Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner.

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.

K63-Ubiquitylation and TRAF6 Pathways Regulate Mammalian P-Body Formation and mRNA Decapping.

Signals and posttranslational modifications regulating the decapping step in mRNA degradation pathways are poorly defined. In this study we reveal the importance of K63-linked ubiquitylation for the assembly of decapping factors, P-body formation, and constitutive decay of instable mRNAs encoding mediators of inflammation by various experimental approaches. K63-branched ubiquitin chains also regulate IL-1-inducible phosphorylation of the P-body component DCP1a. The E3 ligase TRAF6 binds to DCP1a and indirectly regulates DCP1a phosphorylation, expression of decapping factors, and gene-specific mRNA decay. Mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs the interaction with the mRNA decay factors DCP2, EDC4, and XRN1, but not EDC3, thus remodeling P-body architecture. The usage of ubiquitin chains for the proper assembly and function of the decay-competent mammalian decapping complex suggests an additional layer of control to allow a coordinated function of decapping activities and mRNA metabolism in higher eukaryotes.

Cyclin-dependent kinase 6 is a chromatin-bound cofactor for NF-κB-dependent gene expression.

Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer.

The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells.

Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E)-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL)-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF) NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i) an unusual mechanism involving partial degradation of IKKß, NEMO and IκBα and (ii) upregulation of TNFAIP3 (A20), although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1). The data revealed that, in HCoV-infected (but not IL-1-treated) cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKß activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the host cellular chromatin landscape, thereby orchestrating the timely coordinated expression of genes involved in multiple signaling, immunoregulatory and metabolic processes.

Bile acids increase steroidogenesis in cholemic mice and induce cortisol secretion in adrenocortical H295R cells via S1PR2, ERK and SF-1.

BACKGROUND AND AIMS: Bile acids are now accepted as central signalling molecules for the regulation of glucose, amino acid and lipid metabolism. Adrenal gland cortex cells express the bile acid receptors farnesoid X receptor (FXR), the G protein-coupled bile acid receptor (TGR5) and the sphingosine-1-phosphate receptor 2 (S1PR2). We aimed to determine the effects of cholestasis and more specifically of bile acids on cortisol production. METHODS: FXR and TGR5 knockout mice and controls were subjected to common bile duct ligation (CBDL) or chenodeoxycholic acid (CDCA) feeding to model cholestasis. Human adrenocortical H295R cells were challenged with bile acids for mechanistic studies. RESULTS: We found that CBDL and CDCA feeding increased the levels of corticosterone, the rodent equivalent to human cortisol and mRNA and protein levels of steroidogenesis-related enzymes in adrenals independent of FXR and TGR5. Taurine-conjugated CDCA (TCDCA) significantly stimulated cortisol secretion, phosphorylation of extracellular signal-regulated kinase (ERK) and expression of steroidogenesis-related genes in human adrenocortical H295R cells. FXR and TGR5 agonists failed to induce cortisol secretion in H295R cells. S1PR2 inhibition significantly abolished TCDCA-induced cortisol secretion, lowered phosphorylation of ERK and abrogated enhanced transcription of steroidogenesis-related genes in H295R cells. Likewise, siRNA S1PR2 treatment reduced the phosphorylation of ERK and cortisol secretion. Steroidogenic factor-1 (SF-1) transactivation activity was increased upon TCDCA treatment suggesting that bile acid signalling is linked to SF-1. Treatment with SF-1 inverse agonist AC45594 also reduced TCDCA-induced steroidogenesis. CONCLUSIONS: Our findings indicate that supraphysiological bile acid levels as observed in cholestasis stimulate steroidogenesis via an S1PR2-ERK-SF-1 signalling pathway.

Both sarcopenia and frailty determine suitability of patients for liver transplantation-A systematic review and meta-analysis of the literature.

Liver grafts are allocated based on both urgency and utility. Due to a tremendous shortage of suitable organs for liver transplantation (LT), a careful selection of suitable recipients is of utmost importance. While the sickest first principle for organ allocation based on MELD score goes along with poor utility, other parameters reflecting the general health condition like frailty and sarcopenia might be essential to detect suitable patients for the waiting list. Thus, this study was designed to evaluate both frailty and sarcopenia in LT. A systematic review of the literature on sarcopenia and frailty measurements in liver transplant recipients was performed. Thirteen of 238 studies were selected for full paper review. Six of the studies investigating the impact of frailty on waitlist mortality were subjected to a meta-analysis. Despite the different methodologies to assess sarcopenia, reports showed that sarcopenia was highly related to waitlist mortality with a sum of all that highly favored negative outcome in case of sarcopenia. The existing literature clearly underlines that frailty and sarcopenia are important to determine in LT candidates. One unique index for transplant candidates reflecting frailty should be developed and be used as a standard in all transplant centers to facilitate comparability.

Calcification score evaluation in patients listed for renal transplantation.

Based on native CT scans of the pelvic region using a standardized calcification score, evaluation of iliac vascular calcification was performed between 2008 and 2012 prior to listing for renal transplantation in 205 patients with chronic kidney disease. Vascular calcification showed a decrease from proximal to distal. The difference between the degree of calcification in the common iliac artery and in the external iliac artery was significant (P<.001). Risk factors for total iliac vascular calcification were age, smoking, sex, underlying renal disease, and diabetes. Multivariate analysis revealed age to be the most relevant risk factor (P<.001). The duration of hemodialysis correlated significantly with total iliac vascular calcification. As the introduction of the standardized surgical evaluation protocol, no transplantation has had to be broken off and no early graft loss due to calcification has occurred. Thus, careful scoring of vascular calcification prior to transplantation may be a valuable tool to support surgical decisions and to improve patient safety and outcome in increasingly older transplant recipients.

The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-κB.

Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-κB p65 and its deacetylation at various lysines. NF-κB p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-κB p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical use.

The assessment of GFR after orthotopic liver transplantation using cystatin C and creatinine-based equations.

The measurement of kidney function after orthotopic liver transplantation (OLT) is still a clinical challenge. Cystatin C (CysC) has been proposed as a more accurate marker of renal function than serum creatinine (sCr). The aim of this study was to evaluate sCr- and CysC-based equations including the Chronic kidney disease (CKD)-EPI to determine renal function in liver transplant recipients. CysC and sCr were measured in 49 patients 24 months after OLT. The glomerular filtration rate (GFR) was calculated using the MDRD 4, the Cockroft-Gault, Hoek, Larsson, and the CKD-EPI equations based on sCr and/or CysC. As reference method, inulin clearance (IC) was estimated. Bias, precision, and accuracy of each equation were assessed and compared with respect to IC. Forty-five percent had a GFR < 60 ml/min/1.73 m(2) according to the IC. The Larsson, the Hoek and the CKD-EPI-CysC formula identified the highest percentage of patients with CKD correctly (88%, 88%, and 84%, respectively). The sCr-based equations showed less bias than CysC-based formulas with a similar precision. All CysC-based equations were superior as compared with sCr-based equations in the assessment of renal function in patients with an IC < 60 ml/min/1.73 m(2).

Microoptics for efficient redirection of sunlight.

Ray-tracing calculations are employed to identify basic design rules for the configuration of microstructured daylighting systems. The results show the advantage of combinations of lenslike geometries in comparison to conventional microprism arrays regarding the overall light redirection efficiency as well as the producibility and cost efficiency. Measurements at silicone prototypes and large scale industrially produced acrylic panels confirmed the simulation results. Optimization leads to free-form geometries which can further be improved by selective roughening of specific microsurfaces.

Calcium-dependent membrane association sensitizes soluble guanylyl cyclase to nitric oxide.

Nitric oxide (NO) is a ubiquitous, cell-permeable intercellular messenger. The current concept assumes that NO diffuses freely through the plasma membrane into the cytoplasm of a target cell, where it activates its cytosolic receptor enzyme, soluble guanylyl cyclase (sGC). Recent evidence, however, suggests that cellular membranes are not only the predominant site of calcium-dependent NO synthesis, but also the site of its distribution and binding. Here we extend this concept to NO signalling to show that active sGC is partially associated with the plasma membrane in a state of enhanced NO sensitivity. After cellular activation, sGC further translocates to the membrane fraction in human platelets and associates with the NO-synthase-containing caveolar fraction in rat lung endothelial cells, in a manner that is dependent on the concentration of intracellular calcium. Our data suggest that the entire NO signalling pathway is more spatially confined than previously assumed and that sGC dynamically translocates to the plasma membrane, where it is sensitized to NO.

Enhanced myofiber recruitment during exhaustive squatting performed as whole-body vibration exercise.

The purpose of the study was to test the hypothesis that myofiber recruitment is enhanced when whole-body vibration (WBV) is added to squat training. In a randomized cross-over design, 14 recreationally active men were subjected to 2 sessions consisting of 5 sets of 10 squats with external load, performed either on a vibration platform (whole-body vibration squatting [WBVS]) or conventionally without WBV (CON). Electromyographic (EMG) activity of the right vastus lateralis muscle was continuously recorded during WBVS and CON. The integrated EMG values were normalized to the EMG activity recorded during measurement of the maximal voluntary contraction force (MVC) on an isometric leg press at the beginning of each training session. Capillary lactate concentration was determined before and repeatedly after the squatting exercise. Overall mean normalized and integrated EMG (nIEMG) activity during WBVS (62 ± 4% MVC) was significantly (p < 0.001) higher compared with CON (47 ± 2% MVC). There was a tendency for nIEMG to increase during the 5 sets of 10 squats performed as WBVS (p = 0.089), whereas there was a significant (p < 0.001) decrease in nIEMG during CON. Whole-body vibration squatting induced a significantly (p < 0.001) larger increase in capillary lactate than CON (3.03 ± 0.32 vs. 1.60 ± 0.30 mmol · L(-1), p < 0.001). The increased myoelectric activity and the enhanced exercise-induced increase in capillary lactate concentration during WBVS provide evidence for augmented recruitment of muscle tissue when WBV is added to exhaustive squatting exercise.

COVID-19 Pandemic Stress-Induced Somatization in Transplant Waiting List Patients.

Background: The coronavirus disease 2019 (COVID-19) pandemic has resulted in widespread socioeconomic restrictions including quarantine, social distancing and self-isolation. This is the first study investigating the psychological impact of the pandemic on patients waiting for liver or kidney transplantation, a particularly vulnerable group. Methods: Twenty-seven patients on the transplantation waiting list and 43 healthy controls took part in an online survey including the Beck Depression Inventory (BDI-2), the Brief Symptom Inventory-18 (BSI-18), the Pittsburgh Sleep Quality Index (PSQI), the Alcohol Use Identification Test (AUDIT-C), the 12-item Operationalized Psychodynamic Diagnosis Structure Questionnaire (OPD-SQS) and a questionnaire to determine cognitions and beliefs, attitude and fear related to COVID-19. Results: BSI-18 Somatization was increased in waiting list patients compared to controls. Correlation analyses indicated a relationship between Somatization and the fear of contracting the coronavirus in the patient group; however this association was weak. In patients and controls, other psychologicial symptoms (depression, anxiety) correlated highly with emotional distress due to social distancing. There were no differences between patients and controls in depression scores and sleep disturbances. Alcohol consumption and personality structure were not related to COVID-19 fears. Conclusion: In times of the first lockdown during the COVID-19 pandemic, patients on the transplantation waiting list have high somatization symptoms associated with COVID-19 fears. As vulnerable group, they need psychological counseling to improve mental well-being during times of crisis.

Effects of strength training with eccentric overload on muscle adaptation in male athletes.

In classic concentric/eccentric exercise, the same absolute load is applied in concentric and eccentric actions, which infers a smaller relative eccentric load. We compared the effects of 6 weeks of classic concentric/eccentric quadriceps strength training (CON/ECC, 11 subjects) to eccentric overload training (CON/ECC+, 14 subjects) in athletes accustomed to regular strength training. The parameters determined included functional tests, quadriceps and fibre cross-sectional area (CSA), fibre type distribution by ATPase staining, localisation of myosin heavy chain (MHC) isoform mRNAs by situ hybridization and the steady-state levels of 48 marker mRNAs (RT-PCR) in vastus lateralis biopsies taken before and after training. Both training forms had anabolic effects with significant increases in quadriceps CSA, maximal strength, ribosomal RNA content and the levels of mRNAs involved in growth and regeneration. Only the CON/ECC+ training led to significantly increased height in a squat jump test. This was accompanied by significant increases in IIX fibre CSA, in the percentage of type IIA fibres expressing MHC IIx mRNA, in the level of mRNAs preferentially expressed in fast, glycolytic fibres, and in post-exercise capillary lactate. The enhanced eccentric load apparently led to a subtly faster gene expression pattern and induced a shift towards a faster muscle phenotype plus associated adaptations that make a muscle better suited for fast, explosive movements.

The Graz Liver Allocation Strategy-Impact of Extended Criteria Grafts on Outcome Considering Immunological Aspects.

Background: Transplant centers are forced to use livers of extended criteria donors for transplantation due to a dramatic organ shortage. The outcome effect of extended donor criteria (EDCs) remains unclear. Thus, this study was designed to assess the impact of EDCs on outcome including immunological aspects after liver transplantation (LT). Patients and Methods: Between November 2016 and March 2018, 49 patients (85.7% male) with a mean age of 57 ± 11 years underwent LT. The impact of EDCs on outcome after LT was assessed retrospectively using both MedOcs and ENIS (Eurotransplant Network Information System). Results: About 80% of grafts derived from extended criteria donors. Alanine aminotransferase/aspartate aminotransferase (AST/ALT) levels elevated more than three times above normal values in organ donors was the only significant risk factor for primary dysfunction (PDF) and primary non-function (PNF)/Re-LT and early non-anastomotic biliary strictures (NAS). Balance of risk (BAR) score did not differ between EDC and non-EDC recipients. PDF (14.3% of all patients) and PNF (6.1% of all patients) occurred in 23.1% of EDC-graft recipients and in 10.0% of non-EDC-graft recipients (RR 2.31, p = 0.663). The 90-day mortality was 3.6%. There was no difference of early non-anastomotic biliary tract complications and biopsy proven rejections (BPR). There was no correlation of PDF/PNF with BPR and NAS, respectively; however, 66.7% of the patients with BPR also developed early NAS (p < 0.001). Conclusion: With the Graz liver allocation strategy, excellent survival can be achieved selecting livers with no more than 2 not outcome-relevant EDCs for patients with MELD >20. Further, BPR is associated with biliary complications.

Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.

ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc-protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.

Measurement of cardiac output and pulmonary transit time for assessment of pulmonary vascular resistance in domestic piglets.

The degree of elevated pulmonary vascular resistance (PVR) is a crucial clinical parameter. Cardiac output (CO) and pulmonary transit time (PTT) can be ascertained by a number of radiological methods. A close functional relationship between CO, PTT and PVR would facilitate non-invasive PVR measurements. One-hundred and fifty-one measurements were made in six piglets. Pressures in the pulmonary and systemic circulation were measured invasively. Cardic output was determined by the use of a Doppler flow probe around the truncus pulmonalis. Temperature sensors were placed in the pulmonary truncus and left atrium. Elevated PVR was produced by repeated air embolism. After injection of ice-cold saline, the time span between the minimal temperature in the truncus pulmonalis and the left atrium was taken as PTT. The CO and PTT were inserted into a new formula derived from the Hagen-Poiseuille law for the calculation of the PVR model. Numerical constants of the formula were calculated by the robust method of minimization. The PVR values, as calculated from invasively measured mean pulmonary artery pressure, mean left atrial pressure and CO, served as reference. In the six piglets, the PVR model and PVR reference showed a strong linear correlation with r = 0.923. The Bland-Altman plot revealed a standard deviation of -0.64/+0.67 Wood units. Cardiac output, PTT and PVR showed a close functional relationship. With a correction for blood viscosity and body size, this relationship could be used for non-invasive clinical measurements of PVR.

MicroRNA-142-3p improves vascular relaxation in uremia.

BACKGROUND AND AIMS: Chronic kidney disease (CKD) is strongly associated with a high burden of cardiovascular morbidity and mortality. Therefore, we aimed to characterize the putative role of microRNAs (miR)s in uremic vascular remodelling and endothelial dysfunction. METHODS: We investigated the expression pattern of miRs in two independent end-stage renal disease (ESRD) cohorts and in the animal model of uremic DBA/2 mice via quantitative RT-PCR. Moreover, DBA/2 mice were treated with intravenous injections of synthetic miR-142-3p mimic and were analysed for functional and morphological vascular changes by mass spectrometry and wire myography. RESULTS: The expression pattern of miRs was regulated in ESRD patients and was reversible after kidney transplantation. Out of tested miRs, only blood miR-142-3p was negatively associated with carotid-femoral pulse-wave velocity in CKD 5D patients. We validated these findings in a murine uremic model and found similar suppression of miR-142-3p as well as decreased acetylcholine-mediated vascular relaxation of the aorta. Therefore, we designed experiments to restore bioavailability of aortic miR-142-3p in vivo via intravenous injection of synthetic miR-142-3p mimic. This intervention restored acetylcholine-mediated vascular relaxation. CONCLUSIONS: Taken together, we provide compelling evidence, both in humans and in mice, that miR-142-3p constitutes a potential pharmacological agent to prevent endothelial dysfunction and increased arterial stiffness in ESRD.