bluuebunny/crossref_metadata_2025_split huggingface dataset:
The First‐Year Research Advancement Program (FRAP): A research‐centered experience to foster discipline identity, persistence, and agency in under‐represented students in chemistry and biology.
Authors: Mills Kenneth V. , Paxson Julia 
Published: April 2020

The First‐Year Research Advancement Program (FRAP) was started at Holy Cross in 2014 to increase persistence in STEM of academically promising students who belong to groups that traditionally do not persist at the same rate as majority students, including first‐generation students, PELL‐eligible students, and underrepresented minority students. We cofounded FRAP based on evidence that early experiences in research laboratories can increase discipline identity and persistence. We are now in the sixth year of the program, with 58 total first‐year participants, and six former participants serving as mentors. Two first‐year students are paired with both a faculty mentor and a senior student in the lab, and all students are paid for eight hours per week such that their participation replaces campus employment rather than adding an extra burden. Students work in the lab six hours per week, and participate in one‐hour meetings with their lab group and in a one‐hour meeting with all participants in order to create two cohorts of support: one among the research lab who typically are of all class years, and another among their first‐year peers. We have assessed the program in collaboration with the Office of Assessment and Research, and have found that participants show growth in aspects of their attitudes toward science and in their agreement with statements that students like them can succeed in science and that even if they forget facts they can still use thinking skills they learned in science. They did not show as much growth in their confidence to do well in classes. As our total number of participants has grown, we are analyzing persistence data in STEM disciplines. Nine of the 12 students who started in the Mills lab as FRAP students are either still in the lab or stayed in the lab for their four years at Holy Cross.Support or Funding InformationThis work was supported by the National Science Foundation (MCB‐1517138, KVM) and a Henry Dreyfus Teacher‐Scholar Award (KVM).
Investigation of mutants with aberrant transcriptional regulation of OLE1
Authors: Cribbs Lauren , Filippelli Sara , McDonough Virginia 
Published: April 2020

The OLE1 gene in Saccharomyces cerevisiae encodes the Δ9 desaturase. Previously, we isolated mutants that affect the expression of OLE1. In this work, we describe the phenotypes of two of these mutants, arv1 and nro (“no regulation of OLE1”). OLE1 promoter‐URA3 ORF reporter gene assays have shown that both mutants express OLE1 in the presence of the normally repressive unsaturated fatty acid 18:2Δ9,12. Analysis of OLE1 RNA amounts by qPCR, indicate that the nro mutant has similar amounts of mRNA present to the wildtype control, however, the level of OLE1 mRNA is significantly lower in arv1, and confirmed that it is not regulating expression in response to 18:2 feeding. OLE1 promoter deletion analysis resulted in decreased OLE1 transcription in arv1 compared to the wildtype, and did not regulate in response to 18:2 feeding. Surprisingly, OLE1 expression regulation by 18:2 is somewhat recovered with a OLE1 promoter deletion between −488 and −471 in arv1 cells, although this regulation is lost at −255 (same as wildtype). This assay also indicated that the OLE1 promoter region between the −225 and −142 may be important for nro regulation of OLE1 transcription. These results indicate that the arv1 and nro mutants do have an effect on the transcriptional regulation of the OLE1 gene.Support or Funding InformationResearch reported was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM132853
Rapid formation of ER‐PM contacts during early stages of phagocytosis
Authors: Lee Minhyoung , Fairn Greg 
Published: April 2020

Membrane contact sites (MCS) are specialized structures where the endoplasmic reticulum (ER) and other organelle form come in close proximity (~10–30 nm). Functionally, these regions are thought to serve as hubs for cellular processes such as inter‐organellar exchange of lipids, calcium homeostasis, and the access of phosphatases to substrates. Consequently, the spatial‐temporal organization of these structures will impact numerous cellular functions. The formation of ER plasma membrane (PM) contact sites are controlled by a variety of membrane tether proteins as well as the morphology of the ER. We and others have found that the sub‐plasmalemmal cortical actin cytoskeleton, a mesh‐like network that occupies 100–200 nm below the PM, can also limit the formation of ER‐PM contact sites. Yet, how ER‐PM junctions are regulated in circumstances where cells have extensive cortical actin remodeling is not understood. An example of such cellular process involving dynamic cortical actin re‐arrangement is phagocytosis. Phagocytosis is an actin‐dependent processes used to internalize particulate material (≥0.5 μm) that serves both antimicrobial and homeostatic functions. The role of the ER in phagocytosis has been a matter of much controversy. Electron micrographs of macrophages undergoing phagocytosis revealed the presence of ER in extensive, close contact with the forming phagosome. It is speculated that the role for ER in this context is to form ER‐PM contact sites with the PM. My studies have revealed that during phagocytosis, the disassembly of F‐actin from the base of the phagocytic cup allows for the formation of new ER‐PM contact sites. ER‐PM contacts formed promptly and specifically where the polymerized actin has been cleared. As such I have found that the spatial occupancy of ER‐PM junction increased ~3 fold during phagocytosis. Finally, I identified PTP1B, a tyrosine phosphatase, as one of the ER‐PM contact proteins that may be functionally important during phagocytosis.Support or Funding InformationNSERC PGS‐DCIHR Project Grant
Heparan Sulfate Reduction to Enhance Wound Induced Neogenesis
Authors: Zurich Raymond , Phan Anne , Esko Jeffrey 
Published: April 2020

Heparan sulfate regulates cell signaling important in wound healing and regeneration. In normal mammalian wound healing, injuries that penetrate the dermis result in a scar lacking hair follicles and subcutaneous fat because hair and fat do not regenerate. However, if the dermal wound is larger than a critical size (≥20 mm in adult mice) a process termed ‘Wound Induced Neogenesis’ occurs and some neogenic hair and fat is regenerated in the center of the wound. Based on previous observation that a deficiency in heparan sulfate accelerates cutaneous wound healing in mice, we hypothesized that heparan sulfate deficiency can enhance wound induced neogenesis of hair and fat in dermal wounds. Using systemic genetic models of heparan sulfate deficiency (Ext1+/−Ext2+/−), we tested whether mice with less heparan sulfate had enhanced wound induced neogenesis of hair and fat after a subcritical size (15 mm) full‐thickness dorsal skin excisional wound. Although we observed no significant difference in the rate or extent of wound closure, we found increased neogenesis of hair follicles and fat in heparan sulfate deficient mice. Our experiments suggest that inducing heparan sulfate deficiency can be a therapeutic avenue to enhance mammalian regeneration.Support or Funding InformationThis work was supported by the National Institute of Arthritis, Muscular, and Skin Diseases (R61 AR073031).
Reverse transcription and amplification of 2′F DNA by commercially available DNA polymerases
Authors: Thompson Arianna , Barrett Susanna , Weiden Aurora , Leconte Aaron 
Published: April 2020

Modified‐DNA polymerases have been evolved that can synthesize long strands of modified oligonucleotides. The resulting modified DNA (M‐DNA) has many high‐value potential applications, such as clinical diagnostics and therapeutics. To fully apply M‐DNA, it must be reverse transcribed back into natural DNA. Previously, error prone reverse transcriptases have been required for this step, and then the resulting DNA has to be amplified in a separate amplification reaction. Using a synthetic template with 2′F modified nucleotides, we tested a panel of commercially available DNA‐dependent DNA polymerases for their ability to reverse transcribe (RT) and amplify M‐DNA in a one‐pot reaction. We found that four of the six polymerases chosen (Phusion, Q5, Deep Vent, and Vent) were able to RT and amplify synthetic 2′F modified DNA in a single reaction. These products were sent in for high‐throughput sequencing and it was determined that Q5, Deep Vent and Phusion had between 90 and 95% matched reads, while Vent had less than 85%. The conditions of this process (reaction buffer and PCR cycling conditions) were also shown to affect the accuracy, but to a lesser degree. These polymerases were also able to RT and amplify enzymatically synthesized 2′F templates, allowing us to use this as a tool to quantitatively determine error rates of M‐DNA polymerases using high‐throughput sequencing. This demonstrated that P1, one of our labs best modified‐DNA polymerases, was almost four times more accurate than SFM4‐6, the fields previous best enzyme. These discoveries will enable the wider use of 2′F M‐DNA, as well as add to the. evidence supporting the use of commercially available DNA polymerases for reverse transcription reactions.Support or Funding InformationNational Science Foundation
Characterization of Effect of Hydrogen Peroxide on Hydrolysis of Collagen
Authors: Patel Silkey , Chew Charmaine , Keenan Kelly 
Published: April 2020

Americans spend billions of dollars a year on teeth whitening and the active ingredient is usually hydrogen peroxide. The outermost layer of teeth is the enamel and the dentin is underneath: the major protein found in the dentin of teeth is Type I collagen. The goal of this project was to characterize the effect of hydrogen peroxide at concentrations similar to those used in over‐the‐counter‐whitening strips on collagen. Type I collagen was mixed either with hydrogen peroxide, collagenase, both or neither and the amount of protein released was characterized in several ways: dialysis (with a molecular weight cutoff of 3.5 kDa) and gel filtration with an exclusion limit of 10 kDa. The amount of protein or peptide released was measured with a modified Lowry assay, which has been adapted to measure collagen accurately. Collagenase is a metalloprotease that hydrolyzes the peptide bond. When treated with collagenase, no protein was released but 17 mg of protein was released when treated with 7.5% hydrogen peroxide and 18 mg when treated with both. Similar results were obtained with collagen isolated from human teeth. The amounts increased as hydrogen peroxide concentrations increased. For the gel filtration column, untreated collagen emerged in the first fraction after the void volume but when pre‐treated with hydrogen peroxide, there were proteins in later fractions. Type I collagen shows a molecular weight (MW) of 129 kDa when run on 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) that was silver‐stained. This amount matches the expected value based on the subunits of the protein. The addition of hydrogen peroxide caused this band to disappear from the gel and no smaller MW bands were visible. When treated with collagenase, the original band as well as several smaller proteins of 80, 66 and 47 kDa are observed as collagenase produces the occasional hydrolysis with large protein fragments that result. No bands were observed when collagen was treated with both collagenase and hydrogen peroxide. These results suggest that hydrogen peroxide is capable of hydrolyzing type I collagen and produces peptides that are too small to be detected on the gels but have a size of 3.5 kDa or less.Support or Funding InformationAuthors thankfully recognize support by research and professional development funds from Stockton University.
Characterization of Peptides Released from Treatment of Type I Collagen with Hydrogen Peroxide
Authors: Amoah Akua , Ailes Isaiah , Keenan Kelly 
Published: April 2020

Previous research has demonstrated that hydrogen peroxide is capable of hydrolyzing the peptide bond in proteins including collagen. Proteases, like collagenase, are also capable of hydrolyzing peptide bonds at specific amino acid residues producing fragments. The goal of this project was to characterize the size and nature of the peptides produced by treating proteins with hydrogen peroxide. In order to characterize the response of proteins in general to hydrogen peroxide, various albumin proteins were treated with varying concentrations, precipitated using trichloroacetic acid and centrifuged. The amount of peptide small enough to not be precipitated was measured in the supernatant using the Lowry assay. The results demonstrated that increasing concentrations of hydrogen peroxide resulted in increased amounts of peptides too small to be precipitated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to examine the size of these proteins following treatment with hydrogen peroxide treatment or the protease trypsin, which hydrolyzes following the amino acid residues arginine or lysine. Chick albumin showed a band at the expected MW of 43 kDa and treatment with hydrogen peroxide (5.0%) reduced the size slightly. When treated with trypsin, the original band as well and other bands were visible on SDS PAGE. These results suggest that hydrogen peroxide works differently than proteases to hydrolyze proteins. Since trichloroacetic acid cannot precipitate collagen, other methods were used to characterize the peptides released by hydrogen peroxide. Glycine, proline, and hydroxyproline, which are overrepresented in the collagen primary structure, were detectable using thin layer chromatography (TLC). Silica plates were spotted with 1 mg/ml solutions with 70%/30% (v/v) n‐propanol/water as mobile phase. Glycine produced a red spot with an retention factor (Rf ) of 0.37 while proline resulted in a yellow spot (Rf 0.37) and hydroxyproline was pink (Rf 0.46). When the tripeptide Gly‐Pro‐Ala was used, the spot was yellow with an Rf value of 0.18. This result suggests that amino acids and small peptides can be resolved using this method. Type I collagen was mixed with 6.7% hydrogen peroxide or collagenase or both or neither in dialysis tubing and the contents of the fluid outside the tubing was tested using this TLC method as well as by measuring hydroxyproline in a colorimetric test. Reverse phase high pressure liquid chromatography were used to measure Glycine and Proline,Support or Funding InformationAuthors gratefully acknowledge research and professional development funds from Stockton University.
Enzyme polymerization in nucleotide biosynthesis
Authors: Peterson Jeff , Akizu Naiara , Simonet Jacqueline , Foster Maya , O’Reilly Alana 
Published: April 2020

Polymerization of metabolic enzymes into micron‐scale assemblies is an emerging phenomenon. However the role assembly plays in regulating metabolic flux differs between enzymes and in many cases is unknown. Furthermore, the signaling pathways that regulate assembly are often not well understood. Our work focuses on enzymes mediating pyrimidine and purine biosynthetic pathways that undergo regulated assembly. To address the biological function of these assemblies we have developed point mutants of CTP synthase and inosine monophosphate dehydrogenase (IMPDH), that prevent their assembly without eliminating their catalytic activity. We will present new data using these novel tools in mice and Drosophila to elucidate the role of polymerization of these enzymes in native contexts in which they assemble. Our findings demonstrate a critical role for assembly of CTPS and IMPDH in supporting the growth and proliferation of cells with elevated nucleotide demands. Thus, we propose that assembly of these nucleotide biosynthetic enzymes has been harnessed in metazoans as a homeostatic mechanism to ensure that metabolic biosynthetic flux meets demand.Support or Funding InformationNIH GM083025
Insecticides resistance status in Aedes aegypti and Aedes albopictus from Lahore, Pakistan
Authors: Rahman Rafi ur , Costa Monique , Carrara Luana Silva , Lima Josè Bento Pereira , Martins Ademir Jesus 
Published: April 2020

Aedes‐borne disease in Pakistan drew the attention after the dengue outbreak in Lahore in 2011, leaving thousands hospitalized and around 300 deaths. Since then many small and big outbreaks have been reported from various parts of the country. The only available and mostly used mosquito control strategy in Pakistan is the use of neurotoxic chemicals. Pyrethroid deltamethrin and permethrin were extensively used for adults’ control along with organophosphate temephos as a larvicide during and after the outbreaks. This study was designed to find the efficacy of the insecticides in use for Aedes control as well as other insecticides of the same or different classes. Preliminary results after WHO‐like tube tests or CDC‐bottle tests showed that both Aedes aegypti and Aedes albopictus are susceptible to larvicide OP temephos, IGR pyriproxifen and adulticide OP malathion while they are resistant to all classes of pyrethroids tested (deltamethrin, permethrin, etofenprox, alphacypermethrin). Genotyping assays showed low kdr frequencies while assays to find the expression of P450s and other enzymes related to resistance are underway. Sequencing of the voltage gated sodium channel gene will give us a better picture about the mutations in the IIS6 and IIIS6 regions which plays an important role in pyrethroids sensitivity. This study will assist the authorities to design future control strategies.Support or Funding InformationThe study is part of PhD thesis of the presenting author Rafi ur Rahman, who receives monthly scholarship from TWAS and CNPq. No additional funding is provided to the laboratory or the student.
Histone H3 K14 acetylation and H2B SUMOylation help regulate binding of the RSC remodeler complex to the nucleosome
Authors: Wilkins Bryan J. , Jain Neha , Evans Brian , Chaudhry Shereen , Neumann Heinz 
Published: April 2020

Chromatin remodeler (CR) complexes play crucial roles in regulating chromosomal architectural and act to modify nucleosomal DNA contacts by repositioning nucleosomes. The mechanistic details of the CR family of proteins are of importance because each remodeler complex contributes to unique chromatin structural maintenance. Here, we study the RSC complex of yeast to better characterize its dynamics in the living nucleus. Utilizing an expanded genetic code, we incorporate the photo‐crosslinking amino acid, p‐benzoyl‐L‐phenylalanine (pBPA), site‐specifically into histone proteins in order to monitor non‐histone protein interactions at the nucleosomal surface, in vivo. We map the interface between the RSC motor protein, Sth1, and the nucleosome core particle, revealing that crosslinking efficiency to the H3 N‐terminal tail is dependent upon the acetylation of H3 K14. This modification is also a known binding site of the RSC subunit, Rsc4, suggesting that the remodeler favors binding symmetrical H3 K14ac marks on the same nucleosome. Additionally, we reveal that RSC binding is dependent on SUMOylation of histone H2B. Pairing crosslinking with temporal control of the cell cycle we observe that RSC has differential binding modes as the chromatin architectures alter during compaction and passage into mitosis. All together, this data provides greater mechanistic insight to the functional dynamics of the RSC remodeler under true physiological conditions.Support or Funding InformationNational Institute Of General Medical Sciences of the National Institutes of Health under [Award Number R15GM124600] German Research Foundation (DFG) [Grants NE1589/5‐1 and 6‐1]
Epinephrine Responsiveness is Reduced in Livers from Trained Mice
Authors: Dibe Hana A. , Townsend Logan K. , McKie Greg L. , Wright David C. 
Published: April 2020

Catecholamines increase liver glucose production at least in part through increases in glycogenolysis and gluconeogenesis. To date, the effects of prior exercise training on the liver’s response to epinephrine, in vivo, have not been fully elucidated. To examine the role of epinephrine signaling on indices of liver glucose production in trained mice, male C57BL/6 mice were subject to either 12 days of voluntary wheel running (TR) or remained sedentary (SED). Epinephrine (0.5 mg/kg body weight), or vehicle were injected intraperitoneally following 12 days of training and tissues harvested 15 minutes post‐injection. The rise in blood glucose following epinephrine injection was significantly blunted in trained compared to sedentary mice and this was paralleled by a blunted reduction in liver glycogen in trained mice. There was a main effect of epinephrine to increase the phosphorylation of protein kinase‐A substrates (pPKAs), which was driven by increases in the sedentary, but not trained, mice. Similarly, epinephrine‐induced increases in the mRNA expression of α‐ and β‐adrenergic receptor 1 and 2 and glucose‐6‐phosphatase were greater in SED compared to TR mice. Exercise and epinephrine decreased serum insulin levels and increased glucagon levels, however, there were no differences within epinephrine treated groups. Changes in phosphorylated AKT, AMPK, and phosphodiesterase 4B protein content, negative regulators of PKA signaling, do not explain the protective effects of exercise training against epinephrine induced increases in blood glucose and liver glycogen breakdown. Taken together our data suggest that prior exercise training reduces the liver’s response to epinephrine. This could be beneficial in the context of training‐induced sparing of glycogen during exercise.Support or Funding InformationThis work was funded, in part, through a National Sciences and Engineering Research Council of Canada (NSERC) grant to D.C.W., who is a Tier II Canada Research Chair in Lipids, Metabolism, and Health. H.A.D was supported by an Ontario Graduate Scholarship. G.L.M. was supported by an Ontario Graduate Scholarship and subsequently an NSERC Canada Graduate Scholarship. L.K.T. was supported by a Dairy Farmers of Ontario Doctoral Research Assistantship and an NSERC Postgraduate Scholarship.