bluuebunny/crossref_metadata_2025_split huggingface dataset:
BRG1 deletion de‐represses p53 pathways inducing cell cycle inhibition and apoptosis during peri‐gastrulation (541.10)
Authors: Singh Ajeet , Boyle Michael C , Rubino Mark , Tandon Arpit , Shah Ruchir , Archer Trevor 
Published: June 2021

BRG1, a catalytic subunit of the SWI/SNF ATPase chromatin‐remodeling complex, plays a vital role in gene regulation and tumorigenesis. Germline SWI/SNF function loss results in early embryonic lethality at approximately E3.5 in the mouse. BRG1 necessity during development beyond the pre‐implantation stage, however, has not been defined. The myriad temporal genomic changes during development require chromatin remodeling to facilitate differential gene expression. We hypothesized that BRG1 deficiency during post‐implantation development will cause gastrulation failure. Using a tamoxifen inducible conditional mouse model, we show that BRG1 ablation beginning at gastrulation results in arrested growth and early embryonic death. Mutant embryos failed to grow; histologic analysis revealed several developmental defects such as lack of normal cardiac structure and disrupted neural fold architecture. Immunohistochemistry analysis revealed aberrant expression of cell cycle and apoptosis marker proteins in BRG1 mutants. Microarray analysis revealed BRG1 ablation increased expression of cell cycle regulators, particularly within the p53 pathway. Co‐IP analyses reveal BRG1 interacts with CHD4 to suppress p53 pathways in P19 cells. These results suggest BRG1 plays a critical role in genomic surveillance essential for cell survival after the pre‐implantation period for normal cellular survival, proliferation and differentiation.Grant Funding Source: Z01 ES071006‐13
Caffeine induces both short‐term and long‐term effects on gene expression and DNA methylation in the mouse heart (542.3)
Authors: Wendler Christopher , Poulsen Ryan , Fang Xiefan 
Published: June 2021

Introduction: Evidence indicates that disruption of normal prenatal development influences an individual’s risk of developing cardiovascular disease as adults. Thus, understanding how in utero exposure to chemical agents leads to increased susceptibility to adult diseases is a critical health related issue. One substance that fetuses are commonly exposed is caffeine, an adenosine receptor antagonist, with consumption during the first month of pregnancy being reported by 60% of women. Methods: Pregnant CD‐1 dams were injected with 20 mg/kg of caffeine once a day from E6.5‐E9.5. Hypothesis: Altered adenosine action in utero leads to adverse effects on adult heart function and morphology, and these effects are mediated by DNA methylation‐induced changes in cardiac gene expression. Results: Using an outbred strain of mouse, CD‐1, we demonstrated that in utero caffeine exposure induces increased wall thickness and increased left ventricular mass in adult hearts. When we examined gene expression in embryonic hearts following the same caffeine treatment, we demonstrated that enzymes responsible for DNA methylation and demethylation were affected. The DNA methylation genes DNA methyltransferase 1 and 3a were both down regulated and the Tet 1 enzyme which initiates DNA demethylation was up regulated following in utero caffeine exposure. These results are consistent with our observations that in utero caffeine exposure results in global hypomethylation in the adult heart. In addition, the cardiac structural genes Myh7, titan, and troponin I1 had altered gene expression. Conclusion: Caffeine affects the expression level of genes that control DNA methylation in utero, and caffeine leads to altered gene expression and cardiac morphology in adulthood.Grant Funding Source: R01 HD058086
Fibulin‐1 suppresses EMT and apoptosis during semilunar valve morphogenesis (542.6)
Authors: Harikrishnan Keerthi , Cooley Marion , Sugi Yukiko , Kern Christine , Argraves W 
Published: June 2021

Epithelial to mesenchymal transition (EMT) occurring in the cardiac outflow tract (OFT) is a process that is critical to semilunar valve development. Fibulin‐1 (Fbln1), an extracellular matrix protein, is a major component of the OFT that is expressed during OFT EMT (i.e., E9.5‐10.5). The aim of this study was to determine the role of Fbln1 during the earliest events in OFT EMT. At E9.5‐10.5, the proximal OFT cushions of Fbln1 null embryos were hyper cellular (i.e., 82% increase at E9.5, 43% increase at E10.5, n=5 per genotype; p<0.05) as compared to wildtype (WT). Further evidence that Fbln1 normally functions to suppress endocardial cushion EMT came from OFT cushion explant studies. The results showed that OFT explants from Fbln1 null embryos displayed a 137% increase in the number of cells that migrated from the explants as compared to WT (WT n=8, null n=4; p<0.001). Despite the finding that Fbln1 deficiency led to hypercellularity of OFT cushions at E10.5, we observed decreased cell numbers of OFT cushions at E12.5. Further analysis revealed a 172% increase in apoptosis in regions of EMT derived mesenchyme of Fbln1 nulls that accompanied a 35% reduction in cell numbers in the same regions (n=4 per genotype, p<0.05) at E12.5. Decreased cellularity and increased apoptosis persisted in the OFT prevalvular mesenchyme of Fbln1 nulls at E14.5. Morphometric analysis of 3D reconstructions of Fbln1 null OFT valves at E17.5 revealed that the anterior leaflet of the pulmonary valve, and the left and non‐coronary leaflets of the aortic were significantly reduced as compared to WT (30‐40% reduction; p<0.05. The reduced valve sizes at E17.5 correlated with decreased cell numbers (45%, 30% and 30%, respectively; WT n=6, null n=8; p<0.05). Taken together, Fbln1 regulates cellularization of the proximal endocardial cushion and prevalvular mesenchyme. Mechanistically, Fbln1 acts as a suppressor of OFT cushion EMT and apoptosis.Grant Funding Source: Supported by HL095067
Non‐invasive vascular function measurement tools: the correlation between arterial distensibility and venous compliance in young males (546.8)
Authors: Bentley Danielle , Nguyen Cindy , Thomas Scott 
Published: June 2021

Reliable non‐invasive tools for the determination of arterial and venous health permit repeated assessments in intervention studies. While arterial health is commonly estimated using tonometry and venous function estimated using venous plethysmography, no studies have reported on the relation of these measures of vascular function. We assessed the reproducibility (ICC) and correlation (Pearson r) of Augmentation Index (AIx) and Venous Compliance (dV/dP) in a sample (n=8) of healthy (SBP/DBP: 107.1±7.4 / 70.0±5.5mmHg), young (Age: 25.5±3.7 years), physically active (Rapid Assessment of Physical Activity: 6.6±0.5) men. All participants completed an initial familiarization session, followed by two experimental sessions where both arterial Augmentation Index and Venous Compliance were measured. AIx values were accepted only if the operator index > 90%. Participants had good but varied arterial health (AIx = ‐1.9±11.0; range ‐18 to 17) and venous health (.03±.00; range .02 to .05). Reliability between days for AIx was high (Cronbach’s α= .97) while dV/dP scores at 20mmHg were less reliable (Cronbach’s α =.46). There was no correlation between intra‐individual same day AIx and dV/dP variables (r = ‐.31, p >0.05). Based on these results, studies of vascular function should consider the arterial health measurement of Augmentation Index and the venous function measurement of Venous Compliance as independent variables.Grant Funding Source: Supported by CIHR CGS Doctoral Research Award
p38α MAPK mediates saturated fatty acid‐induced cardiomyocyte apoptosis (547.19)
Authors: Oh Charles , Nguy Michael , Migrino Raymond , Thornburg Kent , Schwenke Dawn , Reaven Peter 
Published: June 2021

BACKGROUND: Cardiomyocyte apoptosis mediated by a p38α MAPK‐dependent pathway may be a key step leading to lipotoxic/diabetic cardiomyopathy. Saturated fatty acid (SFA), elevated in type 2 diabetes, induce apoptosis in many cell types including cardiomyocytes. We tested the hypothesis that SFA‐induced cardiomyocyte apoptosis is dependent on p38α activation.METHODS AND RESULTS: Immortalized human adult ventricular cardiomyocytes (AC16 cells) were exposed to high physiological levels of a SFA, palmitate (PA). The apoptotic response was determined by flow cytometry, and the p38α‐dependent pathway evaluated using a p38 inhibitor, and by p38α siRNA knockdown.PA increased apoptosis in AC16 cardiomyocytes dose‐dependently; after 16 h exposure to PA (control‐2.6±0.7%, 150 μM PA‐3.5±1.1%, 300 μM PA‐11.5±3.4%, n=4, p<0.001). Apoptosis rose higher after 24 h treatment (control‐2.3±0.7%, 150 μM PA‐5.9±2.7%, 300 μM PA‐37.6±10.7%, n=3, p<0.02).PA did not change total p38α protein levels, but increased p38α phosphorylation. Specific p38α siRNA reduced the expression of p38α but not p38β, and markedly and dose‐dependently attenuated PA‐induced apoptosis (control siRNA‐7.7±1.0%, 300 μM PA‐34.4±5.0%, 300 μM PA+30 pmol siRNA‐23.7±4.4%, 300 μM PA+60 pmol siRNA‐19.7±2.6%, 300 μM PA+120 pmol siRNA‐17.3±2.8%, n=4, p<0.04). PD169316, which inhibits both p38α and p38β isoforms, tended to reduce PA‐induced apoptosis (n=4, p=0.05).Downstream, AP‐1 transcription factors, c‐fos and c‐jun (n=3, p蠄0.05), and caspase 8 (n=3, p=0.03) mRNA levels, and protein expression were increased by PA. CONCLUSIONS: This study in cardiomyocytes demonstrated that 1) PA induces p38α activation, 2) PA increases AP‐1 and caspase 8 expression, and 3) reduced p38α expression attenuates PA‐induced apoptosis. Weaker results with PD169316 may be due to concurrent inhibition of p38β. Our results suggest a potential mechanism by which high plasma SFA levels may lead to the development of diabetic cardiomyopathy.Grant Funding Source: VA
The Mus81‐Mms4 structure‐selective endonuclease undergoes nicked junction‐induced conformational changes to bend its DNA substrates for cleavage (553.1)
Authors: Mukherjee Sucheta , Wright William , Ehmsen Kirk , Heyer Wolf‐Dietrich 
Published: June 2021

Mus81‐Mms4 is a structure‐selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81‐Mms4 requires inherent flexibility in DNA substrates for optimal catalysis. Förster Resonance Energy Transfer (FRET) experiments further reveal that recognition of 3’‐flap and nicked Holliday junction substrates by Mus81‐Mms4 involves induction of a sharp bend with a 100° angle between the two duplex DNA arms. In addition, thiol crosslinking of Mus81‐Mms4 bound to DNA substrates demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches support a model for catalysis by Mus81‐Mms4 in which initial DNA binding is based on minimal structural requirements and is followed by a rate‐limiting conformational transition of the substrate and protein, which leads to a sharply kinked substrate that may fray the DNA four bp away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotides. These data suggest that mutually compatible conformational changes of Mus81‐Mms4 and its substrates tailor its incision activity to nicked junction molecules, resulting in products that cannot be directly ligated.Grant Funding Source: Supported by NIH and TRDRP
Signaling control of proteasome, immunoproteasome, and PA28 regulator gene expression during adaptation to oxidative stress (555.11)
Authors: Davies Kelvin 
Published: June 2021

Levels of oxidative stress change continuously, depending on environment, metabolism, foods, drugs and medications, and radiation. Young, healthy, mammalian cells can readily adapt to increases in oxidative stress such that they become (temporarily) much more resistant to oxidative damage. Intracellular proteins are major targets of oxidative stress and it is vitally important that cells rapidly and selectively degrade mildly oxidized proteins, before they undergo more severe oxidation, aggregation, and cross‐linking. Our studies show that such adaptive responses include the immediate disassembly of the 26S proteasome (catalyzed by HSP70 and Ecm‐29) to form free 20S proteasome and 19S regulator complexes, at which point ATP/Ubiquitin‐dependent proteolysis is temporarily lost. The additional free 20S Proteasomes (+/‐ 11S or PA28 regulators) are of immediate help in degrading oxidized proteins. The original 26S Proteasomes are re‐assembled, and ATP/Ubiquitin‐dependent proteolysis is restored over a three‐hour period. During this three‐hour period, and during the subsequent 17 hours, new 20S Proteasomes, Immunoproteasomes, and 11S (PA28) proteasome regulators are synthesized, partially under the control of the Nrf2 signal transduction pathway. We have now obtained similar findings in C. elegans worms, and D. melanogaster fruit flies. Proteasome and Immunoproteasome subunit genes, and the 11S (PA28) regulator appear to be true shock/stress genes that provide significant oxidative stress protection. In older cells, Proteasome activities decline, and adaptational responses mediated by Nrf2 become sluggish or even ineffectual. The antagonistic effects of Bach1 and/or Nrf1 may actually blunt adaptive proteolytic responses to stress in older cells and organisms. Studies in animals and humans suggest that declining Proteasome activities and, perhaps, declining responsiveness to stress, may contribute to the ageing process, and to various age‐associated diseases.Grant Funding Source: NIH/NIEHS ES003598
Visualization of actin mono‐ADP‐ribosylation in iota toxin and actin complex (555.4)
Authors: Tsuge Hideaki , Tsurumura Toshiharu , Tsumori Yayoi 
Published: June 2021

Clostridium perfringens iota‐toxin (Ia) mono‐ADP‐ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. We revealed the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole‐4‐carboxamide adenine dinucleotide) (ref1); however, the structures of the NAD+ bound form (NAD+‐Ia‐actin) and the ADP ribosylated form [Ia‐ADP ribosylated (ADPR)‐actin] remain unclear. Here we obtained the good diffraction apo crystals and found that ethylene glycol as cryo‐protectant inhibits ADP ribosylation. We used the apo crystal as the ADP‐ribosylation reaction chamber and applied NAD+‐soaking to this complex in different conditions. Finally, we obtained high‐resolution structures of NAD+‐Ia‐actin and Ia‐ADPR‐actin (ref2). The structures of NAD+‐Ia‐actin and Ia‐ADPR‐actin represent the pre‐ and postreaction states, respectively. The strain‐alleviation model of ADP ribosylation, which we proposed previously, became more plausible with additional snapshots of actin ADP‐ribosylation.(1) Tsuge H, Nagahama M, Oda M, Iwamoto S, Utsunomiya H, Marquez VE, Katunuma N, Nishizawa M, Sakurai J (2008) PNAS 105 (21):7399‐7404.(2) Tsurumura T, Tsumori Y, Qiu H, Oda M, Sakurai J, Nagahama M, Tsuge H (2013) PNAS 110 (11):4267‐4272.
A role for mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (557.1)
Authors: Abeliovich Hagai , Zarei Mostafa , Rigbolt Kristoffer , Youle Richard 
Published: June 2021

Mitophagy, the autophagic degradation of mitochondria, is an important housekeeping function in eukaryotic cells. Defects in mitophagy occur in degenerative disorders such as Parkinson’s disease, and decreases in the levels of mitophagy correlate with aging phenomena. An important goal is to achieve an understanding of the quality control mechanism(s) that specifically cull defective mitochondrial components or compartments, during mitophagy. Specifically, it is not known whether a small cohort of defective molecules will cause the ablation of an entire compartment, or whether an active process segregates defective molecules from functional ones and allows a more efficient culling mechanism. In this study we have combined a proteomic study with a molecular genetic and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We report that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism that couples a weak physical segregation with mitochondrial fission and fusion to achieve a distillation‐like effect. In agreement with this interpretation, the differential rates of mitophagy that we observed for different mitochondrial matrix proteins strongly correlate with the degree of physical segregation observed for these proteins, within the matrix.Grant Funding Source: Supported by the Israel Science Foundation
Dysregulated ubiquitin proteasome system leads to neurodegeneration in a spontaneous obese rat model (559.1)
Authors: Reddy G , Reddy S , Reddy V , Raghu G , Giridharan N 
Published: June 2021

Objective: A spontaneously developed obese rat (WNIN/Ob) was established from the existing WNIN rats by selective breeding. The WNIN/Ob rats show accelerated aging and develop age related diseases including neurodegeneration. The main aim is to investigate the role of ubiquitin proteasome system (UPS) in obesity‐associated neurodegeneration in WNIN/Ob rat model.Methods: Various components of UPS like ubiquitin, UCHs (ubiquitin C‐terminal hydrolases), proteasomal sub‐units and endoplasmic reticulum (ER) stress markers were analysed by qRT‐PCR and immunoblotting. Chymotrypsin‐like activity of proteasome was assayed by fluorometric method. Apoptotis and morphology of neurons in the cerebral cortex was assessed by immunohistochemistry and transmission electron microscopy, respectively.Results: The results indicated dysregulated UPS, increased ER stress and up‐regulation of apoptotic regulators in obese rats. Neurons in the cerebral cortex of obese rats showed swollen mitochondria, disrupted ER and degenerating axons, nucleus and finally neurons. It appears UCHL‐1 mediated apoptosis through stabilizing p53 as a key factor of neurodegeneration.Conclusion: Alterations in the UPS may be causally related to neurodegeneration in WNIN‐obese rat. Further this rat model could be a suitable animal model for investigating the role of UPS in age related neurodegenerative diseases.
Nucleolin‐enhanced deadenylation and bulk translation (566.1)
Authors: Woo Ho Hyung , Lee Sang , Chambers Setsuko 
Published: June 2021

Rate of translation is one of the critical factors, determining protein abundance in a cell. After a pioneer round of translation in which 80S ribosome scans the linear mRNA, a cap‐dependent mRNP closed‐loop is formed for bulk translation.Nucleolin interacts with the PABPC and works together for deadenylation and mRNP closed‐loop formation. Nucleolin interacts to the Pan2/Pan3 and CCR4‐NOT deadenylation complex, indicating nucleolin’s involvement in the mRNA deadenylation. Nucleolin also interacts with the eIF4F translation initiation complex, suggesting another role of nucleolin in translation initiation. We found that an increased level of deadenylated mRNAs is associated with heavy polyribosomes in nucleolin overexpressed cell lines. Together these results indicate that nucleolin‐mediated deadenylation is not followed by mRNA decay, but can lead to the bulk translation by forming poly(A)n‐less mRNP closed‐loop.We propose that a non‐canonical mRNP closed‐loop lacking poly(A)n tail can be formed by nucleolin for bulk translation. In this model, nucleolin enhances deadenylation of the mRNA and induces the formation of poly(A)n‐less mRNP closed‐loop by binding dsRNA formed between the mRNA 5’‐ and 3’‐UTR complementary sequences. Nucleolin is proposed as a mediator for the transition from the canonical to non‐canonical poly(A)n‐less mRNP closed‐loop to enhance the bulk translation.
New structural insights into cap‐independent translation initiation (566.6)
Authors: Brilot Axel , Koh Cha San , Grigorieff Nikolaus , Korostelev Andrei 
Published: June 2021

Conventional initiation of translation of cellular mRNAs depends on the m7G cap located at the 5' untranslated region (UTR) and on extraribosomal proteins called initiation factors. Translation of many viral mRNAs and some cellular mRNAs can be initiated in a cap‐independent manner, with fewer initiation factors, to promote expression under stress conditions when overall cellular translation is downregulated. In these mechanisms, several types of structured RNA at the 5' UTR are employed, called the internal ribosome entry sites (IRES). The structural basis for how an IRES binds to the ribosome and positions the open reading frame of mRNA is not fully understood. Here, we present molecular structures of ribosome complexes, which provide new structural insights into the mechanism of IRES‐dependent translation.Grant Funding Source: Supported by HHMI and National Institutes of Health
Mechanistic studies of ribosome‐dependent toxins active during the stringent response (566.8)
Authors: Dunham Christine , Schureck Marc , Maehigashi Tatsuya , Ruangprasert Ajchareeya , Dunkle Jack , Miles Stacey 
Published: June 2021

Regulation of protein synthesis is one mechanism by which bacteria alter their metabolic rates during times of stress. This process is known as the ‘stringent response’ where the presence of deacylated tRNAs bound to the ribosome is one signal that initiates a cascade of events ultimately resulting in the activation of toxin proteins. Type II toxin proteins halt translation by targeting mRNA bound to the ribosome for degradation in a codon‐dependent manner. Here, we present structural studies of different toxin proteins bound to target mRNAs on the 70S ribosome. Our structures reveal novel interactions between conserved toxin residues with the mRNA backbone in the ribosomal A site that helps to rationalize codon specificity by different toxins. Biochemical and in vivo growth assays complement our structural studies to provide a unified mechanism of toxin‐mediated mRNA degradation.Grant Funding Source: Supported by NSF MCB 0953714 and Pew Scholars Program in the Biomedical Sciences
Functional characterization of natural occurring variants of the 70‐kDa heat‐shock protein, HSPA1A (567.10)
Authors: Siracusa Matthew , Nikolaidis Nikolas 
Published: June 2021

Seventy‐kDa‐heat‐shock proteins (Hsp70s) are molecular chaperones essential for protein homeostasis and cell survival under both physiological and pathological conditions. Given this vital role of Hsp70s, it is important to investigate whether naturally occurring variants of these proteins have altered functional properties. In this study, the functional outcome of four non‐synonymous single nucleotide polymorphisms present in different human populations on HSPA1A was tested. Site directed mutagenesis was used to generate the HSPA1A mutations into both both bacterial and mammalian expression vectors. Through the use of a malachite green colorimetric ATPase assay, the mutant proteins’ ability to hydrolyze ATP at the protein’s ATP‐binding domain was tested. All four proteins were found to be functionally differentiated from each other and the WT. Based on the Km values obtained, the WT had the highest affinity for binding to ATP, but based on the Vmax values, all the mutants were able to hydrolyze ATP at a much faster rate, with the S16P mutant showing nearly a 4‐fold increase from the WT. The mutant proteins’ ability to properly localize intracellularly within HEK‐293 cells was also tested by tagging HSPA1A to GFP, using fluorescent dyes to stain the mitochondria, lysosomes, and plasma membrane, and viewing where the proteins colocalized via confocal microscopy. Under normal growth conditions all four mutants were found to localize to these regions in a similar manner to the WT indicating that these mutations did not affect the protein’s ability to localize within the cell.Grant Funding Source: This project was supported by funds from CSUPERB and CSUF to NN, and HHMI to MS and NN