bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metformin preconditioning protects against myocardial stunning and preserves mitochondrial dynamics and protein translation in a mouse model of cardiac arrest
Authors: Rutledge Cody A. , Lagranha Claudia , Chiba Takuto , Redding Kevin , Stolz Donna B. , Goetzman Eric , Sims-Lucas Sunder , Kaufman Brett A. 
Published: August 2021

AbstractCardiac arrest (CA) causes high mortality due to multi-system organ damage attributable to ischemia-reperfusion injury. Recent work in our group found that among diabetic patients who experienced cardiac arrest, those taking metformin had less evidence of cardiac and renal damage after cardiac arrest when compared to those not taking metformin. To investigate the potential for metformin to impact cardiac arrest outcomes, the current study investigates metformin interventions on cardiac and renal outcomes in a non-diabetic CA mouse model. We found that two weeks of metformin pretreatment protects against reduced ejection fraction and reduces kidney ischemia-reperfusion injury at 24 hours post-arrest. This cardiac and renal protection depends on AMPK signaling, as demonstrated by outcomes in mice pretreated with the AMPK activator AICAR or metformin plus the AMPK inhibitor compound C.At this 24-hour time point, gene expression analysis showed that metformin pretreatment caused changes supporting autophagy, antioxidant response, and protein translation. Further investigation found associated improvements in mitochondrial structure and markers of autophagy. We also found that markers of protein nitrosylation between sham and arrest were not significantly different and thus unlikely to be a driver of gene expression differences. Notably, Western analysis indicated that protein synthesis was preserved in arrest hearts of animals pretreated with metformin. The AMPK activation-mediated protection of protein synthesis was also demonstrated in a hypoxia/reoxygenation cell culture model. Despite the positive impacts of pretreatment in vivo and in vitro, metformin did not preserve ejection fraction when deployed at resuscitation. Taken together, we propose that metformin’s in vivo cardiac preservation occurs through AMPK activation, requires adaptation before arrest, and is associated with preserved protein translation.New and NoteworthyWe leveraged a mouse model of cardiac arrest to study whether metformin pretreatment could protect cardiac function after arrest. We find that AMPK activity is crucial to this protection and characterize several pathways associated with this response. Signaling nodes with strong association may be precise targets to promote cardiac function in ischemia/reperfusion injury or in cardiac stunning.
Using AlphaFold for Rapid and Accurate Fixed Backbone Protein Design
Authors: Moffat Lewis , Greener Joe G. , Jones David T. 
Published: August 2021

AbstractThe prediction of protein structure and the design of novel protein sequences and structures have long been intertwined. The recently released AlphaFold has heralded a new generation of accurate protein structure prediction, but the extent to which this affects protein design stands yet unexplored. Here we develop a rapid and effective approach for fixed backbone computational protein design, leveraging the predictive power of AlphaFold. For several designs we demonstrate that not only are the AlphaFold predicted structures in agreement with the desired backbones, but they are also supported by the structure predictions of other supervised methods as well asab initiofolding. These results suggest that AlphaFold, and methods like it, are able to facilitate the development of a new range of novel and accurate protein design methodologies.
Enterohaemorrhagic E. coli utilizes an AND-OR logic gate to regulate expression of an outer membrane haem receptor
Authors: Sy Brandon M. , Tree Jai J. 
Published: August 2021

ABSTRACTTo sense the transition from environment to host, bacteria use a range of environmental cues to control expression of virulence genes. Iron is tightly sequestered in host tissues and in the human pathogen enterohaemorrhagic E. coli (EHEC) iron-limitation induces transcription of the outermembrane haem transporter encoded by chuAS. ChuA expression is post-transcriptionally activated at 37°C by a FourU RNA thermometer ensuring that the haem receptor is only expressed under low iron, high temperature conditions that indicate the host. Here we demonstrate that expression of chuA is also independently regulated by the cAMP-responsive sRNA CyaR and transcriptional terminator Rho. These results indicate that chuAS expression is regulated at the transcription initiation, transcript elongation, and translational level. The natural dependence of these processes creates a hierarchy of regulatory AND and OR logic gates that integrate information about the local environment. We show that the logic of the chuA regulatory circuit is activated under conditions that satisfy low iron AND (low glucose OR high temperature). We speculate that additional sensing of a gluconeogenic environment allows further precision in determining when EHEC is at the gastrointestinal epithelium of the host. With previous studies, it appears that the chuAS transcript is controlled by eight regulatory inputs that control expression through six different transcriptional and post-transcriptional mechanisms. The results highlight the ability of regulatory sRNAs to integrate multiple environmental signals into a conditional hierarchy of signal input.
Enterotoxigenic Escherichia coli display a distinct growth phase before entry into stationary phase with shifts in tryptophan- fucose- and putrescine metabolism and degradation of neurotransmitter precursors
Authors: Joffré Enrique , Xiao Xue , Correia Mário S. P. , Nookaew Intawat , Sasse Samantha , Globisch Daniel , Zhu Baoli , Sjöling Åsa 
Published: August 2021

AbstractEnterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition between logarithmic to stationary growth. The transient phase during late exponential growth is different from the subsequent induction of stationary phase-induced genes, including stress and survival responses as described earlier. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., L-tyrosine, L-tryptophan, L-phenylalanine, L-leucine, and L-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire transcriptome and metabolome showed activation of pathways involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli.ImportanceWe show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activation and repression of specific pathways. This leads to a rapid decrease of intracellular levels of L-tyrosine, L-tryptophan, L-phenylalanine, L-leucine, and L-glutamic acid due to metabolism into secondary compounds. The resulting metabolic activity leads to an intense but short peak of indole production, suggesting that this is the previously described “indole peak,” rapid decrease of intermediate molecules of bacterial neurotransmitters, increased putrescine and fucose uptake, increased glutathione levels, and decreased iron uptake. This specific transient shift in gene expression and metabolomics is short-lived and disappears when bacteria enter the stationary phase. We suggest it mainly prepares bacteria for ceased growth, but the pathways involved suggest that this transient phase substantially influences survival and virulence.
Systematic investigation of the link between enzyme catalysis and cold adaptation
Authors: Stark Catherine D. , Bautista-Leung Teanna , Siegfried Joanna , Herschlag Daniel 
Published: August 2021

AbstractCold temperature is prevalent across the biosphere and slows the rates of chemical reactions. Increased catalysis has been predicted to be a general adaptive trait of enzymes to reduced temperature, and this expectation has informed physical models for enzyme catalysis and influenced bioprospecting strategies. To broadly test rate as an adaptive trait to cold, we paired kinetic constants of 2223 enzyme reactions with their organism’s optimal growth temperature (TGrowth) and analyzed trends of rate as a function of TGrowth. These data do not support a prevalent increase in rate in cold adaptation. In the model enzyme ketosteroid isomerase (KSI), there was prior evidence for temperature adaptation from a change in an active site residue that results in a tradeoff between activity and stability. Here, we found that little of the overall rate variation for 20 KSI variants was accounted for by TGrowth. In contrast, and consistent with prior expectations, we observed a correlation between stability and TGrowth across 433 proteins. These results suggest that temperature exerts a weaker selection pressure on enzyme rate than stability and that evolutionary forces other than temperature are responsible for the majority of enzymatic rate variation.
Next generation SARM1 knockout and epitope tagged CRISPR-Cas9-generated isogenic mice reveal that SARM1 does not participate in regulating nuclear transcription, despite confirmation of protein expression in macrophages
Authors: Doran Ciara G. , Sugisawa Ryoichi , Carty Michael , Roche Fiona , Fergus Claire , Hokamp Karsten , Kelly Vincent P. , Bowie Andrew G 
Published: August 2021

ABSTRACTSARM1 is an ancient and highly conserved TIR-domain containing protein, with a diverse range of proposed roles in both innate immunity and neuronal death and degeneration. Murine SARM1 has been reported to regulate the transcription of specific chemokines in both neurons and macrophages, however the extent and mechanism by which SARM1 contributes to transcription regulation remains to be fully understood. Here, using RNA sequencing we identify differential gene expression in bone marrow-derived macrophages (BMDM) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared to wild type (WT). However, we show that passenger genes which are derived from the 129 donor strain of mice flank the Sarm1 locus, confounding interpretation of results, since many of the identified differentially regulated genes come from the region containing passenger genes. To re-examine the transcriptional role of SARM1 in the absence of such passenger genes, we generated three different Sarm1-/- mice using CRISPR/Cas9 technology. Vincristine treatment of ex vivo cultured post-natal neurons from these mice confirmed SARM1’s previously identified key function as an executor of axon degeneration. However, using these mice, we show that the absence of SARM1 has no impact on transcription of genes previously shown to be altered in macrophages or in the brainstem. To gain further insight into SARM1 function, we generated and characterized a mouse expressing epitope-tagged SARM1, as it has been difficult to date to confirm which cells and tissues express SARM1 protein. In these mice we see high SARM1 protein expression in the brain and brainstem, and lower but detectable levels in macrophages. Overall, the generation of these next generation SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages but has no general role in transcriptional regulation in these cells, and has provided important new animal models to further explore SARM1 function.
Standard metabolic rate does not associate with age-at-maturity genotype in juvenile Atlantic salmon
Authors: Åsheim Eirik R. , Prokkola Jenni M. , Morozov Sergey , Aykanat Tutku , Primmer Craig R 
Published: August 2021

ABSTRACTAtlantic salmon (Salmo salar) is a species with diverse life-history strategies, to which the timing of maturation contributes considerably. Recently, the genome region including the genevgll3has gained attention as a locus with a large effect on salmon maturation timing, and recent studies on thevgll3locus in salmon have indicated that its effect might be mediated through body condition and accumulation of adipose tissue. However, the cellular and physiological pathways leading fromvgll3genotype to phenotype are still unknown. Standard metabolic rate is a potentially important trait for resource acquisition and assimilation and we hypothesized that this trait, being a proxy for the maintenance energy expenditure of an individual, could be an important link in the pathway fromvgll3genotype to maturation-timing phenotype. As a first step to studying links betweenvgll3and the metabolic phenotype of Atlantic salmon, we measured the standard metabolic rate of 150 first year Atlantic salmon juveniles of both sexes, originating from 14 different families with either late maturing or early maturingvgll3genotypes. No significant difference in mass-adjusted standard metabolic rate was detected between individuals with differentvgll3genotypes, indicating that juvenile salmon of differentvgll3genotypes have similar maintenance energy requirements in the experimental conditions used and that the effects ofvgll3on body condition and maturation are not strongly related to maintenance energy expenditure in either sex at this life stage.Summary statementWe show thatvgll3, a gene known to have significant effects on Atlantic salmon (Salmo salar)life-history strategy, does not associate with standard metabolic rate in salmon juveniles.
Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg2+and ionic strength
Authors: Fleming Aaron M. , Howpay Manage Shereen A. , Burrows Cynthia J. 
Published: August 2021

AbstractThe base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1’s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+and Mg2+and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+found dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ions present showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using Δ33 or Δ61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis. With this knowledge, we found APE1 could remodel the F-containingVEGFpromoter dsDNA→G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.TOC graphic
Repair of APOBEC3G-mutated retroviral DNA in vivo is facilitated by the host enzyme uracil DNA glycosylase 2
Authors: Briceno Karen Salas , Ross Susan R. 
Published: August 2021

AbstractApolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3 (APOBEC3) proteins are critical for the control of infection by retroviruses. These proteins deaminate cytidines in negative strand DNA during reverse transcription, leading to G to A changes in coding strands. Uracil DNA glycosylase (UNG) is a host enzyme that excises uracils in genomic DNA, which the base excision repair machinery then repairs. Whether UNG removes uracils found in retroviral DNA after APOBEC3-mediated mutation is not clear, and whether this occurs in vivo has not been demonstrated. To determine if UNG plays a role in the repair of retroviral DNA, we used APOBEC3G (A3G) transgenic mice which we showed previously had extensive deamination of murine leukemia virus (MLV) proviruses. The A3G transgene was crossed onto an UNG and mouse APOBEC3 knockout background (UNG-/-APO-/-) and the mice were infected with MLV. We found that virus infection levels were decreased in A3G UNG-/-APO-/- compared to A3G APO-/- mice. Deep sequencing of the proviruses showed that there were significantly higher levels of G-to-A mutations in proviral DNA from A3G transgenic UNG-/-APO-/- than A3G transgenic APO-/- mice, suggesting that UNG plays a role in the repair of uracil-containing proviruses. In in vitro studies, we found that cytoplasmic viral DNA deaminated by APOBEC3G was uracilated. In the absence of UNG, the uracil-containing proviruses integrated at higher levels into the genome than did those made in the presence of UNG. Thus, UNG also functions in the nucleus prior to integration by nicking uracil-containing viral DNA, thereby blocking integration. These data show that UNG plays a critical role in the repair of the damage inflicted by APOBEC3 deamination of reverse-transcribed DNA.ImportanceWhile APOBEC3-mediated mutation of retroviruses is well-established, what role the host base excision repair enzymes play in correcting these mutations is not clear. This question is especially difficult to address in vivo. Here, we use a transgenic mouse developed by our lab that expresses human APOBEC3G and also lacks the endogenous uracil DNA glycosylase (Ung) gene, and show that UNG removes uracils introduced by this cytidine deaminase in MLV reverse transcripts, thereby reducing G-to-A mutations in proviruses. Furthermore, our data suggest that UNG removes uracils at two stages in infection – in unintegrated nuclear viral reverse transcribed DNA, resulting in its degradation and second, in integrated proviruses, resulting in their repair. These data suggest that retroviruses damaged by host cytidine deaminases take advantage of the host DNA repair system to overcome this damage.
SOX transcription factors direct TCF-independent WNT/beta-catenin transcription
Authors: Mukherjee Shreyasi , Luedeke David M. , Brown Leslie , Zorn Aaron M. 
Published: August 2021

ABSTRACTWNT/ß-catenin signaling regulates gene expression across numerous biological contexts including development, stem cell homeostasis and tissue regeneration, and dysregulation of this pathway has been implicated in many diseases including cancer. One fundamental question is how distinct WNT target genes are activated in a context-specific manner, given the dogma that most, if not all, WNT/ß-catenin responsive transcription is mediated by TCF/LEF transcription factors (TFs) that have similar DNA-binding specificities. Here we show that the SOX family of TFs direct lineage-specific WNT/ß-catenin responsive transcription during the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and neuromesodermal progenitors (NMPs). Using time-resolved multi-omics analyses, we show that ß-catenin association with chromatin is highly dynamic, colocalizing with distinct TCFs and/or SOX TFs at distinct stages of differentiation, indicating both cooperative and competitive modes of genomic interactions. We demonstrate that SOX17 and SOX2 are required to recruit ß-catenin to hundreds of lineage-specific WNT-responsive enhancers, many of which are not occupied by TCFs. At a subset of these TCF-independent enhancers, SOX TFs are required to both establish a permissive chromatin landscape and recruit a WNT-enhanceosome complex that includes ß-catenin, BCL9, PYGO and transcriptional coactivators to direct SOX/ß-catenin-dependent transcription. Given that SOX TFs are expressed in almost every cell type, these results have broad mechanistic implications for the specificity of WNT responses across many developmental and disease contexts.
Using mass spectrometry to investigate fluorescent compounds in squirrel fur
Authors: Hughes Bryan , Bowman Jeff , Stock Naomi L. , Burness Gary 
Published: August 2021

AbstractWhile an array of taxa are capable of producing fluorescent pigments, fluorescence in mammals is a novel and poorly understood phenomenon. We believe that a first step towards understanding the potential adaptive functions of fluorescence in mammals is to develop an understanding of fluorescent compounds, or fluorophores, that are present in fluorescent tissue. Here we use Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) of flying squirrel fur known to fluoresce under ultraviolet (UV) light to identify potentially fluorescent compounds in squirrel fur. All of the potentially fluorescent compounds we identified were either present in non-fluorescent fur or were not present in all species of fluorescent flying squirrel. Therefore, we consider that the compounds responsible for fluorescence in flying squirrels may also be present in non-fluorescent mammal fur. Some factor currently unexplained likely leads to excitation of fluorophores in flying squirrel fur. A recently suggested hypothesis that fluorescence in mammals is widely caused by porphyrins is consistent with our findings.