bluuebunny/crossref_metadata_2025_split huggingface dataset:
During Ca2+‐dependent gastric epithelial repair, Ca2+ is sourced from both Ca2+ uptake and intracellular Ca2+ release
Authors: Engevik Kristen A , Hanyu Hikaru , Matthis Andrea L , Aihara Eitaro , Montrose Marshall H 
Published: June 2021

BackgroundCalcium (Ca2+) is a known accelerator for gastric repair. However the mechanism by which Ca2+ mobilizes to promote repair remains unclear, and cannot be readily evaluated in vivo. Using gastric organoids derived from transgenic mice expressing a fluorescent Ca2+ reporter (yellow cameleon‐nano15; YC‐Nano), we previously observed intracellular Ca2+ increases in cells directly adjacent to a damaged cell. Using this Ca2+ sensor, we investigate the potential sources of intracellular Ca2+ essential for repair.MethodsGastroids generated from YC‐Nano mouse stomach corpus were cultured 4–5 days prior to experiments. Photodamage and resultant cell death was induced to 1–2 gastroid epithelial cells by ~3 sec high intensity 840 nm light. YC‐Nano reports Förster resonance energy transfer (FRET) from CFP to YFP in response to increased intracellular Ca2+. Change in intracellular Ca2+ was measured as FRET/CFP ratio, in cells adjacent to damaged cells. Inhibitors were used to test roles of Ca2+ channels (10 μM verapamil, 20 μM YM58483), intracellular Ca2+ (50 μM BAPTA/AM), Phospholipase C (10 μM U73122), and IP3R (50 μM 2‐APB).ResultsUnperturbed gastroid cells maintain a constant FRET ratio, indicating stable Ca2+ levels. In response to photodamage of the gastroid epithelium, increased levels of Ca2+ were observed specifically within the lateral membranes of cells neighboring the damaged cell. Chelation of intracellular Ca2+ by BAPTA/AM resulted in significant dampening of Ca2+ response, as well as blocking prompt repair. Inhibition of L‐type channels (verapamil) or store operated Ca2+ entry (YM58483) resulted in delaying repair and dampening intracellular Ca2+ response. Furthermore, inhibition of PLC (U73122) or IP3R (2‐APB) resulted in delayed repair and dampened Ca2+ response.ConclusionThese results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ that stimulates repair. The findings implicate an intracellular Ca2+ raise mediated via Ca2+ uptake via plasma membrane Ca2+ channels and intracellular Ca2+ release from the ER. Collectively this work indicates the usefulness of YC‐Nano to further assess intracellular Ca2+ dynamics and further investigate the signaling cascade behind Ca2+‐mediated repair.Support or Funding InformationThis work was supported by the National Institutes of Health (NIH) R01DK102551 (Montrose; Aihara) and F31DK115126 (Engevik). This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of CD44 Variant Isoforms is an Early Epithelial Response to Helicobacter pylori Infection
Authors: Chakrabarti Jayati , Steele Nina , Holokai Loryn , Broda Taylor , Biesiada Jacek , Pitstick Amy , Mayhew Christopher , Medvedovic Mario , Wells James , Zavros Yana 
Published: June 2021

BackgroundHelicobacter pylori (H. pylori) is the major risk factor for the development of gastric cancer. The progression of H. pylori induced chronic inflammation to gastric cancer may result from the aberrant expression of the cancer stem cell marker, CD44 variant isoform 9 (CD44v9). H. pylori induces reactive oxygen species (ROS) that subsequently leads to cell apoptosis via caspase activation. It is reported that in cancer stem cells, CD44v9 stabilizes the cystine‐glutamate transporter xCT, thus promotes resistance to ROS and subsequently leads to tumor growth. However, it is unknown whether CD44v9 protects the gastric epithelium against H. pylori‐induced apoptosis during early infection. In addition, CD44 variant isoform 6 (CD44v6), is not only a biomarker for diffuse‐type gastric cancer, but also promotes H. pylori‐induced epithelial cell proliferation.HypothesesH. pylori‐induced CD44v9 is an early epithelial response to infection that subsequently promotes resistance to ROS‐induced epithelial cell apoptosis.MethodsAntral and fundic gastric organoids were generated from induced pluripotent stem cell (IPSC) lines derived from the whole blood of normal patients (HGOs). HGOs were microinjected with the H. pylori G27 or a cagA mutant strain. After 72 hr of infection organoids were immunostained for CD44v9, CD44v6 and H. pylori. The expression of IL‐8 and caspase 3 were measured by qRT‐PCR. In a separate series of experiments, infected organoids were treated with either vehicle or 300 mM sulfasalazine (SSZ) for 72 hrs and changes in phosphorylation (activation) of p38MAPK, a downstream target of ROS was measured by western blot. RNA was isolated from uninfected and infected antral and fundic HGOs and RNA‐sequencing was performed to analyze differential expression of genes among those samples.ResultsH. pylori induced the expression of CD44v9 and CD44v6 in HGOs and this correlated with the suppression of caspase 3 and increased IL‐8 gene expression. Despite the Increased expression of ROS observed in H. pylori Infected HGOs, there was increased epithelial cell proliferation within the infected organoids. Proliferation significantly decreased in H. pylori infected HGOs treated with xCT inhibitor SSZ. H. pylori infected fundic HGOs showed 94% dissimilarity in the differentially expressed genes compared to infected antral HGOs, which fall mostly in cancer and apoptotic pathways.ConclusionInduction of CD44v9 and v6 are early epithelial changes in response to H. pylori infection, and may play a role in the protection against H. pylori‐induced apoptosisSupport or Funding InformationThis work was supported by NIH 1U19AI116491‐01 (Weis and Wells, YZ Project Leader),This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neonatal necrotizing enterocolitis alters the development of brainstem neurocircuitry controlling gastrointestinal functions
Authors: Meister Alissa L , Doheny Kim K , Browning Kirsteen N , Travagli R. Alberto 
Published: June 2021

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal (GI)‐related death in premature infants and is most often characterized by a slow progressive onset of symptoms including feeding intolerance and abdominal distention prior to the development of sepsis and necrosis with potentially life‐long effects on both GI health and neurodevelopmental outcomes. The high‐frequency power spectra of heart rate variability (HF‐HRV) is a non‐invasive measure of vagal tone, our laboratory has previously shown that HF‐HRV is decreased in NEC in both human neonates (Doheny et al., Neurogastroenterol. Motil., 2014) and in rodent pups (Meister et al., Neurogastroenterol. Motil., 2018). The present study examined the hypothesis that NEC induces long term alterations in central and peripheral control of the intestine including i) attenuated developmental maturation of brainstem neurocircuits and ii) decreased transit.Mild NEC (stage≤2) was induced in newborn Sprague‐Dawley rats through twice daily exposure to cold stress and hypoxia and HRV was measured using bilateral electrodes to collect an electrocardiogram before and after stress exposure on postnatal days (PD) 1, 5, 10 and 28. Whole cell patch clamp recordings examined whether, in young adult rats (4–8 weeks of age), dorsal motor nucleus of the vagus (DMV) neurons innervating the intestine received inhibitory synaptic currents that were entirely GABAergic (indicating normal development, i.e., unaffected by the glycine antagonist, strychnine; 1mM) or were mixed GABA‐glycine (indicating arrested developmental maturation; inhibitory currents prolonged by strychnine). Intestinal transit was measured using the non‐invasive lactose ureide breath test at the same time‐points.The induction of mild NEC was confirmed by histological grading, these pups showed a significant reduction in HF‐HRV at all time points, except for baseline before stress induction, as compared to controls. Mild NEC rats showed no change in gastric emptying as compared to controls, but a significant increase in gastro‐cecal transit time. In control rats, 0/11 adult DMV neurons showed evidence of inhibitory glycinergic inputs whereas 9/12 DMV neurons from rats in which mild NEC was induced displayed mixed GABA‐glycine currents. Data are summarized in the table below.Our data suggest that mild NEC is associated with adult comorbidities including arrested development of brainstem neurocircuits innervating the intestine as well as delayed intestinal transit. Neonatal insults, such as NEC, may have long term developmental consequences that impair intestinal functions at both central and peripheral sites.Support or Funding InformationNIH grant DK99350 to R.A.T. & K.K.D.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Low‐Dose Dihydrotestosterone Lowers Lipogenic Master Regulator in Liver and Adipose Tissue from Female Mice
Authors: Andrisse Stanley , McWhorter Patrick , Seidu Tina , Alamgir Rabita 
Published: June 2021

Hyperandrogenemia (HA) and insulin resistance (IR) are hallmarks of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects 1 in 10 women. These hallmarks are also integral elements of non‐alcoholic liver disease (NALFD), a disorder that is common in women with PCOS. In lean female mouse models of dihydrotestosterone (DHT)‐induced PCOS, a low dose of (DHT) promotes IR and hepatic lipogenesis via the androgen receptor (AR), thus resulting in NAFLD. However, the molecular mechanism of HA‐induced NAFLD has not been determined. We hypothesized that a low dose of DHT would interrupt hepatic lipid metabolism leading to NAFLD. In this project, we aimed to investigate how androgen and androgen receptor (AR) impact a key player of lipogenesis, SREBP1. We extracted liver tissue, white adipose tissue (WAT), and skeletal muscle from lean, female wild type mice and mice. Next, we analyzed the effect of low dose dihydrotestosterone (DHT) on lipogenic protein and gene expression. We performed Western blot and real‐time quantitative polymerase chain reaction (qRT‐PCR) analysis of lipogenic intermediates on homogenates of energy storage tissues (liver, WAT, and skeletal muscle). Low‐dose DHT lowered the protein expression of the active form of cytosolic SREBP1 in the liver and WAT. Additionally, low‐dose DHT lowered the protein expression of the inactive SREBP1 in the liver. DHT did not alter the levels of the active and inactive forms of SREBP2 in the liver and WAT. Interestingly, the active form of SREBP2 was lowered in skeletal muscle. Further, phospho acetyl co‐A carboxylase (p‐ACC) protein levels were unaltered in liver and WAT. Fatty acid synthase (FAS) levels were unchanged in WAT and skeletal muscle. Taken together, our findings suggest that hepatic and white adipose cytosolic SREBP1 is being decreased due to its translocation to the nucleus, where it regulates lipogenic protein levels.Support or Funding InformationThank you to Howard University College of Medicine, Johns Hopkins Medicine, and Georgetown University Medical Center (GUMC) for supporting this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Integration of Transcriptomics And Proteomics Reveals That miR‐33‐5p Is A Key Player of Lipogenesis Signaling in Fructose‐Induced NAFLD in C57BL/6N Mice
Authors: Redding Mersady , Beane Kaleigh , Tang Jingsi , Lee Jin Hyup , Kong Byungwhi C , Kim Jae Kyeom , Pan Jeong Hoon 
Published: June 2021

Fructose is a strong risk factor of non‐alcoholic fatty liver disease (NAFLD), but limited studies have investigated mechanisms by which microRNAs (miRNA) are interlinked in the etiology of NAFLD. Here, we aimed to investigate the significance of miRNAs in fructose‐induced NAFLD conditions through integration of liver transcriptomics and proteomics. C57BL/6N mice (male and female) were fed with either water (n=14) or 34% fructose (n=14) for 6 wk ad libitum. After, liver transcriptomics and proteomics datasets were integrated to narrow down key signaling pathways and to find upstream regulator(s) that are responsible for fructose induced NAFLD in which srebp1 was found to be the most significant upstream regulator. In an attempt to explore regulatory roles of miRNAs in NAFLD, Ingenuity Pathway Analysis software miRNA database was used to list miRNAs that are known to interact with the upstream regulator, srebp1. Out of seven final miRNA candidates, miR‐33a was selected after our qPCR validation in mice liver and AML12 mouse hepatocytes that were treated with fructose. In order to further validate the interaction between miR‐33a and srebp1, AML12 hepatocytes were transfected with the miR‐33a; in this, srebp1, and its downstream proteins, acc1 and scd1 were all decreased by miR‐33a transfection whereas suppression of miR‐33a, achieved by anti‐miR‐33a transfection, restored those protein expression changes. Altogether, based on our unbiased approaches as well as in vitro validation experiments, fructose induces NALFD conditions via suppressing miR‐33a in liver thereby increasing srebp1 and its downstream lipogenic proteins, acc1 and scd1.Support or Funding InformationThis work was supported by the University of Arkansas, VPRED Start‐up fund (JKK, and JHP). Support has been provided in part by the Arkansas Biosciences Institute, a partnership of scientists from Arkansas Children's Hospital, Arkansas State University, the University of Arkansas‐Division of Agriculture, the University of Arkansas, Fayetteville, and the University of Arkansas for Medical Sciences. The Arkansas Biosciences Institute is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000 (JKK, JHP, KEB, and BCK). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF‐2015R1C1A1A01053746; JHL) as well as the China Scholarship Council (JT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prevalence of Thiamine (B1) deficiency in a cohort diagnosed with Postural Orthostatic Tachycardia (POTS)
Authors: Del Pozzi Andrew Thomas , Blitshteyn Svetlana 
Published: June 2021

Thiamine (vitamin B1), a water‐soluble vitamin that plays a pivotal role in energy metabolism, growth, development and the function of cells. Has been found to be deficient in conditions such as malnutrition, eating disorders, and bariatric surgery complications as well as alcoholism. Previously vitamin B12, vitamin D and Iron deficiencies have been noted in patients with POTS. Yet the prevalence of thiamine deficiency and response to thiamine supplementation in POTS has yet to be determined.PURPOSEto determine the prevalence of Thiamine deficiency in those patients diagnosed with POTS. As well as to indicate if supplementation of thiamine affects outcomes.METHODSSixty‐five consecutive patients (59 women and 6 men) ranging in age from 13 to 54 years old, who met the diagnostic criteria for POTS were evaluated and followed by our clinic. As part of clinic initial diagnostic workup all clinic patients have vitamin panels drawn (Quest Diagnostics Laboratory), thus we reviewed patient medical records noting whole blood vitamin B1. Those patients that were found to be deficient in B1 were given thiamine hydrochloride at 100 mg daily for 4 weeks and then followed up with office exams and self‐report of changes in symptoms.RESULTSSix percent (n = 4) of the patients tested positive for thiamine deficiency. Those that showed deficiency were only mildly deficient (72–77 nm/L vs. Normal 78–185 nm/L). Other vitamin and mineral levels including B6, B9, B12, vitamin E, iron, copper, and magnesium as well as homocysteine level were normal in all deficient B1 patients. All patients were supplemented vitamin B1, however, only 25% (n = 1) responded favorably to the supplementation. Although 100% of those supplemented had vitamin B1 levels returned to the normal range.CONCLUSIONA small subset of POTS patients may have vitamin B1 deficiency. Testing for and correcting the deficiency could prove helpful to those patients. Further research should be done on a larger scale and should include a healthy control group as well as objective measures of improvement such as tilt‐table‐testing as part of the follow‐up.Support or Funding InformationnoneThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Apple pomace influences liver‐adipose tissue inflammatory crosstalk in young female rats consuming a Western diet
Authors: Skinner Roy Christopher , Warren Derek C , Agarwal Garima , Naveed Minahal , Benedito Vagner A , Chantler Paul D , Tou Janet C 
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in Western countries and it has been attributed to poor diet. NAFLD is defined by the presence of hepatic steatosis, which can progress to the more severe non‐alcoholic steatohepatitis (NASH) characterized by inflammation. Apple pomace, a waste byproduct of apple processing, is a rich source of anti‐inflammatory/antioxidant polyphenols. The objective of this study was to determine the effects of caloric replacement with apple pomace on hepatic‐adipose tissue inflammatory crosstalk and oxidative stress status. Growing (age 22–29 days) female Sprague‐Dawley rats were randomly assigned (n=8 rats/group) to consume a purified standard rodent diet (AIN‐93G), AIN‐93G/10% g/kg apple pomace (AIN/AP), Western diet, or Western/10% g/kg apple pomace (Western/AP) diets for 8 weeks. Based on histological evaluation, rats consuming Western diet had the highest hepatic fat infiltration and inflammation. Gene expression of nuclear transcription factor kappa B (NFκB), a key transcription factor of inflammation and reactive oxygen species, was upregulated in liver (p=0.009) and the adipose tissue (p=0.0003) of rats fed Western diet compared to the other diets in the experiment. Gene expression of proinflammatory interleukin‐6 (IL‐6) in both tissues as well as the tumor‐necrosis factor‐α (TNF‐α) in the adipose was upregulated in rats fed Western diet, but not Western/AP compared to rats fed standard diets. Replacing calories in Western and AIN diets with apple pomace upregulated (p<0.01) gene expression of the endogenous antioxidant glutathione (GSH). Additionally, serum total antioxidants were highest (p<0.04) and urine total antioxidants were lowest (p<0.04) in rats fed Western/AP. Collectively the results showed caloric replacement with 10% apple pomace in the Western attenuated risk factors associated with progress to NASH in a rat model suggesting the potential of apple pomace as a sustainable functional food.Support or Funding InformationThis research was funded by Hatch WVA 1017641 and the Davis College Dean's discretionary fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cryo‐EM Structure of the P Element Transposase Strand Transfer Complex
Authors: Kellogg Elizabeth H. , Ghanim George , Nogales Eva , Rio Donald C. 
Published: June 2021

The P element transposon encodes a cut‐and‐paste DNA transposase of the DDE class that has been broadly useful as a tool in Drosophila genetics research. Its mechanism and regulation have been extensively studied in relation to the phenomena of hybrid dysgenesis observed in Drosophila populations, as well as with biochemical in vitro studies. Among the many distinct features of the P element transposase (TNP) are the generation of unusually long (17nt) staggered cuts upon excision from its host genome and the unique requirement for the nucleotide cofactor guanosine triphosphate (GTP) to carry out transposition.Using single‐particle cryo‐EM, we have obtained an atomic model of the P element transposase strand transfer complex (STC), consisting of a dimeric complex of the P element transposase along with two P element ends covalently linked to the target DNA. Severe preferential orientation was circumvented by collecting data on a tilted (40°) stage. The resulting cryo‐EM reconstruction refines to 3.6 Å resolution, and the details of the map are sufficient to interpret atomic details of the nucleic acid substrates and to localize GTP.A helix‐turn‐helix domain of each monomer in the symmetric complex binds one P element end while catalyzing strand transfer on the other end, ensuring fidelity of complex formation. The RNaseH catalytic domain shares common features with other DDE transposases (so named for the catalytic triad) and the catalytic residues are identifiable. The RNaseH domain is interrupted by a non‐canonical GTP‐binding insertion domain which exhibits structural similarities to the insertion domain of other transposases, and in our structure makes contacts with target DNA. Surprisingly, we observe that single‐stranded DNA (the 17nt single‐stranded portion of the terminal inverted repeats) adopts an unusual path in our structure which appears to be stabilized by numerous protein‐DNA contacts but ultimately returns to form distorted A‐form DNA duplex leading into the active site. GTP is located close to the active site and interacts with the terminal guanosine residue of the P element DNA. We postulate that this serves to position the transposon donor in the active site for attack on the target DNA. Ultimately, the structure of the P element strand transfer complex reveals the molecular basis underlying two of the unusual properties of the P element transposition mechanism.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome‐wide Analysis of Enhancers in Development and Disease
Authors: Ren Bing 
Published: June 2021

Transcriptional Enhancers play a crucial role in spatiotemporal expression of genes. Dysregulation of enhancers, due to mutations in transcription factors and chromatin regulators, has emerged as a major cause of human cancer. A thorough understanding of cell type specificity, function and mechanisms of action of enhancers will be valuable for development of new diagnostics and therapeutics targeting cancers. I will present our latest advances in identification and characterization of enhancers in the human and mouse genomes, across multiple stages of development, in normal and disease states, and often in single cell resolution. In addition, I will present progress in mapping distal enhancers to their target genes, via analysis of the 3‐dimensional genome architecture and chromatin loops, in order to access the role of non‐coding variants in human diseases. Finally, I will present results from large‐scale functional screenings of oncogenic enhancers in cancer cells.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.