Bone morphogenetic protein 6-mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro.

Liver sinusoidal endothelial cell-derived bone morphogenetic protein 6 (BMP6) and the BMP6-small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte-endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.

Controlled attenuation parameter and alcoholic hepatic steatosis: Diagnostic accuracy and role of alcohol detoxification.

BACKGROUND & AIMS: Controlled attenuation parameter (CAP) is a novel non-invasive measure of hepatic steatosis, but it has not been evaluated in alcoholic liver disease. Therefore, we aimed to validate CAP for the assessment of biopsy-verified alcoholic steatosis and to study the effect of alcohol detoxification on CAP. METHODS: This was a cross-sectional biopsy-controlled diagnostic study in four European liver centres. Consecutive alcohol-overusing patients underwent concomitant CAP, regular ultrasound, and liver biopsy. In addition, we measured CAP before and after admission for detoxification in a separate single-centre cohort. RESULTS: A total of 562 patients were included in the study: 269 patients in the diagnostic cohort with steatosis scores S0, S1, S2, and S3 = 77 (28%), 94 (35%), 64 (24%), and 34 (13%), respectively. CAP diagnosed any steatosis and moderate steatosis with fair accuracy (area under the receiver operating characteristic curve [AUC] ≥S1 = 0.77; 0.71-0.83 and AUC ≥S2 = 0.78; 0.72-0.83), and severe steatosis with good accuracy (AUC S3 = 0.82; 0.75-0.88). CAP was superior to bright liver echo pattern by regular ultrasound. CAP above 290 dB/m ruled in any steatosis with 88% specificity and 92% positive predictive value, while CAP below 220 dB/m ruled out steatosis with 90% sensitivity, but 62% negative predictive value. In the 293 patients who were admitted 6.3 days (interquartile range 4-6) for detoxification, CAP decreased by 32 ±â€¯47 dB/m (p <0.001). Body mass index predicted higher CAP in both cohorts, irrespective of drinking pattern. Obese patients with body mass index ≥30 kg/m2 had a significantly higher CAP, which did not decrease significantly during detoxification. CONCLUSIONS: CAP has a good diagnostic accuracy for diagnosing severe alcoholic liver steatosis and can be used to rule in any steatosis. In non-obese but not in obese, patients, CAP rapidly declines after alcohol withdrawal. LAY SUMMARY: CAP is a new ultrasound-based technique for measuring fat content in the liver, but has never been tested for fatty liver caused by alcohol. Herein, we examined 562 patients in a multicentre setting. We show that CAP highly correlates with liver fat, and patients with a CAP value above 290 dB/m were highly likely to have more than 5% fat in their livers, determined by liver biopsy. CAP was also better than regular ultrasound for determining the severity of alcoholic fatty-liver disease. Finally, we show that three in four (non-obese) patients rapidly decrease in CAP after short-term alcohol withdrawal. In contrast, obese alcohol-overusing patients were more likely to have higher CAP values than lean patients, irrespective of drinking.

Sensitive and non-invasive assessment of hepatocellular iron using a novel room-temperature susceptometer.

BACKGROUND & AIMS: Liver iron accumulates in various chronic liver diseases where it is an independent factor for survival and carcinogenesis. We tested a novel room-temperature susceptometer (RTS) to non-invasively assess liver iron concentration (LIC). METHODS: Two hundred and sixty-four patients with or without signs of iron overload or liver disease were prospectively enrolled. Thirty-five patients underwent liver biopsy with semiquantitative iron determination (Prussian Blue staining), atomic absorption spectroscopy (AAS, n=33), or magnetic resonance imaging (MRI, n=15). RESULTS: In vitro studies demonstrated a highly linear (r2=0.998) association between RTS-signal and iron concentration, with a detection limit of 0.3mM. Using an optimized algorithm, accounting for the skin-to-liver capsule distance, valid measurements could be obtained in 84% of cases. LIC-RTS showed a significant correlation with LIC-AAS (r=0.74, p<0.001), LIC-MRI (r=0.64, p<0.001) and hepatocellular iron (r=0.58, p<0.01), but not with macrophage iron (r=0.32, p=0.30). Normal LIC-RTS was 1.4mg/g dry weight. Besides hereditary and transfusional iron overload, LIC-RTS was also significantly elevated in patients with alcoholic liver disease. The areas under the receiver operating characteristic curve (AUROC) for grade 1, 2 and 3 hepatocellular iron overload were 0.72, 0.89 and 0.97, respectively, with cut-off values of 2.0, 4.0 and 5.0mg/g dry weight. Notably, the positive and negative predictive values, sensitivity, specificity and accuracy of severe hepatic iron overload (HIO) (grade ≥2) detection, were equal to AAS and superior to all serum iron markers. Depletion of hepatic iron could be efficiently monitored upon phlebotomy. CONCLUSIONS: RTS allows for the rapid and non-invasive measurement of LIC. In comparison to MRI, it could be a cost-effective bedside method for LIC screening. Lay summary: Novel room-temperature susceptometer (RTS) allows for the rapid, sensitive, and non-invasive measurement of liver iron concentration. In comparison to MRI, it could be a cost-effective bedside method for liver iron concentration screening.

Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Return to work and clinical outcome after open wedge HTO.

PURPOSE: The purpose of this study was to examine the incapacity of work related to work load according to the classification that has been introduced by the REFA Association and the clinical outcome after open wedge HTO with autologous bone graft from the iliac crest. METHODS: A total of 32 patients who were employed and able to work at the time of the surgery as well as treated with an open wedge HTO with the LC-DCP and autologous bone wedges from the iliac crest could be included in the radiological and clinical examination (77 months, SD ± 19). Postoperative duration of the incapacity of work and subjective ratings were based on the information provided by the patients themselves. The German classification that has been established by the REFA Association was used to classify the work load. Several clinical scores were used for clinical assessment. RESULTS: The duration of incapacity of work (median, 87 days; range, 14-450) demonstrated a relation to work load according to REFA. The Lysholm score, the HSS score and the score according to Lequesne augmented by 19.2 ± 16.8 (p < 0.0001), 15.6 ± 13.2 (p < 0.0001) and -6.0 ± 5.1 (p < 0.0001), respectively. The Tegner score gained from median 3 (range, 1-5) to 4 (range, 1-8). CONCLUSION: In this study, a relation was found between work load divided into different categories according to the classification established by the REFA Association and the duration of incapacity of work after open wedge HTO. An improvement of all clinical scores was observed. Typical neurological complications after autologous bone transplantation from the iliac crest were observed in 19% of our patient population. LEVEL OF EVIDENCE: IV.

Digital planning of high tibial osteotomy. Interrater reliability by using two different software.

PURPOSE: The purpose of the study was to determine the interrater reliability as well as the correlation of mediCAD(®) and PreOPlan(®) in deformity analysis and digital planning of osteotomies. METHODS: Digital radiographs were obtained from 81 patients planned to undergo an open wedge high tibial osteotomy. The JPEG files of the radiographs were imported to landmark-based software. Deformity analysis and planning of correction were performed by 1 experienced and 2 unexperienced observers. Osteotomy planning was aimed at correction to the predefined mechanical tibiofemoral angle of 3° valgus leg alignment. The interrater reliability of measurements was assessed using intraclass correlation coefficients (ICCs) and the confidence interval. RESULTS: The ICC of PreOPlan(®) was from 0.841 (mechanical lateral distal femur angle) to 0.993 (wedge-angle) and from 0.896 (joint line convergence angle) to 0.995 (mechanical tibiofemoral angle) of mediCAD(®). The ICC of height of wedge-base was 0.979 with PreOPlan(®) and 0.969 with mediCAD(®). Comparing PreOPlan(®) and mediCAD(®), the ICC of the height of wedge-base of the observers was 0.966, 0.956 and 0.969, respectively. CONCLUSIONS: The results show a high interrater reliability of digital planning software. Experience of the observer had no influence on results. Furthermore, a high interrater reliability and correlation of digital planning specific parameters was found. Surgeons need to master limb geometry measurements and osteotomy planning on digital radiographs as digital planning reports are used for intercolleagual correspondence, teaching purposes and as medicolegal documents. The digital planning software tested agrees with the actual demands and could be recommended for deformity analysis and planning of osteotomies. LEVEL OF EVIDENCE: Diagnostic studies, Level I.

Liver stiffness in pregnant women with intrahepatic cholestasis of pregnancy: A case control study.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is a rare but severe complication for both the mother and the unborn child. The diagnosis is primarily based on elevated serum levels of bile acids. In a large ICP cohort, we here study in detail liver stiffness (LS) using transient elastography (TE), now widely used to non-invasively screen for liver cirrhosis within minutes. AIM: To specifically explore LS in a large cohort of women with ICP compared to a control group with uncomplicated pregnancy. METHODS: LS and hepatic steatosis marker controlled attenuation parameter (CAP) were measured in 100 pregnant women with ICP using TE (Fibroscan, Echosens, Paris, France) between 2010 and 2020. In 17 cases, LS could be measured postpartum. 450 women before and 38 women after delivery with uncomplicated pregnancy served as control group. Routine laboratory, levels of bile acids and apoptosis marker caspase-cleaved cytokeratin 18 fragment (M30) were also measured. RESULTS: Women with ICP had significantly elevated transaminases but normal gamma-glutamyl transferase (GGT). Mean LS was significantly increased at 7.3 ± 3.0 kPa compared to the control group at 6.2 ± 2.3 kPa (P < 0.0001). Postpartum LS decreased significantly in both groups but was still higher in ICP (5.8 ± 1.7 kPa vs 4.2 ± 0.9 kPa, P < 0.0001), respectively. In ICP, LS was highly significantly correlated with levels of bile acids and M30 but not transaminases. No correlation was seen with GGT that even increased significantly after delivery in the ICP group. Bile acids were mostly correlated with the liver apoptosis marker M30, LS and levels of alanine aminotransferase, aspartate aminotransferase, and bilirubin. In multivariate analysis, LS remained the sole parameter that was independently associated with elevated bile acids. CONCLUSION: In conclusion, LS is significantly elevated in ICP which is most likely due to toxic bile acid accumulation and hepatocyte apoptosis. In association with conventional laboratory markers, LS provides additional non-invasive information to rapidly identify women at risk for ICP.

Confounders of Serum Phosphatidylethanol: Role of Red Blood Cell Turnover and Cirrhosis.

Purpose: Ethyl glucuronide (EtG), ethyl sulfate (EtS) and phosphatidylethanol (PEth) are considered specific direct biomarkers for detecting alcohol consumption. However, PEth, which is produced in red blood cells (RBC), varies considerably between patients for unknown reasons. We here studied various confounders of PEth elimination including fibrosis after alcohol withdrawal. Patients and Methods: EtG, EtS and PEth together with routine laboratory and clinical parameters were studied in 100 Caucasian heavy drinkers prior and after alcohol detoxification. In addition, fibrosis stage and degree of steatosis were assessed by transient elastography (Fibroscan, Echosens, Paris). Results: All three biomarkers were highly correlated (0.61-0.72) with initial serum alcohol levels, but only PEth correlated with daily alcohol consumption. After alcohol withdrawal, PEth significantly decreased within 6.1 days from 1708 to 810 ng/mL (half-life varied from 1.6 to 15.2 days). Both levels of serum alcohol but also EtG and EtS were higher in patients with liver cirrhosis as compared to patients without fibrosis despite comparable alcohol consumption suggesting a decreased alcohol elimination in patients with cirrhosis. PEth was also elevated in cirrhosis but not significantly. In contrast, PEth elimination rate was significantly higher in patients with enhanced RBC turnover and signs of alcohol-mediated hemolytic anemia with elevated ferritin, LDH and increased mean corpuscular volume (MCV). Conclusion: We here demonstrate that alcohol elimination is decreased in patients with liver cirrhosis. In patients with cirrhosis, PEth levels are both affected in opposite directions by enhanced red blood cell turnover and elevated alcohol levels. Our data have important implications for the use and interpretation of PEth in the clinical setting.

Non-invasive diagnosis of alcohol-related steatohepatitis in patients ongoing alcohol withdrawal based on cytokeratin 18 and transient elastography.

BACKGROUND AND AIMS: The diagnosis of alcoholic steatohepatitis (ASH) is based on liver biopsy, which is costly and invasive with non-negligible morbidity. The aim of this study was to evaluate the accuracy of circulating cytokeratin 18 M65 fragment (K18-M65) alone or in association with other markers for the non-invasive diagnosis of ASH in patients ongoing alcohol withdrawal. METHODS: This study examined the serum level of K18-M65 in a test cohort of 196 patients. All patients underwent liver biopsy, transient elastography (TE) and serum collection. The diagnostic accuracy of K18-M65 alone or combined with clinico-biological data was assessed and the best defined cut-offs were validated in an independent validation cohort of 58 patients. RESULTS: K18-M65 had an area under the curve (AUC) of 0.82 (test cohort) and 0.90 (validation cohort). Using two cut-off decision points, K18-M65 was able to classify 46.9% (test cohort) and 34.5% (validation cohort) of patients with 95% sensitivity or specificity. Combining K18-M65, alpha-2-macroglobulin, TE, body mass index, and age, we created a score allowing accurate diagnosis of ASH with an AUC of 0.93 (test cohort) and 0.94 (validation cohort). This new score was able to rule out or rule in the diagnosis of steatohepatitis for probability ≤0.135 or ≥0.667 respectively in more than two-thirds of patients. CONCLUSIONS: We propose a new validated non-invasive score for the diagnosis of ASH in patients ongoing alcohol withdrawal. This score can help to identify patients that may benefit from potential therapeutics or motivate them to reduce alcohol consumption.

The ileal neobladder in female patients with bladder cancer: long-term clinical, functional, and oncological outcome.

PURPOSE: To assess long-term clinical and oncological outcome in women undergoing radical cystectomy and ileal neobladder reconstruction for invasive bladder cancer. METHODS: From 1995 to 2010, a total of 121 women with clinically organ-confined urothelial carcinoma underwent radical cystectomy with an orthotopic ileal neobladder. Median follow-up was 56 months. Clinical course, functional, pathological, and oncological outcome of these women were analyzed. RESULTS: Seventy-six patients (62.8%) experienced a complication of some type within 90 days of the procedure. 56 patients (46.3%) experienced minor complications, whereas 20 patients (16.5%) experienced major complications. Pathological subgroups included 70 patients (57.9%) with organ confined, lymph node-negative tumors, 24 (19.8%) with extravesical, lymph node-negative disease and 27 (22.3%) patients with lymph node-positive disease. The 5-year overall survival rate in patients with organ-confined (≤pT2, pN0), locally advanced (≥pT3, pN0), and metastatic disease was 80.2%, 81.9%, and 45.1%, respectively. 4 women (3.3%) experienced a local (pelvic) recurrence. One patient presented with a urethral recurrence (0.8%). Daytime and nighttime urinary continence (0-1 pad) was reported by 82.4 and 76.5%, respectively. Clean intermittent self-catheterization was required by 58.0%. The retrospective study design was the major limitation of the study. CONCLUSIONS: Despite a considerable complication rate, radical cystectomy with orthotopic diversion in female patients with bladder cancer may be considered a standard therapeutic option for selected patients with excellent oncological outcome including a low incidence of local and urethral recurrence.

Novel Insights into Alcoholic Liver Disease: Iron Overload, Iron Sensing and Hemolysis.

The liver is the major target organ of continued alcohol consumption at risk and resulting alcoholic liver disease (ALD) is the most common liver disease worldwide. The underlying molecular mechanisms are still poorly understood despite decades of scientific effort limiting our abilities to identify those individuals who are at risk to develop the disease, to develop appropriate screening strategies and, in addition, to develop targeted therapeutic approaches. ALD is predestined for the newly evolving translational medicine, as conventional clinical and health care structures seem to be constrained to fully appreciate this disease. This concept paper aims at summarizing the 15 years translational experience at the Center of Alcohol Research in Heidelberg, namely based on the long-term prospective and detailed characterization of heavy drinkers with mortality data. In addition, novel experimental findings will be presented. A special focus will be the long-known hepatic iron accumulation, the somewhat overlooked role of the hematopoietic system and novel insights into iron sensing and the role of hepcidin. Our preliminary work indicates that enhanced red blood cell (RBC) turnover is critical for survival in ALD patients. RBC turnover is not primarily due to vitamin deficiency but rather to ethanol toxicity directly targeted to erythrocytes but also to the bone marrow stem cell compartment. These novel insights also help to explain long-known aspects of ALD such as mean corpuscular volume of erythrocytes (MCV) and elevated aspartate transaminase (GOT/AST) levels. This work also aims at identifying future projects, naming unresolved observations, and presenting novel hypothetical concepts still requiring future validation.

HYPK promotes the activity of the Nα-acetyltransferase A complex to determine proteostasis of nonAc-X2/N-degron-containing proteins.

In humans, the Huntingtin yeast partner K (HYPK) binds to the ribosome-associated Nα-acetyltransferase A (NatA) complex that acetylates ~40% of the proteome in humans and Arabidopsis thaliana. However, the relevance of HsHYPK for determining the human N-acetylome is unclear. Here, we identify the AtHYPK protein as the first in vivo regulator of NatA activity in plants. AtHYPK physically interacts with the ribosome-anchoring subunit of NatA and promotes Nα-terminal acetylation of diverse NatA substrates. Loss-of-AtHYPK mutants are remarkably resistant to drought stress and strongly resemble the phenotype of NatA-depleted plants. The ectopic expression of HsHYPK rescues this phenotype. Combined transcriptomics, proteomics, and N-terminomics unravel that HYPK impairs plant metabolism and development, predominantly by regulating NatA activity. We demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X2/N-degron. This N-degron targets many nonacetylated NatA substrates for degradation by the ubiquitin-proteasome system.

Cotranslational N-degron masking by acetylation promotes proteome stability in plants.

N-terminal protein acetylation (NTA) is a prevalent protein modification essential for viability in animals and plants. The dominant executor of NTA is the ribosome tethered Nα-acetyltransferase A (NatA) complex. However, the impact of NatA on protein fate is still enigmatic. Here, we demonstrate that depletion of NatA activity leads to a 4-fold increase in global protein turnover via the ubiquitin-proteasome system in Arabidopsis. Surprisingly, a concomitant increase in translation, actioned via enhanced Target-of-Rapamycin activity, is also observed, implying that defective NTA triggers feedback mechanisms to maintain steady-state protein abundance. Quantitative analysis of the proteome, the translatome, and the ubiquitome reveals that NatA substrates account for the bulk of this enhanced turnover. A targeted analysis of NatA substrate stability uncovers that NTA absence triggers protein destabilization via a previously undescribed and widely conserved nonAc/N-degron in plants. Hence, the imprinting of the proteome with acetylation marks is essential for coordinating proteome stability.

Non-invasive Biomarkers of Liver Inflammation and Cell Death in Response to Alcohol Detoxification.

INTRODUCTION: Alcohol-related liver disease (ALD) represents the most common liver disease worldwide, however, the underlying molecular mechanisms are still poorly understood. Namely centrilobular inflammation and programmed cell death are characteristic to ALD and it remains to be elucidated why they persist despite the absence of alcohol. AIMS: To study the effects of alcohol withdrawal in a cohort of heavy drinkers and the role of cirrhosis by using non-invasive biomarkers such as cytokines, apoptotic and angiogenic markers. METHODS: Caspase 3-cleaved M30, M65, cytokines (IL-6, IL-8), tumor necrosis factor alpha (TNF-α), transforming growth factor (TGF-ß) and vascular endothelial growth factor (VEGF) were measured in 114 heavy drinkers. The role of alcohol detoxification was investigated in 45 patients. The liver histology was available in 23 patients. Fibrosis stage and steatosis were assessed by measuring liver stiffness (LS) and controlled attenuation parameter (CAP) in all patients using transient elastography (FibroScan, Echosens, Paris). Mean observation interval between the measurements was 5.7 ± 1.4 days (mean + -SD). RESULTS: Patients consumed a mean of 204 ± 148 g/day alcohol with a heavy drinking duration of 15.3 ± 11.0 years. Mean LS was 20.7 ± 24.4 kPa and mean CAP was 303 ± 51 dB/m. Fibrosis distribution was F0-38.1%, F1-2-31%, F3-7.1 and F4-23.9%. Apoptotic markers M30 and M65 were almost five times above normal. In contrast, TNF- α a, IL-8 and VEGF were only slightly elevated. Patients with manifest liver cirrhosis (F4) had significantly higher levels of M30, M65, IL-6 and IL-8. Histology features such as hepatocyte ballooning, Mallory-Denk bodies, inflammation and fibrosis were all significantly associated with elevated LS, and serum levels of TNF-alpha, M30 and M65 but not with CAP and other cytokines. During alcohol detoxification, LS, transaminases, TGF- ß, IL-6, IL-8 and VEGF decreased significantly. In contrast, no significant changes were observed for M30, M65 and TNF- α and M30 even increased during detoxification in non-cirrhotic patients. Profibrogenic cytokine TGF-beta and pro-angiogenic cytokine VEGF showed a delayed decrease in patients with manifest cirrhosis. CONCLUSION: Patients with alcohol-related cirrhosis have a pronounced apoptotic activity and a distinct inflammatory response that only partly improves after 1 week of alcohol detoxification. Alcohol withdrawal may represent an important approach to better dissect the underlying mechanisms in the setting of alcohol metabolism.

H2O2-mediated autophagy during ethanol metabolism.

BACKGROUND: Alcoholic liver disease (ALD) is the most common liver disease worldwide and its underlying molecular mechanisms are still poorly understood. Moreover, conflicting data have been reported on potentially protective autophagy, the exact role of ethanol-metabolizing enzymes and ROS. METHODS: Expression of LC3B, CYP2E1, and NOX4 was studied in a mouse model of acute ethanol exposure by immunoblotting and immunohistochemistry. Autophagy was further studied in primary mouse hepatocytes and huh7 cells in response to ethanol and its major intermediator acetaldehyde. Experiments were carried out in cells overexpressing CYP2E1 and knock down of NOX4 using siRNA. The response to external H2O2 was studied by using the GOX/CAT system. Autophagic flux was monitored using the mRFP-GFP-LC3 plasmid, while rapamycin and chloroquine served as positive and negative controls. RESULTS: Acute ethanol exposure of mice over 24 h significantly induced autophagy as measured by LC3B expression but also induced the ROS-generating CYP2E1 and NOX4 enzymes. Notably, ethanol but not its downstream metabolite acetaldehyde induced autophagy in primary mouse hepatocytes. In contrast, autophagy could only be induced in huh7 cells in the presence of overexpressed CYP2E1. In addition, overexpression of NOX4 also significantly increased autophagy, which could be blocked by siRNA mediated knock down. The antioxidant N-acetylcysteine (NAC) also efficiently blocked CYP2E1-and NOX4-mediated induction of autophagy. Finally, specific and non-toxic production of H2O2 by the GOX/CAT system as evidenced by elevated peroxiredoxin (Prx-2) also induced LC3B which was efficiently blocked by NAC. H2O2 strongly increased the autophagic flux as measured by mRFP-GFP-LC3 plasmid. CONCLUSION: We here provide evidence that short-term ethanol exposure induces autophagy in hepatocytes both in vivo and in vitro through the generation of ROS. These data suggest that suppression of autophagy by ethanol is most likely due to longer alcohol exposure during chronic alcohol consumption with the accumulation of e.g. misfolded proteins.

The Acquisition of Culturally Patterned Attention Styles Under Active Inference.

This paper presents an active inference based simulation study of visual foraging. The goal of the simulation is to show the effect of the acquisition of culturally patterned attention styles on cognitive task performance, under active inference. We show how cultural artefacts like antique vase decorations drive cognitive functions such as perception, action and learning, as well as task performance in a simple visual discrimination task. We thus describe a new active inference based research pipeline that future work may employ to inquire on deep guiding principles determining the manner in which material culture drives human thought, by building and rebuilding our patterns of attention.

Cardio-microcurrent device for chronic heart failure: first-in-human clinical study.

AIMS: Most devices for treating ambulatory Class II and III heart failure are linked to electrical pulses. However, a steady electric potential gradient is also necessary for appropriate myocardial performance and may be disturbed by structural heart diseases. We investigated whether chronic application of electrical microcurrent to the heart is feasible and safe and improves cardiac performance. The results of this study should provide guidance for the design of a two-arm, randomized, controlled Phase II trial. METHODS AND RESULTS: This single-arm, non-randomized pilot study involved 10 patients (9 men; mean age, 62 ± 12 years) at two sites with 6 month follow-up. All patients had New York Heart Association (NYHA) Class III heart failure and non-ischaemic dilated cardiomyopathy, with left ventricular ejection fraction (LVEF) <35%. A device was surgically placed to deliver a constant microcurrent to the heart. The following tests were performed at baseline, at hospital discharge, and at six time points during follow-up: determination of LVEF and left ventricular end-diastolic/end-systolic diameter by echocardiography; the 6 min walk test; and assessment of NYHA classification and quality of life (36-Item Short-Form Health Survey questionnaire). Microcurrent application was feasible and safe; no device-related or treatment-related adverse events occurred. During follow-up, rapid and significant signal of efficacy (P < 0.005) was present with improvements in LVEF, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, and distance walked. For eight patients, NYHA classification improved from Class III to Class I (for seven, as early as 14 days post-operatively); for one, to Class II; and for one, to Class II/III. 36-Item Short-Form Health Survey questionnaire scores also improved highly significantly. CONCLUSIONS: Chronic application of microcurrent to the heart is feasible and safe and leads to a rapid and lasting improvement in heart function and a near normalization of heart size within days. The NYHA classification and quality of life improve just as rapidly.

Prospective Comparison of Transient Elastography Using Two Different Devices: Performance of FibroScan and FibroTouch.

PURPOSE: Transient elastography (TE) using FibroScan (FS) has been established to non-invasively assess liver fibrosis and steatosis. The aim of this study was to compare the recently introduced FibroTouch (FT) device with the established FS with respect to liver stiffness and CAP. PATIENTS AND METHODS: Thirty-nine patients with and without liver disease were included. All patients were measured three times with FS (FibroScan 530 compact, Echosens, France) and FT (FibroTouch-FT100, Wuxi Hisky Med, China). For FS, M and XL probe were used according to the manufacturer's specifications. For steatosis, CAP and the comparable FT equivalent UAP (ultrasound attenuation parameter) was determined. Finally, FT and FS were explored in liver tissue-mimicking phantoms. RESULTS: LS between FS and FT correlated well with r=0.91. Root-mean-square (RMS) of the coefficient of variation for LS was better in FS (11.1% vs 27.4%). Bland-Altman analysis showed a 3.1 kPa mean overestimation of LS by FT. In addition, UAP strongly and linearly depended on the BMI following UAP=3.02 × BMI+186. In phantoms, a similar relation was found with UAP (phantom)= 3.78 × BMI + 146 suggesting that UAP is directly calculated from entered BMI instead of assessing shear-wave attenuation. Consequently, RMS-CV was lower for FT (6.0% vs 9.7%). However, if using different BMI, CV-RMS for FT increased to 12.7%. LS of a patient with manifest liver cirrhosis and ascites was 38.8 kPa using the FS-XL probe but almost normal with FT (7.2 kPa). CONCLUSION: Although LS by FT shows good correlation with LS-FS, it has larger variation, continuously overestimates LS and completely fails in ascites. Moreover, FT-UAP seems to be a misleading parameter for steatosis assessment because it is at least in part calculated from mandatory entered patient data. In conclusion, novel LS cut-off values need to be defined for LS-FT and usage of UAP is not recommended.

Functional autoantibody diseases: Basics and treatment related to cardiomyopathies.

In the 1970s, autoantibodies directed against G-protein-coupled receptors (GPCR, GPCR-AAB) were discovered. After receptor binding, GPCR-AAB trigger uncontrolled receptor mediated signal cascades, thus producing pathologies. Diseases associated with such functionally active autoantibody type (functional autoantibodies) can be called "functional autoantibody diseases". Here we focus exclusively on GPCR-AAB directed against the GPCR's extracellular loops. The GPCR's role in the pathogenesis and progression is accepted in idiopathic dilated cardiomyopathy and is increasingly considered in diseases such as Chagas' cardiomyopathy, peripartum cardiomyopathy, hypertension, diabetes mellitus and scleroderma and even dementia, complex regional pain syndrome and postural orthostatic tachycardia syndrome. We briefly summarize the mechanistic background of GPCR-AAB induced pathologies, mainly focused on autoantibodies targeting the ß1-adrenergic and muscarinic 2 receptors, due to their importance for cardiomyopathies. Furthermore, treatment strategies for "functional autoantibody diseases", such as for GPCR-AAB removal (therapeutic plasma exchange, immunoadsorption) and in vivo GPCR-AAB attack (intravenous IgG treatment, B-cell depletion, GPCR-AAB in vivo binding and neutralization) are critically reflected with respect to their patient benefits focused on but not exclusive to patients with dilated cardiomyopathy.