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It is no secret that a significant part of scientific research is difficult to reproduce. Here, the authors present a cloud-computing platform called ORCESTRA that facilitates reproducible processing of multimodal biomedical data using customizable pipelines and well-documented data objects.

Abstract DUSP22, an atypical dual-specificity phosphatase enzyme, plays a significant role in regulating multiple kinase signaling pathways by dephosphorylation. Our study demonstrated that decreased DUSP22 expression is associated with shorter disease-free survival, advanced TNM (tumor, lymph nodes, and metastasis), cancer stage, and higher tumor grade in lung adenocarcinoma (LUAD) patients. Exogenous DUSP22 expression reduces the colony-forming capacity of lung cancer cells and inhibits xenograft tumor growth primarily by targeting EGFR and suppressing its activity through dephosphorylation. Knockdown of DUSP22 using shRNA enhances EGFR dependency in HCC827 lung cancer cells and increases sensitivity to gefitinib, an EGFR inhibitor. Consistently, genetic deletion of DUSP22 enhances EGFRdel (exon 19 deletion)-driven lung tumorigenesis and elevates EGFR activity. Pharmacological inhibition of DUSP22 activates EGFR, ERK1/2, and upregulates downstream PD-L1 expression. Additionally, lentiviral deletion of DUSP22 by shRNA enhances lung cancer cell migration through EGFR/c-Met and PD-L1-dependent pathways. Gefitinib, an EGFR inhibitor, mechanistically suppresses migration induced by DUSP22 deletion and inhibits c-Met activity. Furthermore, cabozantinib, a c-Met inhibitor, reduces migration and attenuates EGFR activation caused by DUSP22 deletion. Collectively, our findings support the hypothesis that loss of DUSP22 function in lung cancer cells confers a survival advantage by augmenting EGFR signaling, leading to increased activation of downstream c-Met, ERK1/2, and PD-L1 axis, ultimately contributing to the progression of advanced lung cancer.

Background and purpose: Dose-escalation in rectal cancer (RCa) may result in an increased complete response rate and thereby enable omission of surgery and organ preservation. In order to implement dose-escalation, it is crucial to develop a technique that allows for accurate image-guided radiotherapy. The aim of the current study was to determine the performance of a novel liquid fiducial marker (BioXmark®) in RCa patients during the radiotherapy course by assessing its positional stability on daily cone-beam CT (CBCT), technical feasibility, visibility on different imaging modalities and safety. Materials and methods: Prospective, non-randomized, single-arm feasibility trial with inclusion of twenty patients referred for neoadjuvant chemoradiotherapy for locally advanced RCa. Primary study endpoint was positional stability on CBCT. Furthermore, technical aspects, safety and clinical performance of the marker, such as visibility on different imaging modalities, were evaluated. Results: Seventy-four markers from twenty patients were available for analysis. The marker was stable in 96% of the cases. One marker showed clinically relevant migration, one marker was lost before start of treatment and one marker was lost during treatment. Marker visibility was good on computed tomography (CT) and CBCT, and moderate on electronic portal imaging (EPI). Marker visibility on magnetic resonance imaging (MRI) was poor during response evaluation. Conclusion: The novel liquid fiducial marker demonstrated positional stability. We provide evidence of the feasibility of the novel fiducial marker for image-guided radiotherapy on daily cone beam CT for RCa patients.

Background & Aims: Gallbladder enlargement is common in patients with primary sclerosing cholangitis (PSC). The gallbladder may confer hepatoprotection against bile acid overload, through the sequestration and cholecystohepatic shunt of bile acids. The aim of this study was to assess the potential impact of the gallbladder on disease features and bile acid homeostasis in PSC. Methods: Patients with PSC from a single tertiary center who underwent liver MRI with three-dimensional cholangiography and concomitant analyses of serum bile acids were included. Gallbladder volume was measured by MRI and a cut-off of 50 ml was used to define gallbladder enlargement. Bile acid profiles and PSC severity, as assessed by blood tests and MRI features, were compared among patients according to gallbladder size (enlarged vs. normal-sized) or presence (removed vs. conserved). The impact of cholecystectomy was also assessed in the Abcb4 knockout mouse model of PSC. Results: Sixty-one patients with PSC, all treated with ursodeoxycholic acid (UDCA), were included. The gallbladder was enlarged in 30 patients, whereas 11 patients had been previously cholecystectomized. Patients with enlarged gallbladders had significantly lower alkaline phosphatase, a lower tauro-vs. glycoconjugate ratio and a higher UDCA vs. total bile acid ratio compared to those with normal-sized gallbladders. In addition, gallbladder volume negatively correlated with the hydrophobicity index of bile acids. Cholecystectomized patients displayed significantly higher aspartate aminotransferase and more severe bile duct strictures and dilatations compared to those with conserved gallbladder. In the Abcb4 knockout mice, cholecystectomy caused an increase in hepatic bile acid content and in circulating secondary bile acids, and an aggravation in cholangitis, inflammation and liver fibrosis. Conclusion: Altogether, our findings indicate that the gallbladder fulfills protective functions in PSC. Impact and implications: In patients with primary sclerosing cholangitis (PSC), gallbladder status impacts on bile acid homeostasis and disease features. We found evidence of lessened bile acid toxicity in patients with PSC and enlarged gallbladders and of increased disease severity in those who were previously cholecystectomized. In the Abcb4 knockout mouse model of PSC, cholecystectomy causes an aggravation of cholangitis and liver fibrosis. Overall, our results suggest that the gallbladder plays a protective role in PSC.

Background and Aims: Apolipoprotein A1 (A1) and haptoglobin (HP) serum levels are associated with the spread and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We have constructed and validated a multivariable risk calculator (A1HPV6) integrating A1, HP, alpha2-macroglobulin, and gamma glutamyl transferase to improve the performances of virological biomarkers. Methods: In a prospective observational study of hospitalized patients with nonsevere SARS-CoV-2 infection, A1HPV6 was constructed in 127 patients and validated in 116. The specificity was assessed in 7482 controls representing the general population. The primary diagnostic endpoint was the area under the receiver operating characteristic curve in patients with positive SARS-CoV-2 PCR. The primary prognostic endpoint was the age-and-sex adjusted risk of A1HPV6 to predict patients with WHO-stage > 4 (W > 4) severity. We assessed the kinetics of the A1HPV6 components in a nonhuman primate model (NHP), from baseline to 7 days (D7) after SARS-CoV-2 infection. Results: The area under the receiver operating characteristic curve for A1HPV6 was 0.99 (95% CI 0.97–0.99) in the validation subset, which was not significantly different from that in the construction subset, 0.99 (0.99–0.99; P = .80), like for sensitivity 92% (85–96) vs 94% (88–97; P = .29). A1HPV6 was associated with W > 4, with a significant odds ratio of 1.3 (1.1–1.5; 0.002). In NHP, A1 levels decreased (P < .01) at D2 and normalized at D4; HP levels increased at D2 and peaked at D4. In patients, A1 concentration was very low at D2 vs controls (P < .01) and increased at D14 (P < .01) but was still lower than controls; HP increased at D2 and remained elevated at D14. Conclusion: These results validate the diagnostic and prognostic performances of A1HPV6. Similar kinetics of apolipoprotein A1, HP, and alpha-2-macroglobulin were observed in the NHP model. ClinicalTrials.gov number, NCT01927133.

Background and Aims: The liver cancer risk test (LCR1-LCR2) is a multianalyte blood test combining proteins involved in liver cell repair (apolipoprotein A1, haptoglobin), hepatocellular carcinoma (HCC) risk factors (gender, age, gamma glutamyl transpeptidase), a marker of fibrosis (alpha2-macroglobulin), and alpha-fetoprotein, a specific marker of HCC. The aim was to externally validate LCR1-LCR2 in hepatitis B. Methods: Preincluded patients were from the Hepather cohort, a multicenter, multiethnic prospective study in 6071 patients. The coprimary study outcome was the negative predictive value of LCR1-LCR2 at 5 years for the occurrence of HCC and survival without HCC according to the predetermined LCR1-LCR2 cutoffs, adjusted for risk covariables and for chronic hepatitis B treatment and quantified using time-dependent Cox proportional hazards models. A post hoc analysis compared the number of patients needed to screen one cancer by LCR1-LCR2 and PAGE-B. Results: A total of 3520 patients, 191 (5.4%) with cirrhosis, with at least 1 year of follow-up were included. A total of 76 HCCs occurred over a median (interquartile range) of 6.0 years (4.8–7.3) of follow-up. Among the 3367 patients with low-risk LCR1-LCR2, the 5-year negative predictive value was 99.3% (95% confidence interval = 99.0–99.6), with a significant Cox hazard ratio (6.4, 3.1–13.0; P < .001) obtained after adjustment for exposure to antivirals, age, gender, geographical origin, HBe-Ag status, alcohol consumption, and type-2 diabetes. LCR1-LCR2 outperformed PAGE-B for number of patients needed to screen mean (95% CI), 8.5 (3.2–8.1) vs 26.3 (17.5–38.5; P < .0001), respectively. Conclusion: The performance of LCR1-LCR2 to identify patients with chronic hepatitis B at very low risk of HCC at 5 years was externally validated. ClinicalTrials.gov: NCT01953458.

Summary: Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/β-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.

Abstract Background ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown. Methods To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways. Results ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine1101 in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27S82 and eIF2aS51, and triggers autophagosome formation in lung cancer cells. Conclusions These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.

Abstract Objective Antitumor viral vaccines, and more particularly poxviral vaccines, represent an active field for clinical development and translational research. To improve the efficacy and treatment outcome, new viral vectors are sought, with emphasis on their abilities to stimulate innate immunity, to display tumor antigens and to induce a specific T‐cell response. Methods We screened for a new poxviral backbone with improved innate and adaptive immune stimulation using IFN‐α secretion levels in infected PBMC cultures as selection criteria. Assessment of virus effectiveness was made in vitro and in vivo. Results The bovine pseudocowpox virus (PCPV) stood out among several poxviruses for its ability to induce significant secretion of IFN‐α. PCPV produced efficient activation of human monocytes and dendritic cells, degranulation of NK cells and reversed MDSC‐induced T‐cell suppression, without being offensive to activated T cells. A PCPV‐based vaccine, encoding the HPV16 E7 protein (PCPV‐E7), stimulated strong antigen‐specific T‐cell responses in TC1 tumor‐bearing mice. Complete regression of tumors was obtained in a CD8+ T‐cell‐dependent manner after intratumoral injection of PCPV‐E7, followed by intravenous injection of the cancer vaccine MVA‐E7. PCPV also proved active when injected repeatedly intratumorally in MC38 tumor‐bearing mice, generating tumor‐specific T‐cell responses without encoding a specific MC38 antigen. From a translational perspective, we demonstrated that PCPV‐E7 effectively stimulated IFN‐γ production by T cells from tumor‐draining lymph nodes of HPV+‐infected cancer patients. Conclusion We propose PCPV as a viral vector suitable for vaccination in the field of personalised cancer vaccines, in particular for heterologous prime‐boost regimens.

Background & Aims: The Liver Cancer Risk test algorithm (LCR1-LCR2) is a multianalyte blood test combining proteins involved in liver cell repair (apolipoprotein-A1 and haptoglobin), known hepatocellular carcinoma (HCC) risk factors (sex, age, and gamma-glutamyl transferase), a marker of fibrosis (alpha2-macroglobulin) and alpha-fetoprotein (AFP), a specific marker of HCC. The aim was to externally validate the LCR1-LCR2 in patients with chronic HCV (CHC) treated or not with antivirals. Methods: Pre-included patients were from the Hepather cohort, a multicentre prospective study in adult patients with CHC in France. LCR1-LCR2 was assessed retrospectively in patients with the test components and AFP, available at baseline. The co-primary study outcome was the negative predictive value (NPV) of LCR1-LCR2 for the occurrence of HCC at 5 years and for survival without HCC according to the predetermined LCR1-LCR2 cut-offs. The cut-offs were adjusted for risk covariables and for the response to HCV treatment, and were quantified using time-dependent proportional hazards models. Results: In total, 4,903 patients, 1,026 (21.9%) with baseline cirrhosis, were included in the study. Patients were followed for a median of 5.7 (IQR 4.2–11.3) years. A total of 3,788/4,903 (77.3%) patients had a sustained virological response. There were 137 cases of HCC at 5 years and 214 at the end of follow-up. HCC occurred at 5 years in 24/3,755 patients with low-risk LCR1-LCR2 compared with 113/1,148 patients with high-risk LCR1-LCR2. The NPV was 99.4% (95% CI 99.1–99.6). Similar findings (hazard ratio, 10.8; 95% CI, 8.1–14.3; p

Cytotoxic T lymphocytes (CTLs) are key players to eliminate tumorigenic or pathogen-infected cells using lytic granules (LG) and Fas ligand (FasL) pathways. Depletion of glucose leads to severely impaired cytotoxic function of CTLs. However, the impact of excessive glucose on CTL functions still remains largely unknown. Here we used primary human CD8+ T cells, which were stimulated by CD3/CD28 beads and cultured in medium either containing high glucose (HG, 25 mM) or normal glucose (NG, 5.6 mM). We found that in HG-CTLs, glucose uptake and glycolysis were enhanced, whereas proliferation remained unaltered. Furthermore, CTLs cultured in HG exhibited an enhanced CTL killing efficiency compared to their counterparts in NG. Unexpectedly, expression of cytotoxic proteins (perforin, granzyme A, granzyme B and FasL), LG release, cytokine/cytotoxic protein release and CTL migration remained unchanged in HG-cultured CTLs. Interestingly, additional extracellular Ca2+ diminished HG-enhanced CTL killing function. Our findings suggest that in an environment with excessive glucose, CTLs could eliminate target cells more efficiently, at least for a certain period of time, in a Ca2+-dependent manner.

Molecular photoswitches provide an extremely simple solution for solar energy conversion and storage. Here, the authors report on the assembly of an operational solar energy-storing organic-oxide hybrid interface, which consists of a tailor-made molecular photoswitch and an atomically-defined semiconducting oxide film.

The most typical expression of cystic fibrosis (CF)–related liver disease is a cholangiopathy that can progress to cirrhosis. We aimed to determine the potential impact of environmental and genetic factors on the development of CF‐related cholangiopathy in mice. Cystic fibrosis transmembrane conductance regulator (Cftr)−/− mice and Cftr+/+ littermates in a congenic C57BL/6J background were fed a high medium‐chain triglyceride (MCT) diet. Liver histopathology, fecal microbiota, intestinal inflammation and barrier function, bile acid homeostasis, and liver transcriptome were analyzed in 3‐month‐old males. Subsequently, MCT diet was changed for chow with polyethylene glycol (PEG) and the genetic background for a mixed C57BL/6J;129/Ola background (resulting from three backcrosses), to test their effect on phenotype. C57BL/6J Cftr−/− mice on an MCT diet developed cholangiopathy features that were associated with dysbiosis, primarily Escherichia coli enrichment, and low‐grade intestinal inflammation. Compared with Cftr+/+ littermates, they displayed increased intestinal permeability and a lack of secondary bile acids together with a low expression of ileal bile acid transporters. Dietary‐induced (chow with PEG) changes in gut microbiota composition largely prevented the development of cholangiopathy in Cftr−/− mice. Regardless of Cftr status, mice in a mixed C57BL/6J;129/Ola background developed fatty liver under an MCT diet. The Cftr−/− mice in the mixed background showed no cholangiopathy, which was not explained by a difference in gut microbiota or intestinal permeability, compared with congenic mice. Transcriptomic analysis of the liver revealed differential expression, notably of immune‐related genes, in mice of the congenic versus mixed background. In conclusion, our findings suggest that CFTR deficiency causes abnormal intestinal permeability, which, combined with diet‐induced dysbiosis and immune‐related genetic susceptibility, promotes CF‐related cholangiopathy.

Background & Aims: Low-phospholipid-associated cholelithiasis (LPAC) syndrome, a rare genetic form of intrahepatic cholelithiasis in adults, is still poorly understood. We report the results of the largest-ever case-control study of patients with LPAC syndrome aiming to assess the prevalence, clinical features, and comorbidities of the disease. Methods: We included all LPAC cases diagnosed between 2001 and 2016 in 11 French centres. Controls consisted of all patients who underwent a cholecystectomy for common gallstone disease in a single non-academic centre over 1 year. A logistic regression analysis was used to identify the clinical features associated with LPAC syndrome across several patient strata with increasing levels of diagnostic confidence. The ratio between the incident cases of LPAC syndrome and the total number of cholecystectomies for gallstones was used to assess the relative prevalence of the disease. Results: In this study, 308 cases and 206 controls were included. LPAC syndrome accounted for 0.5–1.9% of all patients admitted with symptomatic gallstone disease. Age at first symptoms

Abstract Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)‐based reporter, PalmGRET, is created to enable pan‐EP labeling ranging from exomeres (200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung‐tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC‐EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP‐specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.

Normothermic perfusion provides a means to rescue steatotic liver grafts, including by pharmacological defatting. In this study, we tested the potential of new drug combinations to trigger defatting in three human culture models, primary hepatocytes with induced steatosis, primary hepatocytes isolated from steatotic liver, and precision-cut liver slices (PCLS) of steatotic liver. Forskolin, L-carnitine and a PPARα agonist were all combined with rapamycin, an immunosuppressant that induces autophagy, in a D-FAT cocktail. D-FAT was tested alone or in combination with necrosulfonamide, an inhibitor of mixed lineage kinase domain like pseudokinase involved in necroptosis. Within 24 h, in all three models, D-FAT induced a decrease in triglyceride content by 30%, attributable to an upregulation of genes involved in free fatty acid β-oxidation and autophagy, and a downregulation of those involved in lipogenesis. Defatting was accompanied by a decrease in endoplasmic reticulum stress and in the production of reactive oxygen species. The addition of necrosulfonamide increased the efficacy of defatting by 8%-12% in PCLS, with a trend towards increased autophagy. In conclusion, culture models, notably PCLS, are insightful to design strategies for liver graft rescue. Defatting can be rapidly achieved by combinations of drugs targeting mitochondrial oxidative metabolism, macro-autophagy and lipogenesis.

Background and study aims Implementation of optical diagnosis of diminutive polyps may potentially increase the efficacy and cost-effectiveness of colonoscopies. To adopt such strategy in clinical practice, the Preservation and Incorporation of Valuable endoscopic Innovations (PIVI) thresholds provide the basis to be met: ≥ 90 % negative predictive value (NPV) for diagnosis of adenomatous histology and ≥ 90 % agreement on surveillance intervals. We evaluated this within the Dutch Bowel Cancer Screening Program (BCSP). Patients and methods Endoscopic and histological data were collected from participants of the national bowel cancer screening program with an unfavorable fecal immunochemical test referred for colonoscopy between February 2014 and August 2015 at four endoscopy centers. The “resect and discard” scenario was studied, resecting diminutive polyps without histological evaluation. Agreement between optical diagnosis and histological diagnosis was measured for surveillance intervals according to Dutch, European and American post-polypectomy surveillance guideline. Results Fifteen certified endoscopists participated in this study and included 3028 diminutive polyps. In 2,330 patients both optical and histological diagnosis were available. Optical diagnosis of diminutive polyps showed NPV of 84 % (95 % CI 80–87) for adenomatous histology in the rectosigmoid. Applying the ‘resect and discard’ strategy resulted in 90.6 %, 91.2 %, 90.9 % agreement on surveillance intervals for the Dutch, European and American guideline respectively. Conclusion Our data representing current clinical practice in the Dutch BCSP practice on optical diagnosis of diminutive polyps showed that accuracy of predicting histology remains challenging, and risk of incorrect optical diagnosis is still significant. Therefore, it is too early to safely implement these strategies.

BackgroundSince 1920, a decrease in serum cholesterol has been identified as a marker of severe pneumonia. We have assessed the performance of serum apolipoprotein-A1, the main transporter of HDL-cholesterol, to identify the early spread of coronavirus disease 2019 (Covid-19) in the general population and its diagnostic performance for the Covid-19.MethodsWe compared the daily mean serum apolipoprotein-A1 during the first 34 weeks of 2020 in a population that is routinely followed for a risk of liver fibrosis risk in the USA (212,297 serum) and in France (20,652 serum) in relation to a local increase in confirmed cases, and in comparison to the same period in 2019 (266,976 and 28,452 serum, respectively). We prospectively assessed the sensitivity of this marker in an observational study of 136 consecutive hospitalized cases and retrospectively evaluated its specificity in 7,481 controls representing the general population.ResultsThe mean serum apolipoprotein-A1 levels in the survey populations began decreasing in January 2020, compared to the same period in 2019. This decrease was highly correlated with the daily increase in confirmed Covid-19 cases in the following 34 weeks, both in France and USA, including the June and mid-July recovery periods in France. Apolipoprotein-A1 at the 1.25 g/L cutoff had a sensitivity of 90.6% (95%CI84.2-95.1) and a specificity of 96.1% (95.7-96.6%) for the diagnosis of Covid-19. The area under the characteristics curve was 0.978 (0.957-0.988), and outperformed haptoglobin and liver function tests. The adjusted risk ratio of apolipoprotein-A1 for survival without transfer to intensive care unit was 5.61 (95%CI 1.02-31.0; P = 0.04).ConclusionApolipoprotein-A1 could be a sentinel of the pandemic in existing routine surveillance of the general population. NCT01927133, CER-2020-14.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

Abstract Lung cancer is the most common cause of cancer-related death worldwide, less than 7% of patients survive 10 years following diagnosis across all stages of lung cancer. Late stage of diagnosis and lack of effective and personalized medicine reflect the need for a better understanding of the mechanisms that underlie lung cancer progression. Quantitative proteomics provides the relative different protein abundance in normal and cancer patients which offers the information for molecular interactions, signaling pathways, and biomarker identification. Here we introduce both theoretical and practical applications in the use of quantitative proteomics approaches, with principles of current technologies and methodologies including gel-based, label free, stable isotope labeling as well as targeted proteomics. Predictive markers of drug resistance, candidate biomarkers for diagnosis, and prognostic markers in lung cancer have also been discovered and analyzed by quantitative proteomic analysis. Moreover, construction of protein networks enables to provide an opportunity to interpret disease pathway and improve our understanding in cancer therapeutic strategies, allowing the discovery of molecular markers and new therapeutic targets for lung cancer.

Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by Mycobacterium tuberculosis (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host's immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. In vitro analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, i.e. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.

The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase.

ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1) domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL), which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.

Chronic hepatitis C is a major cause of liver fibrosis and cirrhosis. It is generally accepted that inflammation that occurs in response to hepatocyte infection by the hepatitis C virus (HCV) is the main mechanism that triggers myofibroblast differentiation and stimulation in chronic hepatitis C. The aim of this study was to determine if HCV might infect human liver myofibroblasts (HLMF) and directly stimulate their fibrogenic activities.We evaluated the expression of the viral entry receptors, levels of HCV-RNA and HCV-protein and the expression of fibrosis markers in HLMF by using quantitative PCR, western blot and immunofluorescence analyses. Pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) were used to study the ability of HLMF to support viral entry, replication and fibrosis induction.We showed that HLMF expressed all known molecules of the HCV receptor complex, i.e. CD81, LDL-R, scavenger receptor-BI, claudin-1 and occludin. These cells were also permissive to HCVpp entry. Inoculation with HCVcc caused short-term infection of these cells, as shown by their content in positive- and negative-strand HCV RNA, in core and NS3 viral proteins, and by their release of core protein levels in the culture supernatants. HCV infection stimulated myofibroblastic differentiation, proliferation and collagen production in these cells. In addition, evidence of in vivo infection was provided by the detection of positive- and negative-strand HCV RNA in preparations of HLMF obtained from HCV-infected patients.These findings indicate that HCV infection of HLMF can occur and trigger extracellular matrix overproduction, thereby contributing to the development of HCV-related liver fibrosis.

Bacille Calmette-Guérin (BCG) vaccination of new born babies can protect children against tuberculosis (TB), but fails to protect adults consistently against pulmonary TB underlying the urgent need to develop novel TB vaccines. Majority of first generation TB vaccine candidates have relied on a very limited number of antigens typically belonging to the active phase of infection. We have designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara virus (MVA). Up to fourteen antigens representative of the three phases of TB infection (active, latent and resuscitation) were inserted into MVA. Using three different strains of mouse (BALB/c, C57BL/6 and C3H/HeN), we show that a single vaccination results in induction of both CD4 and CD8 T cells, displaying capacity to produce multiple cytokines together with cytolytic activity targeting a large array of epitopes. As expected, dominance of responses was linked to the mouse haplotype although for a given haplotype, responses specific of at least one antigen per phase could always be detected. Vaccination of non-human primates with the 14 antigens MVA-TB candidate resulted in broad and potent cellular-based immunogenicity. The remarkable plasticity of MVA opens the road to development of a novel class of highly complex recombinant TB vaccines to be evaluated in both prophylactic and therapeutic settings.

La méthode d'analyse des acides aminés du vin par chromatographie gaz-liquide mise au point en 1979 par LHUGUENOT reste intéressante, bien que la tendance actuelle soit l'utilisation de la chromatographie liquide haute performance (SANDERS,1985; TRICARD, 1986). Cette technique également rapide est toutefois coûteuse. Il nous a semblé opportun d'améliorer la méthode LHUGUENOT, en mettant à profit les développements récents en chromatographie gaz-liquide.

The study of radical stress in the biological environment needs a comprehensive vision of all possible reactive species and their mechanisms. Among them, reductive stress is evaluated for its selective target of sulfur-containing compounds. The selective attack of reducing species like H• atoms or eaq?/H+ to sulfur-containing amino acid residues has been proved in different substrates, peptides and proteins. The transformations include methionine to ?-aminobutyric acid and cysteine/cystine residues to alanine, as recognized in several sequences so far, such as RNase A, lysozyme, Met-enkephalin, amyloid ?-peptide and metallothioneins. The amino acid desulfurization is accompanied by the formation of low-molecular-weight sulfur-centered radicals that may cause geometrical cis–trans isomerization of unsaturated fatty acid residues in lipid bilayer. Thus, tandem protein/lipid damage is accomplished. Progress in research has given us a more comprehensive overview of the protein modifications and their roles, and the chemical biology approach will make its vital contribution to the study of free radical reactions, linking chemistry to biology and medicine.

Methionine is a key amino acid that has numerous roles in essential vital processes. Moreover, methionine oxidation is biologically important during conditions of oxidative stress and represents an important step in the development of some severe pathologies. Considerable work has been performed to understand the mechanisms of one-electron oxidation of the Met-residue as a function of its proteic environment. The most important recent results obtained by means of time-resolved techniques (laser flash photolysis and pulse radiolysis) on model peptides containing single or multiple Met-residues and in selected naturally occurring peptides (Met-enkephalin and ?-amyloid peptide) and proteins (thioredoxin and calmodulin) have been reviewed.

La durée d'activité très limitée dans le temps des protéases du raisin est confirmée en vinification en blanc, mais une nouvelle activité protéolytique peut être caractérisée à 37 °C et à pH 3,7 dans le moût au cours de la fermentation alcoolique. Cette activité doit être attribuée à des protéases exocellulaires libérées dans le milieu des levures. Parmi les souches essayées S. bayanus a une activité protéolytique plus importante que S. cerevisiae, ce qui se traduit par l'obtention de vins plus riches en azote soluble, donc plus stables du point de vue protéique et vraisemblablement d'une meilleure fermentescibilité malolactique. +++ The short time of activity of grape proteolytic enzymes is confirmed in fermentation off skins. However, a new proteolytic activity has been characterized at 37 °C and at pH 3.7, occuring in alcoholic fermentation of the must. This activity comes from exocellular proteolytic enzymes released by the yeasts in the medium. Among tested strains S. bayanus has a proteolytic activity greater than S. cerevisiae; so the soluble nitrogen content of wines is higher. According to this, proteolytic stability is better as, probably, malolactic fermentation capabilities.