bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of Hobo Transposon Excision on DNA repair in Drosophila melanogaster
Authors: Miller Anna , Saville Ken 
Published: June 2021

As the repository of genetic information, it is critical to organisms that DNA damage is repaired quickly and with high fidelity. When DNA damage is improperly repaired, it may cause diseases such as cancer and severe combined immunodeficiency. DNA can be mutated by numerous agents both external and internal. One internal source of DNA damage is from the transposition of transposable elements, including the P and hobo elements. Transposable elements “jump” out from one section of the DNA and reinsert into another, leaving double strand DNA breaks at the excision site. Hobo excisions are preferentially repaired by nonhomologous end‐joining, NHEJ. DNA repair and transposable elements themselves can be studied in Drosophila melanogaster. This study looked at the hobo on P, or HOP, element. The P element is located inside of the Hobo element; HOP8 is nonautonomous and needs hobo transposase to be supplied in trans. Curly winged, glazed eye female flies that have an inducible source of hobo transposase were crossed with HOP8 male flies and heat shocked to trigger transposition, leaving a double strand DNA break requiring repair behind. The resulting mosaic eyed male flies were then crossed with wild type female flies because mosaic eyes mark meiotic recombination in the P element. Female flies with white eyes indicating that hobo transposition occurred, were collected and stored in a freezer. Experiments are underway to determine the original location and new insertion sites of hobo using inverse PCR to further understand DNA repair.Support or Funding InformationAlbion College Foundation for Undergraduate Research, Scholarship, and Creative Activity (FURSCA)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Association of Oxidative Stress with Polycystic Ovarian Syndrome in Oman: A Case‐Control Study
Authors: Sulaiman Maha AH. , Al‐Farsi Yahya M. , Al‐Khaduri Maha M. , Saleh Jumana M. , Waly Mostafa I. 
Published: June 2021

BackgroundPrevious studies in Euro‐American populations have shown that women with polycystic ovarian syndrome (PCOS) have increased levels of serum oxidative stress markers. In the Arab region, there is noticeable lack of research of the association between oxidative stress and PCOS.ObjectiveThis study aims to compare socio‐demographic, clinical characteristics and oxidative stress indices between women diagnosed with PCOS and non‐PCOS women.MethodsThis hospital based case‐control study was conducted among women aged 16–49 years. The study included 51 women diagnosed with PCOS (as per Rotterdam 2003 criteria) and 45 control women who were known not to have PCOS. Serum blood samples were collected to measure biomarkers of oxidative stress including glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), and total antioxidant capacity (TAC) using commercially available kits.ResultsReduced GSH levels (P= 0.006) were significantly lower among PCOS women compared to non PCOS women. GR and GPx activity levels showed higher trends in women with PCOS compared to non PCOS women and the difference was significant for the GPx activity (P= 0.04). PCOS women also showed lower TAC levels (P= 0.08) than the control group with no significant difference between the two groups.ConclusionThe study provides a supportive evidence for the association between oxidative stress and PCOS among Omani women. Oxidative stress might play a role in pathogenesis of PCOS, so these parameters may be suggested as diagnostic markers for early diagnosis and screening of high risk groups.Support or Funding InformationSultan Qaboos UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Different classes of RNA require distinct Mex67 paralogs for processing and nucleocytoplasmic export in trypanosomes.
Authors: Obado Samson , Chait Brian , Field Mark , Rout Michael 
Published: June 2021

The nuclear pore complex (NPC) is at the center of nucleocytoplasmic transport as well as many aspects of gene regulation. A series of steps involving the NPC enable the translation of genes to proteins, starting with the initiation of transcription, through quality control and transport through the NPC, to finally the remodeling of ribonucleoprotein (RNA‐protein) complexes on the cytoplasmic face of the NPC. We recently described the architecture of the trypanosome NPC. Notably absent from the cytoplasmic face of the trypanosome NPC are a host of anchored RNA export and remodeling proteins found in the opisthokont (yeast and human) cytoplasmic RNA export platform. This pointed to a significantly different mode of RNA export and processing in trypanosomes. An evolutionarily conserved group of proteins termed nuclear export factors (NXFs) are responsible for the transport of bulk mRNA from the nucleus to the cytoplasm for translation into proteins, as well as aiding ribosomal subunit export. There is a single major nuclear export factor in yeast (Mex67 or NXF1 in metazoa); although metazoans have additional tissue‐specific NXF variants. All variants of NXF form a heterodimer with Nuclear Transport Factor 2 (NTF2)‐Like Export Factor 1 (NXT1 in humans or Mtr2 in yeast), which facilitates NPC localization and translocation. Orthologs of both NXF and NXT have been identified in trypanosomes, termed TbMex67 and TbMtr2 respectively. Our data suggests that there are in fact three Mex67 paralogs in trypanosomes. We provide evidence strongly indicating they have crucial differing functions. As RNA processing and export are highly integrated events, the export pathway is likely an important step in the regulation of gene expression in trypanosomes.Support or Funding InformationNIAID Exploratory/Developmental Research Grant 1R21AI096069 (to MR)NIGMS GM103314 (to BC), GM103511 and GM109824 (to MR and BC);Wellcome Trust grant 082813/Z/07/Z (to MF and MR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutagenesis of the Human α‐Galactosidase A Active Site, Dimer Interface, and Glycosylation Region
Authors: Stokes Erin 
Published: June 2021

Fabry disease is an X‐linked lysosomal storage disorder caused by the deficiency of the enzyme, α‐galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40–60 years of age. Currently approved enzyme replacement therapy using recombinant human α‐galactosidase A improves patient symptoms but storage of the toxic enzyme substrate remains in the brain and other affected organs. The structure of α‐galactosidase A has been previously determined to be a homodimer with six N‐linked glycosylation sites, and the catalytic mechanism determined to be a ping pong bi bi with the second substrate being water. The purpose of this research is to generate a more efficient enzyme replacement therapy alternative. A more efficient therapy was investigated utilizing Pichia pastoris, as well as site directed mutagenesis of the human enzyme. Three locations on the structure of α‐galactosidase A protein were targeted for mutation: the active site; the dimer interface; and the hydrophobic loop between the active site and the third glycosylation site. Viable candidates for further study into a therapeutic were determined based on catalytic efficiency (kcat/Km), thermostability, and possible glycosylation independence. Three mutations were identified, M208E (hydrophobic loop), W277C (dimer interface), and Y207W (active site) to be of potential therapeutic significance.Support or Funding InformationNIH SBIR Grants and Royalty Income From PatentsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing efficiency of the New England Biolabs Q5® site‐directed mutagenesis kit to produce a library of aminoglycoside N‐acetyltransferase mutants
Authors: Macias Jennifer , Pham Olivia , Vaca Alexis , Pennings Pleuni , Kuhn Misty L. 
Published: June 2021

Our aim was to construct a library of 63 rationally designed aminoglycoside N‐acetyltransferase mutants in a high‐throughput manner using the Q5® site‐directed mutagenesis kit from New England BioLabs Inc. Per the manufacturer's recommendation, we used their NEBaseChanger™ tool to design primers for each mutant and followed their Quick Protocol. Since we wanted to use our 96‐well thermocycler with six different gradient optimization blocks, some of the recommended annealing temperatures were adjusted by ± 0.5°C. Qualitatively, we found that this change did not have a significant impact on obtaining amplified products for most of our samples. We observed 49 PCR products at the correct size of ~6 kB in our first reaction and proceeded with the subsequent steps of the manufacturer's protocol. One colony from each mutant was then sent for sequencing to save funds. Of the 49 constructs tested, 32 were correct, which resulted in a 65% success rate from a single reaction. We sent one additional colony from the remaining 17 constructs and obtained 7 additionally correct mutants, for a total of 39/49 correct constructs. The remaining 10 mutants from this first amplification showed products with premature stop codons, wild‐type template DNA, or incomplete amplification products. Therefore, we performed additional troubleshooting reactions to obtain the remaining 24/63 mutants. We found that adjustments of up to 3°C from the recommended annealing temperature and additional sequencing attempts were required to obtain the remaining mutants. While the Q5 kit is user‐friendly and straightforward, we found it was not as cost‐effective as we had hoped and required several rounds of troubleshooting to complete our library.Support or Funding InformationThis work was supported in part by a Ken Fong Fund Grant (to MLK and PP). Additional support was provided by the Genentech Foundation and NIH MBRS‐RISE: R25‐GM059298 (to OP).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical Investigation of Gastrointestinal Fatty Acid Binding Proteins (FABPs) with Fatty Acid Ethanolamides (FAEs)
Authors: Lai May Poh , Katz Francine , Stark Ruth E. 
Published: June 2021

Fatty acids are the precursors for lipids that make up cellular membranes and have significant roles in signal transduction pathways. In order for fatty acids to be shuttled inside the cytoplasm they bind to fatty acid binding proteins (FABPs). Much interest has been focused on a particular class of lipids known as the fatty acid ethanolamines (FAEs) due to the recent discovery that they are involved in regulating appetite, pain, and inflammation. In this present work, we explore the FAE interaction with the intestinal and liver fatty acid‐binding proteins (I‐ and L‐FABP) that are co‐expressed in the gastrointestinal system. Previously, a striking phenotypic divergence was observed in IFABP and LFABP knock out mice: IFABP−/− mice exhibited lean phenotype while the LFABP−/− mice exhibited obese phenotype after high fat diet. Our direct comparison will elucidate if IFABP and LFABP have distinct ligand preferences to the FAE ligands. Here, we adapt a fluorescence competition assay to characterize the affinity of the FAEs to I‐ and L‐FABP. From our preliminary work, we found that I‐FABP exhibits a lower Kd, i.e. tighter binding, to ligand in all cases examined. We also found that the functional group of the ligands has the most impact on binding affinity: specifically, we found the tightest protein binding for the ligand with the carboxyl end group followed by ligands with amide and then with glycerol ends. In addition, 2D 15N‐1H HSQC NMR was used to reveal significant backbone amide chemical shift changes in the presence of a subset of the ligands we investigated. Further analysis of the HSQC results is expected to identify the perturbed ligand‐binding locations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA‐based molecular tools for monitoring cellular activity
Authors: Söderberg Ola 
Published: June 2021

Methods to determine levels of protein‐protein interactions are essential in analysis of cellular signaling activity. High‐resolution analysis of proteins or protein complexes requires either advanced microscopy, or molecular methods that combine distance dependence with signal amplification. By using DNA molecules as tools, we have over the years developed several molecular methods utilizing a variety of enzymes to modify and amplify the DNA molecules used in the methods. One such method is Proximity Ligation Assay (PLA) where pairs of antibodies equipped with DNA oligonucleotides (so‐called proximity probes) are used to target proteins. Proximal binding of such probes template the creation of a circular DNA molecule, which can be amplified using rolling circle amplification (RCA). The single‐stranded RCA product from a single recognition event will contain several hundreds of repetitive motifs that can be visualized by hybridization of fluorophore‐conjugated oligonucleotides.Information on levels of both free and interacting proteins is important to determine if the interactions monitored are a result of a post‐translational protein modification, e.g. phosphorylations, that changes the conformation of the proteins and subsequently results in a different dissociation constant (KD), or if the amount of interactions reflects mere expression levels of the proteins. New more complex measurements are needed in order to model signaling pathway activity and information flow in single cells. We have hence further developed PLA to provided information on levels of both free and interacting proteins.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 Delivery Method for Poly‐histidine Tagged Proteins and Peptides for Transient Protein Expression with Light Control via Hollow Gold Nanoshells: Successful delivery of CRISPR Cas9 and Apoptotic Peptide NuBCP with NIR light control.
Authors: Morgan Erin , Jain Piyush , Pearce Martin , Bhatia Sangeeta , Kolluri Siva , Reich Norbert 
Published: June 2021

Intracellular delivery of biomolecules is hindered by the size and charge of the biomolecule. While a number of delivery methods are available for genetic material, few are available for larger biomolecules like peptides and proteins. The delivery of these biomolecules are often limited to viral transfections of the genetic material which can lead to off target gene editing due to prolonged expression of a protein within a cell. We have developed a delivery method for poly‐histidine tagged proteins and peptides of variable sizes and charges for transient protein expression with light control via hollow gold nanoshells (HGNs). The construct of our delivery system involves a thiolated DNA strand containing an NTA modification on the 3′ end attached to the gold surface. A his‐tagged protein or peptide of interest is attached to the HGN via copper‐NTA affinity. Endosomal uptake of these particles is mediated through an orthogonal strand containing a cell penetrating peptide (CPP). Protein release and endosomal disruption is achieved after irradiation with a focused femtosecond pulsed‐ laser of biologically benign near‐infrared light. With this technology we have successfully delivered multiple proteins of interest including the gene editing protein Cas9 for efficient knockout of GFP with light control as well as the Nur77 derived apoptotic peptide NuBCP which allowed for an increase in efficiency with our delivery method.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Plasmodium falciparum Glutamic Acid‐Rich Protein (PfGARP) in Recombinant Pichia pastoris for Malaria Vaccine Development
Authors: Martínez‐Muñiz Andrés Enrique 
Published: June 2021

Malaria infections are common occurrences and one of the leading causes of death in developing countries. The World Health Organization reported 212 million malaria cases worldwide in 2016 which resulted in 429,000 deaths. The most affected group of individuals were children under the age of five which accounted for 70% of this death rate. There are no available vaccines for malaria, but the development of this type of treatment would provide a more cost‐effective and efficient way of battling this disease. Within populations affected by malaria, certain children and adolescents have developed protective immune responses against this disease. These protective immune responses decrease the intensity of the infection thus making the individual more resistant against the acquired disease. Through differential screening experiments, malarial‐protein antigens recognized by antibodies from resistant, but not susceptible individuals were discovered. The Plasmodium falciparum Glutamic Acid‐Rich Protein (PfGARP), an antigen that arrests the development of the parasite in its trophozoite stage was discovered using this method. The specific aim of this study was to express functional PfGARP in order to purify and characterize it as a vaccine candidate. The PfGARP gene was inserted into a plasmid vector that was then used to transform the expression system organism: Pichia pastoris. Individual colonies of recombinant P. pastoris were transferred from agar plates to liquid medium cultures. These were then analyzed for protein expression via Western blot every 24 hours after induction of protein expression. 94 colonies of recombinant P. pastoris were analyzed using this method and none showed expression of PfGARP. PCR analyses were then performed to determine the presence of the PfGARP gene in transformed P. pastoris. A set of vector‐specific primers was designed to amplify a region of 2242 base pairs which included the PfGARP gene in the middle of the region. Amplifications from DNA extracted from the recombinant cells showed a band size of roughly 1000 base pairs. Based on results from the Western blot and PCR analyses performed, it can be concluded that P. pastoris deletes a significant amount of the PfGARP gene's sequence and therefore is not a viable protein expression system for PfGARP.Support or Funding InformationResearch reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T34GM008419.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of botanicals and purified phenolic compounds on NFkB pathway in cultured human skin keratinocytes and fibroblasts
Authors: Higgins Jillian Lee , Kraus Aileen , Clark Matthew , Gallant Benjamin , Arruda Dominic , McTigue Christie , Ma Hang , Seeram Navindra P. , Guo Hao , Chen Hong‐duo , Cui Yali , Wan Yinsheng 
Published: June 2021

Our previous studies have demonstrated that ultraviolet radiation‐induced skin photo‐aging is associated with collagen degradation, dehydration, and inflammation. While the cellular and molecular mechanisms through which UV induces skin aging are still being unraveled, active compounds, including those natural products, are being sought to attenuate UV‐induced skin cell damage and inflammation. Recent studies have shown that maple extracts having active ingredients may have beneficial effects for the development of cosmetics products. In this study, five samples including black cumin extracts (BCE), thymoquinone (TQ, a major component of black cumin), Maple syrup extracts (MSX), Maple leave extracts (MAP), gennalin A (GA), were investigated whether those abstracts or compounds have effects on cytokine‐induced activation of NFkB pathway, which is critical for UV‐induced skin inflammation and skin aging. First, we used MTT assay to determine the proper concentration in cultured skin keratinocytes. We found that 5 mg/ml is the concentration that does not affect cellular proliferation. Second, we treated both keratinocytes and fibroblasts with TNFa and IL1b (10 ng/ml) and measured the degradation of IkB and NFkB subunit, p65 translocation from cytoplasm to nucleus, using confocal microscope and Western blot analysis. The results showed that both TNFa and IL1b induce IkB degradation and p65 translocation in both types of skin cells, while fibroblasts are more responsive to TNFa and IL1b. Under UV radiation, keratinocytes are activated and release cytokines that further activate skin fibroblasts. Third, we tested the effects of all five extracts or compounds on TNFa and IL1b p65 translocation and IkB degradation by confocal and Western blot. The results showed that p65 translocation was inhibited after administering 10 mg/ml of the BCE, and inhibition improved as the dosage of BCE increased. IkB level was elevated at the following dosages of BCE 25 and 50 mg/ml. In addition, p65 translocation appeared to be inhibited exclusively at 50 mg/ml in fibroblast cells. IkB level was observed to be consistent as the dosage of BCE increased, suggesting that BCE may block IkB degradation in fibroblast cells, with a result of inhibition of p65 translocation. Other samples are still being further investigated. Taken all attained data together, we conclude that some of those tested extracts or compounds may be added to the cosmetics products to attenuate or prevent from UV‐induced skin cell damage and inflammation.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantifying HNF1A Regulated Gene Expression in Pancreatic Cancer
Authors: Alaniz Anthony A. , Abel Ethan V. , Simeone Diane M. 
Published: June 2021

Hepatocyte nuclear factor 1A (HNF1A) is a transcription factor essential to normal pancreas and liver development and homeostasis. HNF1A has been shown to be upregulated in pancreatic cancer stem cells (CSC), a proposed key driver of pancreatic ductal adenocarcinoma (PDAC), relative to other pancreatic cancer cells. A CSC gene signature consisting of 50 upregulated genes, including HNF1A, was previously identified by a microarray. This signature predicts worse survival compared to HNF1A expression alone. This research aims to establish an HNF1A‐dependent gene signature that could potentially be used as a biomarker for pancreatic cancer. Using quantitative reverse transcriptase PCR, multiple CSC signature genes were shown to respond to HNF1A knockdown and overexpression. Responsive genes varied across cell lines, suggesting a complex pathway significantly affected by HNF1A transcriptional control.Support or Funding InformationThe work was supported by the Pancreatic Cancer Action Network‐AACR Pathway to Leadership Grant (16‐70‐25‐ABEL) and the American Cancer Society Postdoctoral Fellowship (127662‐PF‐15‐033‐01‐DDC) (to EVA) and the Gershenson Pancreatic Cancer Fund (DMS) and SKY Foundation (HC and DMS).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.