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Minecraft : stories from the overworld
\"With tales of witch and pillager rivals finding common ground, a heartless griefer who bit off more than they could chew, and valiant heroes new (or not!) to the Overworld, this anthology tells tales that span the world of Minecraft. Featuring stories from ... writers Hope Larson, Kevin Panetta, Rafer Roberts, and Ian Flynn and ... artists Meredith Gran and more, this collection brings together stories from all realms, leaving no block unturned\"-- Provided by publisher.
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Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly
Volume-regulated anion channels (VRACs) mediate volume regulatory Cl - and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL1 25 ) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL1 25 ) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL1 25 ) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.
Journal Article
The Skp1 Homologs SKR-1/2 Are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK
SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.
Journal Article
Genome-Wide RNAi Screening Identifies Protein Damage as a Regulator of Osmoprotective Gene Expression
The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter ($P_{gpdh-1}::GFP$) is detected only during hypertonic stress but is not induced by other stressors. Using $P_{gpdh-1}::GFP$ expression as a phenotype, we screened ≈16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function.
Journal Article
The protein tyrosine phosphatase 1B inhibitor MSI-1436 stimulates regeneration of heart and multiple other tissues
Regenerative medicine holds substantial promise for repairing or replacing tissues and organs damaged by disease, injury, and degeneration. Much of the field has focused on development of cell-based therapeutics, gene-based therapeutics, and tissue engineering-based therapeutics. In contrast, development of small molecule regenerative medicine therapies is an emerging area. Using the adult zebrafish as a novel screening platform, we identified MSI-1436 as a first-in-class regenerative medicine drug candidate. MSI-1436 is a naturally occurring aminosterol that inhibits protein tyrosine phosphatase 1B. Treatment of adult zebrafish by intraperitoneal injection of MSI-1436 increased the rate of regeneration of the amputated caudal fin, which is comprised of bone, connective, skin, vascular and nervous tissues and also increased the rate of adult zebrafish heart regeneration. Intraperitoneal administration of MSI-1436 to adult mice for 4 weeks after induction of myocardial infarction increased survival, improved heart function, reduced infarct size, reduced ventricular wall thinning and increased cardiomyocyte proliferation. Satellite cell activation in injured mouse skeletal muscle was stimulated by MSI-1436. MSI-1436 was well tolerated by patients in Phase 1 and 1b obesity and type 2 diabetes clinical trials. Doses effective at stimulating regeneration are 5–50-times lower than the maximum well tolerated human dose. The demonstrated safety and well established pharmacological properties of MSI-1436 underscore the potential of this molecule as a novel treatment for heart attack and multiple other degenerative diseases.
The enzyme inhibitor that regenerates multiple tissues
A naturally occurring small molecule shows promise as a drug for tissue and organ repair and regeneration. Viravuth Yin of the Kathryn W. Davis Center for Regenerative Biology and Medicine with colleagues in the US found that treating zebrafish with an intraperitoneal injection of MSI-1436, which inhibits the enzyme ‘protein tyrosine phosphatase 1B’, increased the rate of regeneration of an amputated caudal fin and of partially removed heart muscle without apparent tissue malformation. Intraperitoneal injection of MSI-1436 in adult mice also reduced the size of an induced heart infarction, improved survivability, triggered new heart muscle formation and stimulated regeneration after skeletal muscle injury. Effective doses for tissue regeneration in both animals were much lower than the maximum tolerated doses found for humans in clinical trials for potential treatment of obesity and diabetes.
Journal Article
Abnormal Osmotic Avoidance Behavior in C. elegans Is Associated with Increased Hypertonic Stress Resistance and Improved Proteostasis
Protein function is controlled by the cellular proteostasis network. Proteostasis is energetically costly and those costs must be balanced with the energy needs of other physiological functions. Hypertonic stress causes widespread protein damage in C. elegans. Suppression and management of protein damage is essential for optimal survival under hypertonic conditions. ASH chemosensory neurons allow C. elegans to detect and avoid strongly hypertonic environments. We demonstrate that mutations in osm-9 and osm-12 that disrupt ASH mediated hypertonic avoidance behavior or genetic ablation of ASH neurons are associated with enhanced survival during hypertonic stress. Improved survival is not due to altered systemic volume homeostasis or organic osmolyte accumulation. Instead, we find that osm-9(ok1677) mutant and osm-9(RNAi) worms exhibit reductions in hypertonicity induced protein damage in non-neuronal cells suggesting that enhanced proteostasis capacity may account for improved hypertonic stress resistance in worms with defects in osmotic avoidance behavior. RNA-seq analysis revealed that genes that play roles in managing protein damage are upregulated in osm-9(ok1677) worms. Our findings are consistent with a growing body of work demonstrating that intercellular communication between neuronal and non-neuronal cells plays a critical role in integrating cellular stress resistance with other organismal physiological demands and associated energy costs.
Journal Article
Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans
Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50-70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50-80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70-180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought about by an environmental stressor, demonstrate important differences in aging- versus stress-induced protein damage, and challenge the widely held view that chemical chaperones are accumulated during hypertonic stress to protect protein structure/function.
Journal Article
Leucine‐rich repeat containing protein LRRC8A is essential for swelling‐activated Cl− currents and embryonic development in zebrafish
A volume‐regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl− and small organic solutes from cells. Two groups recently identified the mammalian leucine‐rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B‐E, to reconstitute VRAC activity in LRRC8−/− cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C‐E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling‐activated Cl− current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa. VRAC activity is fully reconstituted in LRRC8−/− human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins. A volume‐regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. Recent studies have identified the mammalian leucine‐rich repeat containing protein LRRC8A as an essential VRAC component. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins.
Journal Article
Regulation of Cell Volume in Health and Disease
Maintenance of a constant volume in the face of extracellular and intracellular osmotic perturbations is a critical problem faced by all cells. Most cells respond to swelling or shrinkage by activating specific metabolic or membrane-transport processes that return cell volume to its normal resting state. These processes are essential for the normal function and survival of cells. This article will outline the cellular and molecular events underlying cell-volume homeostasis and discuss their relevance to medical practice.
Fundamentals of Cell-Volume Regulation
Meeting Osmotic Challenges
Water is in thermodynamic equilibrium across the plasma membrane. In other words, the osmotic concentrations of cytoplasmic . . .
Journal Article