bluuebunny/crossref_metadata_2025 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

Background

Ischemia‐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.

Methods

Male 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.

Results

Post 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.

Conclusion

The 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 Information

Faculty 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 Information

IUPUI New Faculty Start Up Grant

NIH R00 GM98328
Resonance Raman spectroscopy probes the effects of BGP‐15 treatment on Ventilator Induced Diaphragm Dysfunction Myopathy in Rat ICU animal model
Authors: Sriramoju Vidyasagar , Salah Heba , Cacciani Nicola , Akkad Hazem , Larsson Lars , Alfano Robert
Published: November 2023

Ventilator Induced Diaphragm Dysfunction (VIDD) is a clinical condition characterized by the loss of diaphragmatic force‐generating capacity in response to mechanical ventilation (MV). MV is a lifesaving intervention frequently used in critically ill Intensive Care Unit (ICU), but VIDD results in prolonged weaning from the ventilator, negative effects on patient quality of life and increased morbidity and mortality. In this study, the pharmacological intervention with the chaperone co‐inducer BGP‐15 recently shown to increase diaphragm muscle function by more than 100% after 10 days MV in a unique experimental rat ICU model (Salah et al. 2016 Science Transl. Med) was probed using Resonance Raman spectroscopy. The Resonance Raman method with 532nm laser excitation is ideally suited to determine the molecular changes taking place in the muscle tissues under non‐denaturing conditions at ultralow freezing temperature without altering the native chemistry of the constituent molecules. The Raman method helps in the identification of constituent molecular species in the target tissues, their structural alternations and their relative concentrations. The proteins in the muscle‐actin, myosin, and coenzymes like NADH, are high in concentration therefore Raman signals are significantly strong from these molecular targets. The rat diaphragm muscle samples analyzed were from 1. Healthy control group, 2. VIDD Test group without treatment (10 days MV), and 3. Test group with BGP‐15 treatment during 10 days MV. The background subtracted Raman spectra all 3 groups are normalized at 864 cm‐1 for comparison and key molecular fingerprints are compared. The findings from this study suggest that Raman spectroscopy is promising tool for in vivo diagnosis and real time monitoring of clinical progress of VIDD.
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 Information

Center 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 Information

The 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.
Development of Naphthalimide DNA Intercalators as Anticancer Therapeutics
Authors: Skubic Kristin , Adams Miranda , Horn Jill , Priddy Anna , Oelrich Renae , Lewis Michael , Znosko Brent , Arnatt Christopher
Published: November 2023

In the past decade, there has been substantial progress in research of small molecule DNA intercalators as antineoplastic agents. Due to specificity of the DNA‐intercalator interaction, compounds may be tailored to target specific genes to avoid potential side effects. For this research, derivatives of 3‐amino‐1, 8‐naphthalic anhydride were synthesized (initial research by Braña et al) with combination of different amino arms and substituents along the 3‐ or 4‐ position generating a series of naphthalimide derivatives to determine if compound structure influenced DNA intercalation (via DNA melt studies), antiproliferative activities (standard breast cancer cell lines SKBR3, MDAMB231, and MCF‐7), and topoisomerase II inhibition activity. Calculated lethal dose (LD50) values for the antiproliferation assays (ranging from low nM to low μM), DNA melt studies, and topoisomerase II inhibition activities illustrate a clear structure‐activity relationship for this series of naphthalimides.
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 Information

This 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 cardiomyoctes

Support or Funding Information

This 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 Information

RISE Program Grant #R25GM061151‐15
Peak Caspase‐1 p20 Levels During Acute Myocardial Infarction Correlate with Pre‐Existing Hypertension, Age and Post‐Infarction Cardiac Dysfunction
Authors: Greenwood Matthew , Morgan Andrew , Na Sam , Gold Courtney , Liang Brannen , Shapior Linda , Liang Bruce
Published: November 2023

Background

Increased inflammation contributes to the development of myocardial infarction (MI) and adversely impacts infarct healing. Caspase‐1 is an end effector of inflammasome activation and is an important mediator of systemic inflammation. Caspase‐1 is cleaved to produce a 20 kDa (p20) peptide upon activation. Activated Caspase‐1 activates downstream cytokines IL‐1β and IL‐18. Previous work demonstrated that the p20 is elevated in patient serum during and after acute MI.

Objective

The aim of this study was to further elucidate the relationship of Caspase‐1 p20 and risk factors for MI. Between 12/10/2007 and 3/28/2016, 170 eligible patients with acute MI were enrolled that had complete information on p20 levels.

Method

Using a solid phase enzyme‐linked immunosorbent assay, the 20 kDa peptide was detected in serum during ST elevation (STEMI) and non‐ST elevation (NSTEMI) MI as well as after MI. Cardiac function post‐MI was evaluated via standard‐of‐care echocardiography.

Results

There is a positive correlation between age and peak p20 level during acute MI (P=0.01, linear regression, n=170). In STEMI but not in NSTEMI subjects, pre‐existing hypertension (HTN) was associated with a 50.3% higher average circulating peak p20 level during acute MI than those without HTN (P=0.0056, Wilcoxon, n=31 for both groups‐those with HTN and without HTN), suggesting a greater degree of inflammasome activation in HTN patients during STEMI. In these STEMI subjects, the existence of diabetes mellitus or smoking was not associated with a higher peak p20 level during acute MI. There is a trend for all acute MI patients to have a reduced post‐MI cardiac function as quantified by LV fractional shortening in echocardiography (P=0.05, Spearman correlation, r=−0.35, n=31).

Conclusion

Older subjects had more elevated peak p20 levels during acute MI, consistent with the concept of a larger inflammasome response to MI as age increases. Inflammasome activation is more pronounced in HTN patients during STEMI, implying a greater degree of inflammatory response in the presence of HTN. A greater extent of inflammasome activation during STEMI may be associated with a worse LV function subsequently.

Support or Funding Information

The study is supported by the Pat and Jim Calhoun Cardiology Endowment Fund.
Long‐term effects of atenolol use on glycemia
Authors: Camacho‐Narro Carolina , Camacho‐Saavedra Luis Arturo , Bardales‐Zuta Victor Hugo , Fernández‐Rodríguez Lissett Jeanette
Published: November 2023

There is good evidence that the antihypertensive drug atenolol can alter glycemic metabolism in patients. Although these reports offer insight into this undesirable side effect, there currently lacks sufficient information on glycemia over long‐term use of this beta‐blocker. For this reason, we designed a retrospective cohort study in which we measure glycemia in non‐obese patients before and after 6 months of treatment with atenolol (n=36) or a non‐diuretic antihypertensive drug (n=72). We observe that the incidence of hyperglycemia was 47,2% in the atenolol group and 15,3% in the control group after 6 months, which generated RR = 3,091, 95% CI [1,623 to 5,878], p <0,05. Although this increase is significant in the atenolol group, no case achieved glycemia levels that defined diabetes mellitus. However, we recommend that glycemia be measured, and if necessary, controlled during treatment with atenolol to avoid long‐term metabolic complications.

Support or Funding Information

Antenor Orrego Private University
Antidiabetogenic Effects of the Seed Extracts of Picralima nitida
Authors: Ojuola Oluwatosin Moradeke , Abatan Matthew Oluwole
Published: November 2023

Picralima nitida is a plant with medicinal importance including its use for lowering blood sugar levels. The extracts of the seed have been evaluated for possible blood sugar lowering properties using alloxan‐induced diabetes mellitus model in rats. The seed material of P. nitida was extracted using gradient solvent extraction with chloroform, diethyl ether, ethyl acetate and methanol. Pilot toxicity studies using a crude extract indicated that the plant was safe when administered orally at 3000mg/kg. The different solvent extracts were administered at a dose of 750mg/kg, to alloxan induced diabetic rats and their blood sugar levels monitored hourly for four hours post administration. Glibenclamide served as the standard antidiabetic drug. Results show that the different solvent extracts had significant blood lowering effects (P<0.5) compared to the untreated diabetic rats. The extracts also produced decrease in the triglyceride, total cholesterol, low density lipoprotein values and an increase in high density lipoprotein levels.

The chloroform solvent extract produced the highest bioactivity and was therefore subjected to further tests. The extract was subjected to vacuum liquid chromatography (VLC) and the biograded fractions evaluated for their blood sugar lowering effects. The results showed that fraction 5 (30% ethyl acetate and 70% ethanol) produced the highest blood sugar lowering effects. Standard procedures were used to carry out phytochemical analysis which showed that the plant has alkaloids, flavonoids, saponin, tannins and cardiac glycosides. The evaluation substantiates the medicinal use of P. nitida for lowering blood sugar in diabetic patients

Support or Funding Information

Pan African University
Effects of hydroxytyrosol on cardiovascular and neuroprotective biomarkers in experimental diabetes mellitus
Authors: Gonzalez‐Correa Jose A , Rodriguez‐Perez M Dolores , Lopez‐Villodres Juan A , Medina‐Moreno Ursula , de la Vega Jose J Reyes , de la Cruz Jose P
Published: November 2023

The aim of this study was to assess the influence of hydroxytyrosol (HT) on cardiovascular inflammation and neuroprotective effect in streptozotocin‐diabetic rats. Four groups of rats (N=10 per group) were studied for 2 months: nondiabetic rats (NDR), diabetic rats treated with saline (DR) and DR treated with HT (1, and 5 mg/kg/day p.o.). At the end of follow‐up an experimental model of hypoxia‐reoxygenation in brain slices was tested. Vascular variables: DR had higher platelet aggregation values, higher thromboxane B2, plasma lipid peroxidation, 3‐nitrotyrosine, oxidized LDL (oxLDL), myeloperoxidase, vascular cell adhesion molecule 1 (VCAM‐1) and interleukin‐1β (IL‐1β) concentrations, and lower aortic 6‐keto‐prostaglandin F1α and nitric oxide production than NDR. Aortic wall area and smooth muscle cell count were also higher in DR than in NDR. Brain variables: the DR group showed increased cell death, oxidative and nitrosative stress and an increase in brain inflammatory mediators. HT significantly reduced both oxidative and nitrosative stress, oxLDL concentration (35% and 40% of reduction respect untreated animals for 1 or 5 mg/kg/day respectively), VCAM‐1 (63% and 50%), platelet aggregation (22% and 40%) and thromboxane B2 production (22% and 25%). Morphometric values in the aortic wall were reduced to values near those in NDR. Respect the brain variables the result obtained were the following: HT significantly reduced oxidative lipid peroxidation (38% and 52 of reduction respect untreated animals for 1 or 5 mg/kg/day respectively) and nitrosative stress (47% and 50%) and peroxynitrite concentration (43% and 60%) and brain inflammatory mediators (19–30% prostaglandin E2 and 16–50% interleukin 1β concentration). Cell death was reduced by 26% and 38% after the administration of 1 or 5 mg/kg/day.

In conclusion, HT influenced the major biochemical processes leading to diabetic vasculopathy, reduced cell proliferation in the vascular wall and had a neuroprotective effect in this experimental model.
M3 Muscarinic Acetylcholine Receptors Inhibit Autophagy through Activation of mTORC1 in Human Neuroblastoma Cells.
Authors: Onali Pierluigi , Dedoni Simona , Olianas Maria C
Published: November 2023

Activation of Gq/11‐coupled muscarinic acetylcholine receptors (mAChRs) has been shown to promote the growth and survival of different types of cancers derived from epithelial and endothelial cells [1]. Previous studies in human neuroblastoma cells have reported that M3 mAChRs prevent apoptotic cell death induced by a number of cytotoxic agents [2,3]. However, there is little information on the effect of mAChRs on autophagy, an evolutionary conserved self‐degradation process that may act as a tumor‐suppressor pathway. In the present study we show that in human SH‐SY5Y neuroblastoma cells activation of M3 mAChRs inhibits autophagy triggered by serum and L‐glutamine removal from the growth medium. Cell exposure to either the cholinergic agonist carbachol (CCh) (30 μM) or the mAChR agonist oxotremorine‐M (100 μM) inhibited the formation of the autophagosome marker LC3‐II and increased the accumulation of the autophagy substrate p62. These effects were blocked by either atropine or the M3 mAChR‐preferring antagonist darifenacin, but not MT7, a selective M1 mAChR antagonist. The inhibitory effect of CCh on autophagy was prevented by blockade of Gq/11 with YM254890 and by inhibition of protein kinase C (PKC), and was mimicked by the PKC activator phorbol 12‐myristate 13‐acetate (PMA). Both CCh and PMA induced mTORC1 activation and enhanced the phosphorylation/inactivation of ULK1, a mTORC1 substrate and a key initiator of autophagy. Inhibition of mTORC1 by rapamycin prevented CCh‐induced ULK1 phosphorylation and autophagy inhibition. The data indicate that in SH‐SY5Y cells Gq/11‐coupled M3 mAChRs inhibit autophagy by stimulating the PKC‐mTORC1 signaling pathway. The attenuation of the self‐degradation process induced by autophagy may contribute to the growth promoting effects of M3 mAChRs in human neuroblastoma cells.

Support or Funding Information

Supported by Regione Autonoma della Sardegna, L.R.7,‐2012‐CRP‐60567
Ginsenoside Re promotes pancreatic B‐cell proliferation via CREB activation
Authors: Park Ji‐Young , Jun Hee‐Sook , Park Eun Young
Published: November 2023

Proliferation of pancreatic cells is an important mechanism for maintaining islet function to meet physiological demands. We found that ginsenoside Re have a proliferative effect on beta cells. In this study, we investigated the mechanisms of b‐cell proliferation by ginsenoside Re. To examine proliferation of pancreatic beta cell, insulinoma cell line (MIN6 cells and INS‐1 cells) and mouse islets were treated with ginsenoside Re and cell proliferation was detected by CCK or thymidine incorporation assay. The expressions of cyclin A2 and IRS2 protein and mRNA were examined by western blot and qRT‐PCR respectively. To examine whether CREB activation is required in the regulation of these gene expression by ginsenoside Re, we performed luciferase reporter assay in INS‐1 cells transfected with cyclin A2 promoter‐Luc and CRE mutated cyclin A2 promoter‐Luc (cyclin A2‐ΔCRE). In addition, CREB activation was also examined by EMSA. The signaling pathway was determined using H89 (PKA inhibitor). Re treatment increased pancreatic beta cell proliferation compared with vehicle‐treated cells. The expression of both the cyclin A2 and IRS2 mRNA and protein was increased. Re treatment promoted p‐CREB (Ser‐133) nuclear translocation and CREB binding activity. The relative luciferase activity by Re treatment was markedly decreased in INS‐1 cells transfected with cyclin A2‐ΔCRE, as compared to that in cells transfected with cyclin A2 promoter‐Luc. Knockdown of CREB abolished the effect of Re on increased pancreatic beta cell proliferation. Inhibition of PKA attenuated cylinA2 and IRS2 induction and proliferation by EX4 treatment. Re promotes pancreatic beta cell proliferation, via CREB activation which induces cyclinA2 and IRS2 gene expression.
Time‐locked mesolimbic dopamine signals in response to pain onset and to pain offset
Authors: Isakov Bojan , Atcherley Christopher , Gee Taylor , Navratilova Edita , Heien Michael , Porreca Frank
Published: November 2023

Relief of aversive states, including pain, is rewarding and associated with an increase in dopamine (DA) release in the mesolimbic reward circuit. Measurement of DA in the nucleus accumbens (NAc) is usually accomplished with microdialysis but this method does not allow determination of the temporal relationship to pain, and to its relief. In this study, we used isoflurane anesthetized rats to measure NAc DA release on a millisecond to second timescale using fast scan cyclic voltammetry (FSCV). Our approach was to standardize the depth of anesthesia by a reflexive pain response to noxious heat, the tail‐flick test. The depth of anesthesia was standardized to this biological response of approximately 5.0 sec latency. FSCV involves the application of a specific triangular waveform at a high scan rate, allowing for oxidation and reduction of dopamine detected by a specialized electrode previously implanted in the NAc shell. Application of noxious heat to the tail was accompanied by a decrease in the DA signal that peaked at the time of the tail‐flick. Following the tail‐flick, there was a rebound increase in DA signal that peaked after 2.9 s. The data show a time‐locked DA response to pain onset, and to pain offset, likely related to the negative and positive components of pain and its relief.

Support or Funding Information

Supported by DA034975 from the NIH
Effect of Fatty Acid Amide Hydrolase (FAAH) Inhibitors Isolated from Nutmeg on Anxiety
Authors: El‐Alfy Abir T. , Jeon Andrew , An Hea Re , Kendrek Jennifer , Abourashed Ehab
Published: November 2023

The endocannabinoid system (ECS) is a complex neuromodulatory network involved in the regulation of numerous physiological functions. These effects have been substantiated by the use of known exogenous cannabinoid, Δ9‐THC, the active component of marijuana. Thus, the ECS has emerged as an appealing pharmacologic target in drug discovery. Anecdotal reports of Myristica fragrans, commonly known as nutmeg, being used as a substitute for marijuana, suggest that one or more of its components may interact with the ECS. Previous studies in our laboratory demonstrated that administration of nutmeg extracts produced behavioral tetrad effects similar to Δ9‐THC. However, the mechanism of such action requires further exploration. Receptor binding assays revealed that nutmeg extracts did not have direct CB1 or CB2 receptor binding activity. Additional mechanistic studies showed that indirect interaction through the inhibition of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), key endocannabinoid degradation enzymes, was observed. The objective of the current study was to isolate the active components of nutmeg extract responsible for the FAAH inhibition activity and to evaluate them in an animal model of anxiety. Using bioassay guided fractionation and various chromatographic techniques, three compounds were isolated, spectroscopically characterized, and identified (MF 109‐5, MF30‐7, and MF117‐3). Concentration and time inhibition studies were conducted. The IC50 values calculated for the three compounds were 4.57 ± 0.66, 7.50 ± 2.02, and 38.29 ± 6.18 mM, respectively. The compounds were further evaluated in the elevated plus maze animal model of anxiety in comparison to diazepam as the positive control. The three nutmeg compounds were tested at doses ranging from 5 to 120 mg/kg, i.p. injection. While diazepam demonstrated a significant dose‐response increase in open arm entry and permanence time, indicative of anxiolytic action, none of the isolated compounds had a significant effect. In conclusion, the study resulted in the identification of three nutmeg compounds that significantly inhibit FAAH enzyme and hence shed light on potential mechanism of cannabis‐like action of nutmeg. While none of the compounds showed anxiolytic activity in the elevated plus maze model, additional pharmacokinetic studies are needed to optimize the dosing regimen required for significant increase in endocannabinoids that might be needed for elaboration of the anxiolytic effect.

Support or Funding Information

Research reported in this publication was supported by the National Institute on Drug Abuse of the National Institutes of Health under Award Number R24DA036410. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Norclozapine Reduces Beta Amyloid1‐42 Levels and Increases ChAT Levels in 3xTg‐AD Female Mice.
Authors: Burkart Ashley , Potter Pamela E , Jones Douglas C
Published: November 2023

Alzheimer's disease (AD) is a neurodegenerative disease characterized by synaptic dysfunction and cholinergic cell death. Major pathological hallmarks include aggregations of beta‐amyloid (Aβ) proteins, intracellular neurofibrillary tau tangles, and a loss of cholinergic neurons that innervate the hippocampus and cortex. The accumulation of neurotoxic Aβ fragments in AD is caused by the improper processing of amyloid precursor protein (APP). Previous evidence suggests that insoluble Aβ1‐42 oligomers induce synaptic loss and decreases choline acetyltransferase (ChAT) levels in AD brains. In addition, activation of cholinergic receptors, specifically muscarinic M1 receptors, has been shown to reduce the inappropriate and neurotoxic processing of APP. The goal of the current study was to evaluate the effects of M1 stimulation on Aβ levels and cholinergic dysfunction in an animal model of AD, the 3xTg‐AD mouse. 3xTg‐AD female mice (~6 mos old) were randomly divided into 4 experimental groups and administered (i.p.) either saline (control), an M1 agonist (norclozapine) or an M1 antagonist (scopolamine or tolterodine) for 30 days. On day 31, mice were sacrificed and the brain harvested for analysis. ELISA results using whole brain homogenates indicated that norclozapine (8 mg/kg) significantly decreased Aβ protein levels whereas scopolamine (2 mg/kg) and tolterodine (2 mg/kg) had no significant effect on Aβ levels. Analysis of ChAT proteins in brain homogenates of treated 3xTg‐AD mice found that norclozapine significantly increased ChAT levels while neither of the antagonists had an effect. Finally, immunohistochemical (IHC) analysis of sectioned brain tissue using an antibody against Aβ revealed decreased Aβ immunoreactivity in the hippocampus and cortex of norclozapine treated animals. The present results have characterized the effects of M1 stimulation on amyloid levels and cholinergic function in a common animal model of AD. These findings also suggest that (1) the M1 agonistic properties of norclozapine may be effective in reducing the accumulation of Aβ associated with AD and (2) M1 receptor stimulation may be a valuable mechanism for the development of new drugs aimed to inhibit the progression of AD.

Support or Funding Information

This work was supported by Midwestern University Intramural and Biomedical Student funding.
A Humanized Single Chain Fragment Variable Constructed to Use in Breast Cancer Immunotherapy
Authors: Mahgoub Elham Omer
Published: November 2023

The mouse B cell hybridoma line C3A8 was the starting material to generate monoclonal antibody to react specifically toward epidermal growth factor receptors (EGFR) of breast cancer cell line (MCF‐7). Phage display technologies were applied to produce molecular form of those IgG antibodies. The integrated cloning, screening, and phage display system allowed to use extended primers, which were incorporate the entire mouse VH and VL and their linker sequences collected in the Kabat database. The product was called single‐chain fragment variable (scFv) antibodies. The best candidate scFv sequences, based on preliminary enzyme‐linked immunosorbent assay (ELISA) screening data gave 100% identical sequences in genbank data base, while a band of 68kD was appeared in nitrocellose membrane in western blot test had used to characterize the specific reaction of scFv towards MCF‐7 breast cancer cells. Further, immuno‐fluorescence test clearly proved that the scFv recognized the MCF‐7 antigen epitopes which is localized in MCF‐7 nuclear. Also, 99% of the cells numbers were bind to scFv protein as measured by flow cytometry analysis. Herein the recombinant antibody technology is a rapid and effective approach to the development of the next generation of breast cancer diagnosis and immune‐therapy.