bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hypoxia Decreases Wound Stability via Degradation of the Extracellular Matrix
Authors: Caesar Christa , Adesina Sherry , Naylor Petrina Barnett , Woodard‐Grice Alencia 
Published: November 2023

The extracellular matrix (ECM) plays a pivotal role in the healing and repair of wounds. While several studies have established that exposure to hypoxia significantly impairs wound healing, few studies have explored the effects of hypoxia on the extracellular matrix (ECM) and its impact on wound stability. This study aims to determine the underlying mechanism(s) by which hypoxia affects wound stability over a 72 hour time‐course. We utilized a 3D human EpiDermTM wound model (MatTek©) and hypothesize that chronic hypoxia significantly decreases wound stability by promoting degeneration of the ECM.To assess oxygen‐dependent wound stability we exposed 3D EpidermTM wounded samples to Normoxia (21% O2) or Hypoxia (1% O2) for 0, 48, or 72 hours. We then assessed wound closure and stability (confocal microscopy and H&E staining) and measured expression profiles of ECM and adhesion molecules (qRT‐PCR).Hypoxia significantly altered the stability of the wound as observed via immunohistochemistry. Specifically, the leading edge of the wound was observed to recede with 72 hours of hypoxia which resulted in delayed wound closure (~80% decrease in wound re‐epithelialization, n≥3 independent experiments). The re‐epithelializing layer of cells was also observed to peel off the basement membrane in the samples exposed to 72 hours of hypoxia. Furthermore, relative to the untreated control samples, hypoxia‐exposed samples had altered expression of multiple ECM proteins including several collagens (~40% decrease in Col 14A1, ~60% Col 1A, 2–3 independent experiments) and matrix metalloproteinases (MMPs) (~50% decrease in MMP 14, 2–3 independent experiments). Ultimately, these results confirm that chronic exposure to hypoxia impairs wound healing via degradation of the ECM.
Calcium‐handling of Endothelin A and B Receptors in Coronary Artery upon Post Ischemic Reperfusion Injury in Rats
Authors: Kristiansen Sarah Brøgger , Haanes Kristian Agmund , Sheykhzade Majid , Edvinsson Lars 
Published: November 2023

BackgroundIschemia‐reperfusion has been demonstrated to increase coronary artery resistance and increase the sensitivity toward the potent vasoconstrictor peptide endothelin‐1 (ET‐1). The increased sensitivity to ET‐1 is associated with an up‐regulation of vasoconstrictive ETB receptors in the vascular smooth muscle cells. This project aims to investigate any causal link between the ETB receptor‐upregulation and a shift in calcium sensitivity/handling in the vascular smooth muscle cells after experimental myocardial ischemia‐reperfusion injury.MethodsMale SD rats (~320 gram) were either subjected to experimental left anterior descending (LAD) coronary artery occlusion for 30 min followed by 24 hours of reperfusion, or sham operation. Contractile properties of artery segments distal to the ligature were measured with wire‐myography or along with simultaneous measures of the intracellular Ca2+ concentration ([Ca2+]i) using Fura‐2. The Ca2+ sensitivity of the arteries was determined during stimulation of the ETB‐R agonist Sarafotoxin 6c (S6c) followed by cumulative addition of extracellular Ca2+ (up to 2.5 mM). The experiments were subsequent repeated with ET‐1 as stimulating agent to access dual ETB‐R and ETA‐R mediated Ca2+ sensitization. Thapsigargin, 2‐APB and Felodipine were in addition used in order to identify Ca2+ handling mechanisms and their relative contribution for the ETB‐R and ETA‐R mediated contraction following ischemia‐reperfusion injury.ResultsPost ischemic reperfusion injury significantly increased vascular tone of the LAD coronary artery in response to [Ca2+]o. Prior incubation with either 125 mM of K+ or the ETB‐R antagonist BQ788 showed, that this difference in contractility was neither due to changes in membrane depolarization or a constitutive activity of the ETB‐R after ischemia and reperfusion injury. The evoked agonist‐responses of the Endothelin receptor subtypes, confirmed the enhanced ETB‐R mediated tone after ischemia‐reperfusion injury, whereas dual ETA‐R and ETB‐R mediated tone seems unaffected between surgery groups. The enhanced ETB‐R contractility develops an equal [Ca2+]i –tone relationship mainly from extracellular Ca2+ influx, through activation of voltage‐gated Ca2+ channels.ConclusionThe ETA‐R mediated contraction was unaffected between surgery groups and show equal dependence on intracellular store‐release and extracellular Ca2+ influx. The ET‐1 evoked [Ca2+]i –tone balance was leftward‐shifted opposed to the ETB‐R and suggest the ETA‐Rs to additionally stimulate downstream Ca2+ sensitivity mechanisms.Support or Funding InformationFaculty of Health and Medical Sciences, University of Copenhagen, Denmark. Lundbeck Foundation, Grant of Excellence, Denmark. R59‐AR‐5404.
Lysine Acetylation of Nuclear Pif1 Regulates Its Helicase Function
Authors: Ononye Onyekachi E , Sausen Christopher W , Bochman Matthew W , Balakrishnan Lata 
Published: November 2023

Petite integration frequency 1 (Pif1) is a 5′‐3′ helicase which plays a significant role in maintaining the fidelity of nuclear and mitochondrial DNA. Pif1 preferentially unwinds RNA‐DNA hybrids during key metabolic processes such as DNA replication, repair and telomere maintenance. Regulation of Pif1 activity by phosphorylation modulates the role of this helicase in telomere maintenance. Our current work is focused on understanding how lysine acetylation modifies the protein's activity. Lysine acetylation of proteins is a dynamic modification that is regulated by the activity of acetyltransferases and deacetylases. Using biochemical assays we characterized the enzymatic activity of acetylated Pif1, which was modified in vitro using the Gcn5/Ada2/Ada3 and Rtt109/Vps75 acetyltransferase complexes. Helicase assays revealed an enhanced unwinding function of Pif1 upon protein acetylation. Additionally, the ATPase activity of acetylated Pif1 was also significantly stimulated compared to the unmodified form. In concert, the binding function of acetylated Pif1 to its cognate substrates was also improved when assessed using gel electro‐mobility shift assay (EMSA) in tandem with Bio‐layer interferometry technology (BLI). Mass spectrometric analysis of the in vitro acetylated Pif1 protein revealed some acetylated lysine residues on the N‐terminus of the protein. Current work is focused on understanding how acetylation based Pif1 regulation impacts its role in the maintenance of genome fidelity.Support or Funding InformationIUPUI New Faculty Start Up GrantNIH R00 GM98328
Quantitative Real Time PCR Detection of African Swine Fever Virus DNA from Formalin‐Fixed, Paraffin‐Embedded Tissues
Authors: Trujillo Jessie Dorothy , Urbaniak Kinga , Meekins David , Davis Anne Sally , Richt Juergen A. 
Published: November 2023

African Swine Fever (ASF) is a highly contagious and fatal hemorrhagic fever in domestic pigs. ASFV is a large dsDNA virus previously grouped in the Iridoviridae family but reclassified into the Asfarviridae family in which it is the only member of the genus Asfarivirus. Moreover, ASFV is the only arthropod‐borne animal DNA virus. Natural reservoirs in wild and feral pigs exist and this virus is capable of surviving a long time in meat/tissue even following heat, putrefaction, smoking, salt curing, partial cooking, drying and cooling. These are all factors that make control and eradication of this pathogen very challenging.ASFV was first recognized in East Africa in the early 1900's and is now endemic in sub‐Saharan Africa Sardinia (Italy). ASFV spread to Portugal in 1957 and again in 1960, becoming established in the Iberian Peninsula. Subsequently, outbreaks occurred in several European countries, Cuba, the Dominican Republic, Haiti, and Brazil (1970–1980), where it was eventually eradicated but not before causing devastating impacts on the local economy and swine industry. In 2007, ASFV was reported in Georgia, then spread to Russian Federation and Eastern Europe. There is serious concern that ASFv will spread into North America. In the US, ASFV is a BSL‐3Ag Select Agent that requires high containment laboratories for diagnostic and research work. There is no effective vaccine for ASFV. Thus, the current global mitigation strategy against ASF is stringent biosurveillance with molecular and diagnostics playing a key role in prevention and control of virus spread via animals or animal products. Formalin‐fixed, paraffin‐embedded tissues (FFPET) are inactivated, i.e. non‐infectious, specimens that can be safely transported and used for molecular diagnostics and epidemiological studies. The objective of this work is to develop and evaluate a protocol for use of FFPET for the sensitive and specific detection of ASFV DNA.FFPET heart, liver, lung, spleen, tonsil, prescapular and mesenteric lymph nodes were collected from pigs experimentally infected with 4 ASFV strains (Armenia 07, E‐70, Kenya 05 and OURT 88/3). De‐paraffinization of paraffin whorls, tissue lysis with proteinase K, and reversal of nucleic acid cross‐linking in heated alkaline buffer were performed in a single tube. DNA was purified using automated magnetic bead extraction on the Taco MiniTM (GeneReach) or the BioSprint (Qiagen) systems. Real Time PCR (qPCR) for the detection of ASFv was performed using Quanta FastMix II. The ASFv copy number (CN) was calculated via a plasmid‐based standard curve.Support or Funding InformationCenter of Excellence for Emerging and Zoonotic Animal Disease, A Department of Homeland Security Science and Technology Center of Excellence, and the National Pork Board
Utility of Lipoprint® Lipoprotein Subfraction Testing in a Rodent Atherosclerosis Model
Authors: Katavolos Paula , Martzall Angela , Nguyen Trung , Ross Jeff , Zavala‐Solorio Jose , Kolumam Ganesh 
Published: November 2023

Lipid analysis on rodent blood samples has been traditionally constrained by: 1) the need for technically demanding and time‐consuming ultracentrifugation methods requiring large sample volumes and 2) the limited translatability of traditional endpoints (i.e., total High Density Lipoprotein (HDL) or total Low Density Lipoprotein (LDL) cholesterol concentrations) measured using standard clinical chemistry spectrophotometric assays on preclinical studies due to considerable species differences in lipid metabolism and assay cross‐reactivity differences.The Lipoprint® Lipoprotein Subfractions Testing System (Quantimetrix) is a lipid testing option that analyzes lipoprotein subfractions (e.g., Very Low Density Lipoprotein (VLDL), Intermediate Density Lipoprotein (IDL), LDL(1–7), HDL(1–10)) in a small volume serum sample. The technology uses high‐resolution PAGE coupled with digital analysis to provide a profile of the relative distribution of lipoprotein particles within a given sample.We sought to determine if Lipoprint® analysis could be used to investigate alterations in lipid subfractions following administration of a well‐studied pharmaceutical agent in an atherosclerosis rodent preclinical model. Results from the experiments demonstrate the utility of the incorporation of these exploratory lipid subfraction endpoints into this model system in conjunction with conventional total HDL, LDL and triglyceride concentration combined with ex vivo image analysis of plaque burden.
Organoid models to interrogate GPCR regulation of pancreatic tumorigenesis
Authors: Feigin Michael E , Tuveson David A 
Published: November 2023

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with a 5‐year survival rate of 6%, due in part to therapy resistance and late stage at diagnosis. A detailed understanding of the molecular alterations underlying this disease is required to uncover mechanisms of tumorigenesis and enable development of more effective therapies. We recently developed a novel 3D model system for culturing normal and diseased pancreatic tissue from both mice and humans. These pancreatic organoids can be passaged indefinitely in vitro, frozen, thawed, genetically manipulated and transplanted orthotopically into mice where they generate lesions faithfully reflecting their donor phenotype (normal, pre‐malignant, malignant). The ability to culture both normal and tumor‐derived cells in vitro under identical conditions has for the first time allowed a direct comparison of gene expression changes and response to therapeutic agents. We have used this new model to investigate tumor cell‐stroma interactions, screen drug libraries and model genetic alterations found in human tumors.G‐protein coupled receptors (GPRCs) transduce extracellular signals from a variety of ligands through the plasma membrane, resulting in the modulation of intracellular signaling pathways. This is accomplished in large measure by the activation of heterotrimeric G‐proteins and downstream second messengers. Composed of over 900 members in humans, GPCRs are seven‐transmembrane proteins that regulate many physiological processes including vision, olfaction, taste, behavior and autonomic nervous system transmission. This wide array of functions has resulted in the extensive utilization of GPCR targeted therapeutics, accounting for 30–50% of all currently used drugs. The wide use of GPCR drugs can also be attributed to GPCR localization on the cell surface, abrogating the requirement for a drug to be cell permeable, as well as the ability of GPCRs to bind a variety of ligands, including antibodies, peptides and small molecules. Furthermore, GPCR signaling can be tightly regulated through the utilization of agonists, antagonists and inverse agonists. However, drugs targeting GPCRs are rarely utilized in cancer treatment, despite evidence that GPCRs mediate many aspects of tumorigenesis, including cell proliferation, invasion, immune cell recruitment and secondary niche generation. Genomic analyses have uncovered GPCR mutations, copy number alterations and gene expression and methylation changes in a wide variety of cancers. We have utilized the organoid system to identify and characterize GPCRs upregulated in PDA. Furthermore, we have performed high throughout screens to identify GPCR‐targeted FDA approved drugs that specifically reduce proliferation in tumor‐derived organoids. As agonists and antagonists of GPCRs are currently used widely in the clinic, the identification of GPCRs as novel drivers of tumorigenesis may have immediate benefits for human cancer therapy.
Tumor‐induced Factor (CXCL3) suppresses tumor initiation
Authors: Cheung Wing‐Tai , Li Zhou‐Fang , Jin Lili , Wang Da‐Kui , Sun Meina , Qi Wei , Ma Qiang , Zhang Li , Chu Chun , Liu Xing , Lee Susanna S.T. , Wise Helen , To Ka‐Fai , Shi Ying , Zhou Naiming 
Published: November 2023

Previously our lab uncovered a putative chemokine tumor‐induced factor (TIF) from a mouse xenograft model of copy number variation (CNV)‐mediated G protein‐coupled receptor (GPCR) MAS‐driven tumorigenesis. Sequence analysis and chemotactic study suggested that TIF was likely to be a hamster homolog of human GROγ (CXCL3) [IJC 125 (2009): 1316–1327]. Subsequently, genomic study of CHO‐K1 cells indicated that Tif gene consisted of 4 exons, characterized with an antisense B1 element which is embedded in the fourth exon (GenBank Accession No: EU293869). Two Tif transcripts were identified which shared identical sequences except that a string of 71‐nt derived from the antisense B1 element was deficient in the shorter transcript. By using the 71‐nt sequence as a probe, B1‐like RNA ladder was detected in xenografts. Pharmacological studies suggested that TIF activated Gi‐coupled CXCR2 and suppressed forskolin‐stimulated cAMP accumulation. In addition, TIF also induced calcium mobilization and ERK1/2 phosphorylation. Functional studies showed that TIF induced blood vessel formation and chemotaxis of CXCR2‐expressing cells. Overexpression of TIF in CHO‐K1 cells indicated that secreted mature TIF functioned as an autocrine factor and promoted anchorage‐independent growth. Unexpectedly, xenograft studies suggested that TIF delayed the onset of tumor formation but without exerting any inhibitory effect on tumor growth. These results suggest TIF suppresses tumor initiation and could be therapeutically valuable for preventing cancer relapse.Support or Funding InformationThe project was partially funded by a CUHK Direct grant (2041596) and a research grant from HKSAR Health and Medical Research Fund (HMRF 02133116) to WTC, and also supported by grants from the Ministry of Science and Technology of China (2012CB910402 and 2012AA020303), and the National Natural Science Foundation of China (81173106) to NMZ.
Measurement of the Binding of Fluorescent Variants of VEGF165a, VEGF165b and VEGF121a to VEGFR2 Using NanoBRET in HEK293 Cells
Authors: Peach Chloe , Kilpatrick Laura , Robers Matt , Ohana‐Friedman Rachel , Woolard Jeanette , Hill Steve 
Published: November 2023

Vascular endothelial growth factor receptor 2 (VEGFR2) is a key mediator of angiogenesis and vascular permeability (1). Alternative splicing of the Vegfa gene produces isoforms with different signalling and physiological outcomes (2). However, quantitative information on their binding affinities for the full‐length receptor in living cells is currently lacking. In this study, we used bioluminescence resonance energy transfer (BRET) to quantify binding of fluorescently‐tagged VEGF165a, VEGF165b and VEGF121a to N terminal NanoLuc‐tagged VEGFR2 in HEK293 cells. Stably expressing NanoLuc‐VEGFR2 HEK293 cells were seeded 24 hours prior to experimentation in 96 well plates. For competition experiments, cells were co‐stimulated with fixed concentrations of VEGF isoforms labelled at a single cysteine residue with tetramethylrhodamine (TMR) and increasing concentrations of unlabelled VEGF (in Hanks buffered saline solution/0.1% bovine serum albumin). In saturation experiments, increasing concentrations of VEGF165a–TMR, VEGF165b‐TMR or VEGF121a‐TMR were added in the presence and absence of a high concentration of unlabelled VEGF. Following 60min stimulation at 37°C, the NanoLuc substrate furimazine (10μM) was added and BRET ratios were recorded using a BMG Pherastar. Each ligand isoform had a clear saturable component of binding with little evidence of non‐specific binding. Equilibrium binding constants (Kd) derived for each fluorescent ligand from NanoBRET saturation assays show VEGF165a‐TMR, VEGF165b‐TMR and VEGF121a‐TMR bind to NanoLuc‐VEGFR2 with nanomolar affinity (see Table 1). All fluorescent ligands were displaced by unlabelled VEGF, for which comparable affinity values were derived for VEGF165a (pKi = 9.66 ± 0.04; N=5), VEGF165b (pKi = 9.32 ± 0.06; N=5) and VEGF121a (pKi = 9.34 ± 0.08; N=5). Fluorescently tagged VEGF165a, VEGF165b and VEGF121a enabled ligand binding affinities to be quantified for the first time in living cells expressing full‐length VEGFR2 without endogenous co‐receptors present.Support or Funding InformationThis work was supported by the AJ Clark Scholarship (British Pharmacological Society) and a BBSRC link grant.
Binding affinity of VEGF isoforms to NanoLuc‐VEGFR2 expressed in HEK293 cells quantified using NanoBRET.

Kd (nM)

pKi

VEGF165a‐TMR
1.18 ± 0.07 (N=3)
VEGF165a
9.66 ± 0.04 N=5)

VEGF165b‐TMR
7.72 ± 0.47 (N=3)
VEGF165b
9.32 ± 0.06 (N=5)

VEGF121a‐TMR
4.11 ± 0.30 (N=3)
VEGF121a
9.34 ± 0.08 (N=5)
Inhibition of BMP2‐Inducible Kinase (BMP2K) Reduces Hypoxia/Reoxygenation Injury and Decreases Autophagic Flux in H9c2 Cells
Authors: Engman Samuel I , Nelson Nathaniel R , Dong Siwei , Graves Lee M , Wauson Eric Michael 
Published: November 2023

A small percentage of the 518 kinases in the human genome are known to play important roles in the pathophysiology of ischemia/reperfusion (I/R) injury in cardiomyocytes. However, there are many understudied kinases that may impact the damage to cardiomyocytes caused by I/R. Thus, there are likely unmet therapeutic opportunities in targeting kinases to reduce death caused by myocardial infarction. We utilized an unbiased mass spectrometry affinity chromatography method to investigate how simulated ischemia/reperfusion (sI/R) affected the activity of a large number of kinases in a cardiomyocyte cell line. Our data suggested that the activity of bone morphogenic protein 2‐inducible kinase (BMP2K) was elevated in cardiomyocytes exposed to simulated ischemia/reperfusion (sI/R) injury. BMP2K was cloned from a cell line that was induced to differentiate into an osteoblastic phenotype with BMP2. While no physiological substrates have been identified, BMP2K was observed to phosphorylate myelin basic protein and autophosphorylate in vitro. We determined that a reduction in the expression of BMP2K in cardiomyocytes using siRNA reduced apoptosis and overall cell death caused by sI/R injury. In addition, knocking down BMP2K reduced autophagic flux in cardiomyocytes under all the conditions that we tested. To identify potential BMP2K substrates and interacting proteins, we immunoprecipitated Flag‐BMP2K from HEK‐293 cells, performed SDS‐PAGE, and visualized proteins with Coomassie blue stain. We observed a 75 kD protein that was identified by mass spectrometry analysis as mortalin. Mortalin, which is member of the heat shock protein 70 (Hsp70) family, can function to suppress cell death during glucose deprivation. We validated our mass spectrometry results with co‐immunoprecipitation assays followed by immunoblotting with a mortalin antibody. Our future goals are to determine if mortalin is a direct substrate of BMP2K and to identify the functional significance of the BMP2K/mortalin interaction. In addition, we are currently investigating the mechanism by which BMP2K inhibition regulates both cell death and autophagy using both a cardiomyocyte cell line and primary adult rat ventricular cardiomyoctesSupport or Funding InformationThis research was supported by the American Heart Association Award: 15SDG25090279 (EMW) and by The Iowa Osteopathic Education and Research (IOER) Funds (EMW).
Outer Membrane Vesicles from Clinical Strains of Escherichia coli Can Cause Megalocytosis and DNA Damage in HeLa Cells
Authors: Morales‐Lozada Yermary , Gómez‐Moreno Ramón , Baerga‐Ortiz Abel 
Published: November 2023

Outer membrane vesicles (OMV) are proteoliposomes secreted by almost all gram‐negative bacteria with several functions like the transportation of bacterial virulence and survival factors. OMV provide an insoluble secretion pathway for gram‐negative bacteria and are responsible for the release of outer membrane and periplasm content out of the cell. It has been shown that certain OMV promote aberrant growth in mammalian cells upon contact. The pks island gene cluster, a virulence factor in some strains of extraintestinal E. coli, encodes enzymes purported to make a genotoxic compound that we hypothesize is transported towards cells by OMV. The main objective of this investigation is to compare the biological activity of OMV isolated from E. coli strains harboring and lacking the pks island genes. As a preliminary result, we observe megalocytosis, a hallmark of genotoxic activity, on HeLa cells treated with OMV isolated from E. coli IHE3034 (pks+) in comparison with cells exposed to OMV isolated from IHE3034 (ΔclbP), which lacks the pks island. Surprisingly, immunoblotting analysis targeting the phosphorylation of the histone H2AX shows greater DNA damage in cells treated with the IHE3034 wildtype strain, but also, DNA damage was observed in IHE3034 (ΔclbP) mutant strain. Overall, these results show that OMV treated cells exhibit morphological abnormalities similar to those observed in cells exposed to pks positive bacterial cells, thus demonstrating a link between the genotoxic activity of the pks island and the production of OMV by the bacteria.Support or Funding InformationRISE Program Grant #R25GM061151‐15