Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.

Improving prime editing with an endogenous small RNA-binding protein.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

T helper cells exhibit a dynamic and reversible 3'-UTR landscape.

3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.

Discovery of quinazoline HPK1 inhibitors with high cellular potency.

Hematopoietic progenitor kinase 1 (HPK1) is regarded as a highly validated target in pre-clinical immune oncology. HPK1 has been described as regulating multiple critical signaling pathway in both adaptive and innate cells. In support of this role, HPK1 KO T cells show enhanced sensitivity to TCR activation and HPK1 KO mice display enhanced anti-tumor activity. Taken together, inhibition of HPK1 has the potential to induce enhanced anti-tumor immune response. Herein, we described the discovery of highly potent HPK1 inhibitors starting form a weak HTS hit. Using a structure-based drug design, HPK1 inhibitors exhibiting excellent cellular single-digit nanomolar potency in both proximal (pSLP76) and distal (IL-2) biomarkers along with sustained elevation of IL-2 cytokine secretion were discovered.

Evobrutinib pathway to its major metabolite M463-2 and insights from a biotransformation and DDI perspective.

Evobrutinib is a highly selective, covalent, central nervous system-penetrant Bruton's tyrosine kinase (BTK) inhibitor, currently in Phase III trials for the treatment of relapsing multiple sclerosis. One major circulating metabolite of evobrutinib has been previously identified as the racemic dihydro-diol M463-2 (MSC2430422) in a Phase I human mass balance study.Phenotyping experiments were conducted to confirm the metabolic pathway of evobrutinib to M463-2. Ratio of the enantiomers was determined by enantioselective liquid chromatography with tandem mass spectrometry analysis of plasma samples from humans and preclinical species. Drug-drug interaction (DDI) characterisation, evaluation of pharmacological activity on BTK, and off-target screening experiments followed assessing safety of the metabolite.The biotransformation of evobrutinib to M463-2 was determined to be a two-step process with a CYP-mediated oxidation acting to form an epoxide intermediate, which was further hydrolysed by soluble and mitochondrial epoxide hydrolase. Only the (S)-enantiomer was determined to be a major metabolite, the (R)-enantiomer was minor. In vitro studies demonstrated the (S)-enantiomer lacked clinically relevant pharmacological activity, off-target effects and DDIs.The biotransformation of evobrutinib to its major metabolite has been elucidated, with the major (S)-enantiomer being shown to pose no on/off target or DDI risks.

Wheat yield potential can be maximized by increasing red to far-red light conditions at critical developmental stages.

Sensing of neighbours via the Red to Far-Red light ratio (R:FR) may exert a cap to yield potential in wheat. The effects of an increased R:FR inside the canopy were studied in dense wheat mini canopies grown in controlled environments by lowering FR. To distinguish between effects exerted by light sensing and assimilate supply, the treatments were complemented with elevated CO2 , applied between different developmental timepoints to specifically impact tillering, spike growth, floret fertility and grain filling, in different combinations. The yield response to high R:FR was strongly dependent on the developmental stage in all three cultivars and pivoted between positive if applied after the start of stem elongation, and negative or null if applied before. Yield gains of up to 70% and 120% were observed, respectively, in two cultivars, associated with a higher number of tiller spikes and grains per spike in the main shoot. The response to the combination of high R:FR and elevated CO2 or CO2 alone were cultivar dependent. Taken together, our results suggest that R:FR exerts a significant control on yield potential in wheat and achieving a high R:FR from stem elongation to maturity is a promising lever towards a significant increase in grain yield.

PolyASite 2.0: a consolidated atlas of polyadenylation sites from 3' end sequencing.

Generated by 3' end cleavage and polyadenylation at alternative polyadenylation (poly(A)) sites, alternative terminal exons account for much of the variation between human transcript isoforms. More than a dozen protocols have been developed so far for capturing and sequencing RNA 3' ends from a variety of cell types and species. In previous studies, we have used these data to uncover novel regulatory signals and cell type-specific isoforms. Here we present an update of the PolyASite (https://polyasite.unibas.ch) resource of poly(A) sites, constructed from publicly available human, mouse and worm 3' end sequencing datasets by enforcing uniform quality measures, including the flagging of putative internal priming sites. Through integrated processing of all data, we identified and clustered sites that are closely spaced and share polyadenylation signals, as these are likely the result of stochastic variations in processing. For each cluster, we identified the representative - most frequently processed - site and estimated the relative use in the transcriptome across all samples. We have established a modern web portal for efficient finding, exploration and export of data. Database generation is fully automated, greatly facilitating incorporation of new datasets and the updating of underlying genome resources.

Terminal exon characterization with TECtool reveals an abundance of cell-specific isoforms.

Sequencing of RNA 3' ends has uncovered numerous sites that do not correspond to the termination sites of known transcripts. Through their 3' untranslated regions, protein-coding RNAs interact with RNA-binding proteins and microRNAs, which regulate many properties, including RNA stability and subcellular localization. We developed the terminal exon characterization (TEC) tool ( http://tectool.unibas.ch ), which can be used with RNA-sequencing data from any species for which a genome annotation that includes sites of RNA cleavage and polyadenylation is available. We discovered hundreds of previously unknown isoforms and cell-type-specific terminal exons in human cells. Ribosome profiling data revealed that many of these isoforms were translated. By applying TECtool to single-cell sequencing data, we found that the newly identified isoforms were expressed in subpopulations of cells. Thus, TECtool enables the identification of previously unknown isoforms in well-studied cell systems and in rare cell types.

Accelerated white matter lesion analysis based on simultaneous T1 and T2∗ quantification using magnetic resonance fingerprinting and deep learning.

PURPOSE: To develop an accelerated postprocessing pipeline for reproducible and efficient assessment of white matter lesions using quantitative magnetic resonance fingerprinting (MRF) and deep learning. METHODS: MRF using echo-planar imaging (EPI) scans with varying repetition and echo times were acquired for whole brain quantification of T1 and T2∗ in 50 subjects with multiple sclerosis (MS) and 10 healthy volunteers along 2 centers. MRF T1 and T2∗ parametric maps were distortion corrected and denoised. A CNN was trained to reconstruct the T1 and T2∗ parametric maps, and the WM and GM probability maps. RESULTS: Deep learning-based postprocessing reduced reconstruction and image processing times from hours to a few seconds while maintaining high accuracy, reliability, and precision. Mean absolute error performed the best for T1 (deviations 5.6%) and the logarithmic hyperbolic cosinus loss the best for T2∗ (deviations 6.0%). CONCLUSIONS: MRF is a fast and robust tool for quantitative T1 and T2∗ mapping. Its long reconstruction and several postprocessing steps can be facilitated and accelerated using deep learning.

The TGF-b/SOX4 axis and ROS-driven autophagy co-mediate CD39 expression in regulatory T-cells.

The ectonucleotidase CD39 on human regulatory T-cells (Treg) is an important immune regulator which is dysregulated in autoimmune diseases and cancer immunosuppression. We here define that CD39 expression on Treg is independent of the Treg-specific transcription factors FOXP3 and HELIOS and promoted by canonical TGF-b- and mTOR-signaling. Furthermore, the TGF-b mediated upregulation of CD39 is counteracted by reactive oxygen species (ROS)-driven autophagy. In line, CD39+ peripheral blood Treg constitute a distinct lineage with low autophagic flux and absent ROS production. Patients with rare genetic defects in autophagy show supraphysiological levels of CD39+ Treg, validating our observations in vivo. These biological processes rely on a distinct transcriptional program with CD39+ Treg expressing low levels of two genes with putative involvement in autophagy, NEFL and PLAC8. Furthermore, the TGF-b downstream transcription factor SOX4 is selectively upregulated in CD39+ Treg. Overexpression of SOX4 in Treg strongly increases CD39 expression, while Crispr/Cas9-mediated knockout of SOX4 in Treg has the opposing effect. Thus, we identify a crucial role of SOX4 in immune regulation and provide new insights involving the interplay of tolerogenic cues and autophagy in Treg.

Epidemiological trends of HBV and HDV coinfection among Viennese HIV+ patients.

BACKGROUND AND AIMS: Despite vaccination recommendations, hepatitis B (HBV) and D (HDV) coinfections are common in HIV+individuals. METHODS: HBV immunization status (anti-HBs) as well as HBV (HBsAg/HBV-DNA) and HDV (anti-HDV) coinfection rates were assessed in 1870 HIV+individuals at HIV diagnosis (baseline, BL) and last follow-up (FU). RESULTS: Sixty-eight (3.6%) HIV patients were never tested for HBV. At BL, 89/1802 (4.9%) HIV patients were HBV coinfected. Four hundred and fifteen (23.0%) showed virological HBV clearance [HBsAg(-)/anti-HBc(+)/anti-HBs(+)] and 210 (11.7%) presented with anti-HBc(+) only. Seven hundred and ten (39.4%) were HBV naïve [HBsAg(-)/anti-HBs(-)/anti-HBc(-)/HBV-DNA(-)], but only 378 (21.0%) received vaccinations with detectable anti-HBs(+) titres. Among the 89 HBV/HIV-coinfected patients, only 52 (58.4%) were tested for HDV: 11/49 (22.4%) had anti-HDV(+) and 3/12 (25.0%) showed HDV-RNA viraemia. During a median FU of 6.5 (IQR 7.2) years, 44 (4.6%) of the 953 retested BL HBV-negative patients acquired new HBV infection (including 15/304, 4.9% of vaccinated patients). Of the 89 patients, 22 (24.7%) patients cleared their HBsAg, resulting in 60/1625 (3.7%) HIV/HBV individuals at FU: 34 (56.7%) showed HBV-DNA suppression and 15 (25.0%) were HBV viraemic, while 12/89 (13.5%) remained without a FU test. Vaccinations induced anti-HBs(+) in 137 of the retested 649 (21.1%) BL HBV-naïve patients. CONCLUSION: HBV testing is well established among Viennese HIV+patients with HBV coinfection rates around 4%-5%. HBV vaccinations are insufficiently implemented since anti-HBs titres were detected in only 21.1% of HBV-naive HIV(+) patients and new HBV infections occurred in previously vaccinated patients. HDV testing is not systematically performed despite up to 25% of HIV/HBV patients may show HDV coinfection.

Recent outbreaks of severe hepatitis A virus infections in Vienna.

To explore the epidemiology and clinical course of hepatitis A virus (HAV) infections at the Vienna General Hospital. We retrospectively identified patients who were tested positive for HAV-IgM at the Vienna General Hospital form Q1/2008 to Q3/2018. Our definition of severe HAV infection was AST and/or ALT > 5 × above the upper limit of normal (ULN); and liver dysfunction as (i) hepatic encephalopathy or ammonia > 100 µmol/L, (ii) coagulopathy with INR > 1.5, or (iii) jaundice with bilirubin > 5 mg/dL. A total of 578 HAV-IgM (+) were identified, including 31 (5.4%) and 38 (6.6%) without and with liver dysfunction, respectively. A proportional increase in severe HAV cases with and without liver dysfunction occurred in 2016/2017 with (21.5% (vs. 8.0% in the years before; p < 0.001). Thirty-seven (53.6%) patients with severe HAV were hospitalized, 6 (9%) required ICU support, and one patient received liver transplantation within 30 days. Patients with severe HAV and liver dysfunction were more often male (60.5 vs. 43.1%, p = 0.055) and younger (31.5 vs. 63 years, p < 0.001) as compared with other HAV-IgM (+) cases. The observed increase of severe HAV infections in Vienna in 2017 among young males, coincided with a multinational HAV outbreak among MSM. Our data suggests a higher likelihood of severe courses of hepatitis A in MSM. Vaccination against HAV should be recommended for risk groups.

Discovery of potent and selective reversible Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase member of the TEC family of tyrosine kinases. Pre-clinical and clinical data have shown that targeting BTK can be used for the treatment for B-cell disorders. Here we disclose the discovery of a novel imidazo[4,5-b]pyridine series of potent, selective reversible BTK inhibitors through a rational design approach. From a starting hit molecule 1, medicinal chemistry optimization led to the development of a lead compound 30, which exhibited 58 nM BTK inhibitory potency in human whole blood and high kinome selectivity. Additionally, the compound demonstrated favorable pharmacokinetics (PK), and showed potent dose-dependent efficacy in a rat CIA model.

Lesion probability mapping in MS patients using a regression network on MR fingerprinting.

BACKGROUND: To develop a regression neural network for the reconstruction of lesion probability maps on Magnetic Resonance Fingerprinting using echo-planar imaging (MRF-EPI) in addition to [Formula: see text], [Formula: see text], NAWM, and GM- probability maps. METHODS: We performed MRF-EPI measurements in 42 patients with multiple sclerosis and 6 healthy volunteers along two sites. A U-net was trained to reconstruct the denoised and distortion corrected [Formula: see text] and [Formula: see text] maps, and to additionally generate NAWM-, GM-, and WM lesion probability maps. RESULTS: WM lesions were predicted with a dice coefficient of [Formula: see text] and a lesion detection rate of [Formula: see text] for a threshold of 33%. The network jointly enabled accurate [Formula: see text] and [Formula: see text] times with relative deviations of 5.2% and 5.1% and average dice coefficients of [Formula: see text] and [Formula: see text] for NAWM and GM after binarizing with a threshold of 80%. CONCLUSION: DL is a promising tool for the prediction of lesion probability maps in a fraction of time. These might be of clinical interest for the WM lesion analysis in MS patients.

Hepatitis C Virus RNA Is Commonly Detectable in Rectal and Nasal Fluids of Patients With High Viremia.

BACKGROUND: Increasing numbers of hepatitis C virus (HCV) infections among men who have sex with men (MSM) are being observed in the Western world. The actual routes of HCV transmission during high-risk sex practices and associated drug use remain poorly understood. METHODS: Forty-seven patients with HCV were prospectively enrolled. Rectal and nasal swabs were collected to quantify HCV-RNA levels within rectal and nasal fluids. Contamination by occult rectal bleeding was excluded by guaiac paper test. Risk behavior was assessed by standardized questionnaires. RESULTS: Median age was 41.9 years, 89% were HIV positive (+) (42/47) and 85% (40/47) were male, 58% (23/40) of whom were MSM. Acute HCV infection was diagnosed in 32% (15/47) ,with all patients being HIV+MSM and 93% (14/15) having a documented history of sexually transmitted disease. Thirty-three (70%) patients had ≥1 HCV+ swab sample (HCV+SS; 48%, 22/46 rectal; 62%, 29/47 nasal), and contamination with blood was excluded in all patients. Individuals with HCV+SS had significantly higher serum HCV-RNA levels than patients with HCV-negative SS (6.28 [IQR, 0.85] log IU/mL vs 4.08 [2.45] log IU/mL; P < .001). Using ROC-curve analysis, serum HCV-RNA cutoffs for ruling in/out any HCV+SS were established at 6.02 log IU/mL and 4.02 log IU/mL, respectively. CONCLUSIONS: HCV-RNA is commonly detectable in rectal and nasal fluids of both HIV+ and HIV-negative HCV patients with high serum HCV-RNA, independently of the suspected route of HCV transmission. Accordingly, high-risk sex practices and sharing of nasal drug-sniffing "tools" might be important HCV transmission routes, especially in patients with high serum HCV-RNA.

Cytokinin regulates the activity of the inflorescence meristem and components of seed yield in oilseed rape.

The number of flowers and seed-bearing structures formed by the inflorescence meristem and the formation of ovules in the female reproductive part of the flowers are important yield-related traits of crop plants. It has been shown that cytokinin is a pivotal factor regulating these traits. Here, we explore the impact of mutation of CYTOKININ OXIDASE/DEHYDROGENASE (CKX) genes encoding cytokinin-degrading enzymes on these yield-related traits in oilseed rape (Brassica napus L.). We describe the identification of four BnCKX3 and two BnCKX5 genes as regulators of reproductive development in the allotetraploid B. napus. RNA-seq analysis and in situ hybridization showed expression of these genes in reproductive organs. Loss-of-function mutants for each of these CKX gene copies were identified by targeting induced local lesions in genomes (TILLING) and combined by crossing. Sextuple ckx3 ckx5 mutants showed an increased cytokinin concentration and larger and more active inflorescence meristems. They also produced up to 72% more flowers with gynoecia containing 32% more ovules and up to 54% more pods on the main stem. The weight of seeds harvested from the main stem of plants grown in the greenhouse or in the field was increased by 20-32%. Our results show that cytokinin regulates inflorescence meristem and placenta activity in oilseed rape. The work demonstrates the potential to achieve yield enhancement in a dicot crop plant by modulating the cytokinin status through mutagenesis of specific CKX genes.

Epidemiological trends in HCV transmission and prevalence in the Viennese HIV+ population.

BACKGROUND & AIMS: Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection is common in people who inject drugs (PWIDs). Recently, 'high-risk' behaviour among men who have sex with men (MSM) has emerged as another main route of HCV transmission. We analysed temporal trends in HCV epidemiology in a cohort of Viennese HIV+ patients. METHODS: Hepatitis C virus parameters were recorded at HIV diagnosis (baseline [BL]) and last visit (follow-up [FU]) for all HIV+ patients attending our HIV clinic between January 2014 and December 2016. Proportions of HIV+ patients with anti-HCV(+) and HCV viraemia (HCV-RNA(+)) at BL/FU were assessed and stratified by route of transmission. RESULTS: In all, 1806/1874 (96.4%) HIV+ patients were tested for HCV at BL. Anti-HCV(+) was detected in 93.2% (276/296) of PWIDs and in 3.7% (31/839) of MSM. After a median FU of 6.9 years, 1644 (91.0%) patients underwent FU HCV-testing: 167 (90.3%) of PWIDs and 49 (6.7%) of MSM showed anti-HCV(+). Among 208 viraemic HCV-RNA(+) patients at BL, 30 (14.4%) had spontaneously cleared HCV, 76 (36.5%) achieved treatment-induced eradication and 89 (42.8%) remained HCV-RNA(+) at last FU. Among 1433 initially HCV-naive patients, 45 (3.5%) acquired de-novo HCV infection (11.1% PWIDs/80.0% MSM; incidence rate (IR) 0.004%; 95% confidence interval [CI] 0.0%-0.022%) and 14 had HCV reinfections (85.7% PWIDs/14.3% other; IR 0.001%; 95% CI 0.0%-0.018%) during a median FU of 6.7 years (interquartile range 7.4). CONCLUSION: Hepatitis C virus testing was successfully implemented in the Viennese HIV(+) patients. Anti-HCV(+) prevalence remained stable in HIV+ PWIDs but almost doubled in HIV+ MSM. De-novo HCV infection occurred mostly in MSM, while HCV reinfections were mainly observed in PWIDs. HCV treatment uptake was suboptimal with 42.8% remaining HCV-RNA(+) at FU.

Impact of HSD17B13 rs72613567 genotype on hepatic decompensation and mortality in patients with portal hypertension.

BACKGROUND & AIMS: The loss-of-function rs72613567 T > TA-variant in the 17ß-hydroxysteroid dehydrogenase 13 (HSD17B13) gene might protect from alcoholic and non-alcoholic fatty liver disease (ALD/NAFLD) and associated fibrosis/cirrhosis. We investigated the impact of the T > TA-variant on hepatic decompensation and mortality and investigated its implications on retinol and sex steroid metabolism in patients who had already developed advanced chronic liver disease (ACLD). METHODS: Retrospective analysis in prospectively characterized patients with viral hepatitis- and ALD/NAFLD-induced portal hypertension (hepatic venous pressure gradient (HVPG) ≥ 6 mmHg) diagnosed at the Medical University of Vienna. RESULTS: Among 487 patients who were followed longitudinally, 166 (34%) were heterozygous and 24 (5%) were homozygous for the 'protective' TA-allele. Patients harbouring at least one TA-allele had a lower MELD (9 (8-12) vs 10 (8-13) points; P = .003) and showed a trend towards lower HVPG (16 ± 6 vs 17 ± 7 mmHg; P = .067). Interestingly, in competing risk analyses adjusted for age, HVPG and MELD, harbouring the TA-allele was associated with numerically increased risks for mortality (adjusted subdistribution hazard ratio (aSHR): 1.3 (95% confidence interval (95% CI): 0.888-1.91); P = .18), liver-related death (aSHR: 1.34 (95% CI: 0.9-1.98); P = .15) and hepatic decompensation (aSHR: 1.29 (95% CI: 0.945-1.77); P = .11). This might be explained by trends towards worse outcomes (eg liver-related death: aSHR: 1.64 (95% CI: 0.95-2.84); P = .076) in patients with viral hepatitis-induced ACLD. In a cross-sectional analysis of 211 additional patients, serum retinol levels were comparable between HSD17B13 genotypes, but in males, serum testosterone levels numerically decreased with an increasing number of TA-alleles. CONCLUSION: In patients with viral hepatitis- and ALD-induced portal hypertension, the T > TA-variant was not protective of hepatic decompensation and mortality. Further studies should investigate the pathophysiological mechanisms underlying the effects of HSD17B13 genotype at different stages of liver disease.

Processed Eggshell Membrane Powder Is a Promising Biomaterial for Use in Tissue Engineering.

The purpose of this study was to investigate the tissue regenerating and biomechanical properties of processed eggshell membrane powder (PEP) for use in 3D-scaffolds. PEP is a low-cost, natural biomaterial with beneficial bioactive properties. Most importantly, this material is available as a by-product of the chicken egg processing (breaking) industry on a large scale, and it could have potential as a low-cost ingredient for therapeutic scaffolds. Scaffolds consisting of collagen alone and collagen combined with PEP were produced and analyzed for their mechanical properties and the growth of primary fibroblasts and skeletal muscle cells. Mechanical testing revealed that a PEP/collagen-based scaffold increased the mechanical hardness of the scaffold compared with a pure collagen scaffold. Scanning electron microscopy (SEM) demonstrated an interconnected porous structure for both scaffolds, and that the PEP was evenly distributed in dense clusters within the scaffold. Fibroblast and skeletal muscle cells attached, were viable and able to proliferate for 1 and 2 weeks in both scaffolds. The cell types retained their phenotypic properties expressing phenotype markers of fibroblasts (TE7, alpha-smooth muscle actin) and skeletal muscle (CD56) visualized by immunostaining. mRNA expression of the skeletal muscle markers myoD, myogenin, and fibroblasts marker (SMA) together with extracellular matrix components supported viable phenotypes and matrix-producing cells in both types of scaffolds. In conclusion, PEP is a promising low-cost, natural biomaterial for use in combination with collagen as a scaffold for 3D-tissue engineering to improve the mechanical properties and promote cellular adhesion and growth of regenerating cells.