new marker for liver cirrhosis will be further studied.

Liver is unique in its capability to regenerate after an injury. Liver regeneration after a 2/3 partial hepatectomy served as a classical model and is adopted frequently to study the mechanism of liver regeneration. In the present study, semiquantitative analysis of protein expression in mouse liver regeneration following partial hepatectomy was performed using an iTRAQ technique. Proteins from pre-PHx control livers and livers regenerating for 24, 48 and 72 h were extracted and inspected using 4-plex isotope labeling, followed by liquid chromatography fractionation, mass spectrometry and statistical differential analysis. A total of 827 proteins were identified in this study. There were 270 proteins for which quantitative information was available at all the time points in both biologically duplicate experiments. Among the 270 proteins, Car3, Mif, Adh1, Lactb2, Fabp5, Es31, Acaa1b and LOC100044783 were consistently down-regulated, and Mat1a, Dnpep, Pabpc1, Apoa4, Oat, Hpx, Hp and Mt1 were up-regulated by a factor of at least 1.5 from that of the controls at one time point or more. The regulation of each differential protein was also demonstrated by monitoring its time-dependent expression changes during the regenerating process. We believe this is the first report to profile the protein changes in liver regeneration utilizing the iTRAQ proteomic technique.

Efficient repopulation by transplanted hepatocytes in the severely injured liver is essential for their clinical application in the treatment of acute hepatic failure. We studied here whether and how the transplanted hepatocytes are able to efficiently repopulate the toxin-induced acute injured liver. Male dipeptidyl peptidase IV-deficient F344 rats were randomized to receive retrorsine plus D-galactosamine (R+D-gal) treatment or D-galactosamine-alone (D-gal) to induce acute hepatic injury, and retrorsine-alone. In these models, retrorsine was used to inhibit the proliferation of endogenous hepatocytes while D-galactosamine induced acute hepatocyte damage. Wild-type hepatocytes (1 x 10(7)/ml) were transplanted intraportally 24 h after D-galactosamine or saline injection. The kinetics of proliferation and repopulation of transplanted cells and the kinetics of cytokine response, hepatic stellate cell (HSC) activation, and matrix metalloproteinase (MMP2) expression were analyzed. We observed that early entry of transplanted hepatocytes into the hepatic plates and massive repopulation of the liver by transplanted hepatocytes occurred in acute hepatic injury induced by R+D-gal treatment but not by D-gal-alone or retrorsine-alone. The expressions of transforming growth factor-alpha and hepatocyte growth factor genes in the R+D-gal injured liver were significantly upregulated and prolonged up to 4 weeks after hepatocyte transplantation. The expression kinetics were parallel with the efficient proliferation and repopulation of transplanted hepatocytes. HSC was activated rapidly, markedly, and prolongedly up to 4 weeks after hepatocyte transplantation, when the expression of HGF gene and repopulation of transplanted hepatocytes were reduced afterward. Furthermore, the expression kinetics of MMP2 and its specific distribution in the host areas surrounding the expanding clusters of transplanted hepatocytes are consistent with those of activated HSC. Impaired hepatocyte regeneration after acute severe hepatic injury may initiate serial compensatory repair mechanisms that facilitate the extensive repopulation by transplanted hepatocytes that enter early the hepatic plates.

Hepatitis C virus often causes persistent infection and hepatocellular carcinoma. Studies have demonstrated the roles of viral nonstructural protein 5A (NS5A) in the induction of chromosome aneuploidy, but the molecular mechanisms are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by laser capture microdissection. Gene expression profiles of the NS5A-expressing hepatocytes were examined by an Affymetrix oligonucleotide microarray system. Aspm (abnormal spindle-like, microcephaly associated), which encodes the mitotic spindle protein ASPM, was identified to be differentially expressed in the absence and the presence of NS5A. The down-regulation of Aspm mRNA and ASPM protein was confirmed by real time polymerase chain reaction and Western blot analysis, respectively, both in mouse model systems and in viral subgenomic replicon and in vitro transfection culturing systems. In addition, cultured cells that constitutively expressed NS5A protein showed G(2)/M cell cycle block and chromosome aneuploidy. Overexpression of ASPM relieved the G(2)/M cell cycle block. Furthermore, NS5A protein repressed the promoter activity of Aspm gene in a dose-dependent manner. The regulatory effect was abolished when amino acid substitutions P2209L, T2214A, and T2217G known to interrupt the NS5A-PKR interaction were introduced into the NS5A protein. This indicates that the down-regulation of Aspm expression is via the PKR-p38 signaling pathway. These results suggest that NS5A protein down-regulates the expression of the mitotic spindle protein ASPM and induces aberrant mitotic cell cycle associated with chromosome instability and hepatocellular carcinoma.

Purpose: To demonstrate the validity of non-contact holmium:yttrium-aluminum-garnet laser stone fragmentation in an in-vitro model and to characterize the optimal laser settings to perform this technique., Materials and Methods: A caliceal model consisting of a glass test tube filled with 0.9% normal saline was created. Into this system, a 365-mum laser fiber was inserted after stabilization 2 mm above the surface of the stones. Laser experiments were conducted using tubes that contained four soda limestone phantoms with a total stone burden of approximately 0.2 g per tube. The laser energy was varied between 1.0 and 1.5 J, with a firing frequency of 20, 30, or 40 Hz. Ten tubes were tested at each setting combination for a duration of 2 minutes. Thus, 60 tubes were tested. The contents of each tube were filtered through a 2-mm sieve after treatment, and all remaining fragments were dried for 5 days. The fragments were weighed, and the difference from pretreatment was calculated. Differences in weights and laser settings were compared using one-tailed and two-sample t testing., Results: Each experiment resulted in a significant decrease in stone burden from prelaser weight (all P values = 0.00). Laser settings were manipulated between combinations of 1.0 J at a frequency of 20, 30, and 40 Hz, and an energy of 1.5 J and 20, 30, and 40 Hz. This resulted in a respective mean weight loss of -42%, -58%, and -47% at 1.0 J, and -24%, -56%, and -63% at 1.5 J. The t tests were performed to detect differences between weight loss and to determine the optimal settings. Laser settings of 1.5 J and 40 Hz for 2 minutes produced the greatest mean decrease in stone burden (63%). This loss was significantly different from that of all settings at 1.0 J and the setting of 1.5 and 20 Hz (P < 0.05). The mean percentage weight loss for each setting was then normalized to the total energy used. Settings of 1.0 J and 20 Hz were the most efficient, with a change in weight of -18% per kJ., Conclusions: We duplicated the "popcorn effect" of non-contact laser use in vitro. In this model, the technique results in a significant decrease in stone burden (up to 63%) in just 2 minutes. Adequate energy and high frequency seemed to optimize the effectiveness of the method, but excessive energy and frequency produced diminishing returns.

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.

Clathrin-mediated endocytosis is a common pathway for viral entry, but little is known about the direct association of viral protein with clathrin in the cytoplasm. In this study, a putative clathrin box known to be conserved in clathrin adaptors was identified at the C terminus of the large hepatitis delta antigen (HDAg-L). Similar to clathrin adaptors, HDAg-L directly interacted with the N terminus of the clathrin heavy chain through the clathrin box. HDAg-L is a nucleocytoplasmic shuttle protein important for the assembly of hepatitis delta virus (HDV). Here, we demonstrated that brefeldin A and wortmannin, inhibitors of clathrin-mediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly but had no effect on the assembly of the small surface antigen of hepatitis B virus. In addition, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the degradation of epidermal growth factor receptor. These results indicate that HDAg-L is a new clathrin adaptor-like protein, and it may be involved in the maturation and pathogenesis of HDV coinfection or superinfection with hepatitis B virus through interaction with clathrin.

The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1-20, and the central domain from residues 21-34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21-34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.

Objectives: To evaluate the outcomes of hand-assisted laparoscopic nephrectomy in patients with significant complicating clinical factors., Methods: We performed a retrospective review of 322 hand-assisted laparoscopic nephrectomy cases that were completed at a single institution from 1998 to 2004. Patients with a history of extensive abdominal surgery or prior procedures on the affected kidney, evidence of perirenal inflammation, renal lesions 10 cm or more in diameter, or level I renal vein thrombus were included., Results: A total of 42 patients were included in this series. Of these, 16 patients had a lesion 10 cm or larger, 10 had a renal vein thrombus, and 10 had undergone prior major abdominal surgery. Many patients had more than one complicating factor. Another 6 patients had a history of prior renal procedures or chronic inflammatory processes involving the affected kidney. One Stage T4 renal tumor with paraspinous muscle invasion was successfully managed without conversion. The overall mean operative time and estimated blood loss was 235 minutes and 439 mL, respectively, with a mean hospital stay of 4 days. Four patients (9.5%) required open conversion (one renal hilar injury, two failure to progress, and one persistent bleeding from the renal fossa). Postoperative complications included pulmonary embolism in 1, ileus in 1, and chronic obstructive pulmonary disease exacerbation in 1 patient. One patient developed an incarcerated port site hernia requiring reoperation., Conclusions: Hand-assisted laparoscopic nephrectomy is an attractive minimally invasive option in the setting of significant complicating factors. This technique may facilitate the successful laparoscopic completion of these challenging cases with reasonable operative times, blood loss, and complication rates.

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

This study focused on identifying the signalling mediating the effect of magnolol on corticosterone production. Magnolol-induced corticosterone production was completely inhibited by mitogen-activated protein kinase kinase (MEK)-inhibitor PD98059, tyrosine kinase (TK)-inhibitor genistein or Janus tyrosine kinase 2 (JAK2)-inhibitor AG490, suggesting that extracellular signal-regulated kinase (ERK) and JAK2 are both involved in this signaling cascade. Further, magnolol induced the transient phosphorylation of MEK, ERK, cAMP response-element binding protein (CREB) and the expression of 32 and 30 kDa steroidogenic acute regulatory protein (StAR) in a time-dependent manner. Inhibition of TK or JAK2 activities blocked magnolol-induced phosphorylation of MEK and ERK, again supporting the upstream role of JAK2. The activation of JAK2 or MEK apparently mediated the magnolol-induced phosphorylation of CREB and the upregulation of StAR. These findings demonstrate a novel pathway for magnolol to induce the expression of StAR, which regulates the rate-limiting step in sterodiogenesis.

SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations (K(i) = 4 microM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC(50) values ranging from 7.6 to 84.5 microM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.

The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. We have previously identified a chromosome region maintenance 1-independent nuclear export signal (NES) at the C terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) that is required for the assembly of hepatitis delta virus (HDV) (C.-H. Lee et al., J. Biol. Chem. 276:8142-8148, 2001). To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using the GAL4-binding domain fused to the NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI [NES(HDAg-L) interacting protein] that specifically interacted with the wild-type NES(HDAg-L) but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L), in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9-kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of antisense NESI RNAs inhibited the expression of NESI and abolished HDAg-L-mediated nuclear export and assembly of HDV genomic RNA. These data indicate a critical role of NESI in the assembly of HDV through interaction with HDAg-L.

Magnolol stimulates adrenal steroidogenesis and induces the distributional changes of p160 and adipose differentiation-related protein (ADRP) in rat adrenal cells. This study investigated the underlying signaling mechanisms involved in these processes. Magnolol (30 microM) caused a time-dependent increase in the phosphorylation of extracellular signal-related kinase (ERK) in cultured adrenal cells. The following evidence supports a link between ERK activation and p160 translocation. First, the magnolol-induced redistribution of p160 from the lipid droplet surface to the cytosol, resulting in the decrease in the percentages of p160-positive cells, and this decrease in p160-positive cells was completely blocked by pretreatment with either of the MAPK-ERK kinase (MEK) inhibitors PD98059 or U0126. Second, magnolol did not significantly decrease total p160 protein levels but caused an increase in threonine phosphorylation of p160, which reached a maximum after 5 min of magnolol treatment, and this magnolol-induced phosphorylation of p160 was prevented by pretreatment with U0126, suggesting the involvement of ERK. In addition, magnolol decreased both ADRP immunostaining intensity at the lipid droplet surface and the percentage of ADRP-positive cells. This was further confirmed biochemically by the decrease in ADRP levels in total cell homogenates and in lipid droplet fractions. Magnolol-induced decrease in ADRP staining at the lipid droplet surface was not affected by pretreatment with PD98059 or U0126, indicating that ERK signaling was not involved in this event. Furthermore, treatment with 30 microM magnolol for 6 h resulted in about 50% decrease in ADRP protein level. Therefore, decreased protein levels of p160 and ADRP at the lipid droplet surface induced by magnolol were mediated via two different mechanisms: phosphorylation of p160 and downregulation of ADRP expression, respectively.

An antigen detection assay for severe acute respiratory syndrome (SARS) coronavirus was established in this study by an indirect immunofluorescence test, which utilized cells derived from throat wash samples of patients with SARS and a rabbit serum that recognized the nucleocapsid protein of SARS-associated coronavirus (SARS-CoV) but not that of other human coronavirus tested. It detected SARS-CoV in 11 of 17 (65%) samples from SARS patients as early as day 2 of illness but in none of the 10 samples from healthy controls. Compared with other diagnostic modalities for detecting SARS-CoV, this assay is simpler, more convenient, and economical. It could be an alternative for early and rapid diagnosis, should SARS return in the future.

Background: Studies have revealed that losses of chromosome 4q24-25 regions are frequent in cancers including head and neck squamous cell carcinoma. Our previous comparative genomic hybridization analysis showed extensive losses of chromosome arm 4q in oral squamous cell carcinoma (OSCC)., Methods: To be more precise in mapping the potential regions of allelic losses and to understand the microsatellite instability (MSI) on 4q involving in oral pathogenesis, we performed allelotypings using eight polymorphic markers. Microsatellite analyses were first performed on 100 randomly selected controls to confirm the high informative rates of markers. Twenty OSCC tissues were microdissected from surgical specimens for microsatellite alterations (MA) analysis., Results: MA was observed in 95% OSCC cases. The most eminently altered locus was 4q13.1 (75%), followed by 4q22.2 and 4q32.1 (55%). Allelic losses also occurred most frequently on these loci. Thirty-five percent cases had MA spanning 4q13.1 to 4q21.1. MSI occurred in 35% OSCC, at a lesser extent compared with allelic losses. The most common locus for MSI was 4q21.2 (20%). In addition, 4q MSI was significantly associated with the lymph node metastasis of OSCC (P = 0.01). So far, most tumor suppressor genes on 4q have not been specified., Conclusion: Our results were additive to previous findings and proposed novel scenario of suppressor loci located at 4q13.1-21.1 whose inactivation could be important for progression of OSCC.