ORF3c is expressed in SARS-CoV-2-infected cells and inhibits innate sensing by targeting MAVS.

Most SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While the majority of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. One example is the ORF3a sgRNA that also encodes ORF3c, an enigmatic 41-amino-acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2-infected cells and suppresses RIG-I- and MDA5-mediated IFN-ß induction. ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage, and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c, demonstrating that this reading frame is not essential for efficient viral replication in vivo and is likely compensated by other viral proteins. In agreement with this, disruption of ORF3c does not significantly affect SARS-CoV-2 replication in CaCo-2, CaLu-3, or Rhinolophus alcyone cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.

Treatment outcomes of childhood PICALM::MLLT10 acute leukaemias.

The prognostic impact of PICALM::MLLT10 status in childhood leukaemia is not well described. Ten International Berlin Frankfurt Münster-affiliated study groups and the Children's Oncology Group collaborated in this multicentre retrospective study. The presence of the PICALM::MLLT10 fusion gene was confirmed by fluorescence in situ hybridization and/or RNA sequencing at participating sites. Ninety-eight children met the study criteria. T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML) predominated 55 (56%) and 39 (40%) patients, respectively. Most patients received a chemotherapy regimen per their disease phenotype: 58% received an ALL regimen, 40% an AML regimen and 1% a hybrid regimen. Outcomes for children with PICALM::MLLT10 ALL were reasonable: 5-year event-free survival (EFS) 67% and 5-year overall survival (OS) 76%, but children with PICALM::MLLT10 AML had poor outcomes: 5-year EFS 22% and 5-year OS 26%. Haematopoietic stem cell transplant (HSCT) did not result in a significant improvement in outcomes for PICALM::MLLT10 AML: 5-year EFS 20% for those who received HSCT versus 23% for those who did not (p = 0.6) and 5-year OS 37% versus 36% (p = 0.7). In summary, this study confirms that PICALM::MLLT10 AML is associated with a dismal prognosis and patients cannot be salvaged with HSCT; exploration of novel therapeutic options is warranted.

Interactive exploration of adverse events and multimorbidity in CKD.

BACKGROUND AND HYPOTHESIS: Persons with chronic kidney disease (CKD) are at increased risk of adverse events, early mortality, and multimorbidity. A detailed overview of adverse event types and rates from a large CKD cohort under regular nephrological care is missing. We generated an interactive tool to enable exploration of adverse events and their combinations in the prospective, observational German CKD (GCKD) study. METHODS: The GCKD study enrolled 5217 participants under regular nephrological care with an estimated glomerular filtration rate of 30-60 or >60 mL/min/1.73m2 and an overt proteinuria. Cardio-, cerebro- and peripheral vascular, kidney, infection, and cancer events, as well as deaths were adjudicated following a standard operation procedure. We summarized these time-to-event data points for exploration in interactive graphs within an R shiny app. Multivariable adjusted Cox models for time to first event were fitted. Cumulative incidence functions, Kaplan-Meier curves and intersection plots were used to display main adverse events and their combinations by sex and CKD etiology. RESULTS: Over a median of 6.5 years, 10 271 events occurred in total and 680 participants (13.0%) died while 2947 participants (56.5%) experienced any event. The new publicly available interactive platform enables readers to scrutinize adverse events and their combinations as well as mortality trends as a gateway to better understand multimorbidity in CKD: incident rates per 1000 patient-years varied by event type, CKD etiology, and baseline characteristics. Incidence rates for the most frequent events and their recurrence were 113.6 (cardiovascular), 75.0 (kidney), and 66.0 (infection). Participants with diabetic kidney disease and men were more prone to experiencing events. CONCLUSION: This comprehensive explorative tool to visualize adverse events (https://gckd.diz.uk-erlangen.de/), their combination, mortality, and multimorbidity among persons with CKD may manifest as a valuable resource for patient care, identification of high-risk groups, health services, and public health policy planning.

Spot urinary sodium in CKD patients: correlation with 24h-excretion and evaluation of commonly used prediction equations.

BACKGROUND: Salt intake in CKD patients can affect cardiovascular risk and kidney disease progression. Twenty-four hour (24h) urine collections are often used to investigate salt metabolism but are cumbersome to perform. We assessed urinary sodium (U-Na) concentration in spot urine samples and investigated the correlation with 24h U-Na excretion and concentration in CKD patients under nephrological care. Further, we studied the role of CKD stage and diuretics and evaluated the performance of commonly used formulas for the prediction of 24h U-Na excretion from spot urine samples. METHODS: One hundred eight patients of the German Chronic Kidney Disease (GCKD) study were included. Each participant collected a 24h urine and two spot urine samples within the same period. The first spot urine sample (AM) was part of the second morning urine. The second urine sample was collected before dinner (PM). Patients were advised to take their medication as usual without changing dietary habits. U-Na concentrations in the two spot urine samples and their average ((AM + PM)/2) were correlated with U-Na concentration and total Na excretion in the 24h urine collections. Correlations were subsequently studied after stratification by CKD stage and diuretic intake. The usefulness of three commonly applied equations to estimate 24h U-Na excretion from spot urine samples (Kawasaki, Tanaka and Intersalt) was determined using Bland-Altman plots, analyses of sensitivity, specificity, as well as positive (PPV) and negative predictive values (NPV). RESULTS: Participants (42 women, 66 men) were on average (± SD) 62.2 (± 11.9) years old, with a mean serum creatinine of 1.6 (± 0.5) mg/dl. 95% had arterial hypertension, 37% diabetes mellitus and 55% were on diuretics. The best correlation with 24h U-Na total excretion was found for the PM spot U-Na sample. We also found strong correlations when comparing spot and 24h urine U-Na concentration. Correction of spot U-Na for U-creatinine did not improve strength of correlations. Neither CKD stage, nor intake of diuretics had significant impact on these correlations. All examined formulas revealed a significant mean bias. The lowest mean bias and the strongest correlation between estimated and measured U-Na excretion in 24h were obtained using the Tanaka-formula. Also, application of the Tanaka-formula with PM U-Na provided best sensitivity, specificity, PPV and NPV to estimate U-Na excretion > 4g/d corresponding to a salt consumption > 10g/d. CONCLUSION: U-Na concentration of spot urine samples correlated with 24h U-Na excretion especially when PM spot U-Na was used. However, correlation coefficients were relatively low. Neither CKD stage nor intake of diuretics appeared to have an influence on these correlations. There was a significant bias for all tested formulas with the Tanaka-formula providing the strongest correlation with measured 24h U-Na excretion. In summary, using spot urine samples together with the Tanaka-formula in epidemiological studies appears feasible to determine associations between approximate salt intake and outcomes in CKD patients. However, the usefulness of spot-urine samples to guide and monitor salt consumption in individual patients remains limited.

Major cardiovascular events and subsequent risk of kidney failure with replacement therapy: a CKD Prognosis Consortium study.

AIMS: Chronic kidney disease (CKD) increases risk of cardiovascular disease (CVD). Less is known about how CVD associates with future risk of kidney failure with replacement therapy (KFRT). METHODS AND RESULTS: The study included 25 903 761 individuals from the CKD Prognosis Consortium with known baseline estimated glomerular filtration rate (eGFR) and evaluated the impact of prevalent and incident coronary heart disease (CHD), stroke, heart failure (HF), and atrial fibrillation (AF) events as time-varying exposures on KFRT outcomes. Mean age was 53 (standard deviation 17) years and mean eGFR was 89 mL/min/1.73 m2, 15% had diabetes and 8.4% had urinary albumin-to-creatinine ratio (ACR) available (median 13 mg/g); 9.5% had prevalent CHD, 3.2% prior stroke, 3.3% HF, and 4.4% prior AF. During follow-up, there were 269 142 CHD, 311 021 stroke, 712 556 HF, and 605 596 AF incident events and 101 044 (0.4%) patients experienced KFRT. Both prevalent and incident CVD were associated with subsequent KFRT with adjusted hazard ratios (HRs) of 3.1 [95% confidence interval (CI): 2.9-3.3], 2.0 (1.9-2.1), 4.5 (4.2-4.9), 2.8 (2.7-3.1) after incident CHD, stroke, HF and AF, respectively. HRs were highest in first 3 months post-CVD incidence declining to baseline after 3 years. Incident HF hospitalizations showed the strongest association with KFRT [HR 46 (95% CI: 43-50) within 3 months] after adjustment for other CVD subtype incidence. CONCLUSION: Incident CVD events strongly and independently associate with future KFRT risk, most notably after HF, then CHD, stroke, and AF. Optimal strategies for addressing the dramatic risk of KFRT following CVD events are needed.

[Radiofrequency denervation of the spine and the sacroiliac joint : A systematic literature search according to GRADE with new German S3 guideline]. / Radiofrequenzdenervation der Wirbelsäule und des Iliosakralgelenks : Eine systematische Literaturrecherche nach GRADE mit neuer S3-Leitlinie.

BACKGROUND: This article summarizes the results of the German guideline on radiofrequency denervation of the facet joints and the sacroiliac joint. Evidence on the indications, test blocks and technical parameters are presented. OBJECTIVE: The aim is to avoid overtreatment and undertreatment, which is also of socioeconomic importance. MATERIAL AND METHOD: A systematic evaluation of the literature was carried out according to the grading of recommendations assessment, development and evaluation (GRADE) approach. A multidisciplinary guideline group has developed recommendations and statements. RESULTS: Statements and recommendations were given for 20 key questions. There was an 87.5% consensus for 1 recommendation and 100% consensus for all other recommendations and statements. The guideline was approved by all scientific medical societies involved. Specific questions included the value of the medical history, examination and imaging, the need for conservative treatment prior to an intervention, the importance of test blocks (medial branch block and lateral branch block), choice of imaging for denervation, choice of trajectory, the possibility to influence the size of the lesion, stimulation, the possibility of revision, sedation and decision support for patients with anticoagulants, metal implants and pacemakers and advice on how to avoid complications. CONCLUSION: Selected patients can benefit from well-performed radiofrequency denervation. The guideline recommendations are based on very low to moderate quality of evidence.

Comparison of allosteric signaling in DnaK and BiP using mutual information between simulated residue conformations.

The heat shock protein 70 kDa (Hsp70) chaperone system serves as a critical component of protein quality control across a wide range of prokaryotic and eukaryotic organisms. Divergent evolution and specialization to particular organelles have produced numerous Hsp70 variants which share similarities in structure and general function, but differ substantially in regulatory aspects, including conformational dynamics and activity modulation by cochaperones. The human Hsp70 variant BiP (also known as GRP78 or HSPA5) is of therapeutic interest in the context of cancer, neurodegenerative diseases, and viral infection, including for treatment of the pandemic virus SARS-CoV-2. Due to the complex conformational rearrangements and high sequential variance within the Hsp70 protein family, it is in many cases poorly understood which amino acid mutations are responsible for biochemical differences between protein variants. In this study, we predicted residues associated with conformational regulation of human BiP and Escherichia coli DnaK. Based on protein structure networks obtained from molecular dynamics simulations, we analyzed the shared information between interaction timelines to highlight residue positions with strong conformational coupling to their environment. Our predictions, which focus on the binding processes of the chaperone's substrate and cochaperones, indicate residues filling potential signaling roles specific to either DnaK or BiP. By combining predictions of individual residues into conformationally coupled chains connecting ligand binding sites, we predict a BiP specific secondary signaling pathway associated with substrate binding. Our study sheds light on mechanistic differences in signaling and regulation between Hsp70 variants, which provide insights relevant to therapeutic applications of these proteins.

Motorized spiral enteroscopy-assisted ERCP in surgically altered anatomy: early experience from a retrospective cohort study.

BACKGROUND : Motorized spiral enteroscopy (MSE) was recently introduced into clinical practice. The aim of the current study was to evaluate the feasibility and safety of MSE for biliopancreatic interventions in patients with surgically altered anatomy. METHODS : Patients with surgically altered anatomy receiving MSE-assisted ERCP at a single, endoscopy referral center were retrospectively enrolled between January 2016 and June 2021. RESULTS : 36 patients (14 female, 22 male), median age 67 years (range 43-88), with biliary (n = 35) and pancreatic (n = 1) indications for MSE-ERCP, were enrolled. The majority (75.0 %) had relevant comorbidities (American Society of Anesthesiologists class III). Surgical reconstruction included Roux-en-Y (n = 30) and Billroth II (n = 6). Technical success rates for enteroscopy, cannulation, and interventions were 86.1 %, 83.9 %, and 100 %, respectively. The overall MSE-ERCP success rate was 72.2 %. One major complication occurred (2.8 %; delayed post-sphincterotomy bleeding). CONCLUSIONS : This is the first study to demonstrate the feasibility and safety of MSE-assisted ERCP in postsurgical patients with altered anatomy at an expert center. These data justify further evaluation of this new technique, preferably in a prospective multicenter trial.

Digital single-operator pancreatoscopy for the treatment of symptomatic pancreatic duct stones: a prospective multicenter cohort trial.

BACKGROUND: Digital single-operator pancreatoscopy (DSOP)-guided lithotripsy is a novel treatment modality for pancreatic endotherapy, with demonstrated technical success in retrospective series of between 88 % and 100 %. The aim of this prospective multicenter trial was to systematically evaluate DSOP in patients with chronic pancreatitis and symptomatic pancreatic duct stones. METHODS: Patients with symptomatic chronic pancreatitis and three or fewer stones ≥ 5mm in the main pancreatic duct (MPD) of the pancreatic head or body were included. The primary end point was complete stone clearance (CSC) in three or fewer treatment sessions with DSOP. Current guidelines recommend extracorporeal shock wave lithotripsy (ESWL) for MPD stones > 5 mm. A performance goal was developed to show that the CSC rate of MPD stones using DSOP was above what has been previously reported for ESWL. Secondary end points were pain relief measured with the Izbicki pain score (IPS), number of interventions, and serious adverse events (SAEs). RESULTS: 40 chronic pancreatitis patients were included. CSC was achieved in 90 % of patients (36/40) on intention-to-treat analysis, after a mean (SD) of 1.36 (0.64) interventions (53 procedures in total). The mean (SD) baseline IPS decreased from 55.3 (46.2) to 10.9 (18.3). Overall pain relief was achieved in 82.4 % (28/34) after 6 months of follow-up, with complete pain relief in 61.8 % (21/34) and partial pain relief in 20.6 % (7/34). SAEs occurred in 12.5 % of patients (5/40), with all treated conservatively. CONCLUSION: DSOP-guided endotherapy is effective and safe for the treatment of symptomatic MPD stones in highly selected patients with chronic pancreatitis. It significantly reduces pain and could be considered as an alternative to standard ERCP techniques for MPD stone treatment in these patients.

Association of osteopontin with kidney function and kidney failure in chronic kidney disease patients: the GCKD study.

BACKGROUND: Osteopontin (OPN), synthesized in the thick ascending limb of Henle's loop and in the distal tubule, is involved in the pathogenesis of kidney fibrosis, a hallmark of kidney failure (KF). In a cohort of chronic kidney disease (CKD) patients, we evaluated OPN's association with kidney markers and KF. METHODS: OPN was measured from baseline serum samples of German Chronic Kidney Disease study participants. Cross-sectional regression models for estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio (UACR) as well as Cox regression models for all-cause mortality and KF were evaluated to estimate the OPN effect. Additionally, the predictive ability of OPN and time-dependent population-attributable fraction were evaluated. RESULTS: Over a median follow-up of 6.5 years, 471 KF events and 629 deaths occurred among 4950 CKD patients. One-unit higher log(OPN) was associated with 5.5 mL/min/1.73 m2 lower eGFR [95% confidence interval (95% CI) -6.4 to -4.6] and 1% change in OPN with 0.7% higher UACR (estimated effect 0.7, 95% CI 0.6-0.8). Moreover, higher OPN levels were associated with a higher risk of KF [hazard ratio (HR) 1.4, 95% CI 1.2-1.7] and all-cause mortality (HR 1.5, 95% CI 1.3-1.8). After 6 years, 31% of the KF events could be attributed to higher OPN levels (95% CI 3%-56%). CONCLUSIONS: In this study, higher OPN levels were associated with kidney function markers worsening and a higher risk for adverse outcomes. A larger proportion of KF could be attributed to higher OPN levels, warranting further research on OPN with regards to its role in CKD progression and possible treatment options.

Differential Prognostic Utility of Adiposity Measures in Chronic Kidney Disease.

OBJECTIVE: Adipose tissue contributes to adverse outcomes in chronic kidney disease (CKD), but there is uncertainty regarding the prognostic relevance of different adiposity measures. We analyzed the associations of neck circumference (NC), waist circumference (WC), and body mass index (BMI) with clinical outcomes in patients with mild to severe CKD. METHODS: The German Chronic Kidney Disease study is a prospective cohort study, which enrolled Caucasian adults with mild to severe CKD, defined as estimated glomerular filtration rate : 30-60 mL/min/1.73 m2, or >60 mL/min/1.73 m2 in the presence of overt proteinuria. Associations of NC, WC, and BMI with all-cause death, major adverse cardiovascular events (MACE: a composite of nonfatal stroke, nonfatal myocardial infarction, peripheral artery disease intervention, and cardiovascular death), and kidney failure (a composite of dialysis or transplantation) were analyzed using multivariable Cox proportional hazards regression models adjusted for confounders and the Akaike information criteria were calculated. Models included sex interactions with adiposity measures. RESULTS: A total of 4537 participants (59% male) were included in the analysis. During a 6.5-year follow-up, 339 participants died, 510 experienced MACE, and 341 developed kidney failure. In fully adjusted models, NC was associated with all-cause death in women (hazard ratio 1.080 per cm; 95% CI 1.009-1.155) but not in men. Irrespective of sex, WC was associated with all-cause death (hazard ratio 1.014 per cm; 95% CI 1.005-1.038). NC and WC showed no association with MACE or kidney failure. BMI was not associated with any of the analyzed outcomes. Models of all-cause death, including WC offered the best (lowest) Akaike information criteria. CONCLUSION: In Caucasian patients with mild to severe CKD, higher NC (in women) and WC were significantly associated with increased risk of death from any cause but BMI was not.

Apolipoprotein A-IV concentrations and clinical outcomes in a large chronic kidney disease cohort: Results from the GCKD study.

BACKGROUND: Chronic kidney disease (CKD) represents a chronic proinflammatory state and is associated with very high cardiovascular risk. Apolipoprotein A-IV (apoA-IV) has antiatherogenic, antioxidative, anti-inflammatory and antithrombotic properties and levels increase significantly during the course of CKD. OBJECTIVES: We aimed to investigate the association between apoA-IV and all-cause mortality and cardiovascular outcomes in the German Chronic Kidney Disease study. METHODS: This was a prospective cohort study including 5141 Caucasian patients with available apoA-IV measurements and CKD. The majority of the patients had an estimated glomerular filtration rate (eGFR) of 30-60 ml/min/1.73m2 or an eGFR >60 ml/min/1.73m2 in the presence of overt proteinuria. Median follow-up was 6.5 years. The association of apoA-IV with comorbidities at baseline and endpoints during follow-up was modelled adjusting for major confounders. RESULTS: Mean apoA-IV concentrations of the entire cohort were 28.9 ± 9.8 mg/dl. Patients in the highest apoA-IV quartile had the lowest high-sensitivity C-reactive protein values despite the highest prevalence of diabetes, albuminuria and the lowest eGFR. Each 10 mg/dl higher apoA-IV translated into lower odds of prevalent cardiovascular disease (1289 cases, odds ratio = 0.80, 95% confidence interval [CI] 0.72-0.86, p = 0.0000003). During follow-up, each 10 mg/dl higher apoA-IV was significantly associated with a lower risk for all-cause mortality (600 cases, hazard ratio [HR] = 0.81, 95% CI 0.73-0.89, p = 0.00004), incident major adverse cardiovascular events (506 cases, HR = 0.88, 95% CI 0.79-0.99, p = 0.03) and death or hospitalizations due to heart failure (346 cases, HR = 0.84, 95% CI 0.73-0.96, p = 0.01). CONCLUSIONS: These data support a link between elevated apoA-IV concentrations and reduced inflammation in moderate CKD. ApoA-IV appears to be an independent risk marker for reduced all-cause mortality, cardiovascular events and heart failure in a large cohort of patients with CKD.

Heart-Type Fatty Acid Binding Protein, Cardiovascular Outcomes, and Death: Findings From the German CKD Cohort Study.

RATIONALE & OBJECTIVE: Heart-type fatty acid binding protein (H-FABP) is a biomarker that has been shown to provide long-term prognostic information in patients with coronary artery disease independently of high-sensitivity troponin T (hs-TNT). We examined the independent associations of H-FABP with cardiovascular outcomes in patients with chronic kidney disease (CKD). STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 4,951 patients enrolled in the German Chronic Kidney Disease (GCKD) study with an estimated glomerular filtration rate of 30-60 mL/min/1.73 m2 or overt proteinuria (urinary albumin-creatinine ratio > 300 mg/g or equivalent). EXPOSURE: Serum levels of H-FABP and hs-TNT were measured at study entry. OUTCOME: Noncardiovascular (non-CV) death, CV death, combined major adverse CV events (MACE), and hospitalization for congestive heart failure (CHF). ANALYTICAL APPROACH: Hazard ratios (HRs) for associations of H-FABP and hs-TNT with outcomes were estimated using Cox regression analyses adjusted for established risk factors. RESULTS: During a maximum follow-up of 6.5 years, 579 non-CV deaths, 190 CV deaths, 522 MACE, and 381 CHF hospitalizations were observed. In Cox regression analyses adjusted for established risk factors, H-FABP was associated with all 4 outcomes, albeit with lower HRs than those found for hs-TNT. After further adjustment for hs-TNT levels, H-FABP was found to be associated with non-CV death (HR, 1.57 [95% CI, 1.14-2.18]) and MACE (HR, 1.40 [95% CI, 1.02-1.92]) but with neither CV death (HR, 1.64 [95% CI, 0.90-2.99]) nor CHF hospitalizations (HR, 1.02 [95% CI, 0.70-1.49]). LIMITATIONS: Single-point measurements of H-FABP and hs-TNT. Uncertain generalizability to non-European populations. CONCLUSIONS: In this large cohort of patients with CKD, H-FABP was associated with non-CV death and MACE, even after adjustment for hs-TNT. Whether measurement of H-FABP improves cardiovascular disease risk prediction in these patients warrants further studies.

Motorized spiral enteroscopy: results of an international multicenter prospective observational clinical study in patients with normal and altered gastrointestinal anatomy.

BACKGROUND : Motorized spiral enteroscopy (MSE) has been shown to be safe and effective for deep enteroscopy in studies performed at expert centers with limited numbers of patients without previous abdominal surgery. This study aimed to investigate the safety, efficacy, and learning curve associated with MSE in a real-life scenario, with the inclusion of patients after abdominal surgery and with altered anatomy. METHODS : Patients with indications for deep enteroscopy were enrolled in a prospective observational multicenter study. The primary objective was the serious adverse event (SAE) rate; secondary objectives were the diagnostic and therapeutic yield, procedural success, time, and insertion depth. Data analysis was subdivided into training and core (post-training) study phases at centers with different levels of MSE experience. RESULTS : 298 patients (120 women; median age 68, range 19-92) were enrolled. In the post-training phase, 21.5 % (n = 54) had previous abdominal surgery, 10.0 % (n = 25) had surgically altered anatomy. Overall, SAEs occurred in 2.3 % (7/298; 95 %CI 0.9 %-4.8 %). The SAE rate was 2.0 % (5/251) in the core group and 4.3 % (2/47) in the training group, and was not increased after abdominal surgery (1.9 %). Total enteroscopy was achieved in half of the patients (n = 42) undergoing planned total enteroscopy. In 295/337 procedures (87.5 %), the anatomical region of interest could be reached. CONCLUSIONS : This prospective multicenter study showed that MSE was feasible and safe in a large cohort of patients in a real-life setting, after a short learning curve. MSE was shown to be feasible in postsurgical patients, including those with altered anatomy, without an increase in the SAE rate.

The Q41R mutation in the HCV-protease enhances the reactivity towards MAVS by suppressing non-reactive pathways.

Recent experimental findings pointed out a new mutation in the HCV protease, Q41R, responsible for a significant enhancement of the enzyme's reactivity towards the mitochondrial antiviral-signaling protein (MAVS). The Q41R mutation is located rather far from the active site, and its involvement in the overall reaction mechanism is thus unclear. We used classical molecular dynamics and QM/MM to study the acylation reaction of HCV NS3/4A protease variants bound to MAVS and the NS4A/4B substrate and uncovered the indirect mechanism by which the Q41R mutation plays a critical role in the efficient cleavage of the substrate. Our simulations reveal that there are two major conformations of the MAVS H1'(p) residue for the wild type protease and only one conformation for the Q41R mutant. The conformational space of H1'(p) is restricted by the Q41R mutation due to a π-π stacking between H1'(p) and R41 as well as a strong hydrogen bond between the backbone of H57 and the side chain of R41. Further QM/MM calculations indicate that the complex with the conformation ruled out by the Q41R substitution is a non-reactive species due to its higher free energy barrier for the acylation reaction. Based on our calculations, we propose a kinetic mechanism that explains experimental data showing an increase of apparent rate constants for MAVS cleavage in Q41R mutants. Our model predicts that the non-reactive conformation of the enzyme-substrate complex modulates reaction kinetics like an uncompetitive inhibitor.

Motorised spiral enteroscopy: first prospective clinical feasibility study.

OBJECTIVE: Currently available methods for small bowel endoscopy are often time consuming; motorised PowerSpiral Enteroscopy (PSE) is a further development of spiral enteroscopy to facilitate the approach to the small bowel. The aim of this bicentric prospective trial was to study feasibility and yield of peroral PSE. DESIGN: Consecutive patients with suspected small bowel disease and indication for antegrade enteroscopy were included in two tertiary referral centres. Primary objective was diagnostic yield of antegrade PSE. Secondary objectives included technical success (defined as successful endoscope insertion at least to ligament of Treitz), depth of maximum insertion (DMI), median insertion time to DMI, rate of therapeutic procedures and adverse events. RESULTS: During a 30-month period, 140 procedures were performed on 132 patients (58 female, 74 male; median age: 68 (20-100) years) under general anaesthesia. Overall diagnostic yield of PSE was 74.2%; with 68.2% of procedures including some form of endotherapy. Technical success rate of PSE was 97%; median DMI was 450 cm (0-600) with a median insertion time to DMI of 25 min (3-122). Antegrade panenteroscopy to the cecum was achieved in 14 cases (10.6%). Overall adverse event (AE) rate was 14.4%; two major serious AEs occurred (1.5%), one delayed perforation, one bleeding from Mallory-Weiss lesion. CONCLUSION: This pilot clinical trial demonstrates that PSE is effective for diagnostic and therapeutic antegrade enteroscopy and may compare favourably with traditional methods of deep enteroscopy in ease of use and procedural duration. More comparative data are required to assess clinical application and safety of PSE. TRIAL REGISTRATION NUMBER: NCT02965209.

Extended interaction networks with HCV protease NS3-4A substrates explain the lack of adaptive capability against protease inhibitors.

Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168 To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.

Total motorized spiral enteroscopy: first prospective clinical feasibility trial.

BACKGROUND AND AIMS: Motorized spiral enteroscopy (MSE) was recently introduced into clinical practice and shown to be safe and effective for antegrade enteroscopy. The aim of the current trial was to prospectively study the efficacy and safety of MSE for visualization of the entire small bowel. METHODS: All consecutive patients with indications for complete enteroscopy meeting the inclusion criteria were enrolled in a prospective observational bicentric trial, starting with antegrade MSE; a retrograde approach was performed if MSE remained incomplete from antegrade. The primary objective was to ascertain the total enteroscopy rate (TER); secondary objectives were diagnostic yield, procedural success, time, depth of maximum insertion (DMI), therapeutic yield, and adverse events (AEs). RESULTS: Thirty patients (16 women, 14 men; median age 64 years [range, 37-100]) were enrolled. Technical success rate of antegrade MSE (advancement beyond the ligament of Treitz) and retrograde MSE (advancement beyond the ileocecal valve [ICV]) were 100% and 100%, respectively. Overall TER was 70%: 16.6% antegrade approach alone and 53.4% bidirectional approach. Median antegrade DMI distal from the ligament of Treitz was 490 cm (range, 160-600); median insertion time 26 minutes (range, 15-110). The median retrograde DMI beyond the ICV was 120 cm (range, 40-600), and median insertion time was 17 minutes (range, 1-68). Overall diagnostic and therapeutic yields were 80% and 86.7%, respectively. Overall AE rate was 16.7%. No serious AEs occurred. CONCLUSIONS: This prospective study showed that complete enteroscopy is feasible with MSE, either from antegrade alone or bidirectionally, with high success rates and short procedural duration. These results justify further evaluation of MSE in a large prospective multicenter study, preferably with inclusion of a control group. (Clinical trial registration number: NCT03438695.).

The Application of a CMR-B-Scalar Sensor for the Investigation of the Electromagnetic Acceleration of Type II Superconductors.

In this paper, we investigated the behavior of a type II superconducting armature when accelerated by a pulsed magnetic field generated by a single-stage pancake coil. While conducting this investigation, we performed a numerical finite element simulation and an experimental study of the magnetic field dynamics at the edge of the pancake coil when the payload was a superconducting disc made from YBa2Cu3O7-x, cooled down to 77 K. The magnetic field measurements were performed using a CMR-B-scalar sensor, which was able to measure the absolute magnitude of the magnetic field and was specifically manufactured in order to increase the sensor's sensitivity up to 500 mT. It was obtained that type II superconducting armatures can outperform normal metals when the launch conditions are tailored to their electromagnetic properties.

Drugs linked to plasma homoarginine in chronic kidney disease patients-a cross-sectional analysis of the German Chronic Kidney Disease cohort.

BACKGROUND: Elevated plasma concentrations of symmetric and asymmetric dimethylarginine (SDMA and ADMA, respectively) and a lower plasma concentration of the structurally related homoarginine are commonly observed in patients with chronic kidney disease (CKD) and independently predict total mortality as well as progression of renal disease. We aimed to identify drugs that may alter this adverse metabolite pattern in a favourable fashion. METHODS: Plasma ADMA, SDMA, homoarginine and l-arginine were determined by liquid chromatography-tandem mass spectrometry in 4756 CKD patients ages 18-74 years with an estimated glomerular filtration rate (eGFR) of 30-60 mL/min/1.73 m2 or an eGFR >60 mL/min/1.73 m2 and overt proteinuria who were enrolled in the German Chronic Kidney Disease (GCKD) study. Associations between laboratory, clinical and medication data were assessed. RESULTS: Intake of several commonly used drugs was independently associated with plasma concentrations of homoarginine and/or related metabolites. Among these, the peroxisome proliferator-activated receptor alpha (PPAR-α) agonist fenofibrate was associated with the most profound differences in ADMA, SDMA and homoarginine plasma concentrations: 66 patients taking fenofibrate had a multivariable adjusted odds ratio (OR) of 5.83 [95% confidence interval (CI) 2.82-12.03, P < 0.001] to have a plasma homoarginine concentration above the median. The median homoarginine plasma concentration in patients taking fenofibrate was 2.30 µmol/L versus 1.55 in patients not taking the drug (P < 0.001). In addition, fibrates were significantly associated with lower plasma SDMA and higher l-arginine concentrations. In contrast, glucocorticoids were associated with lower plasma homoarginine, with adjusted ORs of 0.52 (95% CI 0.40-0.67, P < 0.001) and 0.53 (95% CI 0.31-0.90, P = 0.018) for prednisolone and methylprednisolone, respectively. CONCLUSIONS: In a large cohort of CKD patients, intake of fenofibrate and glucocorticoids were independently associated with higher and lower plasma homoarginine concentrations, respectively. Effects on plasma homoarginine and methylarginines warrant further investigation as potential mechanisms mediating beneficial or adverse drug effects.