Intracellular antibody targeting HBx suppresses invasion and metastasis in hepatitis B virus-related hepatocarcinogenesis via protein phosphatase 2A-B56γ-mediated dephosphorylation of protein kinase B.

OBJECTIVES: Hepatitis B virus X (HBx) is closely associated with HBV-related hepatocarcinogenesis via the inactivation of tumour suppressors. Protein phosphatase 2A (PP2A) regulatory subunit B56 gamma (B56γ), as a tumour suppressor, plays a critical role in regulating cellular phosphorylation signals via dephosphorylation of signalling proteins. However, the underlying mechanism that B56γ involved in regulating HBx-associated hepatocarcinogenesis phenotypes and mediating anti-HBx antibody-mediated tumour suppression remains unknown. MATERIALS AND METHODS: We used bioinformatics analysis, paired HCC patient specimens, HBx transgenic (HBx-Tg) mice, xenograft nude mice, HBV stable replication in the HepG2.2.15 cells, and anti-HBx antibody intervention to systematically evaluate the biological function of protein kinase B (AKT) dephosphorylation through B56γ in HBx-associated hepatocarcinogenesis. RESULTS: Bioinformatics analysis revealed that AKT, matrix metalloproteinase 2 (MMP2), and MMP9 were markedly upregulated, while cell migration and viral carcinogenesis pathways were activated in HBV-infected liver tissues and HBV-associated HCC tissues. Our results demonstrated that HBx-expression promotes AKT phosphorylation (p-AKTThr308/Ser473 ), mediating the migration and invasion phenotypes in vivo and in vitro. Importantly, in clinical samples, HBx and B56γ were downregulated in HBV-associated HCC tumour tissues compared with peritumor tissues. Moreover, intervention with site-directed mutagenesis (AKTT308A , AKTS473A ) of p-AKTThr308/Ser473 mimics dephosphorylation, genetics-based B56γ overexpression, and intracellular anti-HBx antibody inhibited cell growth, migration, and invasion in HBx-expressing HCC cells. CONCLUSIONS: Our results demonstrated that B56γ inhibited HBV/HBx-dependent hepatocarcinogenesis by regulating the dephosphorylation of p-AKTThr308/Ser473 in HCC cells. The intracellular anti-HBx antibody and the activator of B56γ may provide a multipattern chemopreventive strategy against HBV-related HCC.

Protein phosphatase 2A-B56γ-Drp1-Rab7 signaling axis regulates mitochondria-lysosome crosstalk to sensitize the anti-cancer therapy of hepatocellular carcinoma.

Mitochondria-lysosome crosstalk is an intercellular communication platform regulating mitochondrial quality control (MQC). Activated dynamin-related protein 1 (Drp1) with phosphorylation at serine 616 (p-Drp1Ser616) plays a critical role in mitophagy-dependent cell survival and anti-cancer therapy for hepatocellular carcinoma (HCC). However, the underlying mechanisms that p-Drp1Ser616 involved in regulating mitochondria-lysosome crosstalk and mediating anti-HCC therapy remain unknown. HCC cells and mouse xenograft models were conducted to evaluate the relationship between p-Drp1Ser616 and Ras-associated protein 7 (Rab7) and the underlying mechanism by protein phosphatase 2A (PP2A)-B56γ regulating mitophagy via dephosphorylation of p-Drp1Ser616 in HCC. Herein, we found that Drp1 was frequently upregulated and was associated with poor prognosis in HCC. Mitochondrial p-Drp1Ser616 was a novel inter-organelle tethering protein localized to mitochondrion and lysosome membrane contact sites (MCSs) via interaction with Rab7 to trigger an increase in the mitochondria-lysosome crosstalk, resulting in PINK1-Parkin-dependent mitophagy and anti-apoptosis in HCC cells under the treatment of chemotherapy drugs. Moreover, we demonstrate that B56γ-mediated direct dephosphorylation of p-Drp1Ser616 inhibited mitophagy and thus increased mitochondria-dependent apoptosis. Overall, our findings demonstrated that activation of B56γ sensitizes the anti-cancer effect of HCC chemoprevention via dephosphorylated regulation of p-Drp1Ser616 in inhibiting the interaction between p-Drp1Ser616 and Rab7, which may provide a novel mechanism underlying the theranostics for targeting intervention in HCC.

Silencing HIF-1α aggravates non-alcoholic fatty liver disease in vitro through inhibiting PPAR-α/ANGPTL4 singling pathway / El silenciamiento de HIF-1α agrava la esteatohepatitis no alcohólica in vitro a través de la inhibición de la vía de señalización PPAR-α/ANGPTL4

Objective: Non-alcoholic fatty liver disease (NAFLD) is an aberrant lipid metabolism disease. Hypoxia inducible factor-1 (HIF-1α) is a transcription factor which plays an important part in adapting lower oxygen condition. Here, we aimed to clarify the relationship between HIF-1α and NAFLD. Methods: HepG2 cells was stimulated by oleic acid (OA) and palmitic acid (PA) to establish in vitro model of NAFLD. The expression of lipid metabolism-related genes, the binding of PPARα to HIF-1α promoter, the lipid deposition, and oxidative stress were detected by qRT-PCR, western blot, Chip assay, Oil Red O staining and ELISA assays, respectively. Results: HIF-1α silence promoted lipid accumulation in NAFLD cells, accompanying by the significantly increased contents of TG (triglyceride) and ApoB (apolipoprotein B). In HepG2 cells treated with OA/PA, the expression of lipid metabolism-related genes and proteins, including APOE, A2m, TNFRSF11B, LDLr, and SREBP2, and the intracellular lipid deposition were up-regulated and further aggravated after silencing HIF-1α. In addition, the loss of HIF-1α could remarkably elevate MDA contents while inhibit the activities of beneficial antioxidant enzymes SOD and GSH-Px to activate oxidative stress, and promote the secretion of pro-inflammatory IL-6 and TNF-α to aggravate inflammation in NDFLD cells. PPARα positively bound to HIF-1α promoter. The silence of PPARα aggravated lipid deposition under normal or hypoxic environment in NAFLD cells. In addition, PPAR-α silence could decrease the expression of HIF-1α and ANGPTL4 in NAFLD cell model; moreover, the expression of APOE, A2m and TNFRSF11B and the production of TG and MDA were increased by PPAR-α suppression. Conclusion: HIF-1α plays a crucial role in the regulation of lipid metabolism through activating PPAR-α/ANGPTL4 signaling pathway in NAFLD.(AU)

Objetivo: La esteatohepatitis no alcohólica (EHNA) es una enfermedad del metabolismo aberrante de los lípidos. El factor inducible por hipoxia 1 (HIF-1α) es un factor de transcripción que desempeña una función importante en la adaptación de la afección de nivel de oxígeno bajo. En el presente documento, intentamos aclarar la relación entre HIF-1α y la EHNA. Métodos: Las células HepG2 se estimularon con ácido oleico (OA) y ácido palmítico (PA) para establecer un modelo in vitro de la EHNA. La expresión de los genes relacionados con el metabolismo de los lípidos, la unión de PPARα al promotor HIF-1α, el depósito de lípidos y el estrés oxidativo se detectaron mediante ensayos de qRT-PCR, inmunoelectrotransferencia, ensayo de inmunoprecipitación de cromatina (ChIP), ensayos de tinción de rojo aceite O y ELISA, respectivamente. Resultados: El silencio de HIF-1α promovió la acumulación de lípidos en las células de la EHNA, acompañada de un aumento significativo del contenido de triglicéridos (TG) y apolipoproteína B (ApoB). En las células HepG2 tratadas con OA/PA, la expresión de genes y proteínas relacionados con el metabolismo lipídico, incluidos APOE, A2m, TNFRSF11B, LDLr y SREBP2, y el depósito de lípidos intracelular se regularon al alza y se agravaron aún más después de silenciar HIF-1α. Además, la pérdida de HIF-1α podría elevar notablemente el contenido de MDA e inhibir las actividades de las enzimas antioxidantes beneficiosas SOD y GSH-Px para activar el estrés oxidativo, y promover la secreción de IL-6 pro-inflamatoria y TNF-α para agravar la inflamación en las células de la EHNA. PPARα se unió positivamente al promotor HIF-1α. El silencio de PPARα agravó el depósito de lípidos en un ambiente normal o hipóxico en las células de la EHNA. Además, el silencio de PPAR-α pudo disminuir la expresión de HIF-1α y ANGPTL4 en el modelo de células de la EHNA; por otra parte, la expresión de APOE, A2m y TNFRSF11B, y la producción de TG y MDA aumentaro,

A secondary learning curve in 3D versus 2D imaging in laparoscopic training of surgical novices.

BACKGROUND: Stereoscopic (3D) imaging can be used to facilitate the learning of basic laparoscopic tasks. Its advantages over traditional endoscopic (2D) imaging include better depth perception and spatial orientation. However, the transition between 3D and 2D imaging systems has not been previously studied. This study compares the acquisition of basic laparoscopic skills in a laparoscopic-naïve population using both imaging systems, and explores the possibility of a secondary learning curve in the transition between systems. METHODS: 26 novice learners were randomly allocated into two arms and taught to perform two basic laparoscopic tasks adopted from the fundamentals of laparoscopic surgery (FLS) curriculum, peg transfer (T1) and pattern cutting (T2) using either 2D or 3D imaging systems. These tasks were repeated until proficiency was achieved. Participants in each arm then repeated the tasks in the other viewing system (2D/3D vs 3D/2D). The parameters measured were: (a) time taken to complete the task and (b) number of attempts to achieve proficiency. RESULTS: There was a significant shortening of time required to achieve proficiency in T2 using a 3D system (mean difference-in-differences = - 65.4, 95% CI - 103.6 to - 27.2, t(24) = - 3.5, p value = 0.002) but no difference between 2D and 3D imaging systems for T1, a simpler task. Sub-group analysis of T1 and T2 between the 2D/3D and 3D/2D arms showed the presence of a secondary learning curve in the 2D/3D arm for both tasks, (T1: ß-estimate - 2.68, 95% CI - 3.68 to - 1.68, p value = 0.0003; T2: ß-estimate - 2.45, 95% CI - 3.75 to - 1.14, p value 0.004), but in the 3D/2D arm there was a secondary learning curve only for T2. (ß-estimate 2.60, 95% CI 1.45-3.76, p value 0.001) CONCLUSION: 3D imaging can be an effective tool to speed the acquisition of proficiency in basic laparoscopic tasks for novice learners, especially in more complex tasks such as pattern cutting. The skills learned in 3D imaging can translate into 2D, albeit with a secondary learning curve.

Silencing HIF-1α aggravates non-alcoholic fatty liver disease in vitro through inhibiting PPAR-α/ANGPTL4 singling pathway.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an aberrant lipid metabolism disease. Hypoxia inducible factor-1 (HIF-1α) is a transcription factor which plays an important part in adapting lower oxygen condition. Here, we aimed to clarify the relationship between HIF-1α and NAFLD. METHODS: HepG2 cells was stimulated by oleic acid (OA) and palmitic acid (PA) to establish in vitro model of NAFLD. The expression of lipid metabolism-related genes, the binding of PPARα to HIF-1α promoter, the lipid deposition, and oxidative stress were detected by qRT-PCR, western blot, Chip assay, Oil Red O staining and ELISA assays, respectively. RESULTS: HIF-1α silence promoted lipid accumulation in NAFLD cells, accompanying by the significantly increased contents of TG (triglyceride) and ApoB (apolipoprotein B). In HepG2 cells treated with OA/PA, the expression of lipid metabolism-related genes and proteins, including APOE, A2m, TNFRSF11B, LDLr, and SREBP2, and the intracellular lipid deposition were up-regulated and further aggravated after silencing HIF-1α. In addition, the loss of HIF-1α could remarkably elevate MDA contents while inhibit the activities of beneficial antioxidant enzymes SOD and GSH-Px to activate oxidative stress, and promote the secretion of pro-inflammatory IL-6 and TNF-α to aggravate inflammation in NDFLD cells. PPARα positively bound to HIF-1α promoter. The silence of PPARα aggravated lipid deposition under normal or hypoxic environment in NAFLD cells. In addition, PPAR-α silence could decrease the expression of HIF-1α and ANGPTL4 in NAFLD cell model; moreover, the expression of APOE, A2m and TNFRSF11B and the production of TG and MDA were increased by PPAR-α suppression. CONCLUSION: HIF-1α plays a crucial role in the regulation of lipid metabolism through activating PPAR-α/ANGPTL4 signaling pathway in NAFLD.

Short-Term Changes in Tear Lipid Layer Thickness After Instillation of Lipid Containing Eye Drops.

Purpose: Lipid-containing eye drops is increasingly popular in eye clinics to treat dry eye. Tear lipid layer thickness (LLT) changes after instillation of lipid eye drops have not been characterized. We aim to evaluate these changes of LLT using a noninvasive interferometry-based method. Methods: This prospective clinical study was conducted on staff and patients from Singapore National Eye Centre with ad hoc recruitment. Noninvasive tear break up time was measured using the Keratographer 5M. LLTs were measured using a tear interferometer machine before and at 1, 5, and 15 minutes after instillation of lipid-containing drops, either Cationorm unidose or Artelac Lipids. Fluorescein clearance (tear clearance rate) and Schirmer tests were conducted. The tear clearance rate of fluorescein dye was based on the visual examination of the color of a Schirmer strip after 5 minutes, compared against color standards. Results: This study included a total of 84 participants aged ≥21 years. Many were female (92.8%) and Chinese (89.2%). A tear clearance rate of 1/16 was most common (35.7%), whereas 1/128 and 1/32 were uncommon (3.57% each). Schirmer results were 6.5 ± 8.1 mm, and noninvasive tear break up times were 8.12 ± 6.25 mm. Participants with baseline LLT <60 nm had greater changes in LLT after Cationorm instillation, compared with those with an LLT of >60 nm. LLT changes over 15 minutes were not associated with tear clearance rate. Similar results were obtained when using Artelac Lipids. Conclusions: Our results showed that participants' initial LLT affected their responsiveness to lipid-containing eye drops more than other factors. Translational Relevance: Doctors may choose to measure the baseline LLT of patients before deciding whether to prescribe lipid eye drops to patients.

Screening and treatment of obstructive sleep apnea in acute coronary syndrome. A randomized clinical trial.

BACKGROUND: We evaluated the effects of sleep-study guided multidisciplinary therapy (SGMT) of obstructive sleep apnoea (OSA) in patients presenting with acute coronary syndrome. METHODS: Eligible patients were randomized into (1) SGMT, comprised a sleep study during the index admission and continuous positive airway pressure and behavioral therapy for those with at least mild OSA or (2) standard therapy. The primary end point was the change in the plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) level from baseline to the 7-month follow-up. RESULTS: A total of 159 patients completed the trial. Of the 70 patients randomized to SGMT, 21 (30%), 15 (22%) and 27 (39%) were diagnosed with mild, moderate and severe OSA, respectively. Continuous positive airway pressure and a positional pillow were prescribed to 57 (91%) and 6 (9%) patients with OSA. Although plasma NT-proBNP levels were lower after 7 months compared to the baseline, the levels did not differ significantly between the SGMT and standard therapy groups at baseline (579 ±â€¯1117 vs. 611 ±â€¯899 pg/dL, p = .851) or at 7 months (90 ±â€¯167 vs. 93 ±â€¯174 pg/dL, p = .996). The changes in NT-proBNP levels from baseline to 7 months were similar with SGMT and standard therapy (-489 vs. -518 pg/dL, p = .726). Similar findings were observed for the plasma ST2 and hs-CRP levels. CONCLUSIONS: OSA screening and multifaceted treatment during the sub-acute phase of acute coronary syndrome did not further reduce the levels of cardiovascular biomarkers when compared with standard therapy. CLINICAL TRIAL REGISTRATION: clinicaltrial.gov NCT02599298.

Sleep study-guided multidisciplinary therapy (SGMT) for patients with acute coronary syndrome: Trial rationale and design.

Obstructive sleep apnea (OSA) is an emerging risk marker for acute coronary syndrome (ACS). This randomized trial aims to determine the effects of sleep study-guided multidisciplinary therapy (SGMT) comprising overnight sleep study, continuous positive airway pressure, and behavioral therapy for OSA during the subacute phase of ACS. We hypothesize that SGMT will reduce (1) the plasma levels of N-terminal pro brain natriuretic peptide and suppression of tumorigenicity 2; (2) the estimated 10-year risk of cardiovascular mortality as measured by the European Systematic Coronary Risk Evaluation (SCORE) algorithm; and (3) the cardiovascular event rate during a 3-year follow-up, compared with standard therapy. In the SGMT trial, 180 patients presenting with ACS will be randomly assigned to SGMT (n = 90) and standard therapy (n = 90) groups. Both groups will receive guideline-mandated treatment for ACS. Those assigned to SGMT will additionally undergo a sleep study and, if OSA is diagnosed, attend a multidisciplinary OSA clinic where they will receive personalized treatment including continuous positive airway pressure and behavioral/lifestyle counseling. The primary endpoint is the plasma N-terminal pro brain natriuretic peptide concentration at 7-month follow-up. This report presents the baseline characteristics of 117 patients (SGMT group: n =54; standard therapy group: n =63) who had been enrolled into the study as of August 31, 2017. The results of this trial will help us to understand whether active OSA diagnosis and treatment will improve the physiologic and clinical cardiovascular outcomes of this group of patients.

National survey of outcomes and practices in acute respiratory distress syndrome in Singapore.

INTRODUCTION: In the past 20 years, our understanding of acute respiratory distress syndrome (ARDS) management has improved, but the worldwide incidence and current outcomes are unclear. The reported incidence is highly variable, and no studies specifically characterise ARDS epidemiology in Asia. This observation study aims to determine the incidence, mortality and management practices of ARDS in a high income South East Asian country. METHODS: We conducted a prospective, population based observational study in 6 public hospitals. During a one month period, we identified all ARDS patients admitted to public hospital intensive care units (ICU) in Singapore, according to the Berlin definition. Demographic information, clinical management data and ICU outcome data was collected. RESULTS: A total of 904 adult patients were admitted to ICU during the study period and 15 patients met ARDS criteria. The unadjusted incidence of ARDS was 4.5 cases per 100,000 population, accounting for 1.25% of all ICU patients. Most patients were male (75%), Chinese (62%), had pneumonia (73%), and were admitted to a Medical ICU (56%). Management strategies varied across all ICUs. In-hospital mortality was 40% and median length of ICU stay was 7 days. CONCLUSION: The incidence of ARDS in a developed S.E Asia country is comparable to reported rates in European studies.

Agreement of noninvasive tear break-up time measurement between Tomey RT-7000 Auto Refractor-Keratometer and Oculus Keratograph 5M.

BACKGROUND: It is difficult to standardize assessment of dry eye in different clinical settings. Increasingly, tear stability is recognized to be important for the definition and assessment of patients with dry eye. Recently, two commercially available instruments have been made available for objectively measuring noninvasive tear break-up time (NIBUT), as an indicator of tear stability: the Tomey RT-7000 Auto Refractor-Keratometer and Oculus Keratograph (K)5M. We aim to assess the agreement of NIBUT measurements using these modalities. METHODS: This prospective cross-sectional study was carried out in a tertiary referral eye center and involved 126 consecutive dry eye patients. NIBUT assessment was performed on the right eyes of participants with both the RT-7000 and the K5M techniques, with the order of assessment randomized. The Standardized Patient Evaluation of Eye Dryness (SPEED) questionnaires were administered to assess dry eye symptoms in the 2 weeks before assessment. RESULTS: The age of the participants was 56.0±14.3 years (69.84% females). Measurements for both modalities were non-normally distributed (right-skewed). The median RT-7000 and K5M readings were 4.2 (range 0.1-10.0) and 6.4 (0.1-24.9) seconds, respectively. RT-7000 and K5M readings were poorly correlated (ρ=0.061, P=0.495). Intraclass correlation coefficient between the modalities was 0.187 (95% confidence interval -0.097 to 0.406). The Bland-Altman plot showed no systematic differences between the readings with these machines. The agreement between machines was not different in different SPEED categories. CONCLUSION: While there are theoretical and practical benefits of NIBUT for assessment of tear stability over dye-based methods, the agreement between the two modalities was poor. Hence, studies and trials assessing NIBUT should avoid using these modalities interchangeably for NIBUT assessment. More research is needed to improve consensus on how to determine NIBUT.