bluuebunny/crossref_metadata_2025_split huggingface dataset:
Using CRISPR technology to edit the F508 deletion of the Cystic Fibrosis Transmembrane Regulator gene
Authors: Heavenor Kelsey , Roecklein‐Canfield Jennifer 
Published: November 2023

The genetic disease Cystic Fibrosis is the result of a mutation in the Cystic Fibrosis Transmembrane (CFTR) protein. The CFTR protein is an important chloride ion channel in the cell membrane. The mutant protein results in a terminal disease characterized by breathing problems, digestive issues, and recurring lung infections. The ΔF508 mutation, results in the deletion of a phenylalanine at position 508 in the protein and the failure of the protein to fold into its functional conformation. This mis‐folded protein is targeted for degradation by the endoplasmic reticulum quality control system. The mutant CFTR protein is never transported to the apical membrane to carry out its function leading to severe consequences in the lungs and digestive system. Therapies currently available for Cystic Fibrosis are only able to alleviate symptoms to an extent; however, new strides in genetic technology have allowed researchers to now approach treating the disease on the genetic front, an imperative step to understanding and effectively treating Cystic Fibrosis. The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins) System is a new system derived from the prokaryotic immune system that can be used to precisely edit DNA. The CRISPR system can be designed to knockout or insert new genes into the genome, making it an ideal tool for manipulating the CFTR gene to learn more about the gene and the role of mutations in the progression of disease. We attempted to apply CRISPR technology in a gene knockout experiment and homology directed repair (HDR) of the mutant CFTR gene, using two MDCK cell lines, one expressing wt CFTR and the other expressing ΔF508 mutant. Confirmation of cellular expression of the proteins was confirmed by visualization of the Green Fluorescent Protein (GFP) fused to the N‐terminus. We used Cl‐ ion output to estimate how much of the protein is reaching the cell membrane as a measure of protein function. Regain of function in ΔF508 mutant will determine if the gene editing was a success. The data will determine which CRISPR design is most effective at repairing the gene, for example determining what is the ideal length for homologous arms in HDR repair that yields best results. Perfecting the art of CRISPR design in the laboratory is essential to the eventual creation of a functional CRISPR design for use in humans. Many diseases cannot be fully controlled through drugs; the editing of the genome offers an extraordinary pathway for eliminating disease.Support or Funding InformationSimmons SURE Grant
Molecular Genotyping of Transposable Element Insertions in a Population of Uniform Mu Events
Authors: Martinez Gerardo Antonio Arroyo , Springer Nathan 
Published: November 2023

Uniform Mu is a maize strain that was developed as a model for genetic studies. Mutator transposable elements (TE) have been activated within an inbred W22 background and the movement of these transposons results in novel mutations. Researchers can identify Uniform Mu events that contain transposon insertions within a gene of interest. TE insertion events within the organism's genome often result in loss‐of‐function mutant alleles. This study aimed to genotype the presence of a TE insertion in several genes predicted to be involved in the regulation of gene expression or chromatin structure using Uniform Mu crops. Molecular genotyping was performed by using Polymerase Chain Reaction (PCR) and gel electrophoresis. We determined that 43.89% of the population contained a homozygous wild‐type genotype. On the other hand, 7.24% of the individual scored had a homozygous mutant genotype, while 11.08% of the rows assessed presented heterozygous alleles. In future experiments, we expect to analyze these genes further and identify their possible role in causing abnormalities in the chromosomes. As an integral part of people's diet around the world, these results will lead to a better understanding of the agriculture of maize crops.Support or Funding InformationI would like to thank the support of Springer Lab at the University of Minnesota for all the help and guidance this summer. Also, I would like to acknowledge the RISE program in the University of Puerto Rico at Ponce for their funding. NIH‐NIGMS #2R25GM096955
Characterization of a Plasmid Isolated from Enterococcus faecalis Found in the Fecal Material of a Blue Whale
Authors: McLaughlin Richard , Kopanic Robert 
Published: November 2023

Enterococcus faecalis is widely distributed in nature being found in soil, water, plants, and foods. It is also found in the gastrointestinal tract of humans and animals including Blue Whales. Despite its well‐reported existence in animal species, very few plasmids isolated from E. faecalis have been described in the literature. Therefore, the purpose of this study was to more completely describe the genomics of E. faecalis. The complete 52325 bp sequence of the plasmid pGTC2 was determined using Next‐Generation sequencing technology. Harbored by a strain of E. faecalis present in the fecal material of a Blue Whale, this plasmid does not contain any antibiotic or heavy metal resistance genes. Instead multiple mobile element proteins, relaxase and transposon sequences are found throughout. Since these genes are present it may indicate that horizontal gene transfer may occur frequently in pGTC2.Support or Funding InformationThis project was supported by funding provided by Gateway Technical College and by the Gateway Foundation.
Liver and Serum miRNAs in Toxic Liver Fibrosis
Authors: Permenter Matthew Grant , Ippolito Danielle L , Trabosh Valerie A 
Published: November 2023

Toxic chemical exposure is a risk factor for toxic liver injury in the military, chemical plant workers, and the general public. Early indicators of disease would facilitate medical and strategic decisions involving treatment, protective equipment, and/or relocation. Here, we map liver miRNA to their predicted mRNA targets in a rodent model of hepatotoxicity. We report a panel of serum miRNA that may serve as early, blood‐based indicators of toxic liver injury or fibrosis.Rats were orally administered vehicle control or the hepatotoxicant 4, 4′‐methylenedianiline (MDA; 22, 60 or 162 mg/kg) for 1 or 5 consecutive days. Serum and liver tissue were collected at necropsy, 24 hours after the last exposure. Liver sections were stained with hematoxylin and eosin for histology. Liver tissue and serum miRNA expression was determined by next‐generation sequencing for selected doses of MDA. mRNA gene expression in the liver was measured by microarray. Known and novel miRNAs were mapped to the rat genome using miRDeep2 and differential expression was determined by DESeq (FDR<0.05). mRNA changing by at least 1.5 fold were used for the target analysis using Ingenuity Pathway Analysis (IPA) software. Pathway enrichment analysis was also conducted in IPA.MDA poisoning caused moderate liver fibrosis and bile duct hyperplasia after 5 consecutive doses, as shown by histological analysis. One day after the final dosing, we identified 0, 7, and 38 differentially expressed liver miRNA at 22, 60, and 162 mg/kg, respectively. After 5 doses, 0, 126, and 127 liver miRNA were differentially expressed at 22, 60, and 162 mg/kg, respectively. Sixteen miRNAs were modulated in the serum of the rats exposed to 162 mg/kg of MDA. We also identified novel, previously undescribed differentially expressed miRNAs which may play a role in liver injury progression. We predicted mRNA targets of each differentially expressed miRNA. Pathway enrichment analysis of the predicted targets identified pathways involved in the liver fibrosis adverse outcome pathway, including inflammation, collagen accumulation, and stellate cell activation. Many of the modulated mRNAs targeted by these miRNAs have been previously associated with liver fibrosis.In conclusion, we identified liver miRNAs that may target and regulate gene expression contributing to liver fibrosis. The miRNAs in the serum may serve as prognostic indicators of disease progression.Support or Funding InformationDisclaimer: Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.
High Resolution Melting Analysis for the Study of Single Nucleotide Polymorphisms
Authors: Beuschel Grant , Diez Christina 
Published: November 2023

Pharmacogenetics is the study of how inherited genetic variations correspond to an individual's specific reaction to drugs and their metabolism. An ideal pharmacogenetic test would provide information about an individual's genotype which would then inform the the doctor and patient about drug efficacy, provide specific dosage recommendations, and warn of the risk of adverse side effects.The purpose of this project is to determine how to use high resolution melting analysis to characterize the different alleles caused by single nucleotide polymorphisms (SNPs). The SNPs being tested are in the VKORC1 and CYP2D6 genes which code for enzymes involved in the metabolism of warfarin and codeine, respectively. In addition, to refine our DNA extraction and amplification method and to provide support for an existing introductory biology laboratory exercise, we are also working on the SNP for PTC (Phenylthiocarbamide) tasting as well. The PTC SNP is currently being genotyped by students using restriction enzyme digestion. However, results of the restriction enzyme digest have conflicted with direct observations of PTC tasting ability. Therefore, we are interested in developing a reliable genotyping method using high resolution melting analysis. We were able to extract DNA from cheek cells and amplify the SNP alleles. We are presently developing a protocol for high resolution melting analysis of SNPs using the Roche LightCycler 480. The high resolution melting analysis should be sensitive enough to distinguish between the different SNPs. We will verify our SNPs results with DNA sequence analysis. Once we have developed a reliable assay, we will explore the possibility of creating an introductory biology laboratory module to expose students to pharmacogenetics.The overall goal is to optimize a protocol to make such testing more effective and frequent. This way when a patient is in need of medication a simple analysis of their genes can be made to determine the correct type and dosage of drug that will be most beneficial to them. Perfecting a pharmacogenetic testing using high resolution melt curve analysis would extinguish the current and less effective method of trial and error.Support or Funding InformationLawrence Technological University, Department of Natural Sciences
Conditional Loss of Pten in Myogenic Progenitors Leads to Postnatal Skeletal Muscle Hypertrophy But Age‐dependent Exhaustion of Satellite Cells
Authors: Yue Feng , Bi Pengpeng , Wang Chao , Li Jie , Liu Xiaoqi , Kuang Shihuan 
Published: November 2023

Skeletal muscle stem cells (satellite cells, SCs) are normally maintained in a quiescent (G0) state. Muscle injury not only activates SCs locally, but also alerts SCs in distant uninjured muscles via circulating factors. The resulting GAlert SCs are adapted to regenerative cues and regenerate injured muscles more efficiently, but whether they provide any long‐term benefits to SCs is unknown. Here we report that embryonic myogenic progenitors lacking Pten exhibit enhanced proliferation and differentiation, resulting in muscle hypertrophy but fewer SCs in adult muscles. Interestingly, Pten‐null SCs are predominantly in the GAlert state even in the absence of injury. The GAlert SCs are deficient in self‐renewal and subjected to accelerated depletion during regeneration and aging, and fail to repair muscle injury in old mice. Our findings demonstrate a key requirement of Pten in G0‐entry of SCs, and provide functional evidence that prolonged GAlert leads to stem cell depletion and regenerative failure.Support or Funding InformationThis work was supported by a grant from the US National Institutes of Health (R01AR060652 to S.K.) and Purdue incentive grant from Purdue University Office of Vice President for Research (OVPR) to S.K.
Modulation of MEK/ERK and mTOR pathways by Na+‐K+‐ATPase and AMPK inhibitors in ovarian cancer cells
Authors: Clark Matthew P , Madigan Audrey , Gallant Benjamin Paul , Zhang Wei , Lu Hongguang , Wan Yinsheng 
Published: November 2023

In addition to ion transport activities, Na+‐K+‐ATPase acts as a cellular signal transduction molecule. Its inhibitors, such as Ouabain, induce the EGFR/ERK pathway as well as the PI3K/AKT pathway. AMPK is involved in cellular metabolism and inhibiting the mTOR pathway. Activation of AMPK leads to cell growth inhibition and apoptosis in cancer cells. Reports have demonstrated that activation of the AMPK stimulates Na+‐K+‐ATPase. In this study, we investigated the effects of both the Na+‐K+‐ATPase inhibitor Ouabain and the AMPK inhibitor Compound C on ovarian cancer cells. MTT assay results showed that both Ouabain and Compound C alone decrease cell viability. The combination of both yields a synergistic effect. Western blot analysis results indicated that Ouabain induces AKT/mTOR and ERK activation in a time and dose dependent manner. Interestingly, while Compound C inhibits Ouabain‐induced mTOR activation as measured by S6 phosphorylation in a dose dependent manner, Compound C enhances Ouabain‐induced ERK activation. Compound C alone induces ERK activation. Further studies showed that the MEK/ERK inhibitor U0126, but not the mTOR inhibitor Rapamycin, inhibits Ouabain‐induced S6 phosphorylation. ER redox assay data showed that Compound C treatment alters the redox level. Mito‐Tracker test data showed that both Ouabain and compound C increase mitochondria activities. Taken together, our data indicates that both Na+‐K+‐ATPase and AMPK are important for MEK/ERK and mTOR pathways in ovarian cancer cells. Modulation by inhibitors of both may reveal molecular targets for cancer treatments.Support or Funding InformationRI‐INBRE
Activation of the E3 ligase Cbl by Neutrophil Cathepsin G Impairs CXC chemokine receptor 4 Signaling in Cardiomyocyte Degeneration.
Authors: Shukla Sanket , Sikder Kunal , Sarkar Amrita , Liu Weijing , Rafiq Khadija 
Published: November 2023

ObjectiveInflammatory cells and their proteases contribute to tissue repair at the site of inflammation. Although beneficial in the early stage, excessive inflammation leads to cell death and tissue damage. Increasing evidence demonstrates the role of inflammatory cells and their proteases in cardiac remodeling during the progression to heart failure; however the mechanisms by which inflammation contribute to myocyte death and subsequent alteration in the geometry and mechanical properties of the heart are still largely unknown. Therefore, we hypothesized that neutrophil‐derived protease Cat.G regulates CXC chemokine receptor (CXCR) 4 protein stability and turnover in myocytes through activation of Casitas b‐lineage lymphoma (c‐Cbl), an adaptor protein with an intrinsic E3 ubiquitin ligase activity.Methods and ResultsWe have previously shown that neutrophil serine protease Cat.G promotes cardiomyocyte death through c‐Cbl activation and interaction with tyrosine receptors that result in their ubiquitination and degradation. We next assessed whether Cbl activation was involved in CXCR4 signaling downregulation and myocyte apoptosis secondary to exposure of Cat.G. Acute stimulation with Cat.G (5 min to 30 min) induced CXCR4 phosphorylation along with its interaction with Cbl. However, chronic stimulation with Cat.G (1–4hrs) increased both CXCR4 ubiquitination and degradation. Inhibition of the ubiquitin proteasome system, with MG132 or lactacystin, or adenoviral expression of dominant negative mutant Cbl significantly reduced CXCR4 degradation induced by Cat.G. In contrast, adenoviral expression of wild type Cbl enhanced CXCR4 ubiquitination and degradation in response to Cat.G. Restoration of CXCR4 signaling, by adenoviral expression of CXCR4, attenuated CXCR4 downstream signaling downregulation, myofibril degeneration and myocyte apoptosis induced by Cat.G.ConclusionThese data support a model in which neutrophil derived serine proteases promote c‐Cbl interaction with CXCR4 protein, resulting in enhanced c‐Cbl‐mediated CXCR4 protein degradation, myofibril degradation and subsequent down‐regulation of myocyte survival signaling.
CARMA2sh and Its Psoriasis‐Linked Variants Regulate Inflammatory Pathways in Human Keratinocytes
Authors: Scudiero Ivan , Mazzone Pellegrino , Telesio Gianluca , Pizzulo Maddalena , Vito Pasquale 
Published: November 2023

OBJECTIVEThe molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain‐containing adapter molecule BCL10, and MALT1 represent a central hub in regulating activation of the pleiotropic transcription factors NF‐κB. Recently, dominant germline mutations in CARMA2sh have been shown to cause with high penetrancy psoriasis and pityriasis rubra pilaris, two related skin inflammatory disorders. Aim of our work was to dissect the molecular mechanisms by which CARMA2sh and its variants associated with psoriasis control NF‐κB activity in the skin.METHODSMolecular partners of CARMA2sh were searched by a yeast two‐hybrid screening and tested by co‐immunoprecipitation in HEK293T cells. Immunoblot analysis, in vitro kinase and phosphatase assays were used to monitor the phosphorylation status of CARMA2sh. Deletion mutants of CARMA2sh served to map the phosporylation region and its functional significance. Murine strains expressing psoriasis‐linked mutants of CARMA2sh in their keratinocytes were generated via homologous recombination in the Rosa26 locus. NF‐κB activation signaling and the expression of inflammatory genes were investigated by biochemical and genetic assays in human and murine keratinocytes. A BCL10 inhibitory peptide was delivered by liposome in NHEK cells exposed to heat‐killed bacteria or fungi and NF‐κB target genes expression levels were assessed by Real Time RT‐PCR.RESULTS1) We identified a serine/threonine kinase, termed CIK (CARMA2sh Inhibitory Kinase), which binds to and phosphorylates CARMA2sh. Phosphorylation of CARMA2sh is required to promote lysosomal degradation of BCL10 and to turn off CARMA2sh‐mediated NF‐κB signaling. 2) We demonstrate that CARMA2sh mutants associated with psoriasis escape CIK inhibition. 3) We show that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway connecting Pathogen Associated Molecular Patterns recognition to NF‐κB activation. 4) A peptide blocking CARD‐mediated BCL10 interactions reduces the capacity of psoriasis‐linked CARMA2sh mutants to activate NF‐κB.CONCLUSIONSOur work elucidates a basic signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.Support or Funding InformationQatar National Research Fund NPRP 7‐466‐3‐119
Chronic Low‐grade Inflammation in Obesity and Diabetes associated Colorectal Cancer Development.
Authors: Kumar Narendra , Mishra Jayshree , Kumar Premranjan 
Published: November 2023

Epidemiological studied identified that colorectal cancer (CRC) is a worldwide health problem, being the third most commonly identified cancer in males and the second in females. Increasing CRC incidence trends are associated with life‐style and calorically excessive high‐fat diet and obesity. Recent, studied have evidenced an important correlation between obesity and diabetes‐associated inflammations in colorectal cancer development. The regulatory pathway by which metabolic disorders induce inflammation in obesity leading to development and progression of CRC is remain largely unknown. Here, we demonstrate the intestinal specific chronic low grade inflammation is associated with obesity and diabetes, which impact CRC development. Our data shows that Jak3 facilitates prevention of CLGI through multiple mechanism that involves down‐regulation of the receptors of innate immunity, the toll like receptors (TLR), promotion of regulatory T‐cell expansion and suppression of Th17 effector functions in both in human and mouse model of obesity. Jak3 expression also suppresses TLR‐mediated activation of NF‐kB through activating PI‐3K‐AKT axis.Support or Funding InformationHealth‐SBIR (GM109528)
Melatonin modifies peripheral blood cell oscillators in humans
Authors: Kostovski Emil , Frigato Elena , Dahm Anders , Skretting Grethe , Mowinkel Marie‐Christine , Sandset Per Morten , Iversen Per Ole , Bertolucci Cristiano 
Published: November 2023

ObjectiveThe blood concentrations of melatonin are highly associated with daylight patterns, with peak concentrations late at night. In healthy adults, the average daytime and peak night‐time values range from 10 to 200 pg/ml, however in tetraplegic persons the melatonin levels is blunted, ranging from 2 to15 pg/ml. Mounting evidence suggests that biological clocks, exist outside the central circadian pacemaker. We here explored how melatonin may influence oscillators in peripheral blood mononuclear cells (PBMCs) in tetraplegic and control persons.Study design and settingA double‐blind, randomized, placebo‐controlled cross‐over study, was conducted at Sunnaas Hospital, Nesoddtangen, Norway.MethodsSix subjects with long‐standing complete tetraplegia received 2 mg of melatonin or placebo 4 days before sampling. We also included six able‐bodied men sleeping or being kept awake during the night. Plasma samples were collected from 4 to 10 times during 24‐hours. Expression of the period circadian clock‐1 (PER1) and −2 (PER2) genes, aryl hydrocarbon receptor nuclear translocator‐like protein 1 gene (BMAL1) and the nuclear receptor subfamily 1, group D, member 1 (REV‐ERB) gene was quantified in PBMCs using quantitative RT‐PCR (qRT‐PCR). The data were analysed using linear mixed models.ResultsMelatonin substitution increased night time melatonin plasma levels from 10 to 500 times in tetraplegic persons. The 24‐h profiles of all clock genes tended to differ (P < 0.09) or differed (P < 0.05) between tetraplegic patients receiving melatonin and tetraplegic patients receiving placebo, shifting and normalizing the mean maximum from a delayed peak (0400 h) in the tetraplegic patients receiving placebo towards the time of melatonin substitution (2200 h) in the tetraplegic persons receiving melatonin. There were no differences in the 24 h profile‐ or level‐expressions in able‐bodied staying awake compared to those sleeping or in between able‐bodied (all groups) compared to tetraplegic persons (all groups).ConclusionsWe here show differences in circadian variation of several clock genes related to melatonin concentration fluctuations in tetraplegic persons, suggesting that melatonin play a role in regulation of peripheral oscillators in humans.Support or Funding InformationSponsorship: Financial support was provided from the Throne Holst Foundation.