DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the ...interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.
Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone ...modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.
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•Modules of correlated molecular phenotypes represent inter-individual chromatin variation•Variable chromatin modules (VCMs) are embedded within chromosomal contact domains•VCMs are orchestrated by cis-acting genetic variation•VCMs rationalize chromatin state changes that are independent of local DNA sequence
Spatially defined chromosome regions, termed variable chromatin modules, exhibit coordinated chromatin state changes across cis-regulatory elements. Within these modules, genetic changes distal from the regulatory element itself can induce variation in chromatin patterns between individuals.
•A brief overview of principles of TGE using mammalian cells.•Description of TGE processes for HEK293 and CHO cells.•Description of orbitally shaken bioreactors for suspension cell ...cultivation.•Description of polyethylenime-based transfection processes.
Transient gene expression (TGE) from mammalian cells is an increasingly important tool for the rapid production of recombinant proteins for research applications in biochemistry, structural biology, and biomedicine. Here we review methods for the transfection of human embryo kidney (HEK-293) and Chinese hamster ovary (CHO) cells in suspension culture using the cationic polymer polyethylenimine (PEI) for gene delivery.
To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic ...peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K i = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody–drug conjugates.
In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI‐ APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a ...probe. We find that GPI‐APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft‐like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI‐APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI‐APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI‐APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI‐APs in different cell types depend on the residence time of GPI‐APs in lipid rafts, and hence that raft partitioning regulates GPI‐APs sorting in the endocytic pathway.
Since 2014, the Swiss Hepatitis Strategy (SHS) has targeted the elimination of Hepatitis C Virus (HCV) in Switzerland. The epidemiology of HCV is diverse across Swiss cantons, therefore ...cantonal-level screening and treatment strategies should be developed. This study aimed to identify scenarios to achieve HCV elimination in the canton of Bern by 2030. A preexisting Markov disease burden model was populated with data for Bern, and used to forecast the current and future prevalence of HCV, annual liver-related deaths (LRDs), and incidence of hepatocellular carcinoma and decompensated cirrhosis until 2030. Scenarios were developed to assess the current standard of care and potential long-term impact of the COVID-19 crisis on the HCV infected population. Additionally, potential scenarios for achieving the WHO 2030 targets and the SHS 2025 and 2030 targets (reduction of new cases of HCV, HCV-related mortality and viremic HCV cases) were identified. In 2019, there were an estimated 4,600 (95% UI: 3,330-4,940) viremic infections in the canton of Bern and 57% (n = 2,600) of viremic cases were diagnosed. This modelling forecasted a 10% increase in LRDs (28 in 2020 to 31 in 2030) with the current standard of care and a 50% increase in LRDs in a scenario assuming long-term delays. To achieve the WHO and SHS targets, the canton of Bern needs to increase the annual number of patients diagnosed (from 90 in 2019 to 250 per year in 2022-2024 WHO, or 500 per year in 2022-2025 SHS) and treated (from 130 in 2019 to 340 per year in 2022-2024 WHO or 670 per year in 2022-2025 SHS). The SHS goals and the WHO targets for HCV elimination can be achieved in the Swiss canton of Bern by 2030; however, not at the current pace of screening, linkage to care and treatment.
Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively ...investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.
ABSTRACT
Background and objective
Daily physical activity (PA) is reduced in patients with COPD. Previous cross‐sectional analyses indicate various predictors for a low level of PA including airway ...obstruction, exacerbations and co‐morbidities. However, information from longitudinal studies evaluating PA in the context of disease progression, survival and co‐morbidities is scant.
Methods
In a heterogeneous cohort of COPD patients, we annually assessed the number of steps per day over 1 week and potential determinants including lung function, exacerbations and co‐morbidities. Univariable and multivariable mixed effect models were used to investigate associations between the change in steps per day (dependent variable) and possible predictors and their annual changes.
Results
A total of 177 COPD patients (46% GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage 1/2, 38% stage 3 and 16% stage 4) with a mean (min/max) follow‐up time of 2.7 (1/5) years were annually assessed. The number of steps per day decreased significantly over time (P < 0.001) with a mean annual change of −508 steps. The decrease in activity was significantly associated with forced expiratory volume in 1 s (FEV1
) % predicted (P = 0.020) but not with annual changes in FEV1
. Hyperinflation, exacerbations, co‐morbidities and their annual changes, and survival did not significantly affect change in PA.
Conclusion
COPD patients have a substantial decrease of PA over time. This decrease seems to be determined by the degree of airflow limitation. However, patients with a greater annual decline in lung function did not show a greater decrease in PA. The rate of decline in PA did not differ between survivors and non‐survivors in this cohort.
COPD patients have a substantial decrease of physical activity (PA) over time mainly determined by the degree of airflow limitation. The rate of decline in PA did not differ between survivors and non‐survivors in this cohort.
This study investigated adherence and associated factors among people with recent injection drug use (IDU) or current opioid agonist therapy (OAT) and compared once-daily to twice-daily hepatitis C ...virus (HCV) direct-acting antiviral (DAA) therapy.
SIMPLIFY and D3FEAT are international, multicenter studies that recruited participants with recent IDU (previous 6 months; SIMPLIFY, D3FEAT) or current OAT (D3FEAT) between March 2016 and February 2017 in 8 countries. Participants received sofosbuvir/velpatasvir (once daily; SIMPLIFY) or paritaprevir/ritonavir/ombitasvir, dasabuvir (twice daily) ± ribavirin (D3FEAT) for 12 weeks administered in electronic blister packs. We evaluated overall adherence (proportion of prescribed doses taken) and nonadherence (<90% adherent) between dosing patterns.
Of 190 participants, 184 (97%) completed treatment. Median adherence was 92%, with higher adherence among those receiving once-daily vs twice-daily therapy (94% vs 87%, P = .005). Overall, 40% of participants (n = 76) were nonadherent (<90% adherent). Recent stimulant injecting (odds ratio OR, 2.48 95% confidence interval {CI}, 1.28-4.82), unstable housing (OR, 2.18 95% CI, 1.01-4.70), and twice-daily dosing (OR, 2.81 95% CI, 1.47-5.36) were associated with nonadherence. Adherence decreased during therapy. Sustained virologic response was high in nonadherent (89%) and adherent populations (95%, P = .174), with no difference in SVR between those who did and did not miss 7 consecutive doses (92% vs 93%, P = .897).
This study demonstrated high adherence to once- and twice-daily DAA therapy among people with recent IDU or currently receiving OAT. Nonadherence described did not impact treatment outcomes, suggesting forgiveness to nonadherence.
Background
Currently, Roux-en Y gastric bypass (RYGB) is the most efficient therapy for severe obesity. Weight loss after surgery is, however, highly variable and genetically influenced. Genome-wide ...association studies have identified several single nucleotide polymorphisms (SNP) associated with body mass index (BMI) and waist-hip ratio (WHR). We aimed to identify two genetic risk scores (GRS) composed of weighted BMI and WHR-associated SNPs to estimate their impact on excess BMI loss (EBMIL) after RYGB surgery.
Methods
Two hundred and thirty-eight obese patients (BMI 45.1 ± 6.2 kg/m
2
, 74 % women), who underwent RYGB, were genotyped for 35 BMI and WHR-associated SNPs and were followed up after 2 years. SNPs with high impact on post-surgical weight loss were filtered out using a random forest model. The filtered SNPs were combined into a GRS and analyzed in a linear regression model.
Results
An up to 11 % lower EBMIL with higher risk score was estimated for two GRS models (
P
= 0.026 resp.
P
= 0.021) composed of seven BMI-associated SNPs (closest genes:
MC4R
,
TMEM160
,
PTBP2
,
NUDT3
,
TFAP2B
,
ZNF608
,
MAP2K5
,
GNPDA2
, and
MTCH2
) and of three WHR-associated SNPs (closest genes:
HOXC13
,
LYPLAL1
, and
DNM3
-
PIGC
). Patients within the lowest GRS quartile had higher EBMIL compared to patients within the other three quartiles in both models.
Conclusions
We identified two GRSs composed of BMI and WHR-associated SNPs with significant impact on weight loss after RYGB surgery using random forest analysis as a SNP selection tool. The GRS may be useful to pre-surgically evaluate the risks for patients undergoing RYGB surgery.
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