bluuebunny/crossref_metadata_2025 huggingface dataset:
A High Fat Diet Induces Decreased Levels of Colonic Tight Junctions and Inflammatory Cytokine Expression in Sprague Dawley Rats
Authors: Sambolin‐Escobales1 Lubriel , Nazario Madeline , Torres‐Aguiar Roberto , Cruz Myrella L , Yamamura Yasuhiro , Appleyard Caroline B , Chompre Gladys
Published: November 2023

High fat diet consumption is associated with a variety of metabolic disorders such as diabetes, hyperlipidemia, and cardiovascular diseases among others. Also, an intake of high fat is associated with inflammation of the intestinal epithelial barrier, and characterized by increased intestinal permeability. This increased permeability is characterized by the loss of tight junctions due to decreased expression of claudin‐5 and occludin. Our preliminary data found increased colonic macroscopic damage and a trend towards increased microscopic damage in rats fed a High Fat Diet (HFD) compared with a Low Fat Diet (LFD). In addition, metagenomic analysis from HFD feces demonstrated increased bacteriodes with higher serum levels of lipopolysaccharide (LPS) in the HFD group, however it is still unclear if tight junction expression is altered.

HYPOTHESIS

High fat diet will cause a lower expression of tight junction proteins, which increases membrane permeability allowing the entrance of LPS contributing to intestinal inflammation.

METHOD

We purified colon RNA samples from rats fed with LFD (n=14) or HFD (n=14) for 30 days. Amplification of mRNA levels of chemokine ligand (CCL2), interleukin1β (IL‐1β), tumor necrosis factor receptor alpha (TNF‐α receptor), claudin‐5 and occludin were measured to determine their total expression.

RESULTS

We found a decreased expression of both claudin‐5 and occludin in the HFD rats compared with the LFD (p<0.05) suggesting damage in the epithelial membrane as expected. On the other hand, there was a trend towards lower levels of CCL2 (p=0.08), IL‐1β (p=0.06), and TNF‐α receptor (p=0.05) in the HFD compared with the LFD. In conclusion, colon is constantly exposed to bacterial components such as LPS, without triggering pro‐inflammatory immune responses suggesting an alternate molecular mechanism of evasion.

CONCLUSIONS

This study helps to understand the consequences in the colon following consumption of a high fat diet.

Support or Funding Information

PUCPR seed grant G12RR003050/G12MD007579, P20 GM103475‐14
Assessing Muscle Protection with Pharmacological Post‐Conditioning Agents in an Acute Ischemia Reperfusion Model
Authors: Thomas Kathryn N , Soule Charles L , Mattison Lars M , Iaizzo Paul A
Published: November 2023

Background

Cardiorespiratory complications remain as a primary source of morbidity in elective aortic surgeries and heart transplantations. This is in part due to induced tissue ischemia, resulting in an increased risk in reperfusion injuries. In particular, diaphragmatic dysfunction and/or atrial fibrillation produced by ischemia reperfusion (I/R) injuries contributes to clinical complications after cardiothoracic surgeries. Therefore, post‐conditioning of the diaphragm and cardiac muscles with pharmacological agents may play a critical role in conferring protection and restoring tissue function in such patients.

Methods

Portions of muscle were removed from swine diaphragm (n=5) and from a human heart deemed non‐viable for transplant (n=1). The diaphragm or ventricular endocardial surface (trabeculae) were dissected into bundles, attached to force transducers, and submersed into tissue baths. Electrical stimulation was applied to the bundles, and peak forces of muscle contractions were recorded. These tissues were exposed to hypoxia followed by reoxygenation to assess resultant damage throughout a recovery period. Randomly, after the hypoxic periods, bundles were treated with given concentrations of either Omegaven fatty acid emulsions, delta‐opioid (DPDPE), and tauroursodeoxycholic acid (TUDCA).

Results

Percent changes in peak forces of the swine diaphragm and human trabeculae muscle contractions post‐conditioned with these agents and various concentrations showed no significant changes from controls (p>0.05). Non‐toxic agents with potential therapeutic benefits could be applied to various in situ animal models to provide further insights as to the dosing paradigms for full‐organ effects.

Conclusion

This experimental approach with isolated muscle bundles within tissues baths has the potential to be both a time‐and cost‐effective means for screening pre‐ and post‐conditioning pharmacological agents. Further work is needed to find the drugs, dosages, and hypoxia levels necessary to determine whether the tissue baths mimic an ideal I/R model in vitro.

Support or Funding Information

This project was funded in part by IEM and Medtronic, Inc. Support for summer student research was provided by the Clinical and Translational Science Institute Advanced Research Program 2015 at the University of Minnesota.
Human Skeletal Muscle Glyoxalase‐1 Protein Expression is Attenuated with Type 2 Diabetes
Authors: Mey Jacob T , Mahmoud Abeer M , Haus Jacob M
Published: November 2023

INTRODUCTION

Individuals with type 2 diabetes mellitus (T2DM) present with increased levels of advanced glycation endproducts (AGEs), which have been associated with progression and complications of the disease. AGEs are known to contribute to the disruption of insulin signaling in skeletal muscle and are primarily produced in vivo by the damaging glycolytic byproduct, methylglyoxal (MG). MG is effectively detoxified by the enzyme, glyoxalase‐1 (GLO1), whose protein expression has been previously shown to be inversely related to MG‐mediated protein‐damage and AGE formation. Despite the role of skeletal muscle insulin resistance in the pathogenesis of T2DM, GLO1 protein expression has not been fully elucidated in skeletal muscle. Additionally, the metabolic effects of insulin stimulation on GLO1 protein in human skeletal muscle has not been described.

PURPOSE

Therefore, the purpose of this study was to examine skeletal muscle GLO1 protein content in individuals with T2DM compared to lean, healthy controls (LHC). Further, we sought to examine the effects of insulin stimulation on GLO1 protein expression through in vivo and in vitro approaches that included the hyperinsulinemic‐euglycemic clamp and primary human skeletal muscle cell culture experiments, respectively.

METHODS

Skeletal muscle biopsies from the vastus lateralis were obtained from 7 individuals with T2DM (age: 55 ± 4 yrs, BMI: 35.6 ± 2.4 kg/m2) and 10 LHCs (age: 27 ± 1 yrs, BMI: 22.3 ± 0.8 kg/m2) during basal and insulin stimulated states of the hyperinsulinemic (40mU/m2/min)‐euglycemic (5 mM) clamp. To further investigate the effect of insulin, primary human skeletal muscle cells were incubated with 300 pM insulin (a concentration similar to that experienced during the hyperinsulinemic‐euglycemic clamp) for 0, 30, and 180 mins. GLO1 protein content in human tissue and cell culture was assessed via western blot.

RESULTS

Individuals with T2DM displayed a 5.6 fold reduction (p<0.0001) in GLO1 compared to LHC at baseline. Clamp‐induced hyperinsulinemia had no significant effect on GLO1 protein expression in either group (p>0.05). In cell culture, 30 minutes of insulin stimulation increased GLO1 protein expression by 37 % (p=0.0093). Interestingly, continued insulin stimulation for 180 mins returned GLO1 protein expression to levels comparable to baseline (0 min vs 180 min, p>0.05).

CONCLUSION

These data suggest that the skeletal muscle of T2DM may be susceptible to MG‐mediated protein damage and accumulation of AGEs. Further, prolonged exposure to hyperinsulinemia may attenuate early insulin mediated increases in GLO1 expression profiles. Further study is need to elucidate the exact mechanisms of these observations.
Elevated Intramuscular Calcium Effects on Mitochondrial Biogenesis
Authors: PinaJr Dagoberto , Zhang Rui , López Jose R. , Pessah Isaac , Baar Keith
Published: November 2023

Caffeine is known to raise intracellular calcium (Ca2+) levels in the brain; however, the effects of caffeine consumption on intramuscular Ca2+ and subsequent mitochondrial biogenesis have not been addressed in vivo.

Purpose

To determine whether caffeine can increase intracellular calcium levels in skeletal muscle and stimulate mitochondrial biogenesis.

Methods

Mice were administered caffeine (0.2 mg/mL) through their water for 7 days. At the end of seven days of caffeine treatment, intramuscular calcium levels were determined through a modified patch clamp technique. Following determination of intramuscular calcium, muscles were excised and rapidly frozen in liquid nitrogen prior to analysis of mitochondrial protein levels by western blot. Antibodies used include p‐CAMKII Thr286 as a measure of CAMK activity, COX5, COX4, HAD, LCAD, MCAD, VLCAD, Cytochrome‐C, CD36, CPT‐1, citrate synthase, and PGC1‐α.

Results

Caffeine treatment increased intracellular Ca2+ levels (CON=113nM, CAF=156nM; p<0.05). However, the increased intramuscular Ca2+ was not sufficient to modify the levels of mitochondrial proteins (p>0.05).

Conclusion

Caffeine treatment increases intramuscular Ca2+ levels but is not sufficient to drive mitochondrial biogenesis. However further studies are needed to determine whether caffeine could augment the effects of endurance training on mitochondrial mass.
Generation Of A New Renal Nephrotoxicity Model By Using A Microfluidic System
Authors: Sanchez‐Romero Natalia , Arias Cristina , Martinez Laura , Torcal Pilar , Sanchez‐Zalabardo Manuel , Iñigo Pablo , Gimenez Ignacio
Published: November 2023

Aim

To generate a biomimicking cell culture system to study the toxicity caused by exposure to cisplatin and the process of reparation‐regeneration after the injury, in proximal tubule cells grown in microfluidic devices.

Methods

Primary cultures of human proximal tubule cells (hPTPC) are generated from healthy tissue isolated from nephrectomy specimens. The hPTPC are cultured on conventional cell culture containers or inside the channels in polysterene microfluidic devices. Morphological and biochemical characterization of the cultured cells is performed by using PCR, immunofluorescence (IF) and immunocytochemistry (ICC) to verify the expression of selected proximal tubule and epithelial markers.

Confluent hPTPC monolayers are exposed to six cisplatin concentrations in the 0–300 uM range. Cisplatin citotoxicity is assessed by determining three parameters in two different optical reactions: the first assay combines PrestoBlue reagent, a cell viability indicator, with a gamma‐glutamyl transferase 1 (GGT1) substrate. GGT1 is a specific luminal border enzyme in hPTPC and is responsible for the bioactivation of cisplatin into a more potent toxin. In the second assay, cell number is estimated through nuclear DNAstaining with crystal violet.

Results

The phenotyping techniques have been optimized to work with the small volumes and cell numbers present in microfluidic devices. Biochemical characterization of hPTPC through multiplex analysis of phenotypic markers shows these cells retain the most relevant PT markers when cultured in microfluidic channels.

Cisplatin exhibits cytotoxicity on hPTPC when grown on multiwell plates or microfluidic channels. The EC50 was found to be 30–50 μM for the three parameters determined in our assay. Maximal effects were observed in approximately 48–72h after exposure to cisplatin. Our data suggest that hPTPC cultures do not possess autoregenerative capacity. The cytotoxicity model has been successfully adapted for its use in microfluidic devices, with preliminary data showing no change in hPTPC sensitivity to cisplatin.

Conclusions

The phenotypic characterization of our hPTPC confirms typical characteristics of proximal tubule, and its preservation in cells cultured on microfluidic channels.

The effects of cisplatin are time and concentration dependent. Simultaneous reading of assays for cell viability, enzymatic activity, and cell numbers in hPTPC allow us to perform studies of renal toxicity and repair in cells grown inside microfluidic devices.

This is the first step towards a cell culture model for the study of proximal tubule physiology and physiopathology under biomimicking conditions that will include exposure to flow‐mediated shear stress.

Support or Funding Information

Spain's Ministerio de Economía, grant DPI2011‐28262/C02 and fellowship BEP‐2012‐059562 (to NS‐R)
The Effects of Fasting and Refeeding on Regulating NLRP3 Inflammasome Activation in Asthmatic Subjects
Authors: Nguyen An , Li Jessica , Kwarteng‐Siaw Miriam , Traba Javier , Han Kim , Sack Michael
Published: November 2023

The NLRP3 inflammasome is linked to inflammatory diseases including asthma. Interestingly, we have shown that fasting (24 hours) blunts and refeeding (3 hours after a meal) activates the NLRP3 inflammasome in peripheral blood mononuclear cells (PBMCs) from normal volunteers and that the serum extracted at the fasted and refed time points can recapitulate this effect in THP‐1 macrophage cells. At the same time it has also been found that intermittent fasting reduces asthma exacerbations in a clinical study. Based on this we have begun to evaluate whether fasting and refeeding regulate the NLRP3 inflammasome in mild to moderate asthmatic subjects exposed to a 24 hour fasting and 2.5 hour refeeding protocol. Preliminary studies in the first 8 study participants have been analyzed. Interestingly, the asthma subjects (n=3) who were not treated with inhaled steroids showed a 24% increase in refeeding activation of the NLRP3 inflammasome as measured by IL‐1β release as was found in the normal volunteers. However, the asthma subjects (n=5) who were treated with inhaled corticosteroids did not show an induction of the inflammasome with refeeding (rather a 39% reproduction in IL‐1β release compared to fasting). When comparing the serum from the asthmatic subjects, the results were opposite to the normal controls where serum from fasting subjects showed increased IL‐1β release in response to inflammasome triggering compared to serum from the fed state. Interestingly, the baseline IL‐1β levels were markedly elevated in the asthmatic subjects serum compared to the normal controls. These preliminary data suggest that: (i) inhaled steroids have systemic effects and disrupt the immune modulatory effects of fasting and refeeding; (ii) the basal inflammatory milieu evident in asthma disrupts the nutrient‐deprivation effect on blunting the NLRP3 inflammasome.

Support or Funding Information

Cardiovascular and Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD USA.Biomedical Research Training Program for Individuals from Underrepresented Groups (BRTPUG), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD USA.
Comparison of Hypoglossal Motoneuron Transgene Expression following Single vs Dual Tongue Injection of AAV9‐CBA‐GFP
Authors: Doyle Brendan M. , Turner Sara M.F. , Johnson Bobbi , Streeter Kristi A. , Falk Darin J. , Mandel Ronald J. , Byrne Barry J. , Fuller David D.
Published: November 2023

Retrograde transport of viral vectors following intramuscular injection allows for targeted gene delivery to motoneuron pools. Gene delivery to the hypoglossal (XII) motoneurons is of potential interest in disorders involving impaired tongue muscle control such as dysphagia, dysarthria, and possibly obstructive sleep apnea. Using a murine model of Pompe disease, our group has shown that a single intramuscular injection of 1.00e11 vector genomes (vg) of recombinant adeno‐associated virus serotype 9 (AAV9) into the base of the tongue produces sustained transgene expression in a subset of XII motoneurons. Here we investigated whether distributing the virus over a broader portion of the tongue via two near‐simultaneous injections would increase XII motoneuron transduction as compared to a single injection. Four week old 129SVE mice were anesthetized and administered either a unilateral, single 20μL injection (n=2) or bilateral, 10μL injections (n=2) to the anterior base of the tongue targeting the insertion point for the genioglossus muscle. In both cases, a total of 1.00e11 vg of AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken β‐actin promotor (AAV9‐CBA‐GFP) was given. The medulla was histologically evaluated 6 weeks after tongue injection; GFP was detected using immunohistochemistry. Robust XII motoneuron GFP expression was observed in the caudal XII nucleus after either single or dual AAV9 injection; initial observations suggest no differences in XII motoneuron transduction between the two delivery methods. With both approaches, GFP expression was primarily restricted to XII motoneurons, but was also sporadically present in neurons throughout the medulla. The data confirm that tongue injection of AAV9 drives robust transgene expression in the XII motoneuron pool, and suggest that expression is similar whether using the single or dual injection approach.

Support or Funding Information

2R01HD052682‐06A1
Effect of SCI on Learning and Memory; Age, Gender and Species Differences?
Authors: Kempisty Rachel A , Zimmer M. Beth
Published: November 2023

Spinal cord injury (SCI) results in damage to neurons that connect the brain to all areas of the body leading to disturbances in sensory and motor signals below the injury site. A few studies also show that neural changes occur above the site of injury and memory is affected. Preliminary data in our lab showed that the acquisition phase of learning was altered 2 weeks after injury in young male rats. This study hypothesized that older rats may not recover as quickly as young rats from SCI and therefore SCI may lead to more significant alterations in learning and memory. Older, female Sprague Dawley rats were anesthetized with ketamine and xylazine and a C2 hemisection (n=14) or a sham surgery (n=10) was performed. Rats were allowed to recover for 2 weeks before learning and memory was assessed with the Morris Water Maze. Rats completed 4 trials a day for 5 days followed by a probe trial. Results obtained were analyzed and compared to preliminary data that used young, male Long Evans rats. The results showed that SCI injured rats took longer to acquire new memories compared to sham controls, however this was only significant in young male Long Evans rats. The differences between the two groups of animals may be due to age, gender or species. Current studies are aimed at determining why SCI did not affect learning and memory in older, female Sprague Dawley rats.

Support or Funding Information

This work was funded by a Ferris Student Research Fellowship (RAK), the Dean's Student Research Grant (RAK), a Ferris Foundation Merit Award (MBZ) and a Ferris Faculty Grant (MBZ).
Teaching Muscle Recruitment using Applied Technology in High School Students
Authors: Cole Shelby N , Hayslip Rachel , Jarrett Steven , Miller Breena , Doades Shelby , Gurovich Alvaro N
Published: November 2023

This project was designed to educate High School students on the importance of understanding muscle physiology. Forty‐seven students from 2 upper anatomy/biology classes at South Vermillion High School in Clinton, IN were exposed to the importance of muscle recruitment and how the number of motor units increases in response to an increase in weight. The primary information was presented in a PowerPoint presentation. Then, 6 Biopac MP40 were available to both classes and 4 students per system collected data. The Biopac MP40 is an interactive device used to display data using electrodes to pick‐up electrical signals released by the muscle cells (electromyography or EMG). Three electrodes were placed on each subject's dominant and then non‐dominant anterior forearm and the EMG was measured during three conditions: 1) wrist flexion without load, 2) wrist flexion with 5 Lb., and 3) wrist flexion with 10 Lb. The students were challenged to set up the Biopac MP40 system, perform the experiment, and analyze the data. Finally, during a second day of this activity, students were asked to analyze and present the collected data. The students enjoyed learning the physiological effects behind motor unit recruitment and they have ‘PhUn’ during the process.

Support or Funding Information

Supported by IU AHEC 15/16 #PIU535 and APS PhUn week program
Increased muscle myofibrillar protein breakdown rates using 3‐methyl‐histidine (tau‐mHIS) stable isotopes in a Pseudomonas aeroginosa (PM) induced hyperdynamic sepsis pig model
Authors: Ten Have Gabriella A , Engelen Marielle P , Wolfe Robert R , Deutz Nicolaas E
Published: November 2023

Rationale

Sepsis is characterized by severe muscle breakdown. Tau‐methyl‐histidine (mHIS) is generally considered a valuable marker of muscle myofibrillar protein breakdown. Quantitative measurement of the production of mHIS in urine is complicated due to factors like timing and correct sampling, meat‐free diet requirement, and differences in mHIS metabolism between species. Therefore, we measured whole body mHis production (WbRa) using stable isotope technology during acute sepsis in the pig. We also examined organ mHis metabolism to establish the site of mHIS production.

Method

In 13 conscious post‐absorptive pigs (±25 kg) with intravenous PM induced hyper‐dynamic sepsis and 9 healthy controls, we measured mHIS‐WbRa during a primed, constant and continuous infusion of L‐[tau‐2H3]‐mHIS stable isotope. Two hours before and between 14 and 18 hours during sepsis, we collected arterial plasma, and at 18 hours we obtained jejunum, jejunal mucosa scraping, ileum, liver, muscle and lung tissue. Enrichments of mHIS in plasma and intracellular pools were analyzed by LC‐MS/MS. WbRa was calculated with the tracer infusion rate and plasma mHIS steady state enrichment. Low mHIS enrichment in tissue in comparison to plasma enrichment defines a mHIS production site. Data are mean (SE) in nmol/kg bw/min; Fitting of plasma steady state enrichment curves, ANOVA and unpaired t‐tests were done by GraphPad Prism6.

Results

We found increased WbRa values of mHIS 16 to 18h after sepsis induction in jejunal mucosa biopsies: 53.7(2.1) vs 37.2(1.9), p<0.0001. At t=18 h, we found low mHIS enrichments (25 (2.4)% of plasma enrichment, P<0.0001) only in muscle. Jejunum, jejunal mucosa scraping, ileum, liver, and lung tissue show higher enrichments than plasma (112(1.6)%, NS between tissues).

Conclusion

During acute severe sepsis in the pig, the increased whole body myofibrillar protein breakdown as measured in plasma during mHIS tracer infusion is fully related to muscle myofibrillar protein breakdown. We hypothesize that the use of mHIS stable isotope technology is a reliable tool to specifically measure changes in muscle myofibrillar protein breakdown in multiple species, including humans.

Support or Funding Information

This study was supported by NIH R01GM084447 and S10RR027047
The Effect of Placental Ischemia‐Induced Hypertension on Circulating Copeptin Levels of Pregnant Rats
Authors: Palei Ana Carolina , Warrington Junie P. , Granger Joey P.
Published: November 2023

Although the etiology of preeclampsia (PE) remains unclear, evidence indicates that impaired trophoblast invasion followed by placental ischemia/hypoxia promotes the release of placental anti‐angiogenic and inflammatory factors into the maternal circulation that cause endothelial dysfunction and ultimately hypertension. Interestingly, recent reports have shown that circulating copeptin (a surrogate measurement for arginine vasopressin (AVP) synthesis and release) levels are increased in PE patients and in pregnant women who subsequently developed PE, suggesting first AVP as a potential pathogenic factor in PE and second copeptin as a predictive biomarker for PE. However, the role of AVP/copeptin in the pathophysiology of PE is unclear. The aim of this study was to evaluate the effect of placental ischemia‐induced hypertension on circulating copeptin levels. Age‐matched, timed pregnant Sprague Dawley rats were randomly assigned to the normal pregnant (NP; n=7) group or to the group that underwent reduction of uterine‐placental perfusion pressure (RUPP; n=7). On gestational day (GD) 14, RUPP rats had a silver clip (0.203 mm) placed on the abdominal aorta above the iliac bifurcation and two silver clips (0.100 mm) placed on the right and left ovarian arteries. On GD 19, mean arterial pressure (MAP) was measured consciously via carotid catheters, rats were sacrificed, individual fetuses and placentas were weighted, and blood was collected. After centrifugation of blood, serum was obtained in order to determine circulating copeptin concentration by ELISA. MAP was elevated in RUPP rats compared with NP controls (124.3±4.4 vs. 105.4±1.4 mmHg; P<0.05). In addition to reduced number of viable fetuses per rat (4.7±1.5 vs. 14.7±0.5; P<0.05), fetuses and placentas of the RUPP group were lighter than those of the NP group (1.95±0.07 vs. 2.29±0.04 g and 0.44±0.02 vs. 0.55±0.01 g, respectively; both P<0.05). However, although there was a trend for serum copeptin levels to be decreased in RUPP rats compared with NP rats, this comparison did not reach statistical significance (128.6±15.2 vs. 78.8±18.4 ng/mL; P=0.0584). We have repeated the measurement of circulating copeptin levels in another cohort of NP and RUPP rats and similar results were observed (162.4±10.9 vs. 137.5±29.2 ng/mL, respectively; P>0.05). In conclusion, our findings suggest that increased AVP/copeptin in the circulation of PE patients does not result from placental ischemia. However, our findings do not rule out the possibility that AVP may contribute to the mechanisms leading to placental ischemia in PE.

Support or Funding Information

AHA 14POST18970005, NIH HL051971, and NIH HL108618.
Alterations in Cytochrome P450 Expression and Circulating 20‐HETE Levels Contribute to Maternal Hypertension and Decreased Trophoblast Migration
Authors: Murphy Sydney , Lee Nicole , Spencer Shauna‐Kay , Wallace Kedra , LaMarca Babbette
Published: November 2023

Preeclampsia (PreE) results from an impaired vascular remodeling of the placental spiral arteries. Recent data suggests cytochromeP450 (CYP450) vasoactive eicosanoids in the etiology of the disease. Therefore, we investigated whether CYP450 expression and 20‐HETE production are elevated in preeclamptic populations and if 20‐HETE plays a role in hypertension and trophoblast migration. CYP450 enzymatic activity and circulating eicosanoids were measured in normal pregnant (NP) and PreE patients by LC/MS. In addition, the proliferative actions of 20‐HETE, a major CYP450 metabolite were investigated in an in vivo model of trophoblast syncytialisation. PreE women displayed a significant increase in CYP450 enzyme expression and 20‐HETE production relative to NP women (0.192±0.04 vs 0.119±0.06 pg/min/mg, p<0.05). Circulating 20‐HETE:EETs were statistically increased in women with PreE (1.4±0.5 vs 0.1± 0.04 mg/ml, p<0.05). BeWo cell cultures displayed an increase (45%, p<0.05) in proliferation in response to exogenous 20‐HETE (1μM), while inhibition of endogenous 20‐HETE with 17‐ODYA (10uM) and HET0016 (1nM) displayed a 10% (ns) and 75% (p<0.05) inhibition. Moreover, trophoblast migration and invasion were decreased in the presence of exogenous 20‐HETE (1μM, p<0.05). Therefore, alterations in CYP450 enzyme activity and 20‐HETE may contribute to the altered vascular remodeling seen in women PreE.

Support or Funding Information

American Heart Association 14SDG20160020
Organ and Tissue Perfusion Subsequent to Short‐Term Intermittent PTH 1–34 Administration in Mature and Middle‐Aged Mice
Authors: Lee Seungyong , Guderian Sophie , Prisby Rhonda
Published: November 2023

Introduction

Intermittent parathyroid hormone (PTH) administration is used to treat osteoporosis; however, the secondary benefits of PTH treatment are under explored. PTH elicits vasodilation in the systemic vasculature, implying improved delivery of blood flow to various tissues. Intermittent administration of PTH 1–84 and PTH 1–34 for 15 days augmented and tended to augment vasodilation of the femoral principal nutrient artery in young (p<0.05; Prisby et al., 2013) and old (p=0.07; Lee et al., ASBMR 2014) rats, respectively. Further, intermittent PTH 1–84 administration enhanced skeletal tibial perfusion in mice (Roche et al., 2014). Given the influence of PTH on the vascular system, we sought to determine if short‐term intermittent PTH administration alters organ and tissue perfusion.

Methods

Mature (6–8mon) and middle‐aged (10–12mon) male and female C57BL/6 mice were equally divided into three groups (i.e., CON, 5dPTH and 10dPTH). Mice were given either PTH 1–34 (43 μg/kg/d) or PBS (50 μl) for 5 and 10 consecutive days. Under anesthesia, fluorescent microspheres were injected into the left ventricle. At sacrifice, the myocardium, left and right gastrocnemii, solei, and kidneys were dissected. Tissue fluorescent density (TFD, AU/g) was determined as tissue fluorescence ÷ tissue mass. Data were excluded if organs and tissues contained <200 fluorescent beads (Prinzen et al., 2000, Cardinal et al., 2007).

Results

Body and tissue masses were similar among groups. There were age‐related declines (p<0.05) in perfusion measured by TFD to the right gastrocnemius (6–8mon: 20273 ± 473 AU/g vs. 10–12mon: 17181 ± 520 AU/g), and left gastrocnemius (6–8mon: 18993 ± 473 AU/g vs. 10–12mon: 16699 ± 676 AU/g). There was a tendency (p<0.10) for lower perfusion to the right kidney (6–8mon: 19409 ± 276 AU/g vs. 10–12mon: 18498 ± 385 AU/g) and left kidney (6–8mon: 19073 ± 567 AU/g vs. 10–12mon: 17503 ± 480 AU/g) in middle‐aged mice. TFD in the myocardium and right and left solei were not altered (p>0.05) by age. In contrast, TFD in other organs and soft tissues was not altered (p>0.05) by either 5 or 10 days of PTH.

Conclusion

Organ and tissue perfusion is reduced in middle‐aged vs. mature mice. However, organ and muscle perfusion did not change following short‐term intermittent PTH 1–34 administration. Additional studies are required to determine the influence of intermittent PTH administration on organ and tissue perfusion or blood flow in conscious animals.

Support or Funding Information

Supported by the University of Delaware Research Foundation Strategic Initiative Grant and NIH NIAMS (7R15AR062882‐02).
Small Molecule HIF‐2α Antagonists and Their Therapeutic Applications
Authors: Wallace Eli M.
Published: November 2023

HIF‐2α is activated in several cancers where it has been implicated in initiation, progression and metastasis. In particular, HIF‐2α plays a critical oncogenic role in von Hippel‐Lindau (VHL) disease associated kidney cancer where it drives proliferation, angiogenesis and metastasis. Additionally, HIF‐2α is activated in the tumor microenvironment where it has been demonstrated to support an immunosuppressive milieu. Human and mouse genetic data have also implicated HIF‐2α in the pathophysiology of non‐oncology diseases, most notably the development and progression of pulmonary vascular disease. Utilizing structure‐based design employing the PAS‐B* domains of HIF‐2α and ARNT (aryl hydrocarbon receptor nuclear translocator, HIF‐1β), we have discovered potent, selective, orally bioavailable HIF‐2α antagonists, including PT2385, which is in clinical development for the treatment of clear cell renal cell carcinoma. In this presentation the characterization of these small molecule antagonists will be summarized, including the molecular mechanism of HIF‐2α inhibition. The biological activity of HIF‐2α antagonists in cancer and other disease models will also be described.
Genotoxic Effect of Long‐term Cigarette and Water‐pipes Smoking on DNA Damage/Repair and Oxidative Stress Genes in Healthy Subjects
Authors: Belali Osamah M , Attafi Ibraheem M. , Korashy Hesham M.
Published: November 2023

The present study was designed to evaluate the influence of long‐term cigarette and water‐pipe (shisha) smoke on the expression of detoxifying and DNA repair genes in human subjects. The study groups consisted of 45 subjects who were divided into three groups; healthy non‐smoker group (Control), cigarette‐smoker group (Cigarette), and Shisha‐smoker group (Shisha). A questionnaire and consent form was created according to the International Union of Pure and Applied Chemistry commission (IUPAC) and distributed to all groups. The study was approved by the College of Pharmacy Research Center Ethics Committee. Fresh blood samples were collected, and the total RNA was extracted from blood using the PAXgene Blood RNA Kit for isolation and purification (PreAnalytiX, Qiagen) and mRNA expression levels of target genes were quantified by RT‐PCR. The levels of several trace elements were measured using ICP‐MS method. The demographic data showed that among the cigarette‐smoker group, 80% smoke 20–39 cigarettes/day, whereas 12% smoke more than 40 cigarette/day. With regard to water pipe smoke, the majority (46%) smoke more than 5 times/week. Blood analysis showed that a significant increase (50%) in the plasma levels of 8‐hydroxy‐2‐deoxyguanosine, a DNA damage marker, was observed in cigarette‐smoker group whereas no significant changes were demonstrated in shisha–smokers. In addition, the mRNA expression levels of DNA repair genes (OGG1 and XRCC1) were significantly inhibited in cigarette and shisha‐smoker by approximately 30% and 60%, respectively. This was associated with a marked decrease (50%) in the expression of detoxifying genes such as GST. In addition, both cigarette‐ and shisha‐smoker volunteers exhibited significant increase in the plasma concentrations of several toxic heavy metals such as Cd (130%), Pb (47%), and Ni (30%). In conclusion: these findings clearly explore the genotoxic effect of cigarette and shisha smoking on human DNA.

Support or Funding Information

National Plan for Science, Technology and Innovation. Award Number: 12‐MED3131‐02 to HMK
Ursolic acid from shea butter tree (Vitellaria paradoxa) leaf extract synergizes with β‐lactams against methicillin‐resistant Staphylococcus aureus
Authors: Catteau Lucy , Olson Joshua , Van Bambeke Françoise , Leclercq Joelle , Nizet Victor
Published: November 2023

Given the increasing emergence of antibiotic resistance, the discovery of novel therapeutic options for the treatment of infections is urgently needed. One strategy for overcoming bacterial resistance mechanisms is the use of drugs in combination, such as β‐lactams together with β‐lactamase inhibitors. Here, via a screen of direct and indirect antimicrobial activities of plants used in traditional medicine, we show that a dichloromethane extract of leaves from the shea butter tree (Vitellaria paradoxa) exhibited potent antimicrobial activity against methicillin‐resistant Staphylococcus aureus (MRSA), both alone and in synergistic combination with β‐lactams. Using dichloromethane extraction and an HPLC‐MS method, we detected ursolic (UA) and oleanolic acids (OA) in leaves and twigs of this species and further quantified the abundance of the two triterpenic acids by HPLC‐UV. We then compared the antimicrobial activity of crude V. paradoxa extracts against different clinical and reference strains of MRSA to that of purified UA and OA. Direct antimicrobial activity was determined as minimal inhibitory concentrations (MICs), and potential synergy with β‐lactams was assessed using checkerboard titration and calculation of fractional inhibitory concentration index (FICI), with FICI values ≤ 0.5 indicative of synergy and ≤ 1 defined as additivity. High UA (21%) and OA (6%) content correlated with the antimicrobial activity of the plant extract. UA was more active than OA, with anti‐staphylococcal MICs ranging from 8 – 32 compared to 64 – 256 μg/mL, respectively. Both triterpenic acids synergized with all three β‐lactams tested (ampicillin, oxacillin and nafcillin), indicating an activity targeting PBP2a +/− β‐lactamases. Analysis of the effects of UA and OA on host cells revealed that they inhibited reactive oxygen species production and chemotaxis of human neutrophils, with the plant extract only mimicking the inhibition of chemotaxis. UA, OA and the leaf extract also exhibited a protective effect against red blood cells hemolysis caused by MRSA and purified alpha‐toxin. Finally, we showed that local administration of UA provided a synergistic benefit to nafcillin in reducing lesion size and inflammatory cytokine (IL‐1β) production in a murine model of subcutaneous MRSA infection. Together, our findings reveal both antimicrobial and immune regulatory activities of shea butter tree leaf extract indicating its potential as a therapeutic agent for the treatment of antibiotic resistant infections and source of original lead compounds.

Support or Funding Information

The authors gratefully thank the Belgian Fonds de la Recherche Scientifique, the Faculty of Pharmacy and Biomedical Sciences of UCL and the Nizet lab of UCSD for financial support. Lucy Catteau has been a visiting graduate student at UCSD from June to November 2016 thanks to a Fulbright fellowship.
Development of In Vitro Screening Assay to Detect Inhibition of Mitochondrial RNA Polymerase by Anti‐viral Drugs
Authors: Sharapova Tatiana , Liguori Michael , Lai‐Zhang Jie
Published: November 2023

Many antiviral agents have been shown to have an off‐target inhibitory effect on mitochondrial DNA (mtDNA) and RNA (mtRNA) polymerases. Quantification of mitochondrial DNA relative to nuclear DNA content by qPCR has been proven as a reliable and fast approach to screen for mitochondrial DNA toxicity. Methods used to assess the function of mitochondrial RNA polymerase (POLRMT) include biochemical and cell‐based assays, and are not optimal for routine screening. Here we studied the utility of RT‐qPCR for the analysis of mitochondrial gene expression as a function of POLRMT in a standard cell culture model. HepG2 cells were treated with 5 and 10mM 2′C‐methyladenosine and 10 and 50uM balapiravir (RG1626, prodrug of 4‐azidocytosine) every 48 hours for 7 and 14 days. Both test compounds have previously been shown to be chain terminators upon mtRNA transcription. 2′ 3′‐dideoxycytidine (ddC) and fialuridine were used as positive controls and 2‐epi‐fialuridine was used as negative control for the study. Upon harvest cells were counted and extracted RNA and DNA were used for gene expression and mtDNA copy number analysis. RT‐qPCR was performed for five mitochondrially encoded (MT‐ND1, MT‐ND4L, MT‐ND5, MT‐CYB, MT‐RNR2) and two nuclear (GAPDH, ACTB) genes. Gene expression analysis in cells treated with the test compounds revealed a dose dependent decrease in the expression of mitochondrial genes when compared to untreated controls, while mtDNA copy number remained unchanged. The degree of expression of each gene had a reverse correlation with the distance between the gene positon in the mitochondrial genome and the initiation transcription site ITH2. The mRNA levels of the housekeeping genes, GAPDH and ACTB, transcribed by RNA Pol II remained unchanged across all samples and could be used for data normalization. As a conclusion, analysis of mitochondrial gene expression using RT‐qPCR can be utilized as a simple and sensitive first pass method to screen for POLRMT inhibition in vitro and would aid in identification of mitochondrial toxicity of anti‐viral compounds prior to in vivo testing.

Support or Funding Information

TS, JLZ, and ML are employees of Abbvie.

The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication
ANTIMICROBIAL ACTIVITY OF CARESOO® ELECTROLYZED WATER AGAINST BURN ASSOCIATED INFECTION
Authors: Atanacio Ronald Comedia , Mondoy Melisa , Bigol Ursela , Atanacio Jayson Reiner , Malbas Fedelino , Galit Salvacion Rosario , Dancel Manuel
Published: November 2023

Burns are a global public health problem, accounting for an estimated 265 000 deaths annually. The majority of these occur in low‐ and middle‐income countries and almost half occur in the Southeast Asia Region like the Philippines1. Infection is one of the common causes of burn death. The vulnerability to pathogens depends on different factors including the severity of burn and the mode of treatment. This study is intended to determine the in‐vitro antimicrobial activity of Caresoo®Hypochlorous Acid Water Generator (Delight Factory Korea) against pathogenic microorganisms.

Electrolyzed water produced from Caresoo®Hypochlorous Acid Water Generator was subjected against gram (+)/(−) bacteria and a fungus in 3 different concentration of salt and distilled water (1:300,3: 300, 5:300), 3 different concentration of Mac Farland and electrolyzed water(1:1, 1: 3, 3:1,), and different exposure time interval (15, 30, 60, 120, 180seconds). The 3 concentration of electrolyzed water were also used in 24 hours incubation period under disc diffusion method to determine if it can hold the inhibitory zones.

Electrolyzed water exhibited bioactivity against Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA), Methicillin Resistant Staphylococcus epidermidis, (MRSE) Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus mirabilis and Candida albicans in all concentrations < 15 seconds. No antimicrobial activity noted in 24 hrs. incubation but secondary inhibitory zones were noted in each sample concentration.

Electrolyzed water produced from Caresoo®Hypochlorous Acid Water Generator is potent on test microorganisms regardless of the concentration of salt solution, and MacFarland and electrolyzed water concentration. The bioactivity of electrolyzed water cannot sustain bacterial and fungal growth in 24 hours incubation time. This is because of its low stability; HOCl rapidly decomposes into its original state (saline solution).

This study affirms the antimicrobial activity of the electrolyzed water from Caresoo®Hypochlorous Acid Water Generator. The inapplicability of this electrolyzed water for human use as recommended by the manufacturer is the definitive intention of this study. That is to make electrolyzed water suitable in wound care and infection control. It is recommended that the electrolyzed water produce by Caresoo®Hypochlorous Acid Water Generator should be used to other pathogenic test organisms. Toxicity test is also recommended to determine its medical significance to man.

Support or Funding Information

This was initiated and financed by the authors.
Toxicological evaluation of L‐histidine methyl ester of Amphotericin B in vivo.
Authors: Sánchez‐Peña Walfred , Ortega‐Blake Ivan , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Lourdes
Published: November 2023

Amphotericin B is the most potent antimycotic known to date. However due to its large colateral toxicity, its use, although long standing, had been limited. Many attempts have been made to produce derivatives with reduced collateral damage. The molecular mechanism of polyene has also been closely studied for this purpose and understanding it would contribute to the development of safe derivatives. The aim of present work was to evaluate the toxicity of L‐histidine methyl ester of Amphotericin B in vivo. Fort he study we used male and female Wistar rats. Animals received a dose of 2 mg/kg during 26 weeks by i.p. After treatment animals were sacrificed under an anesthesia, blood and tissues samples were obtain in order to evaluate biochemical and hematological parameter, liver and renal function. Lymphocytes populations in blood, thymus and spleen were also evaluated. Tissue samples were fixed in 4% formaldehyde solution; sections were stained with H& E and subjected to histopathological analysis. In general chronic toxicity studies, L‐histidine methyl ester of Amphotericin B administered by i.p., showed no sign of toxicity in any of the analyzed blood parameters involved in rodent repeated‐dose studies. Histopathological analysis revealed the presence of morphological testicular alterations. Toxicological evaluation of L‐histidine methyl ester of Amphotericin B in vivo showed that this molecule did no produce hematological, metabolic, biochemical, immunological, renal and hepatic alterations. Solely testicular alterations were observed. The results of the toxicological research confirmed the safety profile of L‐histidine methyl ester of Amphotericin B.
Antiviral Modalities of Multivalent Ag4O4 Nanoparticles for Clinical Infectious Disease Applications
Authors: Mack Leigh Jennings , Einstein George P , Tulp Orien L
Published: November 2023

The purpose of this study was to demonstrate the modality of action, efficacy and safety of Ag4O4 nanoparticles in the application of hindering the targeting and acquisition of viruses on host cells. This study also illustrates how molecular frequency can change the dynamics of viral load and illustrate through both bench and clinical trials an empirical log reduction in viruses. In regard to medical devices, the new world of nanomedicine is just in it's infancy; over the next 10 years large jumps should occur connecting theory to actual medical devices and treatment protocols. To this end, we hope to add a small part to medicine as a whole by researching as well as developing new nanosized particle approaches and devices that can clearly make a difference in improving the effectiveness in wound healing, and in the lives of people and animals. We have already contributed in some specific areas of antibacterial applications but feel we have only begun to scratch the surface in this emerging field of medicine. The process of developing effective nanoparticles is an ever changing area of research. It is a series of thousands of small steps to reach an option for treatment. However, as technology increases at a rate that is exponential in nature, we will see faster and shorter development periods for both pharmaceuticals as well as medical devices. We will see computer processing and applications give us advantages that were never dreamed of previously. Today, we live in a world in which we can use technology productively to build faster and better new technology. In summary, this research was designed to pull together current research of the Ag4O4 nanoparticle and add another small study on how it eliminates specific viruses. Many researchers have alluded to this aspect of the particle, but none have directly addressed the modalities in which it works. To this end, we have illustrated two specific modalities of the Ag4O4 as antiviral in nature. The application of Ag4O4 nanoparticles in wound healing resulted in an apparent misleading of the targeting system on the virus and hindering glycoproteins from attaching to the host cells.

Support or Funding Information

Supported by Institutional Resources of USAT and Einstein Medical Institute