Fatal familial insomnia (FFI) is a rare neurodegenerative disease caused by a dominantly inherited single amino acid substitution (D178N) within the prion protein (PrP). No in vitro human brain ...tissue model for this disease has previously been available. Consequently, how this mutation exerts its damaging effect on brain cells is still unknown. Using CRISPR-Cas9 engineered induced pluripotent stem cells, we made D178N cerebral organoids and compared these with isotype control organoids. We found that, in the absence of other hallmarks of FFI, the D178N organoids exhibited astrogliosis with cellular oxidative stress. Abnormal post-translational processing of PrP was evident but no tissue deposition or propagation of mis-folded PrP isoforms were observed. Neuronal electrophysiological function was compromised and levels of neurotransmitters, particularly acetylcholine and GABA, altered. Underlying these dysfunctions were changes in cellular energy homeostasis, with substantially increased glycolytic and Krebs cycle intermediates, and greater mitochondrial activity. This increased energy demand in D178N organoids was associated with increased mitophagy and depletion of lipid droplets, in turn resulting in shifts of cellular lipid composition. Using a double mutation (178NN) we could confirm that most changes were caused by the presence of the mutation rather than interaction with PrP molecules lacking the mutation. Our data strongly suggests that shifting biosynthetic intermediates and oxidative stress, caused by an imbalance of energy supply and demand, results in astrogliosis with compromised neuronal activity in FFI organoids. They further support that many of the disease associated changes are due to a corruption of PrP function and do not require propagation of PrP mis-folding.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective To evaluate whether male fertility status and/or embryo quality during in vitro fertilization (IVF) therapy can be predicted based on genomewide sperm deoxyribonucleic acid (DNA) ...methylation patterns. Design Retrospective cohort study. Setting University-based fertility center. Patient(s) Participants were 127 men undergoing IVF treatment (where any major female factor cause of infertility had been ruled out), and 54 normozoospermic, fertile men. The IVF patients were stratified into 2 groups: patients who had generally good embryogenesis and a positive pregnancy (n = 55), and patients with generally poor embryogenesis (n = 72; 42 positive and 30 negative pregnancies) after IVF. Intervention(s) Genomewide sperm DNA methylation analysis was performed to measure methylation at >485,000 sites across the genome. Main Outcome Measure(s) A comparison was made of DNA methylation patterns of IVF patients vs. normozoospermic, fertile men. Result(s) Predictive models proved to be highly accurate in classifying male fertility status (fertile or infertile), with 82% sensitivity, and 99% positive predictive value. Hierarchic clustering identified clusters enriched for IVF patient samples and for poor-quality–embryo samples. Models built to identify samples within these groups, from neat samples, achieved positive predictive value ≥94% while identifying >one fifth of all IVF patient and poor-quality–embryo samples in each case. Using density gradient prepared samples, the same approach recovered 46% of poor-quality–embryo samples with no false positives. Conclusion(s) Sperm DNA methylation patterns differ significantly and consistently for infertile vs. fertile, normozoospermic men. In addition, DNA methylation patterns may be predictive of embryo quality during IVF.
In mammalian embryos, proper zygotic genome activation (ZGA) underlies totipotent development. Double homeobox (DUX)-family factors participate in ZGA, and mouse Dux is required for forming cultured ...two-cell (2C)-like cells. Remarkably, in mouse embryonic stem cells, Dux is activated by the tumor suppressor p53, and Dux expression promotes differentiation into expanded-fate cell types. Long-read sequencing and assembly of the mouse Dux locus reveals its complex chromatin regulation including putative positive and negative feedback loops. We show that the p53-DUX/DUX4 regulatory axis is conserved in humans. Furthermore, we demonstrate that cells derived from patients with facioscapulohumeral muscular dystrophy (FSHD) activate human DUX4 during p53 signaling via a p53-binding site in a primate-specific subtelomeric long terminal repeat (LTR)10C element. In summary, our work shows that p53 activation convergently evolved to couple p53 to Dux/DUX4 activation in embryonic stem cells, embryos and cells from patients with FSHD, potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD.
Human brain has proven difficult to model, and this tissue is not widely available for study. ...the application of organoid technology to produce 3D cultures of self-patterning brain tissue, human ...cerebral organoids (hCOs), offers great promise for investigating how brain cells become dysfunctional and die in neurodegenerative conditions. Organoids permit investigation into various aspects of prion disease including (1) different sCJD subtypes; (2) neuronal dysfunction and death; (3) cellular cross-talk; (4) conversion initiating triggers; and (5) efficacy of putative therapeutics. hCO, human cerebral organoid; PrP, prion protein; sCJD, sporadic Creutzfeldt–Jakob disease. https://doi.org/10.1371/journal.ppat.1009747.g001 Organoids become infected with and accumulate human prions Until 2017, no reproducible human cell model of prion infection existed. There was precedent in the literature from mouse models that neural stem cells (NSCs), and mature cultures differentiated from NSCs, would be permissible to infection 1–3. ...iPSCs were a prime technology to apply to this problem. Other cells are also involved in maintaining brain health, including microglia and endothelial cells, but these do not originate from the neuroectoderm lineage used to differentiate the neural lineage cells. ...different approaches are being developed to study these cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prion diseases are a group of rare, transmissible, and invariably fatal neurodegenerative diseases that affect both humans and animals. The cause of these diseases is misfolding of the prion protein ...into pathological isoforms called prions. Of all human prion diseases, 10–15% of cases are genetic and the E200K mutation, which causes familial Creutzfeldt-Jakob disease (CJD), is the most prevalent. For both sporadic and genetic disease, it remains uncertain as to how initial protein misfolding is triggered. Prior studies have linked protein misfolding with oxidative stress insults, deregulated interactions with cellular cofactors, and viral infections. Our previous work developed a cerebral organoid (CO) model using human induced pluripotent stem cells containing the E200K mutation. COs are three-dimensional human neural tissues that permit the study of host genetics and environmental factors that contribute to disease onset. Isogenically matched COs with and without the E200K mutation were used to investigate the propensity of E200K PrP to misfold following cellular insults associated with oxidative stress. Since viral infections have also been associated with oxidative stress and neurodegenerative diseases, we additionally investigated the influence of Herpes Simplex Type-1 virus (HSV1), a neurotropic virus that establishes life-long latent infection in its host, on E200K PrP misfolding. While COs proved to be highly infectable with HSV1, neither acute nor latent infection, or direct oxidative stress insult, resulted in evidence of E200K prion misfolding. We conclude that misfolding into seeding-active PrP species is not readily induced by oxidative stress or HSV1 in our organoid system.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this work we quantify and characterise the effects of air-oxidation on nitrogen-vacancy defect luminescence in both high-temperature-high-pressure and detonation synthesized nanodiamonds using ...Raman and luminescence spectroscopies. We find that oxidation treatments result in an increased nitrogen-vacancy centre excited state lifetime from 13
ns to 25
ns and in 5-nm diamonds the intensity of this luminescence increases by at least 5-fold. At the same time, in 5-nm diamonds, short lived surface-defect related luminescence is reduced by 10-fold. Furthermore we find that air oxidation reduces the sp
2 and disordered carbon fraction of nanodiamonds by up to 5-fold in 5-nm nanodiamonds. Based on these results, the authors suggest that the disordered-carbon and graphite shell of 5-nm nanodiamonds quenches nitrogen-vacancy luminescence, and that this quenching can be partially reduced by surface oxidation. These findings provide useful insights into the role of the graphite and disordered carbon shell in quenching luminescence, and have implications for the applicability of 5-nm nanodiamonds to bio- and quantum physics applications.
OBJECTIVE:--The purpose of this article was to determine the relationships among total body fat, visceral adipose tissue (VAT), fat cell size (FCS), ectopic fat deposition in liver (intrahepatic ...lipid IHL) and muscle (intramyocellular lipid IMCL), and insulin sensitivity index (Si) in healthy overweight, glucose-tolerant subjects and the effects of calorie restriction by diet alone or in conjunction with exercise on these variables. RESEARCH DESIGN AND METHODS--Forty-eight overweight volunteers were randomly assigned to four groups: control (100% of energy requirements), 25% calorie restriction (CR), 12.5% calorie restriction +12.5% energy expenditure through structured exercise (CREX), or 15% weight loss by a low-calorie diet followed by weight maintenance for 6 months (LCD). Weight, percent body fat, VAT, IMCL, IHL, FCS, and Si were assessed at baseline and month 6. RESULTS:--At baseline, FCS was related to VAT and IHL (P < 0.05) but not to IMCL. FCS was also the strongest determinant of Si (P < 0.01). Weight loss at month 6 was 1 ± 1% (control, mean ± SE), 10 ± 1% (CR), 10 ± 1% (CREX), and 14 ± 1% (LCD). VAT, FCS, percent body fat, and IHL were reduced in the three intervention groups (P < 0.01), but IMCL was unchanged. Si was increased at month 6 (P = 0.05) in the CREX (37 ± 18%) and LCD (70 ± 34%) groups (P < 0.05) and tended to increase in the CR group (40 ± 20%, P = 0.08). Together the improvements in Si were related to loss in weight, fat mass, and VAT, but not IHL, IMCL, or FCS. CONCLUSIONS:--Large adipocytes lead to lipid deposition in visceral and hepatic tissues, promoting insulin resistance. Calorie restriction by diet alone or with exercise reverses this trend.
Kidney donation results in reductions in kidney function and lasting perturbations in phosphate homeostasis, which may lead to adverse cardiovascular sequelae. However, the acute effects of kidney ...donation on bone mineral parameters including regulators of calcium and phosphate metabolism are unknown. We conducted a prospective observational controlled study to determine the acute effects of kidney donation on mineral metabolism and skeletal health. Biochemical endpoints were determined before and after donation on days 1, 2 and 3, 6 weeks and 12 months in donors and at baseline, 6 weeks and 12 months in controls. Baseline characteristic of donors (n = 34) and controls (n = 34) were similar: age (53±10 vs 50±14 years, p = 0.33), BMI (26.3±2.89 vs 25.9±3.65, p = 0.59), systolic BP (128±13 vs 130±6 mmHg, p = 0.59), diastolic BP (80±9 vs 81±9 mmHg, p = 0.68) and baseline GFR (84.4±20.2 vs 83.6±25.2 ml/min/1.73m2, p = 0.89). eGFR reduced from 84.4±20.2 to 52.3±17.5 ml/min/1.73m2 (p<0.001) by day 1 with incomplete recovery by 12 months (67.7±22.6; p = 0.002). Phosphate increased by day 1 (1.1(0.9-1.2) to 1.3(1.1-1.4) mmol/L, p <0.001) but declined to 0.8(0.8-1.0) mmol/L (p<0.001) before normalizing by 6 weeks. Calcium declined on day 1 (p = 0.003) but recovered at 6 weeks or 12 months. PTH and FGF-23 remained unchanged, but α-Klotho reduced by day 1 (p = 0.001) and remained low at 6 weeks (p = 0.02) and 1 year (p = 0.04). In this study, we conclude that kidney donation results in acute disturbances in mineral metabolism characterised by a reduced phosphate and circulating α-Klotho concentration without acute changes in the phosphaturic hormones FGF23 and PTH.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In contrast to other cancers, survival rates for pancreatic ductal adenocarcinoma (PDAC) patients have improved but minimally over the past thirty years. The aim of this study was to perform a ...meta-analysis of clinical trials published since 1986 to determine trends in median overall survival in primarily metastatic PDAC.
All Phase 2-4 clinical trials published during or after 1986 investigating first-line systemic chemotherapy in metastatic PDAC were included in the meta-analysis. Publications obtained through PubMed and www.ClinicalTrials.gov were cross-referenced to identify additional trials. Trials enrolling fewer than 50% of study participants with metastatic disease were excluded.
Of 19,488 patients enrolled in 151 clinical trials, 84% had metastatic disease and 16% had locally advanced pancreatic cancer. In clinical trials published from 1986 to 2016, the weighted median overall survival (wMOS) increased by 3.0 months. The median wMOS was higher in combination therapy (7.31 months, IQR 5.4 to 8.5) compared to non-gemcitabine, single-agent therapy (4.76 months, IQR 3.5 to 6.0), gemcitabine monotherapy (6.48 months, IQR 5.9 to 7.2), and gemcitabine plus single-agent therapy (7.09 months, IQR 6.3 to 8.2). Of all regimens used in more than one study arm, FOLFIRINOX had the highest wMOS (10.9 months).
Regardless of treatment regimen, survival rates in PDAC have minimally improved over time. Of drugs used in two or more study arms, only FOLFIRINOX has a wMOS greater than ten months. Emphasis should, therefore, be placed on identification of novel targets that promote early diagnosis and intervention.
The authors on this manuscript are in parts, supported by grants from the National Institutes of Health (EDRN U01 CA200466, SPORE P50 CA127297, R01 CA183459, R21 AA026428 and R01 CA 195586).
ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the ...nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of ∼13 bp/s and generate forces up to ∼12 pN, producing DNA loops of a broad range of sizes (20–1200 bp, average ∼100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.
PDF
