Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The ...antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress–induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis.
•Gpx4 is essential for preventing anemia in mice via inhibiting RIP3-dependent necroptosis in erythroid precursor cells.•ROS accumulation and lipid peroxidation in erythroid precursor cells trigger receptor-independent activation of necroptosis.
Differentiation alters molecular properties of stem and progenitor cells, leading to changes in their shape and movement characteristics. We present a deep neural network that prospectively predicts ...lineage choice in differentiating primary hematopoietic progenitors using image patches from brightfield microscopy and cellular movement. Surprisingly, lineage choice can be detected up to three generations before conventional molecular markers are observable. Our approach allows identification of cells with differentially expressed lineage-specifying genes without molecular labeling.
The accumulation of the human tumor suppressor 53BP1 at DNA damage sites requires the ubiquitin ligases RNF8 and RNF168. As 53BP1 recognizes dimethylated Lys20 in histone H4 (H4K20me2), the ...requirement for RNF8- and RNF168-mediated ubiquitylation has been unclear. Here we show that RNF8-mediated ubiquitylation facilitates the recruitment of the AAA-ATPase valosin-containing protein (VCP, also known as p97) and its cofactor NPL4 to sites of double-strand breaks. RIDDLE cells, which lack functional RNF168, also show impaired recruitment of VCP to DNA damage. The ATPase activity of VCP promotes the release of the Polycomb protein L3MBTL1 from chromatin, which also binds the H4K20me2 histone mark, thereby facilitating 53BP1 recruitment. Consistent with this, nematodes lacking the VCP orthologs CDC-48.1 or CDC-48.2, or cofactors UFD-1 or NPL-4, are highly sensitive to ionizing radiation. Our data suggest that human RNF8 and RNF168 promote VCP-mediated displacement of L3MBTL1 to unmask 53BP1 chromatin binding sites.
Due to the development in team handball, there is a need to optimize the physical capacities of team handball players for which knowledge of the physical match demands is essential. The aim of this ...study was to investigate the physical match demands of four LIQUI-MOLY Handball-Bundesliga (HBL) teams across three seasons with respect to the effects of season, team, match outcome, playing position, and halftime.
A fixed installed local positioning system (Kinexon) was used, collecting 2D positional and 3D inertial measurement unit data at 20 and 100 Hz, respectively. The physical match demands were operationalized by basic (e.g., distance, speed, and acceleration) and more advanced variables (e.g., jumps, throws, impacts, acceleration load, and metabolic power). A total of 347 matches (213 with an additional ball tracking) were analyzed from four teams (one top, two middle, and one lower ranked) during three consecutive seasons (2019-2022). One-way ANOVAs were calculated to estimate differences between more than two groups (e.g., season, team, match outcome, playing position). Mean differences between halftimes were estimated using Yuen's test for paired samples.
Large effects were detected for the season (
), team (
), and playing position (
). Medium effects were found for match outcome (
) and halftime (
).
For the first time, we provide a comprehensive analysis of physical match demands in handball players competing in the LIQUI-MOLY Handball-Bundesliga. We found that physical match demands differ on that top-level with up to large effect sizes concerning the season, team, match outcome, playing position, and halftime. Our outcomes can help practitioners and researchers to develop team and player profiles as well as to optimize talent identification, training, regeneration, prevention, and rehabilitation procedures.
Two possible future biofuels, 2-butanone also referred to as methyl ethyl ketone (MEK) and 2-methylfuran, identified within the Cluster of Excellence “Tailor-Made Fuels from Biomass” (TMFB), have ...been evaluated as pure fuels in the present study. Investigations of the autoignition tendency were carried out on a rapid compression machine (RCM), whereas thermodynamic investigations were conducted on a direct injection spark ignition single cylinder research engine. 2-Methylfuran and 2-butanone were compared against the present benchmark biofuel for spark ignition engines ethanol and conventional RON95 gasoline. A similar autoignition tendency compared to ethanol was found for 2-methylfuran. In case of 2-butanone very high ignition delay times were measured, even higher than for ethanol and 2-methylfuran. For 2-butanone and 2-methylfuran, the lower heat of vaporization in combination with higher vapor pressure and better primary breakup compared to ethanol are beneficial for mixture formation. During the engine testing for both fuels, superior characteristics compared to conventional gasoline and ethanol were identified. In case of 2-methylfuran, an increased combustion stability, especially at low engine load and cold boundary conditions, could be found at a higher knock resistance than conventional gasoline. In combination with increased compression ratio this enables an efficiency increase of up to 19%, whereas for ethanol an even further increase of up to 21% is possible. 2-Butanone shows increased combustion stability at low engine load and cold boundary conditions compared to ethanol and also conventional gasoline as well as highest knock resistance equal to ethanol. However, for both 2-butanone and 2-methylfuran increased emissions of nitrogen oxides were found when compared to ethanol. For both possible future biofuels and also ethanol, a significant reduction of particle emissions compared to conventional gasoline was found.
Human organoids allow the study of proliferation, lineage specification, and 3D tissue development. Here we present a genome-wide CRISPR screen in induced pluripotent stem cell (iPSC)-derived kidney ...organoids. The combination of inducible genome editing, longitudinal sampling, and endpoint sorting of tubular and stromal cells generated a complex, high-quality dataset uncovering a broad spectrum of insightful biology from early development to “adult” epithelial morphogenesis. Our functional dataset allows improving mesoderm induction by ROCK inhibition, contains monogenetic and complex trait kidney disease genes, confirms two additional congenital anomalies of the kidney and urinary tract (CAKUT) genes (CCDC170 and MYH7B), and provides a large candidate list of ciliopathy-related genes. Finally, identification of a cis-inhibitory effect of Jagged1 controlling epithelial proliferation shows how mosaic knockouts in pooled CRISPR screening can reveal ways of communication between heterogeneous cell populations in complex tissues. These data serve as a rich resource for the kidney research community and as a benchmark for future iPSC-derived organoid CRISPR screens.
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•Whole-genome pooled CRISPR screen in iPSC-derived kidney organoids by an inducible Cas9•ROCKi facilitates mesoderm induction and early differentiation into the kidney lineage•Nomination of relevant genes for childhood/chronic kidney diseases and ciliopathies•Mosaic knockout leads to discovery of a cis-inhibitory effect of Jag1 in tubular cells
Ungricht et al. use a doxycycline-inducible Cas9 for whole-genome pooled CRISPR screening in kidney organoids. Cross-comparison of longitudinal samples nominates hits between early development and late lineage-specific phenotypes. Hit follow-up confirms that ROCK inhibition facilitates differentiation, suggests numerous disease-relevant genes, and describes a Jag1 cis-inhibitory effect in tubular epithelial cells.
It was suggested that intestinal mucosal secretion is enhanced during muscle relaxation and contraction. Mechanisms of mechanically induced secretion have been studied in rodent species. We used ...voltage clamp Ussing technique to investigate, in human and porcine colonic tissue, secretion evoked by serosal (Pser) or mucosal (Pmuc) pressure application (2-60 mmHg) to induce distension into the mucosal or serosal compartment, respectively. In both species, Pser or Pmuc caused secretion due to Cl- and, in human colon, also HCO3- fluxes. In the human colon, responses were larger in proximal than distal regions. In porcine colon, Pmuc evoked larger responses than Pser whereas the opposite was the case in human colon. In both species, piroxicam revealed a strong prostaglandin (PG) dependent component. Pser and Pmuc induced secretion was tetrodotoxin (TTX) sensitive in porcine colon. In human colon, a TTX sensitive component was only revealed after piroxicam. However, synaptic blockade by ω-conotoxin GVIA reduced the response to mechanical stimuli. Secretion was induced by tensile rather than compressive forces as preventing distension by a filter inhibited the secretion. In conclusion, in both species, distension induced secretion was predominantly mediated by PGs and a rather small nerve dependent response involving mechanosensitive somata and synapses.
Diabetes worsens atherosclerosis progression and leads to a defect in repair of arteries after cholesterol reduction, a process termed regression. Empagliflozin reduces blood glucose levels via ...inhibition of the sodium glucose cotransporter 2 (SGLT-2) in the kidney and has been shown to lead to a marked reduction in cardiovascular events in humans. To determine whether glucose lowering by empagliflozin accelerates atherosclerosis regression in a mouse model, male C57BL/6J mice were treated intraperitoneally with LDLR- and SRB1- antisense oligonucleotides and fed a high cholesterol diet for 16 weeks to induce severe hypercholesterolemia and atherosclerosis progression. At week 14 all mice were rendered diabetic by streptozotocin (STZ) injections. At week 16 a baseline group was sacrificed and displayed substantial atherosclerosis of the aortic root. In the remaining mice, plasma cholesterol was lowered by switching to chow diet and treatment with LDLR sense oligonucleotides to induce atherosclerosis regression. These mice then received either empagliflozin or vehicle for three weeks. Atherosclerotic plaques in the empagliflozin treated mice were significantly smaller, showed decreased lipid and CD68
macrophage content, as well as greater collagen content. Proliferation of plaque resident macrophages and leukocyte adhesion to the vascular wall were significantly decreased in empagliflozin-treated mice. In summary, plasma glucose lowering by empagliflozin improves plaque regression in diabetic mice.
Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further ...increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target
was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases.
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