bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating the Role of DNA Polymerase IV in the Resolution of R‐loops
Authors: Nguyen Brian , Tashjian Tommy , Godoy Veronica 
Published: June 2021

As the replication fork progresses through the DNA template, it may encounter a number of obstacles, one of which is the RNA transcription machinery. This encounter occurs because both processes utilize the same template and because the rate of replication (~1000 nt/s) is much faster than the rate of transcription (40–80 nt/s). The encounter of the two machineries, in either a co‐directional or a head‐on orientation, results in replication‐transcription collisions (RTCs). Failure to resolve these collisions leads to replication fork stalling and eventually cell death. One of the events required to resolve RTCs is the removal of RNA: DNA hybrids, known as R‐loops. In E. coli, RNase H1, coded by rnhA, is a member of a family of highly conserved endonucleases mediating resolution of R‐loops by cleaving the RNA strand on RNA: DNA hybrids. If not resolved, R‐loops cause genomic instability in the form of double‐stranded breaks through the collapse of the replication fork and accumulation of DNA lesions.In E. coli, both replication stalling and accumulation of DNA lesions leads to induction of the SOS response. Among the first genes to be upregulated is dinB, encoding DNA polymerase IV (DinB), becoming the most abundant DNA polymerase in the cell (~2500 nM). DinB main role is to synthesize DNA over template lesions. There is evidence to support the notion that DinB is involved in recombination, a mechanism that is used to repair double‐stranded breaks. In stressful conditions, DinB, due to its abundance, outcompetes other polymerases for the recombination intermediate and synthesizes DNA from it. Because of its abundance during SOS induction and its role in recombination, DinB may also be involved in the resolution of R‐loops. Here, we investigate the possible role of DinB in R‐loop resolution in a rnhA‐deletion strain. Loss of rnhA results in slow growth that is rescued by expression of DinB from a low copy plasmid. This data suggest DinB is multicopy suppressor of the rnhA deletion and that DinB plays a role in the resolution of R‐loops.Support or Funding InformationNational Institute of General Medical Sciences (RO1GM088230 to VG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ataxia Telangiectasia and Rad3‐Related Kinase (ATR) May Prevent Replication Stress In Planarian During Regeneration
Authors: Tirgar Reyhaneh , Shamoon Ushna , Akpati Lois , Sawyer Nathaniel , Nguyen Lillian , Nam Edward 
Published: June 2021

S. mediterranea is a planarian with extensive regenerative ability which requires substantial cell division. However, DNA replication stress occurs every cell division and must be resolved. From yeast to man, an evolutionary conserved DNA replication stress response (RSR) is essential for life. The RSR is a signal transduction pathway regulated by Ataxia telangiectasia and Rad3‐related kinase (ATR) and Checkpoint 1 kinase (CHK1) to respond to replication stress. However, the RSR has not been established in planarian nor do we know if it functions in regeneration. We hypothesize that ATR may be essential for regeneration. Preliminary results show reduced regenerative ability in the presence of hydroxyurea (HU), a replication stress inducing agent. However, low concentrations (2 mM) did not attenuate regeneration, indicating that ATR and the RSR may be present and able to overcome replication stress. To test this, we treated amputated tails with HU, ATR inhibitor (ATRi), and a combination of both. In 2mM HU, tails regenerated similarly to control. We observed only a one day delay in regeneration with ATRi alone. However, ATRi and 2mM HU in combination prevented planarian regeneration completely. This synergistic effect suggests that ATR is significant in the response to replication stress during regeneration. To obtain genetic evidence, we are in the process of knocking down ATR as well as other RSR genes like CHK1, WRN, etc. Further determining the importance of RSR genes in planarian will elucidate the conserved components and allow us to understand how ATR and the RSR contributes to the incredible regenerative abilities in planarian.Support or Funding InformationU.S. Department of Education HSI STEM Articulation Grant P031C160118University of St. Thomas Committee of Student ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐resolution Maps of Genome‐wide Human Damage and Repair
Authors: Adebali Ogun , Hu Jinchuan , Sancar Aziz 
Published: June 2021

Our DNA is continually damaged by cellular and environmental factors. Some of the most common mutagens, including ultraviolet light and platinum‐based drugs, cause bulky adduct formation; these are almost exclusively corrected through nucleotide excision repair. Although the basic mechanisms of excision repair are reasonably well understood, it was previously not known which genomic loci are preferentially damaged and repaired. Using the novel technologies, Damage‐seq and XR‐seq, we were able to decipher the genome‐wide kinetics of damage formation and excision repair following ultraviolet irradiation and cisplatin treatments. The single‐nucleotide resolution maps revealed cellular components affecting the DNA damage formation and repair. Despite some local exceptions, the overall damage formation profiles at initial time points exhibited a uniformity. However, the heterogenous repair resulted in an imbalanced distribution of the remaining damages after time. The repair bias is mostly caused by the transcription‐coupled repair as well as the chromatin structure of the DNA.Support or Funding InformationNational Institutes of Health Grants GM118102 and ES027255.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insights into the molecular mechanism of alkylation‐induced mutagenesis
Authors: Lee Seongmin 
Published: June 2021

The genomic DNA is persistently attacked by endogenous and exogenous alkylating agents (e.g., S‐adenosyl methionine, cisplatin), which gives rise to a wide variety of alkylated DNA lesions such as N7‐MeG, N3‐MeA, O6‐MeG, and cisplatin‐GG intrastrand cross‐links. These lesions, if not removed by DNA repair pathways, can be bypassed by error‐prone translesion synthesis DNA polymerases, which could cause mutations and cancers. The structural basis for promutagenic replication past these lesions remains elusive. To elucidate the alkylation‐induced mutagenesis mechanism, we conducted kinetic and structural studies of the bypass of these lesions by various human DNA polymerases. N7‐MeG formed a Watson‐Crick‐like base pair, rather than a wobble base pair, with thymine, suggesting promutagenicity of N7‐MeG. N3‐MeA formed a Watson‐Crick base pair with thymine but strongly deterred nucleotide incorporation opposite the lesion. O6‐MeG formed a Watson‐Crick‐like base pair with thymine, which was consistent with the reported high mutagenicity and carcinogenicity of O6‐MeG. Cisplatin‐GG intrastrand cross‐links engage in favorable interactions with adenine in non‐instructional fashion, suggesting that the predominant cisplatin‐induced G to T mutations may follow an “A‐rule”. Taken together, these studies revealed the features of promutagenic base pairings of N7‐MeG, O6‐MeG and cisplatin‐GG cross‐links, thereby providing new insights into the molecular mechanisms of alkylated‐induced mutagenesis and carcinogenesis.Support or Funding InformationWelch Foundation F‐1741National Institutes of Health ES26676This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The tumor suppressor phosphatase PHLPP1 suppresses inflammatory signaling by regulating the phosphorylation state and activity of STAT1
Authors: Katsenelson Ksenya Cohen , Stender Joshua D. , Uchiyama Satoshi , Nizet Victor , Glass Chris K. , Newton Alexandra C. 
Published: June 2021

Post‐translational modifications, such as phosphorylation, play a key role in regulating gene expression. Although much is known about the kinases that control epigenetic mechanisms, much less is known about the phosphatases. Here we report that the PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) regulates the transcription of a large number of genes by a mechanism that depends, in part, on the dephosphorylation of the transcription factor STAT1. Using non‐biased high‐throughput RNA‐sequencing, we analyzed genome‐wide mRNA levels of genes in lipopolysaccharide (LPS)‐treated bone marrow‐derived macrophages from wild‐type and Phlpp1−/− mice and found that approximately 1600 transcripts were up or down‐regulated 2‐fold or more in the Phlpp1−/− macrophages. Gene ontology analysis revealed that many of the genes that were upregulated upon loss of PHLPP1 are involved in proinflammatory signaling. De novo motif analysis of the promoters for those genes revealed that 46% of them have recognition motifs for STAT1, a key regulatory transcription factor of inflammatory signaling. Biochemical analysis revealed that PHLPP1 regulates the phosphorylation of STAT1 on Ser727, a site that is important for full transcriptional activity of STAT1, but not Tyr701, a site that recruits STAT1 to the nucleus. Importantly, PHLPP1 deletion resulted in enhanced downstream inflammatory signaling as assessed by a luciferase reporter assay of the Interferon‐Gamma Activated Sequence (GAS) promoter. In addition to the new finding that PHLPP1 is controlling inflammatory signaling, Phlpp1−/− mice are protected from sepsis‐induced death. Our data support a model in which PHLPP1 plays a previously undescribed role in regulating inflammation which has potential therapeutic implications for sepsis‐induced death.Support or Funding InformationNIH/NCI T32 CA009523 (K.C.K.)NIH Grant R01 GM067946 (A.C.N.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Zika Viral Polymerase: a promising therapeutic target
Authors: Wells Grace S. , Bernatchez Jean , Coste Michael , Luna Lucas , Neto Jair Siqueira , Purse Byron , Sohl Christal State 
Published: June 2021

Zika virus, a member of the Flaviviridae family, has garnered global attention due to the link of recent outbreaks with increased risk of fetal microcephaly and Guillan‐Barré syndrome in adults. As of yet, there are no approved therapies for treatment or prevention of Zika virus. In this research, we analyze inhibition of Zika virus replication by nucleoside analogs – a therapeutic strategy that has been successful levied against other members of the Flaviviridae family. We have recombinantly expressed and purified the RNA‐dependent RNA polymerase (RdRP) domain of the ZIKV NS5 protein. The purified protein is used in an in vitro primer extension assay to characterize incorporation of native nucleotides and inhibitory analogs opposite a Zika specific template. Of a pool of nucleoside analogs, we have identified a compound that incorporates at approximately 1/5 the rate of native nucleoside. We plan to combine biochemical, cellular, and animal model studies to determine efficacy of these compounds in inhibiting Zika viral replication and infection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Biophysical Analysis of Malate Dehydrogenase and Citrate Synthase Protein‐Protein Interaction
Authors: Ghebreigziabher Daniel , Provost Joseph 
Published: June 2021

The metabolon theory describes the protein interactions of enzymes regulating a metabolic pathway. Through these interactions proteins can share substrates through direct channeling. One set of enzymes that are involved in metabolon complexes are citrate synthase and malate dehydrogenase. This study attempts to understand the interactions regulating the metabolon of malate dehydrogenase (MDH) and citrate synthase (CS), which are integral intermediate metabolic enzymes. Using size exclusion chromatography and sedimentation, previous studies identified the interaction between CS and MDH to be isoform specific, but no further work has been conducted to obtain affinity values or the biophysical nature of the interaction. Codon optimized His tagged recombinant proteins were expressed and purified using affinity chromatography. These enzymes include: glyoxysomal malate dehydrogenase (gMDH), human cytosolic malate dehydrogenase (MDH1), human mitochondrial malate dehydrogenase (MDH2), and human mitochondrial citrate synthase (CS). We analyzed the protein interactions using fast thermal melts which further explain the thermal stability of the the dimerization of the metabolons. We first show that protein concentration did not induce oligomerization of CS or any MDH isoform, indicating that any changes are not from MDH or CS homodimerization. Next, we investigated specific CS‐MDH interactions using fast thermal melts. The results show that from the initial melting temperature of CS, the addition of mitochondrial MDH had a greater shift in its melting temperature than the addition of cytosolic MDH, by 3–4°C. The melting point of CS was not altered in the presence of the plant MDH isoform. This indicates a specific and dramatic set of contacts for mitochondrial MDH and CS. However unlike the information found in the literature there was still a measurable shift in melting point when cytosolic MDH was incubated with CS. Overall this shows for the first time a quantifiable interaction between proteins in a well‐established pathway that will allow the further dissection of these interactions.Support or Funding InformationSupport for this project was from NSF REU 146045This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
E‐cadherin interact with Ajuba‐LIM during Drosophila larval brain development
Authors: Tadros Tiffany , Viera Astrid , Underwood Taylor , Fung Siaumin 
Published: June 2021

Brain development is an orderly process, but the fundamental steps at its earliest stages remain unclear. Drosophila is an ideal model to characterize these critical maturational mechanisms because they possess neuroblasts that generate neurons and glia throughout the developing brain. These neuroblasts are capable making new cells through a process known as the asymmetrical cell division. In each division, neuroblasts creating one cell that continues to be a neuroblast (maintaining/renewing themselves) and the other cells, the ganglion mother cells will differentiation into neurons and glia (differentiation).E‐cahderin expression can be visualized by Western blot throughout all stages of pupal development. The pattern of E‐cadherin expression switches from dividing cells to the glial cells at 72 hrs APF. A screen was done to identify proteins interact with E‐cadherin. This screen was done with co‐IP of GFP fusion proteins with E‐cadherin from Drosophila larval brain. Preliminary results showed that Ajuba‐LIM interact with E‐cadherin in the larval brain of DrosophilaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of PEA‐15 allosterically induces conformational change suited for FADD binding and negatively regulates apoptosis
Authors: Hassan Sherouk , Crespo Sergio , Wei Yufeng 
Published: June 2021

Phosphoprotein Enriched in Astrocytes, 15 kD (PEA‐15) is a small non‐catalytic protein, with an N‐terminal death effector domain (DED) and an unstructured C‐terminal tail. PEA‐15 appeals to many biochemists because it regulates two important pathways. Unphosphorylated PEA‐15 interacts with extracellular regulated protein kinase (ERK) 1 and 2, preventing ERK‐dependent cell proliferation. When it is doubly phosphorylated at the two C‐terminal serine residues, PEA‐15 is recruited to the death inducing signaling complex (DISC) when it interacts with Fasassociated death domain (FADD) protein, blocking the normal recruitment and activation of procaspase‐8, which triggers the signal for apoptosis. In our lab, PEA‐15 and FADD are cloned into pET‐28b(+) vector, and are expressed in BL21(DE3) E. Coli competent cells. To mimic the phosphorylation, S104D/S116D mutant protein (PEA‐15DD) was also expressed. The proteins are purified using metal affinity (Ni2+ or Co2+) chromatography followed by ion‐exchange chromatography. The protein structures and interactions between PEA‐15DD and FADD are determined using NMR spectroscopy. Our results showed that the phosphorylation of PEA‐15 changes the conformation of the folded DED, and FADD binding does not further modulate the overall conformation. We concluded that the binding specificity of PEA‐15 is determined by the DED conformation, which is allosterically induced by the C‐terminal phosphorylation states. We further proposed that the balance of various phosphorylation states of PEA‐15 should be strictly regulated in different tissues and cell types in order to maintain the healthy conditions of the cells.Support or Funding InformationNIH AREA R15CA179410This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Cia1:Cia2 Sub‐Complex of the Cytosolic Fe‐S Cluster Assembly Pathway
Authors: Esonwune Stephanie , Vo Amanda , Marquez Melissa , Perlstein Deborah 
Published: June 2021

Iron‐sulfur (FeS) clusters are essential protein cofactors required for numerous biological functions including iron regulation, DNA synthesis and DNA repair. The Cytosolic Iron‐Sulfur Cluster Assembly (CIA) pathway is responsible for the metallocofactor assembly of extra mitochondrial FeS proteins. The CIA pathway culminates with the targeting complex, which contains the proteins Met18, Cia1 and Cia2. This complex is crucial for the last step in the CIA pathway, which is the recognition of apo‐FeS targets and insertion of their cofactors. To begin understanding the structure of this complex, we wanted to identify the residues responsible for the complex formation. We began by identifying the clusters of conserved residues required for the formation of the Cia1‐Cia2 sub‐complex since both of these proteins are essential for yeast viability, whereas the Met18 subunit is not. We alanine scanned these residues, then determined how the mutation of these residues affect Cia1‐Cia2 complex formation and the ability to support viability via genetic complementation assays. If the interactions are important in vitro, then there could be phenotypic changes in yeast cells that can alter CIA function. Our lab has previously determined that Glutamate208 within one of Cia2's conserved motifs was vital for Cia2's ability to bind to Cia1. Therefore, we scanned the surrounding conserved residues to identify additional residues important for Cia1‐Cia2 interaction. Several residues close to E208 also disrupted the Cia1‐Cia2 interaction in vitro, confirming that this conserved motif in Cia2 is the docking site for Cia1. Previously, E208A supported yeast viability, whereas, an adjacent residue, Aspartate 206 (D206), appears to be particularly important as it was the only one that failed to support viability in the complementation assay. This shows that this residue does not allow for the targeting complex to form and identify targets, negatively impacting CIA function. Now that we understand which residues are required for Cia1‐Cia2 binding, this allows for further investigation of which specific targets the complex binds to for proper CIA function.Support or Funding InformationBoston University Undergraduate Research Opportunities Program (UROP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Carnosol increases skeletal muscle cell glucose uptake via AMPK‐dependent GLUT 4 glucose transporter translocation
Authors: Baron David C. , Vlavcheski Filip , Vlachogiannis Ioannis , Tsiani Evangelia 
Published: June 2021

Skeletal muscle is highly important in glucose homeostasis since it is quantitatively a major insulin‐target tissue. Insulin action in muscle cells activates the phosphatidylinositol‐3 kinase (PI3K)/Akt signaling pathway causing the translocation of intracellularly stored GLUT4 glucose transporters to the plasma membrane leading to increased glucose uptake. Impaired insulin action in muscle leads to insulin resistance and type 2 diabetes mellitus (T2DM).AMP‐activated kinase (AMPK) is a cellular energy sensor and its activation increases glucose uptake by skeletal muscle cells. Finding AMPK activators is viewed as an effective approach to combat insulin resistance and T2DM. Rosemary extract (RE) has been shown to increase muscle glucose uptake and AMPK activity but the components responsible for these effects have not been identified yet. In the current study, we investigated the effect of carnosol, a polyphenol found in high concentrations in RE. L6 rat muscle cells were used to measure uptake of [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting. Carnosol stimulated glucose uptake in L6 myotubes in a dose‐ and time‐dependent manner. A response comparable to maximum insulin stimulation (196±9.2 % of control) was seen with 50μM of carnosol (2h) (182±7.8 % of control). Carnosol did not affect Akt phosphorylation while it significantly increased AMPK phosphorylation. Furthermore, the increase in glucose uptake in the presence of carnosol was significantly reduced by the AMPK inhibitor compound C (CC) while it was not affected by the PI3K inhibitor wortmannin. Carnosol increased plasma membrane GLUT4 glucose transporter levels in GLUT4myc overexpressing L6 cells and this response was abolished by the AMPK inhibitor CC. Our study is the first to show a significant increase in muscle glucose uptake by carnosol via a mechanism that involves AMPK.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.