bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidant and Angiotensin AT1 Receptor Antagonist Treatment Reduced the Hypertension Induced by Chronic Intermittent Hypoxia in rats, but had Different Effects on Endothelial Dysfunction
Authors: Iturriaga Rodrigo , Krause Bernardo , Casanello Paola , Dias Ana CR , Arias Paulina , Del Rio Rodrigo
Published: November 2023

Chronicintermittent hypoxia (CIH) produces sympathetic hyperactivity and oxidative stress, leading to endothelial dysfunction and systemic hypertension. Concomitant administration of the antioxidant N‐acetylcysteine (NAC) and the AT1 receptor blocker Losartan®, before and during CIH prevented the hypertension in rats. However, it is not known if treatments with NAC and/or Losartan® are able to reduce arterial blood pressure and improve the endothelial function when rats became hypertensive due to CIH exposure. Thus, we studied if NAC and Losartan® may revert the hypertension and endothelial dysfunction in CIH‐hypertensive rats. Male Sprague‐Dawley rats (200 g) were exposed to CIH (5%O2, 12 times/h 8 h/day) or sham condition for 28 days. After 14 days, CIH‐rats were treated with NAC (400 mg/kg/day in drinking water) or Losartan® (3mg/kg/day with osmotic pump) until the day 28. Arterial blood pressure (BP) was continuously measured with radio‐telemetry in conscious rats, and at day 28 the external carotid arteries (2mm) were isolated to measure vasoactive responses to KCl (10–125 mM) and acetylcholine (ACh, 10−10–10−5 M) using wire‐myography. CIH increased mean BP (≈ 10 mmHg) after 3 days of exposure. NAC reduced the elevated BP towards normal values (100.0±2.2 mmHg) in 6–8days despite of CIH exposure, while Losartan® reduced the elevated BP in 2 days below the normal values(89.0±2.0 mmHg). External carotid arteries from CIH‐rats showed higher active contraction induced by KCl (3.8±0.4 vs. 2.3±0.3 N/m2) and reduced relaxation to ACh (11.4±1.2 vs. 24.8±3.9 %max) related to sham rats. NAC did not reverse the increased contraction induced by KCl (3.9±0.3 N/m2), and failed to restore the impaired ACh‐induced relaxation in carotid arteries (9.9±1.1%max). Losartan® also failed to reverse the increased vessel contraction induced by KCl (3.7±0.4 N/m2), but improved the ACh‐induced relaxation (40.6±5.1 %max). Present results suggest that NAC and Losartan® reverts the CIH‐induced hypertension, but have different effects on the altered endothelial and contractile artery properties in rats exposed to CIH.

Support or Funding Information

Supported by FONDEF D11I1098.
The Role of Dbx1‐Derived Cells in the Generation of Inspiration In Vivo
Authors: Baertsch Nathan Andrew , Ramirez Jan Marino
Published: November 2023

The preBötzinger Complex (preBötC) in the ventral medulla generates the inspiratory phase of breathing. Cells expressing the transcription factor Dbx1 during prenatal development have been proposed to give rise to the excitatory neurons that form the rhythmogenic core of the preBötC. When isolated in brainstem slices from neonatal DBx1/Ai27 mice, optogenetic stimulation of Dbx1‐derived cells reliably elicits inspiratory population bursts from the preBötC. However, it has recently been reported that presynaptic depression occurring during endogenous preBötC population bursts results in a refractory period lasting ~2 seconds, during which Dbx1 cell stimulation cannot elicit a burst (Kottick and Del Negro, 2015). In vivo, anesthetized mice breathe at a frequency that is typically 5 times faster (~1Hz) than that observed in vitro (~0.2Hz). We hypothesized that higher respiratory frequencies in vivo would minimize the effect of Dbx1 cell stimulation in anesthetized Dbx1/Ai27 mice. Using a ventral approach, the occipital bone was removed to expose the medulla, and the hypoglossal (XII) nerve was isolated to record inspiratory activity while mice were allowed to spontaneously breathe 100% O2. An optical fiber was placed unilaterally on the ventral surface of the medulla over the preBötC. During a 10 second continuous light pulse, a modest laser power‐dependent increase in respiratory frequency was observed (15–20% change from baseline at ~9mW). Dbx1 cell stimulation entrained the inspiratory rhythm when 200ms light pulses were delivered at or near the endogenous respiratory frequency (~1Hz); however entrainment was lost when the stimulation frequency was altered by ~ ±0.3Hz. Dbx1 cell stimulation had varied effects when light pulses were delivered at random time points in the inspiratory cycle. If a light pulse coincided with inspiration, the cycle duration was lengthened. When stimulation occurred during the first ~3/4 of the inspiratory cycle there was no effect on cycle duration. A burst could only be elicited and the cycle duration was shortened if stimulation occurred during the final ~1/4 of the cycle. To test if respiratory frequency influences the effect of Dbx1 stimulation, animals were placed on a ventilator and hyperventilated to slow and eventually stop breathing during random light stimulation. As breathing slowed, the probability of eliciting inspiration with Dbx1 stimulation increased; during apnea, inspiratory activity was reliably evoked with each stimulation. We conclude that Dbx1 stimulation in vivo can alter the respiratory pattern, but the effects are subtle compared to stimulation of Dbx1 in vitro. We hypothesize that the decreased Dbx1 influence is due to the elevated respiratory drive and frequency present in the intact animal.

Support or Funding Information

NIH RO1 HL 126523
Recruitment of Eosinophils to Lungs and Their Role in Vagally Mediated Airway Hyperreactivity Depends upon TNFa and Sensitization Status
Authors: Wicher Sarah A. , Jacoby David B. , Fryer Allison D.
Published: November 2023

Background

Fifty‐percent of Americans are routinely exposed to high levels of ozone. Inhaled ozone transiently reduces lung function in healthy individuals, and induces severe exacerbations of airway disease in asthmatics. Since many asthmatics have airway eosinophilia, we evaluated the interaction of ozone and eosinophils in lungs of guinea pigs. We found that eosinophils mediate ozone‐induced airway hyperreactivity 1 day after exposure, but were paradoxically protective against airway hyperreactivity at 3 days. This protective function of eosinophils was lost in guinea pigs with airway eosinophilia induced by ovalbumin sensitization.

Objective

Since ozone is known to induce tumor necrosis factor‐a (TNFa) production, we characterized the role of TNFa on eosinophil production, function, and migration in non‐sensitized and sensitized guinea pigs exposed to ozone.

Methods

Non‐sensitized and ovalbumin‐sensitized (4.2 mg i.p., day 1, 3 and 5) guinea pigs were treated with 5‐bromo‐2′‐deoxyuridine (BrdU; 100mg/kg i.p., day 21) to label newly dividing cells. Animals were then exposed to air or ozone (2.0ppm, 4h) and treated with BrdU daily thereafter (50mg/kg i.p.). Some animals were given the TNFa antagonist, etanercept (3mg/kg i.p.) 3 hours before ozone exposure. Three days later, guinea pigs were anesthetized, paralyzed, and ventilated at constant volume and flow. Bronchoconstriction was measured as an increase in pulmonary inflation pressure (mmH20) in response to electrical stimulation of the distal ends of cut vagus nerves (2–25hz, 10V, 0.2msec 5sec on 60sec off). Inflammatory cells were harvested from bone marrow, blood, and lungs.

Results

Ozone caused eosinophil hematopoiesis in bone marrow, recruited newly divided eosinophils to lungs, and induced airway hyperreactivity to vagal stimulation in non‐sensitized guinea pigs. Sensitization potentiated ozone‐induced airway hyperreactivity, however, eosinophil hematopoiesis and recruitment of newly divided eosinophils to lungs failed to occur. Blocking TNFa inhibited eosinophil hematopoiesis and recruitment to the lungs in non‐sensitized animals, loss of eosinophils further potentiated ozone‐induced airway hyperreactivity. Blocking TNFa had no effects in sensitized guinea pigs exposed to ozone.

Conclusions

These data demonstrate a novel protective role for eosinophils in vagally‐mediated bronchoconstriction in non‐sensitized animals. In non‐sensitized guinea pigs TNFa mediates ozone induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs where they inhibit vagally mediated bronchoconstriction. These beneficial eosinophils are lost with sensitization.

Support or Funding Information

Health Effects Institute (ADF), ES017593 (ADF), HL113023 (DBJ), AR061567 (DBJ), HL124165 (DBJ)
DNA Mismatch Repair Protein Interactions on Mismatch‐Containing DNA
Authors: Dos Santos Nilse Lene Goncalves , Gupta Dipika , Heinen Christopher D
Published: November 2023

Lynch syndrome (LS) is the most common form of hereditary colon cancer. Stemming from the inheritance of mutations in the DNA mismatch repair (MMR) genes, LS increases lifetime risk for colonic and certain extra‐colonic cancers. Unlike sporadic cancers, LS cancers have an average age of onset of 40 years‐old. MMR improves replication fidelity by recognizing and coordinating the repair of single base‐pair mismatches and small insertion/deletion loops. When deficient for MMR, cells acquire a mutator phenotype that increases the rate of mutation in other important oncogenes and tumor suppressors. This eventually leads to cancer development. The MMR pathway is composed of four main genes: MSH2, MSH6, MLH1 and PMS2. MSH2 and MSH6 proteins form a heterodimer that recognizes mismatches on DNA and recruits a heterodimer of MLH1 and PMS2. Together, they signal for Exonuclease I to remove the erroneous region on the nascent strand. Previous studies have indicated that MSH2‐MSH6 bound to ATP forms a sliding clamp on the DNA that is required for its interaction with MLH1‐PMS2. However, the nature of this interaction for downstream signaling remains elusive. Current models propose that MSH2‐MSH6 and MLH1‐PMS2 form a quaternary structure on DNA and function as a unit to promote repair, but there is no direct evidence for this structure. We therefore explored the hypothesis that the two heterodimers form stable quaternary structures that function as a unit to signal excision. Using an in vitro system with biotinylated mismatch‐containing DNA and streptavidin coated magnetic beads, we isolated MMR protein‐DNA complexes and studied their interaction and stability. We confirmed that MSH2‐MSH6 can form sliding clamps on mismatch‐containing DNA in the presence of ATP. We next found that while initial loading of MLH1‐PMS2 on mismatch‐containing DNA requires these MSH2‐MSH6 sliding clamps, MLH1‐PMS2 remains stably bound to DNA even after MSH2‐MSH6 has dissociated. We also found that MSH2‐MSH6 is less stable on DNA in the presence of MLH1‐PMS2. These results suggest that MSH2‐MSH6 loads MLH1‐PMS2 onto mismatch‐containing DNA and that a stable, quaternary unit may not necessarily be required. These studies will improve our knowledge of the mechanism by which MMR maintains genomic stability and, ultimately, improve prevention and treatment of MMR‐defective cancers.

Support or Funding Information

Connecticut Institute for Clinical and Translational Science (CICATS)
Development of Physiologically Relevant Methods for Assessing Inhibition of Glycoprotein Virulence Factors by Small Molecules in Human Pathogen Campylobacter jejuni
Authors: Zamora Cristina Y. , De Schutter Joris W. , Chen Kelly W. , Griffith Linda G. , Imperiali Barbara
Published: November 2023

Campylobacter jejuni is a major cause of gastroenteritis, often resulting in death of the young, elderly and immunocompromised. Campylobacteritis also may result in complications leading to autoimmune and neurological disorders such as Guillain‐Barre syndrome. In this organism, a consistent structure‐function relationship has emerged in recent years: the importance of N‐linked protein glycosylation for bacterial pathogenic and colonization behaviors in avians and mammals. However, the biochemical roles and molecular mechanisms of C. jejuni N‐glycans in the infection of human intestinal epithelium are not understood. Our laboratory has developed small molecule inhibitors of critical biosynthetic enzymes in the N‐glycosylation pathway (pgl pathway), acting on their respective targets with up to nM affinity in vitro. Assessing the efficacy of small molecule inhibitors of protein glycosylation in vivo with microaerophilic Gram‐negative organisms has proven difficult. To this end, we have pursued several efficient, quantitative and broadly applicable strategies for assessing glycosylation inhibition and any downstream effects on bacterial pathogenicity. We have developed conditions for C. jejuni culture in the presence of small molecule inhibitors and subsequent quantification of protein glycosylation via immunoassay. In addition, inhibitors of biosynthetic enzymes in the pgl pathway are also assessed in vivo utilizing E. coli strains expressing C. jejuni protein glycosylation pathway enzymes, providing additional insight into the effects of outer membrane composition on inhibitor permeability. Lastly, in collaboration with the Griffith lab, we are developing methods for the characterization of C. jejuni pathogenicity using a unique co‐culture platform bearing polarized human gut epithelial monolayers comprised of enterocyte (Caco2), mucin‐producing goblet cells (HT29‐MTX), and immune cells (dendritic cells). This “gut chip” platform, providing histological, fluorometric and immunological readouts, will be critical for monitoring the adhesion and invasion of C. jejuni strains with attenuated glycosylation patterns and can be easily be turned toward the investigation of pathogenicity in other organisms. These multifaceted approaches will provide useful molecular detail and phenotypic characterization toward the further development of this new class of inhibitors and a greater understanding of mechanisms behind pathogenesis.

Support or Funding Information

This work is supported by the National Institutes of Health (R01‐‐GM097241 and R03‐MH096549 to B.I.) and the Belgian American Exchange Fellowship.
Antioxidant Capacity of Selected Vegetables, Nuts and Berries and Their Effects on Interferon Gamma Produced By Salmonella Typhi Parasitized Female Albino Rats
Authors: AKANDE IDOWU SULE , OLA MOJOYINOLA JOANNA , SALAKO FAITH OLUGBEMILEKE
Published: November 2023

Almost all synthetic drugs possess adverse side effects in human body. Natural processes by which the immune system can be improved to combat diseases apart from vaccination remain the best options of preventing and terminating diseases. We investigated the antioxidant capacity and effects of selected vegetables, nuts and berries (Vernonia amygdalina, Telfairia occidentalis, Amaranthus hybridus, Juglans regia, Anacardium occidentale, Elaeis guineensis, Fragaria ananassa and Vitis vinifera) on antioxidant system and potentials to improving the immune system responses in a typhoid fever model disease in rat. Extracts of selected vegetables, nuts and berries obtained by the use of the sohxlet extractor were orally pre‐administered to adult female albino rats (n=30), of Wistar strain weighing between 120 to 190 g and divided into 6 groups, before inoculation of Salmonella typhi. Widal test was done to ascertain typhoid induction. Blood samples were collected and used for haematological parameters count and determination of oxidative stress biomarkers activities (GSH, CAT, SOD and MDA), liver and kidney function biomarkers activities using automated analyzer and concentration of interferon gamma (INF γ) using Enzyme linked immunosorbent assay (ELISA). Salmonella typhi colony count was done using faecal samples. Phytochemical screening and the antioxidant activities of the extracts were also determined. Pre‐administration of the extracts maintained steady weight of the parasitized albino rats. Extracts of berries and nuts had the highest in vitro antioxidant capacity compared to the controls. Vegetables improved quality of red cell indices. In vivo antioxidant assay indicated a statistically significant value (p≤0.05) for the extracts of nuts and berries in the levels of GSH compared to the controls. There were no statistically significant difference (p≥0.05) in the levels of liver and kidney function biomarkers between the test and the control groups. Nuts and berries reduced S.typhi colonies and recorded lowered production of INF γ compared to other groups. We conclude that the combined consumption of vegetables, berries and nuts improve immune responses and reduction of oxidative stress.

Support or Funding Information

SELF SPONSORED
Anti‐inflammatory effect of caffeine by regulating NF‐κB activation in murine macrophage
Authors: Hwang Ji‐Hyun , Koh Eun‐Jeong , Lee Yeon‐Joo , Chio Jia , Song Ji‐hyeon , Seo Young‐Jin , Lee Boo‐Yong
Published: November 2023

Caffeine is white crystalline xanthine alkaloid and found in the seeds, the coffee plant and the leaves of the tea bush. In this study, we determined whether caffeine exerts anti‐inflammatory effects on lipopolysaccharide (LPS)‐induced inflammation in RAW264.7 cells.

To examine the effects of caffeine on LPS‐induced inflammation, RAW 264.7 cells were treated with various concentrations of caffeine in the presence or absence of LPS.

Our data showed that caffeine decreased the LPS‐induced inflammatory mediator, nitric oxide (NO). Caffeine treatment also reduced the expression of pro‐inflammatory genes inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), interleukin (IL)‐3, IL‐6 and IL‐12, and decreased IL‐6 secretion and phosphorylated p38MAPK expression in LPS‐treated RAW264.7 cells. Caffeine inhibited the nuclear translocation of nuclear factor κB (NF‐κB) via IκBα phosphorylation. In addition, caffeine inhibited LPS‐induced NO production in zebrafish.

Caffeine may inhibit LPS‐induced inflammatory responses in murine macrophage by regulating NF‐κB activation and MAPK phosphorylation.
Exploring the Potential for Technology‐Based Nutrition Education for Alaska Native WIC Recipients in Rural Southwest Alaska
Authors: Power Julianne Mary , Bersamin Andrea
Published: November 2023

Alaska Native People are disproportionately impacted by nutrition‐related diseases, and those living in remote communities (e.g. off the road system) are particularly hard to reach with traditional nutrition education. Media technologies, including the Internet and cell phones, provide novel ways to reach rural populations; however, these technologies have only been recently introduced to remote Alaska Native communities. The purpose of this study was to quantify media technology use among Alaska Native WIC recipients in Southwest Alaska to understand the feasibility of technology‐based nutrition education. Surveys were mailed to 967 WIC authorized representatives in the Bethel Census Area using the Tailored Design Method. The response rate was 37.4% (N=362). Most participants were Alaska Native (99.1%) women (97.5%) between 18–29 years old (56.6%) with a high school education (61%). Use of media technology was extremely common; 80.2% of respondents use the internet, 78.1% of respondents have a smart phone, and 89.5% of respondents use Facebook. A multiple regression analysis found older respondents had lower media technology use scores (β = −.25, t(323) = −4.74, p < .05), while more educated respondents had higher media technology use scores (β = .18, t(323) = 3.40, p < .05). Additionally, online health information seeking was prevalent; 48.2% of respondents look for nutrition information online every few months or more frequently, and 73.7% of respondents are somewhat or very likely to search for answers to nutrition‐related questions online. Potential barriers to receiving nutrition information on a phone or computer include slow internet (51.0%), no computer access (42.1%), and the high cost of internet (35.1%). These data demonstrate that the prevalence of media technology use is similar to national estimates and that technology is a viable way for WIC and other Federal Food Assistance Programs to deliver nutrition education to Alaska Native People living in remote communities.
Enhanced carotid body chemoreflex function drives respiratory‐sympathetic coupling in systolic heart failure
Authors: Del Rio Rodrigo , Marcus Noah J , Schultz Harold D
Published: November 2023

Enhanced carotid body (CB) chemoreflex sensitivity in systolic heart failure (HF) has been linked to oscillatory breathing patterns and autonomic imbalance. Importantly, breathing pattern alterations and heightened chemoreflex‐mediated sympathetic nerve activity are both associated with higher morbidity and mortality in HF. Therefore, the objective of this study was to determine whether CB denervation (CBD) would improve respiratory stability and decrease sympathetic drive in HF. In rabbits with pacing‐induced HF, we measured resting breathing and renal sympathetic nerve activity (RSNA), before and after CBD. In addition, we assessed respiratory‐sympathetic coupling using power spectral analysis and the magnitude of the peak coherence function between tidal volume (Vt) and RSNA frequency spectra. Minute ventilation and RSNA responses to acute hypoxia were significantly augmented in HF compared to controls and were abolished by CBD. Resting RSNA was greater in HF compared to control animals (43 ± 5% max vs. 23 ± % max, p<0.05). HF animals after CBD displayed resting RSNA levels similar to control animals. HF animals displayed altered breathing patterns characterized by oscillatory breathing and increases in the incidence of apneas/hypopneas. Remarkably, CBD totally normalized oscillatory breathing patterns in HF animals. Peak coherence between RSNA and Vt signals was increased in HF animals (control 0.49 ± 0.05; HF 0.79 ± 0.06), and was significantly attenuated by CBD (0.59 ± 0.05, p<0.05 for all comparisons). Our data show that enhanced CB‐mediated chemoreflex drive contributes to tonic elevations in RSNA and breathing instability in HF. Additionally, a tonic increase in CB afferent discharge may also play an important role in strengthening a link between respiratory and sympathetic neural outflow in HF.

Support or Funding Information

Supported by Fondecyt 1140275, NIH PO1‐HL62222 and NIH F32 HL108592‐01A1.
Learning About Physiology is PhUn at an Elementary School in Ponce, Puerto Rico
Authors: Cruz Myrella L , Hernandez Siomara , Isidro Raymond A , Hernandez Anixa , Vazquez Bethzaly , Diaz Hector , Lopez Abdon , Torres‐Reveron Annelyn , Colon Maria , Appleyard Caroline B
Published: November 2023

Since 2010, members of the Puerto Rico Physiological Society at PHSU‐PRI have been involved in PhUn week, the annual K‐12 outreach program sponsored by The American Physiological Society. As part of the PhUn week 10th anniversary celebration, graduate students and staff from Ponce Health Sciences University ‐ Ponce Research Institute (PHSU‐PRI) took the “PhUn” to the elementary school students at Academia Santa María Reina in Ponce, Puerto Rico.

Aim

To assess the impact of a PhUn week activity in 3rd and 6th graders.

Methods

A team of graduate students, staff and faculty from PHSU‐PRI organized a half‐day workshop for a group of thirty‐eight 8–11 year old students and two science teachers. The workshop consisted of three short presentations (gastrointestinal system, circulatory system and nervous system) which included integrated videos, optical illusions, experiments and demonstrations. Next, the children were divided into smaller groups to rotate through five interactive stations featuring: histology, anatomy, emulsification, a life‐size neuron model and DNA extraction. At each station the PHSU‐PRI helpers explained what the purpose of the activity was and were available to answer questions. All activities were conducted in Spanish. Pre‐ and post‐tests evaluated student learning. Data was analyzed using an unpaired Student t‐test and chi‐square test.

Results

Students were clearly excited, demonstrated lots of enthusiasm, and were very proactive in all of the activities asking a lot of questions. A significant increase in the number of correct answers for all questions occurred following the workshop (gastrointestinal system p<0.001, circulatory system p<0.05 and nervous system p<0.001).

Conclusions

Short presentations and engagement of students from 3rd and 6th grade in interactive hands‐on activities in their native language helped to significantly increase their knowledge of basic physiological concepts.

Support or Funding Information

Supported in part by R25GM082406
Developing Integrated Learning Objectives in Physiology and Pathology for a Spiral System Based Curriculum
Authors: Benoit Joseph N , Selinfreund Richard , Osborne David L , Rodenbaugh David W , Woods Michael E , Das Mita
Published: November 2023

Current trends in preclinical medical education are moving away from traditional discipline based courses and towards integrative learning sessions centered around organ or system based courses. Elaborate mapping approaches are often developed to demonstrate linkages between and within courses. Yet curriculum design in preclinical medical education often fails to model interdisciplinary reasoning or provide sufficient time for students to practice integrative reasoning skills. The purpose of this report is to describe an approach to developing an integrated preclinical physiology/pathology curriculum by a faculty at a new college of osteopathic medicine. The Burrell College of Osteopathic Medicine will welcome its inaugural class in Fall 2016 into an educational environment based upon a spiral curriculum. The preclinical years will consist of two passes through the all organ systems of the human body. Faculty in our department are working to develop integrated sessions that form clear connections between normal physiology and the pathologic mechanisms of disease. In doing so, we are identifying core principles that can be revisited throughout in the organ system courses(Selinfreund, et al, 2016). Fabricated longitudinal clinical patient profiles guide the integration of course content (Osborne, et al, 2016). To assist students with learning within the integrated physiology and pathology framework, we are incorporating core principles of physiology, pathology and osteopathic medicine into spirally integrated learning objectives that function to guide student learning and ensure congruent faculty participation throughout preclinical curriculum. In this model, core principles are derived from discipline specific learning objectives (e.g., American Physiological Society [APS]; Group for Research in Pathology Education [GRIPE]]; National Board of Osteopathic Medical Examiners [NBOME]) and merged into session objectives that focus learning outcomes on application of knowledge. The objectives are designed to promote scaffolding within the spiral curriculum and are linked to the principles of osteopathic medicine. Session objectives in first year system courses are built around weekly themes that integrate core concepts centered on physiology and general pathology. Session objectives in the second year are built around systemic pathology and relevant physiological material in clinical medicine textbooks. The multi‐organ nature of systemic pathology will be underscored using longitudinal virtual patients. The result is a fully integrated physiology/pathology curriculum within a program of osteopathic medical education.
Blocking Peripheral Opioid Receptors with Naloxone Methiodide Prevents Acute and Chronic Training‐Induced Analgesia in a Rat Model of Fibromyalgia
Authors: Williams Emily R , Ford Caleb M , Simonds J. Grant , Leal Anna K
Published: November 2023

Data from our lab and others has shown that chronic treadmill training decreases mechanical hyperalgesia in a rat model of fibromyalgia. Opioid receptors have been shown to contribute to this training effect. Our study investigated the contribution of peripheral opioid receptors to acute and chronic exercise‐induced analgesia using naloxone methiodide, a nonspecific opioid receptor antagonist that does not cross the blood‐brain barrier. Fibromyalgia, characterized by chronic widespread pain, was induced in female Sprague Dawley rats (n=10) by acidic saline injections into the right gastrocnemius muscle (0.1 mL; pH 4). Trained + treated rats received injections of naloxone methiodide (120 μg) into both gastrocnemius muscles prior to moderate‐intensity treadmill running (4 weeks; 5 days/week; 30 min/day). Hindpaw pain withdrawal threshold (PWT), a measure of mechanical hyperalgesia, was quantified with von Frey filaments before and after each running bout. PWT was also measured daily in a subgroup of sedentary control rats. Selectively blocking peripheral opioid receptors prior to running prevented acute and chronic training‐induced increases in PWT (p > .05). Further, PWT was significantly lower in the trained + treated group (week 1: 30 ± 6 mN; week 2: 26 ± 4 mN; week 3: 22 ± 4 mN; week 4: 21 ± 3 mN) compared to sedentary rats (week 1: 64 ± 6 mN; week 2: 57 ± 5 mN; week 3: 51 ± 4 mN; week 4: 40 ± 5 mN; all p < .05). Results from sedentary rats suggest that preventing endorphin release by limiting physical activity maintains increased pain sensitivity. Results from trained + treated rats also show peripheral opioid receptors contribute to acute and chronic exercise‐induced analgesia.
Effect of Core Temperature during Exercise on Markers of Muscle Injury and Oxidative Stress
Authors: Starnes Joseph , Herberg Timothy , Lee Kuan‐Ting , Hixson Lindsay , Vervaecke Lauren , Goldfarb Allan
Published: November 2023

Exercise of sufficient intensity carried out at room temperature significantly increases core temperature (Tc) in rats. In this study we tested the hypothesis that preventing the exercise‐induced rise in Tc will decrease markers of skeletal muscle injury and oxidative stress that are associated with prolonged exercise bouts. Following one week of familiarization to a motor driven treadmill in a 23°C room, male, 10‐wk‐old Sprague‐Dawley rats were assigned to a sedentary (S) group (n = 15) or to groups that ran on a treadmill for 90 min at a speed of 20 m/min up a 6° grade in either a 23°C room (23R) (n = 10) or an 8°C room (8R) (n = 9). Tc was monitored via telemetry. Blood was collected from the tail vein at rest or immediately post exercise followed by removal of gastrocnemius and plantaris muscles. An aliquot of blood was immediately acidified to stabilize oxidized (GSSG) and reduced (GSH) glutathione and these markers of oxidative stress were subsequently measured by HPLC with electrochemical detection. Serum was prepared in the remainder and used to measure lactate dehydrogenase activity (LDH) as an indicator of muscle injury. Glycogen content and additional markers of oxidative stress (TBARS and aconitase activity) were measured in skeletal muscles. Tc was unchanged from rest in 8R and increased 2.5°C to 40.5±0.2°C in 23R (P<0.05). Compared to S, serum LDH in 23R increased 105% (P<0.05) but did not change in 8R (P>0.05). Similarly, blood GSSG content and GSSG/total glutathione increased 132% and 180%, respectively, in 23R (P<0.05) and did not change in 8R compared to S. TBARS and aconitase activity in muscles were not different among groups (P>0.05). Gastrocnemius glycogen was depleted approximately 40% in both 23R and 8R compared to S (P<0.05). We conclude that elevated Tc plays a major role in aerobic exercise‐induced increases in oxidative stress and muscle injury.

Support or Funding Information

Supported by UNC‐Greensboro Safrit Research Measurement Fund
Effects of intrarenal afferent stimulation by Bradykinin on Renal Sympathetic Nerve Activity: Tonic Inhibition facilitating Sodium and Water Excretion
Authors: Hindermann Martin , Ditting Tilmann , Rodionova Kristina , Loosen Sonja , Ott Christian , Schmieder Roland E , Amann Kerstin , Veelken Roland
Published: November 2023

Bradykinin may increase renal sodium excretion by decreasing ENaC activity in tubuli. Renal afferent nerves (RANs) play an important role in the modulation of renal sympathetic nerve activity (RSNA) involved in renal sodium handling. We recently reported tonic sympatho‐ inhibitory action due to intrarenal afferent nerve stimulation by the TRPV1 agonist capsaicin. Since BK is known to augment TRPV1 effects we hypothesized that intrarenally applied BK would rather inhibit than excite RSNA thus also facilitating sodium and water excretion by neurogenic pathways.

3 groups of anesthetized rats (n=8, each) were equipped with arterial and venous catheters for blood pressure (BP) and heart rate (HR) recording and drug application; left sided renal arterial catheter for intrarenal administration (IRA) of Bradykinin (BK; group 1) or the TRPV1 agonist capsaicin (CAP, pos. control; group 2) to stimulate RANs (CAP 3.3, 6.6, 10 and 33*10−7M, 10μl each; BK 10*10−6M, 20μl and BK 10*10−5 M; 2.5, 5, 10μl at 30, 45, 60, 75min). Normal saline (IRA NS) served as neg. control (group 3); right sided electrode for RSNA recording; After 105min the NK1‐receptor blocker RP67580 (10*10−3M, 15μl). IRA BK and IRA CAP decreased RSNA from 4.2±0.8 to 1.3±0.2μV*sec (BK, P<0.01) and 3,6±0,5 to 0.9±0.2μV*sec (CAP, P<0.01) while maximum inhibition was reached within 70 minutes. IRA NS had no effect. Tonically suppressed RSNA (CAP & BK group) was transiently but completely unmasked by systemic (IV) administration of the NK1‐blocker (1.6±0.5 to 8.6±2.9μV*sec; P<0.01 (BK); 1.0±0.2 to 6.1±1.5μV*sec; P<0,01 (CAP)). IRA BK was associated with short lived (1–2 sec) increases of RSNA (up to 13.3±2.4μV*sec; P<0.01). The effect of intrarenal BK was similar to CAP. Thus, the net effect of intrarenal stimulation of RANs by BK is sympatho‐inhibitory in nature. The similarity of NK1‐blocker effects indicates a common final pathway of intrarenal afferent stimulation. Bradykinin by inducing renal sympathodepression rather increases sodium and volume excretion likely enhancing the effect of salt‐sensitive inhibition of ENaC activity by bradykinin in the kidney.

Support or Funding Information

Deutsche Forschungsgemeinschaft
Effects of Transforming Growth Factor‐Beta (TGF‐β) Receptor I Inhibition on Renal Biomarkers and Fibrosis in Unilateral Ureteral Occluded (UUO) Mice.
Authors: Richards Toni L. , Minor Kenneth , Plato Craig F.
Published: November 2023

Renal fibrosis is a characteristic of progressive renal diseases leading to end‐stage renal failure. Induction of renal fibrosis via the mouse unilateral ureteral occlusion (UUO) model occurs within 5 days making it a high throughput model to evaluate novel therapeutic targets. Transforming growth factor‐beta (TGF‐β) is a key signaling molecule in the pathogenesis of renal fibrosis mediated through its actions on its receptor, Activin A Receptor‐Like Kinase (ALK5). Previously, we have shown the anti‐TGF‐β antibody (1D11; clone 1D11.16.8) attenuates UUO‐induced renal fibrosis. Here, we evaluated and compared the dose‐dependent ability of an orally‐available ALK5 inhibitor (SB‐525334) to attenuate UUO‐induced tissue biomarkers. To minimize the number of necessary control groups and ensure all mice were handled similarly, all mice received intraperitoneal injections of PBS or 3 mg/kg 1D11 QD d‐1 through d4 and oral gavage administration of vehicle or SB‐525334 (10, 30 or 100 mg/kg) QD d‐1 through d5. On d0, mice (male, CD‐1, 8–9 weeks age) underwent sham (n=8) or permanent total ligation of the right ureter (UUO, n=12–13/group) via aseptic laparotomy. Five days post‐UUO or sham surgery, renal tissue from sham or obstructed kidney was collected for analysis of profibrotic mRNA biomarker expression, TGF‐β content, hydroxyproline content and renal fibrosis as expressed as collagen volume fraction (CVF; quantitated via histological image analysis of Picrosirius Red stained mid‐transverse kidney sections). Consistent with a pathophysiological response to ureteral obstruction, UUO reliably (100%) elicited hydronephrosis. UUO increased renal cortical mRNA expression of several fibrotic markers—fibronectin‐1 (FN‐1; 3.4±0.5 fold induction from sham), alpha‐smooth muscle actin (α‐SMA; 0.9±0.2 fold), collagen 1A1 (Col1A1; 12.7±4.2 fold) and collagen 3A1 (Col3A1; 5.8±0.8 fold) as well as increased renal cortical TGF‐β content (2.8±0.2 fold), hydroxyproline content (1.6±0.1 fold) and CVF (8.8±0.9 fold). 1D11 attenuated UUO‐induced increases in FN‐1 (45.5±5.6%), Col1A1 (61.3±9.6%) and Col3A1 (48.6±8.2%) mRNA expression, TGF‐β content (38.8±5.9% inhibition), hydroxyproline content (23.4±6.5%), and CVF (42.6±5.7%). High dose (100 mg/kg) SB‐525334 attenuated UUO‐induced increases in FN‐1 (77.8 ±6.4%), α‐SMA (111.0±23.6%), Col1A1 (77.1±4.1%) and Col3A1 (56.0±7.8%) mRNA expression, as well as TGF‐β content (35.2±6.3%), hydroxyproline content (46.5±4.4%), and CVF (73.7±4.8% inhibition). The current data demonstrate the importance of TGF‐β signaling in fibrotic consequences of ureteral obstruction. Additionally, UUO‐induced renal injury can be modulated by a parenterally administered biological agent (1D11) as well as by an orally administered small molecule (SB‐525334) thus eliminating the need for multiple dosing routes in future UUO studies where a reference agent is utilized.
Effects of Electronic Cigarette Vapor on Body Mass, Food Intake, and Body Composition
Authors: Breit Matthew , Hoskinson Hannah , Pitzer Chris , Wu Zhongxin , Bryner Randall , Olfert I.M
Published: November 2023

Nicotine and cigarette smoking is known to promote weight loss and suppress appetite. Since becoming available in 2007, the use of electronic cigarettes (E‐cig) has increased dramatically in the US, however there are still very few studies that examine the long‐term consequences of e‐vapor, particularly in the context of appetite regulation or weight management. The present study was devised to compare the effects of cigarette smoke and E‐cig vapor exposure on food intake, body weight, and body composition in mice. We hypothesized that E‐cigs would elicit similar changes on body mass, adiposity, and food intake as conventional cigarettes (i.e. 3R4F reference cigarette). Female C57BL/6 mice were exposed to filtered room air (n=15), mainstream smoke from 3R4F reference cigarette (n=15), or cappuccino E‐cig vapor (n=15). We report assessments in body mass, food intake, and body composition following daily exposure (4 h/d, 5d/wk) for up to 6 months. Food and water were administered ad libitum. 3R4F, E‐cig and control mice increased body mass by 15%, 30%, and 31%, respectively, over 6 months. 3R4F mice had 15–16% lower body mass (24.9±0.55 g, p<0.01) compared to E‐cig (28.2±0.77 g) and controls (27.7±0.72 g). 3R4F exposed mice also exhibited reduced total body fat (2.74±0.25 g, p<0.01) compared to E‐cig (4.10±0.42 g) and controls (4.18±0.40 g). E‐cig mice food consumption was significantly increased compared to 3R4F (12.4±0.8 vs 9.5±0.7 g, respectively; p<0.05) but not significantly different compared to controls (12.4±0.8 vs 10.5±0.8 g, respectively, p=0.12). Unlike conventional cigarettes, we found that our E‐cig exposed mice did not elicit reductions in total body or adipose mass. This suggests the effects of E‐cig may not be the same as that occurring with traditional tobacco cigarettes, or that the exposure to nicotine and/or other chemicals in the E‐cig liquid elicits a different response on appetite or feeding behavior. Further studies are needed to evaluate the effect that flavorings and/or the compounds produced in E‐cig vapor exert on metabolism and the hypothalamic appetite neurosystems.

Support or Funding Information

NIH Grant P20GM103434 (West Virginia IDeA Network for Biomedical Research Excellence) and Marshall‐WVU Health Initiative Award
Inhibiting G protein bg signaling blocks prostate cancer stem cell‐like properties and enhances the efficacy of paclitaxel
Authors: Vaddi Prasanna K. , Paudyal Prakash , Xie Qing , Chen Songhai
Published: November 2023

Aberrant activation of G protein‐coupled receptors (GPCRs) is implicated in prostate cancer progression. As a treatment strategy, however, targeting GPCR has been challenging because prostate cancer cells overexpress many GPCRs, which have redundant roles in cancer progression. To address this redundancy, we tried to block signaling via a hub through which multiple GPCRs converge — the G‐protein Gβγ subunits. Inhibiting signaling via Gβγ by overexpressing the scavernger, Gαt, or the Gβγ inhibitor, gallein, in several castration‐resistant prostate cancer cell lines (i.e. PC3, DU145 and 22Rv1), impaired cell growth and migration in vitro, and halted tumor growth and metastasis in nude mice. The blockade of Gβγ signaling also diminished prostate cancer stem cell‐like activities, by reducing tumorsphere formation in vitro and tumor formation in a limited dilution assay in nude mice. Furthermore, Gβγ blockade enhanced the sensitivity of prostate cancer cells to paclitaxel treatment, both in vitro and in vivo. Together, our results identify a novel function of Gβγ in regulating prostate cancer stem‐cell‐like activities, and demonstrate that targeting Gβγ signaling is an effective approach in blocking prostate cancer progression and augmenting response to chemotherapy.

Support or Funding Information

This work was supported in part by grants from NIH grant GM094255 (S. Chen) and DOD PCRP breakthrough award PC111628 (S. Chen).
Response Surface Methodology as a Model for Combination Drug Therapy in Human Prostate Cancer Cells
Authors: Oblad Richard , Kenealey Jason
Published: November 2023

Combining chemotherapeutic drugs to treat malignant tumors has been proven to be effective at preventing drug resistance, tumor regression, and reducing tumor size. Most combinations come from a regimen of treatments that were used in the past, or were devised using synergistic drugs that were identified using the Chou Talalay method. We modeled combination drug therapy in PC‐3 human prostate cancer cells using conventional chemotherapeutics (Mitoxantrone Hydrochloride, Cabazitaxel, and Docetaxel) and natural bioactive compounds (Resveratrol, Piperlongumine, and Flavopiridol). We measured cell viability via the Alamar Blue assay to identify effective concentrations and combinations of drugs. Three drugs were used per combination at varying concentrations. Those points were then used to plot a response surface using the statistical software JMP. The response surface generated provided us with an area of effective combinations which allowed us to differentiate between combinatorial and synergistic combinations. Strong synergistic effects between resveratrol, piperlongumine, cabazitaxel, and flavopiridol were observed, with weaker interactions between the aforementioned drugs and mitoxantrone. Cabazitaxel and docetaxel, both microtubule synthesis inhibitors, showed additive effects when used in combination, but were both effective when used in combination with other drugs. We propose response surface methodology as an effective tool in devising combination treatments based on the response of tumorigenic cell lines.

Support or Funding Information

BYU Life Science Startup Grant
Identification of Pharmacogenetic Risk Factors for Zileuton‐Induced Liver Injury Using Diversity Outbred Mice
Authors: Harrill Alison , Gatti Daniel M , Luo Shaoke , Lyn‐cook Lascelles , Churchill Gary
Published: November 2023

Zileuton is an orally active inhibitor of leukotriene synthesis that is currently used for the maintenance treatment of asthma, although there has been interest in expanding its indications to other inflammatory diseases involving leukotrienes, such as rheumatoid arthritis and inflammatory bowel disease. A limitation for expanding clinical usage is an association with rare (idiosyncratic), but serious liver injury. In this study, we hypothesized that genetic sequence variants could be identified that influence risk of zileuton‐induced hepatotoxicity. Diversity Outbred (DO) mice were used as a surrogate for the human population because they harbor a high degree of genetic diversity and this diversity is well‐randomized throughout the genome, providing an ideal platform for complex trait analysis. In this study, female DO mice were administered zileuton (300 mg/kg, N=400) or vehicle (N=50) and dosed daily for seven days (i.g.). Serum alanine aminotransferase (ALT) was significantly elevated in the zileuton group, with significant interindividual variability in response (mean ± SD and range ‐ vehicle: 38 ± 53, 16–71 U/L; zileuton: 48 ± 42, 14–481 U/L; P<0.05). Zileuton treatment‐induced histopathological findings included microvesicular steatosis (37.5%, 150/400 mice), increased mitosis (3.5%, 14/400 mice), and focal and multifocal hepatocellular necrosis (2%, 8/400 mice). Quantitative trait locus mapping of the terminal ALT level revealed a significantly associated gene region on chromosome 3 (log of odds (LOD)=6), with smaller suggestive peaks on chromosomes 5, 11, 13, 16, and 18, suggesting multiple genetic contributors to the toxicity response. Within the Chr 3 support interval lies several promising candidate genes, including: 1) Tpmt (thiopurine methyltransferase) for which genetic variants have been used to select dosages of several drugs known to cause adverse reactions in the clinic, and 2) Ltc4s (leukotriene C4 synthase), for which variant rs730012 is used to predict an increase in asthma exacerbations for patients administered the leukotriene receptor inhibitor drug montelukast. Taken together, our data provide a starting point for identifying the underlying genetic sequence variants that contribute to zileuton‐induced liver injury in susceptible patients. Follow up studies are ongoing to confirm a functional role and human relevance of the candidate pharmacogenetic risk alleles identified in the present study.

Support or Funding Information

This work was supported by the Food and Drug Administration [HHSF223201400183C], Burroughs Wellcome Fund Innovation in Regulatory Sciences Award, Arkansas Biosciences Institute, NIH [Translational Research Institute grant UL1TR000039 and T32 GM106999].
Attenuation of polycyclic aromatic hydrocarbon (PAH)‐mediated pulmonary carcinogenesis in mice by omega‐3‐fatty acids
Authors: Moorthy Bhagavatula , Jiang Weiwu , Zhou Guodong
Published: November 2023

3‐Methylcholanthrene (MC) and benzo[a]pyrene (BP) are potent polycyclic aromatic hydrocarbons (PAHs) carcinogens. PAHs are present in cigarette smoke, diesel exhausts, and charcoal broiled meats, etc. Cytochrome P450 (CYP) 1A/1B1 enzymes play key roles in the activation of PAHs to carcinogenic metabolites, which initiate carcinogenesis by binding covalently to DNA, and the adducts, if not repaired, could lead to tumorigenesis. In this study we tested the hypothesis that pre‐treatment of mice with omega‐3‐fatty acids, i.e. [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) will lead to attenuation of PAH‐mediated pulmonary DNA adduct formation and pulmonary tumorigenesis. Twelve week old male and female A/J mice received EPA (60 mg/kg) and DHA (40 mg/kg) from day 1 to day 24. Control mice were treated with vehicle corn oil. On day 3, mice were treated with BP (40 μmol/kg) or 3‐methylcholanthrene (MC, 40 μmol/kg) by i.p. In the short‐term experiment (DNA adduct studies), 3–5 mice from each group were terminated at day 10 (7 days after BP or MC treatment). EPA/DHA significantly suppressed formation of BP‐DNA and MC‐DNA adducts in lung and liver of both male and female mice. In the long‐term experiment (tumor studies), 8 mice for each group continued to receive EPA/DHA twice per week till end of experiment (16 weeks). Pulmonary tumor incidence and multiplicity was significantly suppressed in mice given EPA+DHA, compared to those given the PAHs, BP or MC alone. These studies suggest that the omega 3‐fatty acids could be developed as novel candidates for the prevention of PAH‐mediated lung cancers in humans.