bluuebunny/crossref_metadata_2025_split huggingface dataset:
Follistatin‐BMP Signaling Regulated by Isl2/CoupTFIb is Critical for Vascular Development
Authors: Li Ru‐Fang , Chang‐Yi Wu 
Published: June 2021

While many regulators and signaling molecules controlling the process of vascular development are in zebrfish, genetic regulating the vein identity, intersegmental vessels (ISV) and cardinal vein plexus (CVP) patterning is still not fully understood. We previously identified the transcription factors isl2/coupTFIb required for specification of the vein and ISV growth mediated by Notch pathway in zebrafish. Microarray analysis showed highly overlapping downstream targets controlled by isl2/coupTFIb, suggesting that cooperatively regulate vascular development. In this study, we analyze the functional relevance of one potential target from array hits, follistatin 1a (fst1a). Fst1a is a bone morphogenetic protein (BMP) antagonist related to axis formation during the development and shown involved in venous angiogenesis. However, no description of fst1a functioning in vascular so far. The interaction of notch and BMP signalings to regulate vasculization is unknown.We first examine amino acid sequence comparison and show fst1a is highly conserved among the vertebrates. Our in‐situ hybridization results showed fst1a is expressed in the lateral plate mesoderm and vessels. Overexpressing fst1a driven by fli1 promoter in zebrafish embryos, we observe the vascular defects in the growth of ISV and CVP. We further address the genetic interaction between fst1a and isl2, we found fst1 expression is increased in isl2 morphants by in‐situ hybridization. In addition, knockdown of fst1a can partial rescue the vascular defects in isl2 morphants. We suggest fst1a likely regulated by isl2. Since fst1 involved in BMP and isl2 functions downstream of notch, we conclude fst1a‐BMP signaling play an important role to regulate vascular patterning and interacts with isl2/coupTFIb‐Notch signaling.
Inhibition of DNA Methyltransferase 1 Promotes Proliferation and Alters Gene Expression in Mouse Embryonic Cardiomyocytes
Authors: Fang Xiefan , Rivkees Scott , Wendler Christopher 
Published: June 2021

IntroductionOur previous research demonstrated that in utero caffeine exposure from embryonic day (E) 6.5‐10.5 down‐regulates the expression of DNA methyltransferases (Dnmts) and enhances cardiac gene expression in embryonic ventricles.HypothesisWe hypothesize that inhibition or down‐regulation of Dnmts promotes proliferation and differentiation of embryonic cardiomyocytes.MethodVentricles were dissociated from mouse E13.5 embryos and treated with 0.5‐1 mM 5‐azacytidine, a non‐specific DNMT inhibitor, or Dnmt1 siRNA. Immunofluorescence staining was performed to identify cardiomyocytes and illustrate morphological changes. Total RNA was isolated and used for quantitative real‐time PCR (qPCR).ResultsKnock down of Dnmt1 significantly decreased global DNA methylation in cultured cells. Immunofluorescence staining showed that the percentage of α‐actinin and connexin 43 positive cells was increased in cultures treated with Dnmt1 siRNA compared to control. Dnmt1 knocked down cardiomyocytes formed larger beating units compared with control cells. 5‐azacytidine treatment increased the expression of cardiac structural genes (Myh7 and Myh7b), hormonal genes (Bnp), and transcription factors (Gata4 and Nfatc1) in HL‐1 cardiomyocytes. Dnmt1 knock down by siRNA increased the expression of Myh6, Tnni3, Tnnt2, Tnnc1, Bnp, Gata4, Mef2c, Nfatc1, Camta1, and Camta2 in primary embryonic cardiomyocytes. Ongoing studies will evaluate cardiomyocyte function after Dnmt1 knock down and identify CpG sites that regulate cardiac gene expression.ConclusionReduced Dnmt1 expression increases cell proliferation and cardiac gene expression of embryonic cardiomyocytes in culture.
Fueling the Interdisciplinary Flame: Exploring Plant‐Based Alternative Fuels in the Undergraduate Laboratory
Authors: Gregorzak Gabriel , Fajardo Jacqueline , Baillie Mark , Hlousek‐Radojcic Alenka 
Published: June 2021

A laboratory module intended for an integrated course was developed to highlight relevant and interrelated concepts described in both introductory biology and general chemistry with an enrollment of nearly 500 students. The ever‐present depletion of petroleum energy reserves is an ongoing societal concern. Concomitantly, there is growing interest in identifying natural sources of alternative fuels and optimizing the efficiency of their use. To enhance student awareness of alternative fuel availability from natural resources, we have developed a lab centered on the production of biofuels produced from seed‐oil extracts. Oil was physically extracted from a variety of seed types including pumpkin, sunflower, walnut, & flax, and utilized an efficient, small‐scale, and green approach. The physical extraction technique replaced commonly used organic solvent extraction methods used in many academic and educational labs. Extracted oil was then subject to transesterification to yield the biofuel product. The energy content of this biofuel was measured and compared to ethanol using calorimetric analysis. This distinctive laboratory experience will allow large numbers of freshmen students to recognize the broader implications of their curriculum beyond the boundaries of the classroom.This project was made possible through funding by the Howard Hughes Medical Institute.
The Q Motif is Involved in DNA Binding in ChlR1 (DDX11) Helicase
Authors: Ding Hao , Wu Yuliang 
Published: June 2021

Helicases are molecular motors that couple the energy of nucleoside triphosphate (NTP) hydrolysis to the unwinding and remodeling of structured DNA or RNA. The conversion of energy derived from NTP hydrolysis into unwinding of double‐stranded nucleic acids is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Previous studies have suggested that the Q motif is involved in ATP binding. Mutations in the human ChlR1 (DDX11) gene are associated with Warsaw Breakage Syndrome characterized by cellular defects in genome maintenance. ChlR1 is known to play essential roles to preserve genomic stability, particularly in sister chromatid cohesion; however, its molecular mechanism remains unknown. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. The ChlR1‐Q23A mutant abolished helicase activity of ChlR1, and further DNA binding assays suggested the Q motif affected the DNA binding ability of ChlR1. ATP hydrolysis assays revealed that the ChlR1‐Q23A had impaired ATP hydrolysis activity. However, further ATP binding assays showed that the Q motif is not essential for ATP binding. The Q motif in FANCJ helicase, a ChlR1 sequence homology protein, regulates FANCJ's dimerization, while our size exclusion chromatography indicated that ChlR1 protein functions as monomer. Taken together, our results suggest that the Q motif in ChlR1 helicase is involved in DNA binding but not ATP binding.This work was supported in part by the Natural Sciences and Engineering Research Council of Canada (YW)
Investigating the Importance of DinB‐RecA Interactions to the Regulation of DinB's Activity and the Fidelity of the DNA Damage Response
Authors: Tashjian Tommy , Lin Ida , Cafarelli Tiziana , Godoy Veronica 
Published: June 2021

The error prone DNA Polymerase IV, DinB, specializes in synthesizing DNA from a lesion‐containing DNA template, an activity that allows cell survival during DNA‐damaging conditions at a mutagenic cost. We have shown that there is direct physical interaction between DinB and Escherichia coli's main recombinase, RecA. We also have in vitro evidence suggesting that DinB•RecA interaction is conserved across many bacterial species, including human pathogens. Circular dichroism spectroscopy experiments show that the DinB•RecA complex undergoes a drastic conformational change compared to the single proteins alone. This is supported by the fact that DinB(C66A), a derivative with tight binding to RecA, and RecA copurify by size exclusion chromatography at a size that is much smaller than the size of the individual proteins, suggesting that compaction and tertiary structure changes are likely involved upon complex formation. Alteration of the physical interaction between DinB and RecA leads to changes in the mutagenic outcome in E. coli cells that are exposed to DNA damaging agents. Our current model is that the DinB•RecA complex not only alters the activity of DinB, but results in the sequestration of RecA from the lowest fidelity polymerase, DNA Polymerase V. The DinB•RecA complex, therefore, is central in controlling the fidelity of the DNA damage response.Source of Funding: R01 and its supplement from the National Institute of Health (NIH) Institute of General Medicine to V. Godoy
RTT105 in Ty1 Retrotransposition: a Possible Role in Virus‐like Particle (VLP) Assembly
Authors: Scales Jessica , Peterson Mark , Keeney Jill 
Published: June 2021

Retrovirus‐like transposon mRNA encodes envelope and replication proteins (gag and pol), and serves as genomic material for virus‐like particles (VLPs), a cytosolic structure in which the element's genome is reverse transcribed. In Saccharomyces cerevisiae, there are several families of such retrotransposons, including the abundant Ty1 element. Previously, genome‐wide screens have identified candidate genes that mediate Ty1 mobility. One candidate, RTT105, was identified as a negative regulator of Ty1 mobility. Because RTT105 contains no conserved domains, the nature of RTT105's role in Ty1 retrotransposition remains unknown. Rtt105p localizes at Ty1 RNA and Gag foci, suggesting a role in ribonucleoprotein complexes. Here, we use RNA‐Seq analysis of isogenic WT and RTT105 knockout strains to determine changes in gene expression that may explain RTT105's regulation of Ty1. In rtt105Δ, we identified significant down regulation of signal recognition particle (SRP), a conserved chaperone that binds ribosome‐nascent chain complexes for post‐transcriptional processing. SRP aids in translocating Gag to the ER where it encapsulates Ty1 RNA in trans at presumptive VLP assembly sites. Because endogenous Ty1 mobility is maintained at low levels by the host cell, our data suggest that RTT105 may support SRP‐dependent processing of Ty1. Deletion of RTT105 may induce down regulation of SRP‐dependent processing of Ty1, thereby decreasing genome‐wide mobility of the element. This result illuminates an important step in retrotransposon processing and regulation, and suggests at least one route by which Ty1 activity is actively maintained by the cell.This research was funded by HHMI, NIH, and the Huck Institutes of the Life Sciences.
Role of ATP synthase as a molecular drug target against bacterial infections
Authors: Ahmad Zulfiqar 
Published: June 2021

BackgroundF1Fo ATP synthase is the main source of cellular energy in almost all organisms from bacteria to man through oxidative phosphorylation. Binding of ADP and Pi in the catalytic sites of ATP synthase is essential for the formation of ATP. Inhibition of ATP synthase deprives cells of required energy in the form of ATP resulting in cell death. Here, we propose that ATP synthase can be used as a molecular drug target against microbial infections in general and bacterial infections in particular. This can be achieved by targeting the bacterial ATP synthase. A wide variety of phytochemicals and peptides are known to potently in inhibit the ATP synthase. Thus, identification of natural phytochemicals or antibacterial peptides is an important goal towards using ATP synthase as a molecular drug target.MethodsPhytochemicals or peptide induced inhibition of wild type purified F1 or membrane bound F1Fo ATP synthase were performed. Many polyphenolic compounds were structurally modified to increase the extent of inhibition and potency on molar scale. Mutagenic analysis of polyphenol binding and peptide binding site was done to understand the mechanism of inhibition. Growth yield in limiting glucose and non‐fermentable carbon source succinate in presence or absence of inhibitors was done to prove the ATP synthase related cell death.ResultsLately, we found that structural medication of polyphenolic compound such as resveratrol resulted in ~60% additional inhibition of ATP synthase and caused complete abrogation of wild type E. coli cells while mutant cells along with null cells were not affected. Currently, we are studying the role of βDELSEED‐motif in peptide binding to elucidate the mechanism of inhibition of ATP synthase.
rLosac, a Hemolin, Prevents Human Chondrocyte Aging and Degeneration of Articular Cartilage
Authors: Chudzinski‐tavassi Ana , Alvarez‐Flores Miryan , Bosch Rose , Antonoili Eliana , Pereira‐Junior Edgard , Ferreti Mario , Cohen Moises 
Published: June 2021

Chrondrocyte aging is associated with the degeneration of articular cartilage such as rheumatoid arthritis and osteoarthritis (OA). One of the most prominent changes in cartilage affected by these diseases is loss of extracellular matrix due to its elevated degradation and lowered synthesis. Moreover, pro‐inflammatory cytokines such as TNF‐a and IL‐1b play roles in the progression of these degenerative changes.The aim was to investigate of rLosac as an anti‐inflammatory/chondroprotective agent and to analyze its capacity to stimulate the production of extracellular matrix molecules. Primary cultures of human chondrocytes from cartilage of osteoarthritic knee joints was cultured in the absence or in the presence of IL‐1b and treated with rLosac. Specific senescence‐associated β‐galactosidase activity, cell viability, production of extracellular matrix molecules, cell cycle, expression of collagen I receptor and IL‐1 β were analysed. rLosac inhibited cellular death and reduced the IL‐1β‐induced expression of β‐galactosidade and stimulated the production of collagen type I, laminin and fibronectin. Increased expression of Col‐I and decreased expression of IL‐1β, proposing a down‐regulation of the inflammatory condition, suggesting that rLosac may have the potential to prevent inflammation, aging and cellular death induced in pro‐inflammatory chondrocytes.Supported by FAPESP (CETICs), Capes, INCTTox‐CNPq.
Investigating the Interaction between the Dopamine Transporter and the Sigma 1 Receptor
Authors: Rodriquez Meghan , Perley Danielle , Henry Keith , Khoshbouei Habibeh , North Ashley , Sambo Danielle 
Published: June 2021

Addictive psychostimulants such as cocaine and amphetamines bind to the dopamine transporter (DAT) and alter its ability to recycle dopamine (DA), resulting in adverse psychological and physiological effects. Several of these stimulants also bind the sigma 1 receptor (S1R), which modulates signal amplification of neurotransmitters, calcium and potassium through protein‐protein interactions at the plasma membrane. The S1R is expressed in dopaminergic neurons however; a physiological relationship between DAT and S1R has not been identified. Here we investigate physical interaction between DAT and S1R through co‐immunoprecipitation techniques in COS7 cells expressing the two proteins. Our data support that DAT and S1R are at least involved in a single protein complex as antibodies against DAT also purify S1R and vice versa.Grant Support: This work is supported by an NIH Grants DA026947 and NS071122 awarded to HK.
Lipid Metabolism Regulation by Hyperosmolarity in Renal Cells
Authors: Casali Cecilia , Weber Karen , Morel Gómez Emanuel , Fernández María del Carmen 
Published: June 2021

Physiologically, renal medullary cells are immersed in an unusual extracellular matrix that shows a high osmolarity and plays a major role in the urine concentrating system. To survive and work in such environment, cells have developed adaptive and protective mechanisms. Various works from our laboratory showed that the preservation of membrane structure helps to maintain cell architecture and viability. We showed that changes in environmental osmolarity regulate membrane phospholipid (PL) synthesis and turnover. PL de novo synthesis requires fatty acids which usually come from cellular storage in triglycerides (TG) molecules.The aim of the present work was to evaluate whether hyperosmolarity modulates lipid metabolism through the activation of the transcription factor sterol response element binding protein (SREBP). For this purpose, MDCK cells, derived from renal structures, were incubated in isosmolar and hyperosmolar media. Then, SREBP participation in TG synthesis and storage, and in the expression of the main TG biosynthetic enzymes, was evaluated. Results showed that hyperosmolarity increased TG storage and synthesis, and also the expression of fatty acid synthase (FAS), acetyl‐CoA carboxylase and lipin‐2 mRNA levels. Fatostatin (20 mM), a SREBP inhibitor, blocked hyperosmolar‐increased TG content and synthesis, and the expression of FAS enzyme. Fatostatin treatment also decreased the number of cells that survive to the treatment. These results suggest that hyperosmolarity activates SREBP which in turn modulates lipid synthesis. Thus, SREBP would play a key role in cell adaptation to osmotic changes by ensuring the substrates for PL synthesis that leads to membrane homeostasisSupported by CONICET 0327 and PICT 2013‐1132.
Interaction of Ceramide Synthase with Long Chain Fatty Acyl‐CoA Synthase 5 Channels de novo Ceramide to Acylceramide Generation by Diacylglycerol Acyltransferase 2 on Lipid Droplets
Authors: Senkal Can , Hannun Yusuf , Obeid Lina 
Published: June 2021

The central molecule of the sphingolipid pathway, ceramide, has been shown to regulate multiple cellular responses including growth arrest, senescence, apoptosis and autophagy. Fatty acyl‐CoA (FA‐CoA) dependent ceramide generation is catalyzed by ceramide synthases in the de novo pathway. Importantly, six CerS isoforms displaying different preferences to generate ceramides with distinct fatty acyl chains have been identified. Currently, there is little insight into the regulation of the distinct CerS enzymes. In an approach aimed at defining interacting partners with CerSs, we found that that fatty acyl CoA synthase ACSL5 interacts with CerS. Contrary to our initial hypothesis, ACSL5 generated FA‐CoA was not a direct substrate utilized by CerS to generate ceramide as measured by LC/MS. Rather, ACSL5 generated FA‐CoA was utilized with de novo ceramide for the generation of acylceramides, poorly studied ceramide metabolites. Moreover, inhibition of ceramide channeling to acylceramide enhanced accumulation of de novo ceramide and resulted in augmentation of ceramide‐mediated apoptosis. Mechanistically, we show that acylceramide generation is catalyzed by DGAT2 and involves the formation of an ACSL5‐CerS‐DGAT2 complex on lipid droplets. In summary, this study identifies the molecular mechanism of a novel metabolic pathway of acylceramide generation and implicates the importance of this novel pathway in ceramide‐mediated apoptosis.This study is supported by NIGMS, NIH 9R01GM097741‐13A1
O‐GlcNAcylation Stabilizes Nod2, an Innate Immune Receptor Involved in Crohn's Disease
Authors: Hou Ching‐Wen , Zachara Natasha , Grimes Catherine 
Published: June 2021

Nucleotide‐binding oligomerization domain2 (Nod2) is an intracellular receptor that can sense bacterial components, such as, Muramyl dipeptide (MDP). MDP is a peptidoglycan fragment from bacterial cell wall that can activate NF‐kB, a transcriptional factor that can induce the production of various inflammatory molecules. Nod2 mutations have been linked to Crohn's disease with a decreased ability to activate NF‐kB. Recently, using the classic cycloheximide stabilization assay, we demonstrated that Nod2 variants have a lower half‐life compared to wild type Nod2 in cells. To further characterize this protein, the objective of this study is to determine if Nod2 is post‐translationally modified. O‐GlcNAcylation is a post translational modification in which the O‐GlcNAc transferase (OGT) transfers N‐acetylglucosamine (GlcNAc) from UDP‐GlcNAc to selected serine and threonine residues of a target protein. As GlcNAc is a major component of peptidoglycan of bacterial cell wall and a large amount of GlcNAc is released from bacterial cell wall during cell wall remodeling, we hypothesized that Nod2 could be O‐GlcNAcylated. Preliminary data show that wild type Nod2 and Nod2 variants are O‐GlcNAcylated. In addition, increasing the O‐GlcNAcylation level can increase the half‐life of wild type Nod2 or Nod2 variants. In future experiments, we will determine which residue of Nod2 is O‐GlcNAcylated and investigate if this modification affects Nod2 mediated NF‐kB pathway in wild type Nod2 or Nod2 variants.Funding of this project is provided by University of Delaware.
Impact of Environmental Conditions on the Pathogenesis of the Common Cold
Authors: huang Chao 
Published: June 2021

The common cold is a worldwide health issue, and is associated with severe complications in patients with chronic illnesses and disability, which can even be life‐threatening. More than 200 types of viruses are associated with the common cold. However, until now, there is still no effective protection against the cold. The fact that a cold cannot be prevented is reflective of a poor understanding of the etiology of the disease. The present study identified a relationship between the incidence of the common cold and environmental conditions, namely low temperature and air humidity. Three basic etiological factors of the common cold were identified: (1) Dry air, defined as relative humidity of <40%, challenges the lungs to maintain exhaled air humidity of approximately 100%. (2) Low temperature (<5°C) in combination with excessive relative humidity (>90%), challenges the lungs to maintain exhaled air temperature of approximately 37°C. (3) Common cold induced by lung chilling during sleep.ConclusionExtreme weather reduces airway defenses against viral infections. Therefore, adequate regulation of indoor air humidity (55%–70%) is expected to prevent most colds.
Triphosphate Re‐orientation of the Incoming Nucleotide as A Fidelity Checkpoint in Viral RNA‐dependent RNA Polymerases
Authors: Liu Xinran , Yang Xiaorong , Musser Derek , Boehr David 
Published: June 2021

The fidelity of the virus‐encoded RNA‐dependent RNA polymerase (RdRp) plays an essential role in generating sufficient genetic diversity to escape the bottlenecks of the host's anti‐viral response. For many nucleic acid polymerases, a pre‐chemistry conformational change is at least partially rate‐limiting and represents a key fidelity checkpoint. There has been disagreement in the polymerase field about the structural rearrangements important in this step. One suggestion has been that this step represents a rearrangement of the triphosphate of the incoming nucleotide that will facilitate nucleophilic attack by the primer 3′‐hydroxyl group. We have now gained insight into the triphosphate re‐orientation in the poliovirus (PV) RdRp via 31P NMR. Modeling studies suggest that Lys167 and Arg174 on structural motif F make important interactions with the triphosphate. 31P NMR results demonstrate that WT RdRp interacts with “correct” nucleotide in a way different from “incorrect” nucleotide; R174K RdRp is unable to rearrange correct nucleotide like WT enzyme and instead interacts with correct nucleotide in a similar fashion as incorrect nucleotide; The R174K substitution also substantially reduces enzyme efficiency and leads to very high fidelity. The K167R variant has similar catalytic efficiency as WT when correct nucleotide is incorporated, however, incorrect nucleotide is more readily incorporated. Altogether these results suggest that triphosphate re‐orientation is an essential fidelity checkpoint and interactions with motif F residues are important for this alignment.The research is supported from Penn State start‐up funds and NIH R01 AI104878 to DDB.