bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of mitochondrial energy production in the diabetic renal proximal tubule by the β2‐adrenergic receptor agonist formoterol
Authors: Cleveland Kristan , Brosius Frank C. , Schnellmann Rick G.
Published: April 2020

While
diabetic kidney disease is the leading cause of end stage renal disease, the pathophysiology of this disease remains poorly understood. Diabetes causes mitochondrial dysfunction and changes in cellular energy production in renal proximal tubules. Therefore, we examined the effect of formoterol, a β2‐adrenergic receptor (AR) agonist previously demonstrated to induce mitochondrial biogenesis and promote recovery from acute kidney injury, on renal cortical mitochondrial function and energy production in diabetic db/db mice and in renal proximal tubule cells (RPTC) treated with high glucose. Db/db and db/m (control) mice were treated with formoterol or vehicle daily for 3 wks beginning at 10 wks of age. Compared to controls, 13‐wk‐old diabetic mice showed in
creased renal cortex expression of electron transport chain (ETC) proteins in complexes I, II, III and V that was reversed to control levels with formoterol treatment. Conversely, we found that renal cortex from db/db mice had lower ATP levels than that from db/m controls and that these levels were restored with formoterol treatment. Similarly, we found that RPTC exposed to 17 mM glucose for 96 hr exhibited increased expression of ETC complex I–V proteins and reduced ATP content vs. cells cultured in
in the absence of glucose or osmotic controls.
ETC proteins and ATP levels were unaffected by high glucose when cells were co‐treated with formoterol. Additionally, exposure to 17 mM glucose decreased uncoupled oxygen consumption rate in the cells, suggesting that glucose reduced electron transport chain function. Together, these results demonstrate that diabetes and exposure to high glucose alters the mitochondrial bioenergetic profile in proximal tubule cells in vivo and in vitro, and that treatment with formoterol can reverse these effects.
Effects of Vacuolar H+‐ATPase Inhibition and the a1 and a3 Isoforms on Secretion and Activation of Cathepsins in MDA‐MB231 Breast Cancer Cells
Authors: Hinton Ayana , El Sayed Mohamed , Henry Abhijeet , Swickard Jamie
Published: April 2020

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH in a localized environment which promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells cleave extracellular matrix proteins and activate other proteases, facilitating tumor cell invasion. Our lab is investigating a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but not cathepsin L. However, although there was a trend, the difference in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data supports the conclusion that there is a significant difference in the effects of V‐ATPase inhibition on secreted cathepsins. Inhibiting V‐ATPases does not appear to affect the activity of secreted cathepsin L, however it reduces the activity of secreted cathepsins B and D To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for two of the four V‐ATPase “a” subunit isoforms, a1 and a3. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. The a1 isoform is found in intracellular compartments. We hypothesize that the a3 isoform knockout cells will have reduced activation of secreted cathepsins B and D while not effecting activation of cathepsin L. We predict that the a1 isoform knockout will affected the activity of intracellular cathepsins, but not affect the activity of secreted cathepsin B, D or L. Separate abstracts submitted by our lab are focused on the a1 and a3 isoform knockouts.

Support or Funding Information

Denison University Anderson AssistantshipDenison University Research Fund
Hypercapnia Attenuates Ventricular Ectopy during Wakefulness in a Young Man with Heart Failure
Authors: Keir Daniel A. , Duffin James , Floras John S.
Published: April 2020

Ventricular premature beats (VPBs) are common in heart failure (HF). Patients with HF often exhibit oscillations in breathing pattern (or Cheyne‐Stokes Respiration, CSR) that lead to increased VPBs during the hyperpneic phase. In such patients, CO2 inhalation during sleep eliminates CSR and reduces VPB prevalence. Whether this VPB‐lowering effect of CO2 occurs in awake HF patients without CSR remains unexplored. In a young CSR‐free HF patient (26 years; LVEF=20%; BMI=30 kg·m−2) with mild obstructive sleep apnea (AHI=17 events/h) and frequent ectopy, we compared the incidence of VPBs during: 1) resting breathing; 2) volitional hyperventilation; 3) and at standardized intervals (quartiles) during two CO2 rebreathing tests with end‐tidal O2 tension (PETO2) maintained at either 50 mmHg (hypercapnic‐hypoxia) or 150 mmHg (hypercapnic‐hyperoxia). Breath‐by‐breath ventilation, PETO2, and PETCO2 were measured by flowmeter and rapid gas analyzer and ECG and O2 saturation were monitored continuously. At rest (PETCO2=39 mmHg) and during hyperventilation (PETCO2=28 mmHg), VPBs occurred with 15% and 12% of all heart beats, respectively. With hyperoxic CO2 rebreathing (PETO2=148mmHg), as PETCO2 increased from 40, 46, 52, 57 mmHg in the first through fourth quartile, VPB incidence decreased from 11% to 6%, 3%, and 0%, respectively. Hypoxic rebreathing (PETO2=51mmHg) showed a similar trend: VPB incidence fell from 9% at a PETCO2 of 40 mmHg to 0% at 46 mmHg and remained at 0% thereafter. The hypercapnia‐induced reduction in VPB frequency was not dependent on heightened ventilation: VPBs were evident during volitional hyperventilation (12%) but not during the CO2 rebreathing quartiles with matched ventilation (0% for both hypoxia and hyperoxia). Furthermore, blood O2 also did not appear to modulate this response: VPB incidence at rest (15%) was similar to both hypoxic (11%; O2 saturation=86%) and hyperoxic (13%; O2 saturation=99%) CO2 rebreathing quartiles with matched ventilation (~9 L·min−1) and PETCO2 (~39 mmHg). These observations suggest that in HF, acute hypercapnia attenuates ventricular ectopy. The mechanism appears to be unrelated to increased respiratory or sympathetic drive, myocardial stretch accompanying large tidal volumes, or altered O2 availability. How hypercapnia exerts anti‐arrhythmic effects remain uncertain; we hypothesize that CO2 mediated increases in coronary flow may be involved.

Support or Funding Information

Experiments were funded by a Project Grant from the Canadian Institutes of Health Research (PJT 159491).
Collective epithelial cell migration in secondary palate fusion
Authors: Teng Teng , Bush Jeffrey
Published: April 2020

Cleft lip with or without cleft palate (CL/P) is the most common congenital craniofacial anomaly with considerable morbidity in affected children. Though extensive studies have established the basic histological and genetic mechanisms of palatal development, the cellular mechanisms that regulate secondary palate development are still not completely understood. Palate fusion is the crucial final step of palate development, in which the medial edge epithelial cells (MEE) make contact to form a midline epithelial seam (MES), which is subsequently removed to allow mesenchymal continuity. Though much progress has been made on understanding the cellular mechanisms of secondary palate fusion, major questions remain in part due to limitations in imaging technologies. Here we utilized new processing and imaging methods to visualize dynamic cell behaviors underlying secondary palate fusion temporally and spatially. We apply these methods to the study of mouse mutants in which apoptosis, or actomyosin contractility have been conditionally disrupted in the secondary palate epithelium. Our results indicate that apoptosis and cell extrusion contribute to MES removal, but that cellular compensation can overcome their loss. Our studies also reveal a unique form of collective epithelial cell migration that is critical for proper completion of secondary palate fusion and suggesting that secondary palate fusion may be a valuable model for understanding how collective epithelial migration occurs in other developmental and disease contexts.
Insulin Does Not Decrease the Effectiveness of Resveratrol or Curcumin in Promoting Cell Death in Melanoma Cells
Authors: Oliver Sally , Hill Braeden , West Lori
Published: April 2020

The effectiveness of phytochemicals such as curcumin and resveratrol in promoting cell death in various cancer cell lines has been well established in the literature. Given the increased incidence of type II diabetes mellitus and associated insulin therapy for insulin resistance in the United States, our goal was to determine whether insulin treatment would abrogate the cytotoxic effects of curcumin and resveratrol in a melanoma cell line. Cell death was measured using a trypan blue dye exclusion assay while increased caspase activity was qualitatively measured using a Caspase 3/7 Green ReadyProbe. Although both resveratrol and curcumin treatments significantly increased cell death, insulin treatment did not significantly affect cell death in either treatment group. Therefore, insulin therapy is not expected to interfere with curcumin or resveratrol in patients choosing homeopathic options.
Maternal Exposure to Electronic Cigarettes Causes Body Composition Alterations in Pups
Authors: Coblentz Tyler , Reppert Sarah , Cheuvront Tristen , Chantler Paul D , Olfert I. Mark , Bryner Randy
Published: April 2020

Electronic cigarette (E‐cig) usage is promoted as a safe alternative to traditional smoking, even during pregnancy, but long‐term effects of vaping are still unknown. Maternal smoking during pregnancy has been well studied and shown to increase both body mass index and obesity within children later in life. However, the effects of vaping on the offspring of maternal E‐cig users remains poorly studied. We hypothesized that maternal vaping during pregnancy will also increase body mass and obesity in the mature offspring with E‐cig exposed rat dams.

Pregnant Sprague Dawley dams were exposed to nicotine‐free E‐cig aerosol (E‐cig0), E‐cig aerosol containing 18 mg/ml of nicotine (E‐cig18), or ambient air (control). Maternal E‐cig exposure (Joyetech eGrip OLED using 5‐sec puff @ 17.5 W) consisted of 60 puffs over 1‐hour each day, 5 days/week, starting on gestational day 2 and continued until pups were weaned. Progeny themselves were never directly exposed to E‐cig aerosol. Dual energy X‐ray absorptiometry (DXA) was used for body composition analysis in male and female rat offspring at 31±1 weeks of age (Air n=16, E‐cig0 n=21, E‐cig18 n=19).

In females, total body mass and lean body mass (LBM) was not different between the exposure groups, however fat mass (FM) was greater in E‐cig0 compared to air and E‐cig18 exposed groups (125±9 vs. 103±12 and 100±5 g, respectively, ANOVA p<0.05). Percentage BF (%BF) was also greater in E‐cig0 compared to air and E‐cig18 (33±2 vs. 28±2 and 29±1 %, respectively, ANOVA p<0.05). As expected, total body mass, LBM, and FM values were significantly lower in females than males (ANOVA p<0.001), but no differences in body composition was observed within the males across the exposure groups.

In conclusion, maternal vaping without nicotine may increase risk for elevated body fat in adult life and the development of obesity in female progeny with fetal exposure in utero.

Support or Funding Information

WVU Cancer Institute Philip R Dino Innovative Research Grant (IMO), NIHGMS 5U54GM104942‐03 (PDC)
NSF S‐STEM Program Updates: Cohort 3 of iMASS
Authors: Jackson Alissa , Daft Sheila , Kasper Sherry
Published: April 2020

Lee University’s Integrated Math and Science Scholars (iMASS) program began in the spring of 2015 as a scholarship funded by the National Science Foundation (NSF) for students interested in Science, Technology, Engineering, and Math (STEM) fields. The purpose of the iMASS program is to introduce students to the vast opportunities for research, industry, and continued education in the fields of science and mathematics while focusing on local and regional opportunities. The iMASS program aims to create a learning community of students, peer mentors, and faculty mentors that aid in the retention of students in the STEM fields. The program seeks to recruit academically talented, financially eligible students with a focus on underrepresented minorities and first‐generation students. We provide a seminar class including resume building, interview tips, and speakers from local graduate programs (the University of Tennessee). We also provide tours of regional facilities (Oak Ridge National Labs and Bayer). For our third cohort, 87% of students receiving the scholarship were retained both at Lee and in STEM to junior year compared with 53% retention of our alternates and associates and 44% retention among overall STEM. We recruited diligently throughout both the fall and spring semesters and we had the students spread the word to others about their experience. This greatly improved the program with a total of 40 applications for the 3rd cohort. In addition, we have built our mentoring program by having the mentors trained before the semester and giving adequate time in class to build these relationships. According to our student surveys, this has significantly improved our mentoring interactions and has now produced a culture of mentoring. In addition, there was an increase in summer research and internships in cohort 3 compared with cohort 2. We believe that this is due to their exposure to the seminar speakers and sessions on REUs that raise the awareness of the students to research opportunities. Therefore, we feel that the iMASS program has begun to be recognized by our students as an enriching opportunity and this has helped with the success of cohort 3.
Exploring the Mechanisms of Drug Resistance with a 3D Molecular Model of HIV Protease
Authors: Gilmore Keviyanah , Craft Alexis , Lauderdale Tra’Mya , Jernigan Joy , Billings Ryan , Jones Candace , Van Stry Melanie
Published: April 2020

Human immunodeficiency virus (HIV) affects 37 million people worldwide. HIV causes Acquired Immunodeficiency Syndrome (AIDS) by impairing the immune functions in the human body and is transmitted through contact of body fluid. HIV attacks the body’s immune system, specifically CD4 T cells which help the immune system fight off infection. Antiretroviral therapy (ART) has prolonged the life expectancy of those under the treatment process. ART is a combination of antiretroviral drugs that keep the virus from replicating. The drug combination “cocktail” combines the power of multiple drugs to block multiple targets in HIV replication. HIV‐1 protease is an essential enzyme for the life cycle of HIV. Therefore, HIV‐1 protease is an important target used in antiretroviral therapy. However, under the selective pressure of protease inhibitors, mutations may occur at the active site, crippling inhibitor binding and leading to drug resistance. Umass6 is an analog of the HIV protease inhibitor, duranovir, with a bulky P1′ moiety that allows enhanced overall van der Waals interactions at the active site. Here, we design a 3D model of the drug resistant HIV protease mutant I50V bound to UMass6 using Jmol based on the crystal structure described by Gordon J. Lockbaum and colleagues
(1). HIV protease is a homodimer with the active site located in the interface between the subunits. Our model highlights the location of the I50V mutation within the active site and the resulting structural changes that cause the loss of inhibitor potency. Our 3D model also highlights the critical residues involved in inhibitor binding and the catalytic residue, D25, within the active site. Designing 3D model exposes students to protein structure, mutations, and tools for understanding drug resistance.

Support or Funding Information

This work is supported by NSF‐DUE1725940 and NSF‐HRD1623340.
Sex Differences in an Fmr1 Knock‐out Mouse Model of Fragile X Syndrome
Authors: Armstrong Jessica L. , Chen Yiming , Saraf Tanishka S. , Canal Clinton E.
Published: April 2020

Fragile X Syndrome (FXS) is the leading inherited, monogenetic cause of intellectual disabilities and autism spectrum disorder (ASD). Epilepsy, sensory hypersensitivities, and several other neuropsychiatric symptoms are also common in FXS and ASD. FXS is caused by a >200 CGG‐repeat on the FMR1 gene, located on the X‐chromosome, that silences its transcription, preventing expression of the protein, FMRP. Possessing a single X‐chromosome, FXS occurs more frequently in males than females. Males with FXS typically have more severe clinical symptoms than females with FXS. For example, males are more than twice as likely to have epilepsy. Genetic mosaicism (X‐inactivation) in females with FXS is the presumed, exclusive reason for the lower symptom severity. Our results presented here challenge this presumption. During preclinical testing of Fmr1 knock‐out mice, we observed a statistically significant, higher prevalence of audiogenic seizures in juvenile (P23–P25), male Fmr1 knock‐out mice (89%) compared to juvenile, female Fmr1 knock‐out mice (50%) (P=0.01, N=19 males and N=18 females), closely paralleling the sex difference in epilepsy prevalence in FXS. Importantly, however, female Fmr1 knock‐out mice are homozygous for the mutated Fmr1 gene, and therefore, like male Fmr1 knock‐out mice, do not express FMRP. We also found that expression of the serotonin receptor, 5‐HT1AR, was significantly elevated in whole brains of adult, male Fmr1 knock‐out mice, compared to wild‐type mice (P=0.04, N=8 each); we did not observe this difference in female mice (P=0.24, N=7 each). We are currently investigating whether 5‐HT1ARs contribute to the sex difference in audiogenic seizure susceptibility. In conclusion, our preclinical data suggest that the reduced symptom severity in females with FXS may not be solely attributed to X‐inactivation of the mutated FMR1 allele.

Support or Funding Information

Funded by: FRAXA Fellowship and DOD W81XWH‐17‐1‐0329
Evaluation of Antimicrobial Activity & Phytochemical Analysis of Leaf Extracts of Acacia rigidula and Acacia berlandieri
Authors: Cavazos Peter Daniel , Lanorio Jocelyn , Ynalvez Ruby A.
Published: April 2020

The emergence and spread of antibiotic resistance, as well as the evolution of new strains are of great concern to the global health community. The search for novel agents to combat the rapid evolution of bacterial pathogens becomes ever more important as current methods are proving ineffective. Plant species within the Fabacae family have been reported to exhibit antimicrobial activity; therefore, we hypothesize that these two species will display antibacterial properties. The objectives of this study were to (1) determine the presence of antimicrobial activities, (2) qualitatively determine secondary metabolites of plant extracts from Acacia berlandieri (Guajillo) and Acacia rigidula (Blackbrush), (3) analyze extracts using Nuclear Magnetic Resonance spectroscopy (NMR), infrared spectroscopy (IR), and ultraviolet‐visible spectroscopy (UV‐Vis) to identify different secondary metabolites within a test sample, and (4) determine antioxidant activities within both plant species.

Plant leaves were sequentially extracted using acetone, methanol, and acetic acid that were then subjected to susceptibility testing against nine microorganisms. Results provided evidence of antimicrobial activity from the crude extracts of both species. The activity was significantly higher (p<0.001) from the leaves of species A. rigidula. Among the nine microorganisms, two gram‐negative and two gram‐positive bacteria were susceptible. Qualitative phytochemical testing revealed important secondary metabolites: phenols, flavonoids, saponins, terpenoids, and tannins. Each of which have been shown to possess antimicrobial properties and exert different modes of action. NMR, IR, and UV‐Vis spectroscopy revealed the basic structures within our unknown samples that are commonly found in flavonoids, saponins, and tannins, reinforcing the results of phytochemical testing. Preliminary results revealed some antioxidant activity within selected A. rigidula extracts. Analysis of the leaves extracts of A. berlandieri and A. rigidula have revealed the presence of medicinally valued bioactive components. This study confirms the efficacy of selected plant extracts as natural antimicrobials and suggests the possibility of employing them in drugs for the treatment of infectious diseases caused by the test organisms.
Using the CLASS‐Bio Survey to Assess Engagement through Forensic Science in General Biology
Authors: Jones Candace
Published: April 2020

Lane College is a historically black college committed to educating underserved minority students. My objective was to give students different pedagogical strategies to meet students where they are and moving away from the lecture based format to improve student success. In General Biology I chose to engage students through forensic sciences. I implemented forensic science in the laboratory in one of my two sections of General Biology I in the Fall 2018 semester. Students determined the items that needed to be analyzed from a mock crime scene after being presented with some basic forensic science techniques in the first week of laboratory. Each additional week the students participated in analysis of hair, fiber, soil, synthetic blood tests, chromatography, and DNA fingerprinting. To assess the intervention, I used the CLASS‐Bio Survey which measures student attitudes and beliefs about learning science (n=32) that was given pre and post to both sections as a way to compare the effect of the intervention. In the section that I gave the Forensic Science intervention the CLASS‐Bio Survey perceptions were higher in all categories Enjoyment, Real World, Problem Solving Synthesis and Application, Problem Solving Effort, Conceptual, Problem Solving Strategies, and Reasoning in the pre survey. The class that received the forensic science intervention had a 19.1% increase in Enjoyment, 18.4 % increase in Problem Solving Strategies and a 15.9 % increase in Problem Solving Effort than compared with the class that did not receive the intervention in the post survey.

Support or Funding Information

National Science Foundation Award HRD # 1623340 to MVS
Myoblast Mitochondrial Adaptation to Chronic Alcohol Administration
Authors: Levitt Danielle E. , Simon Liz , Molina Patricia E.
Published: April 2020

Skeletal muscle adaptation to chronic alcohol consumption involves disruption of anabolic, catabolic, and oxidative metabolism pathways and is associated with decreased muscle mass and impaired metabolic function. These maladaptive responses may contribute to increased risk of metabolic disorders, particularly in people living with human immunodeficiency virus (PLWH) on combination antiretroviral therapy (ART). Aging, increased survival of PLWH on ART, and lifestyle choices including unhealthy alcohol use are complicated by metabolic comorbidities. We tested the hypothesis that mitochondrial homeostasis is critically impacted by chronic binge alcohol (CBA) administration. Specifically, we hypothesized that CBA impairs skeletal muscle expression of genes underlying mitochondrial metabolic function and alters myoblast function in SIV‐infected, ART‐treated rhesus macaques. Female rhesus macaques (n=10) were assigned to receive either CBA (50–60 mM peak blood alcohol/day, 5 days/week) or isovolumetric water (VEH) for 3 months before SIVmac251 infection. After 2.5 months, ART was initiated, and skeletal muscle samples were collected from all animals at study endpoint (~12 months post‐SIV infection). mRNA was extracted and analyzed for genes related to mitochondrial biogenesis and metabolic function [peroxisome proliferator‐activated receptor‐gamma coactivator (PGC)‐1α, PGC‐1β, mitochondrial transcription factor A (TFAM), estrogen related receptor (ERR)α, nuclear respiratory factor (NRF)1 and 2]. Naïve (no alcohol or SIV) primary myoblasts were isolated from skeletal muscle and incubated with alcohol (50 mM) or control (media only) for three days. To assess mitochondrial metabolic function, oxygen consumption rate was determined at baseline in alcohol‐treated and control myoblasts after the addition of oligomycin, carbonyl cyanide‐4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone and antimycin A using Agilent Seahorse technology. CBA reduced expression of PGC‐1β, TFAM, ERRα, NRF1, and NRF2, but not PGC‐1α mRNA in skeletal muscle from SIV‐infected, ART‐treated female rhesus macaques. In vitro, alcohol treatment reduced basal and maximal oxygen consumption rate of naïve myoblasts. Together, our findings demonstrate maladaptive changes in myoblast mitochondrial gene expression and function. These results identified potential therapeutic targets to improve skeletal muscle metabolic health in PLWH. Specifically, the role of PGC‐1β as a potential mechanism by which alcohol impairs skeletal muscle mitochondrial function warrants further investigation on its responsiveness to pharmacotherapeutic intervention to restore its levels and improve mitochondrial metabolic health in PLWH with unhealthy alcohol use.

Support or Funding Information

This research was supported by grants from the National Institute on Alcohol Abuse and Alcoholism, 1F32AA027982‐01A1, 5P60AA009803‐25, K01AA024494‐03, and 5T32AA007577‐20.
HRH1 in Human and Mouse Pancreatic Cancer
Authors: Salmerón Cristina , Sriram Krishna , Baird Alyssa , Insel Paul
Published: April 2020

Pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), is a highly lethal cancer (5‐year survival rate <10%). New, effective therapies are thus an important, unmet need. We recently found that many G protein‐coupled receptors (GPCRs), the largest and most diverse group of membrane receptors in eukaryotes and targets of a third of FDA‐approved drugs, are overexpressed in PDAC compared to normal pancreatic tissue. One such receptor, histamine receptor H1 (HRH1; ~32‐fold increase in PDAC), is targeted by many FDA‐approved antihistamines and high HRH1 expression is an unfavorable prognostic marker for PDAC. Moreover, increased mast cell (histamine‐secreting cells) in PDAC correlates with angiogenesis and tumor progression and a recent epidemiologic study showed that atopic allergic conditions and antihistamines protect subjects with age >70 from developing PDAC. We hypothesize that HRH1 enhances the malignant phenotype of PDAC and that antihistamines might be repurposed as a therapy for PDAC.

We found that HRH1 mRNA is highly expressed in multiple human PDAC cell lines and is the highest expressed among the 4 HRH subtypes. Histamine via HRH1 increases Ca2+ levels in PDAC cancer cell lines, an effect blocked by multiple antihistamines (clemastine, fexofenadine, loratadine and diphenhydramine). We also observed that histamine can promote PDAC cell proliferation and migration via HRH1. Using the KPfC mouse (KrasLSLG12D/; p53fl/fl; Ptf1a‐Cre/), a genetically engineered mouse model of PDAC that spontaneously develops PDAC tumors after 6 weeks of age, we isolated PDAC tumors and generated five separate PDAC cancer cell lines. We found that KPfC mice PDAC tumors have higher HRH1 mRNA expression than does the normal pancreas of p53fl/fl littermate controls. As in human PDAC, KPfC PDAC cancer cell lines expressed higher HRH1 mRNA levels than the other HRH subtypes. Moreover, histamine increases intracellular Ca2+ in KPfC cancer cells, an effect blocked by antihistamines (clemastine and loratadine).

Taken together, these data strongly suggest that HRH1 regulates Ca2+ signaling and functions associated with the malignancy of PDAC cancer cells and tumors. HRH1 is thus a potential therapeutic target for PDAC, perhaps by the repurposing of FDA‐approved HRH1 antihistamines.

Support or Funding Information

Supported by an HHMI post‐bac fellowship and an ASPET David Lehr Award
Addition of retinoic acid‐inducible gene 1 to enhance Ebola virus‐like particle vaccine
Authors: Heath Arden , Fogarty Julia , Shrestha Ramila , Martinez Osvaldo
Published: April 2020

Ebola virus (EBOV), a filovirus family member, is a highly pathogenic virus that causes Ebola hemorrhagic fever (EHF) resulting in documented mortality rates in humans as high as 50%. Currently, the basic EBOV virus‐like particle (VLP) vaccine contains the Ebola virus (EBOV) matrix VP40 and attachment glycoprotein (GP). VLPs are morphologically and biochemically similar to parental virus, yet because they lack a genome and cannot replicate, are safe enough to be used as vaccines. We hypothesize that addition of a constitutionally active retinoic acid‐inducible gene 1 (RIG‐I) would enhance the ability of the vaccine to induce interferon‐dependent immune functions yielding an improved vaccine. Expression of EBOV VP40 in 293T cells induces the spontaneous production of VLPs into the media supernatant and if expressed with EBOV GP, will produce VLPs studded with the attachment GP. Recombinant chimeric constitutively active (ca)RIG‐I‐VP40 matrix and a nonfunctional mutant L58A (mu)RIG‐I‐VP40 matrix genes were constructed to produce VLPs containing constitutively active and nonfunctional RIG‐I. Supernatant from 293Ts transfected with caRIG‐I‐VP40, muRIG‐I‐VP40 or VP40 along with GP expression plasmids were tested for the presence of VLPs. Western blotting of purified VLPs confirmed the presence of RIG‐I in caRIG‐I‐VP40 and muRIG‐I‐VP40, but not VP40 containing VLPs. Monocyte‐like and PMA‐differentiated macrophage‐like THP‐1 Dual cells were treated with nothing, VP40+GP, caRIG‐I‐VP40+GP, muRIG‐I‐VP40+GP VLPs as well as LPS and a Vaccinia virus (VACV‐70) positive controls and tested for induction of interferon (IFN) signaling. CaRIG‐I containing, but not muRIG‐I containing VLPs induced interferon signaling from both macrophages and monocytes. In Future studies we will also test whether addition of RIG‐I to EBOV VLPs can activate human peripheral blood monocytes.

Support or Funding Information

Winona State University student research and travel grants

Winona State University Foundation Special Projects
Sex‐Related Differences in Rapid‐Onset Vasodilation with Aging
Authors: Hanson Brady , Joyner Michael , Casey Darren
Published: April 2020

Rapid‐onset vasodilation (ROV) in response to a single muscle contraction is attenuated with aging. Moreover, sex‐related differences in muscle blood flow and vasodilation during dynamic exercise have been observed in young and older adults. The purpose of the present study was to explore if sex‐related differences in ROV exist in young (n=36; 21 male/15 female; 25 ± 1 yr) and older (n=32; 19 male/13 female; 66 ± 1 yr) adults. Subjects performed single forearm contractions at relative intensities of 10%, 20%, and 40% maximal voluntary contraction (MVC). Brachial artery blood velocity and diameter were measured with Doppler ultrasound, and forearm vascular conductance (FVC; ml·min−1·100 mmHg−1) was calculated from blood flow (ml·min−1) and blood pressure (mmHg) and used as a measure of peak ROV (peak change in FVC from baseline). Due to significant differences in MVC and consequent differences in weight used for each relative intensity between males and females, the data were analyzed two additional ways. First, responses were normalized for workload (i.e. peak FVC per kg of weight lifted). Second, the peak ROV responses in a subset of subjects (n=44; 12 young male, 10 young female, 11 older male, 11 older female) with similar absolute workloads (10–12kg) were compared. Lastly, the magnitude of change in peak ROV with increasing intensity (calculated as the slope of a linear regression line between 10, 20, and 40% MVC) was calculated and compared between groups. Peak ROV was attenuated in females across all relative intensities in the young and older groups (P < 0.05). When normalized for workload, peak ROV was attenuated with age in both males and females at all intensities (P<0.05) except at 10% for females (P=0.47). However, sex‐related differences were not observed within age when normalized by workload at any intensity (P>0.05). In the subset of subjects with a similar absolute workload (~11kg), age‐related differences in ROV remained in both the female and male groups (P<0.05). Older females demonstrated an attenuated peak ROV compared to their older male counterparts (91 ± 6 vs. 121 ± 11 ml·min−1·100 mmHg−1, P<0.03), whereas no sex differences in peak ROV were observed between young females and males (134 ± 8 vs. 154 ± 11 ml·min−1·100 mmHg−1, P=0.15). Examining the slope of the peak ROV response across contraction intensities revealed a main effect of age (P<0.05), with the slope being blunted in older compared to young subjects. When separated by sex, the slope was smaller in older compared to young females (5.5 ± 0.7 vs. 8.9 ± 0.9 FVC units/intensity, P<0.05) but not between older and young males (7.0 ± 0.50 vs. 8.2 ± 1.0 FVC units/intensity, P=0.38). Our data suggest that sex‐related differences in the rapid vasodilatory response to single muscle contractions exist in older but not young adults, such that older females have a blunted response compared to older males.
Sex Differences in Urinary Angiotensinogen (uAGT) Excretion, Renal Function, and Systolic Blood Pressure in 2‐Kidney, 1‐Clip Hypertensive Rats
Authors: Bell Annie L. , Shao Weijian , Katsurada Akemi , Sato Ryosuke , Navar L. Gabriel
Published: April 2020

Intrarenal renin‐angiotensin system (RAS) activation and tissue injury in nonclipped kidneys of 2‐kidney, 1‐clip (2K1C) hypertensive rats have been characterized in previous studies of male but not female rats. To evaluate possible sex differences in response to renovascular hypertension, urinary angiotensinogen (uAGT) excretion, arterial blood pressure, urinary protein excretion, and renal function were assessed in 2K1C female rats.

Female (n=8) and male (n=6) Sprague Dawley rats underwent placement of a 0.2 mm silver clip on the left renal artery to simulate unilateral renal artery stenosis. BP was measured by tail‐cuff plethysmography, and 24‐hour urine volume and water intake were monitored via metabolic cages over 21 days. After this period, renal clearance studies were conducted in anesthetized rats, and urine was collected from each kidney separately over 30 minute intervals. Urine protein concentration was determined by pyrogallol red method, and uAGT was measured by ELISA as an index of intrarenal RAS activity. Inulin and PAH concentrations were measured by spectrophotometry for calculation of glomerular filtration rate (GFR), renal plasma flow (RPF), and renal blood flow (RBF).

21 days after renal artery clipping, systolic BP had risen to 176±8 mmHg from a baseline pressure of 120±1 mmHg, and urinary protein excretion increased to 20±5 mg/day in females. uAGT excretion was significantly increased from the baseline of 13 ng/day to 74 ng/day (507±127% increase). The nonclipped kidney showed 90±29% higher uAGT excretion compared to the clipped kidney, consistent with our previous findings in males. GFR and RPF were higher in nonclipped kidneys than in clipped kidneys but were greater than in corresponding measurements in males. GFR, RPF, and RBF were significantly higher in control rats compared to both clipped and nonclipped kidneys. Male rats showed significantly higher increases in uAGT, BP, and protein excretion.

The present study demonstrates that BP, uAGT, and urinary protein excretion substantially increase after renal artery clipping in females, but the magnitude of the changes is markedly lower than in males. Additionally, the nonclipped kidney showed higher uAGT excretion compared to the clipped kidney. Although GFR, RPF, and RBF were significantly lower than control values for both clipped and nonclipped kidneys, these values were higher for females than males. These results demonstrated that female rats may be protected against AngII‐mediated renal injury and hypertension.

Support or Funding Information

Supported by the ASPiRE Program at Tulane Univ Sch Medicine, New Orleans, LA and the Warren R. Bourgeois III, MD and Usha Ramadhyani Bourgeois, MD Student Research Endowed Fund at Tulane Univ Sch Medicine, New Orleans, LA
Commensal microbiota are essential for vascular contractility mediated by actin polymerization
Authors: Furtado Janara , Bearss Nicole R. , Edwards Jonnelle M. , Mell Blair , McCarthy Cameron G. , Vijay-Kumar Matam , Joe Bina , Wenceslau Camilla F.
Published: April 2020

Introduction

Commensal microbiota within a holobiont contribute to the overall health of the host via mutualistic symbiosis. Disturbances in such symbiosis are prominently correlated with a variety of diseases affecting humans, including cardiovascular diseases, which are the number one contributors to mortality. Given that a hallmark of all cardiovascular diseases is changes in vascular function, we hypothesized that depleting microbiota from a holobiont would induce vascular dysfunction.

Methods

To test this hypothesis we used male inbred Sprague Dawley (SD) rats that were either germ‐free (GF) or re‐conventionalized germ‐free (GFC) rat (7 weeks‐old). Re‐conventionalization of GF rats were performed by co‐housing GF rats with conventionally‐raised SD rats for 10 days (1:1 ratio); this allows GF rats to receive a natural, continuous exposure to gut microbiota via coprophagy. Mesenteric resistance arteries were used to evaluate vascular function using a wire myograph. Vascular smooth muscle cells (VSMC) were cultured from aorta to evaluate cell migration via scratch assay; proliferation via IncuCyte; and cofilin phosphorylation (inactive form) via Western blot. We quantified cardiac hypertrophy by dividing the left ventricle weight by 26.73 + tibia length3. (Statistics: t‐test: p<0.05: *vs. GF, n=5–6/group)

Results

GF rats had a lower LV mass compared to GFC (GF: 6.9±0.2; GFC: 7.5±0.2*). Intriguingly, GF rats presented a reduction in both KCl‐and phenylephrine‐induced contraction and GFC ameliorated these responses [KCl: Emax (mN): GF: 4.8±1.0 vs. GFC: 8.5±1.1* and phenylephrine: Emax (mN): GF: 6.9±1.3 vs. GFC: 11.7±0.7*]. Given that no differences were observed in acetylcholine‐induced relaxation, we hypothesized that the hyporesponsiveness of the GF arteries were due to cytoskeleton dynamics. Indeed, we observed that GF rats present a reduction in cofilin phosphorylation (phosphoCofilin/TotalCofilin; AU: GF: 0.3±0.03 vs. GFC: 0.6±0.11*). Decreased phospho‐cofilin leads to its active form and subsequently actin depolymerization in VSMCs.

Conclusion

This study shows that commensal bacteria are essential for normal structure and function of the vascular system of the host.

Support or Funding Information

NIH: R00GM118885
Tissue distribution studies of the peptide‐bilayer coated magnetic nanobeads and delivery of an adducted peptide, to retard melanoma growth, in vivo
Authors: Natarajan Pavithra , Martinez Baltazar , Lingo Joshua D. , Fleming Sherry D. , Tomich John M.
Published: April 2020

Self‐assembling peptides based delivery systems are gaining increased importance as they are highly biocompatible, biodegradable and tunable i.e. structure and function can be modified. Branched amphiphilic peptides (BAPs) self‐assemble to form bilayer delimited vesicles, called the Branched Amphiphilic Peptide Capsules (BAPCs), whose molecular architecture is similar to liposomes. BAPCs have been used to date to deliver encapsulated radionuclides and dsRNA and DNA electrostatically bound to the exterior of the capsules. Recently, we have synthesized magnetic nanoparticles with the BAP bilayer coating for studying their interaction with a biological system and for exploring applications in delivery. The magnetic nanoparticles are being used as quantification tools to study some aspects of the peptide bilayer interactions such as their route of uptake by cells in culture and to study their tissue distribution in mice, employing several routes of delivery. Recent in vitro studies using widely used techniques such as confocal imaging and flow cytometry alongside the quantification method using the newly developed BAP‐MNBs, demonstrate that they are readily taken up by cells in culture via more than one endocytic pathway. This study suggests that quantification using BAP‐MNBs is a reliable tool for studying BAPCs delivery system and associated molecules. Current studies involve studying the tissue distribution of BAP‐MNBs when injected intravenously in mice. The BAP‐MNBs are alongside being tested as delivery systems to deliver a therapeutic retro‐inverso D‐peptide (RD‐p9) known to retard the growth of melanoma tumors in mice. Our current studies in mice will provide insight into the fate of BAPCs in vivo, aiding future studies which aim to tailor and use BAPCs as well as BAP‐MNBs for specific applications.

Support or Funding Information

Phoreus Biotechology Inc., Olathe, Kansas
Characterizing the role of POLQ variants in genomic instability and cancer
Authors: Ray Sreerupa , Sheld Francesca , Moen Avery , Avalos Lisbeth , Victorino Jorge , Towle-Weicksel Jamie
Published: April 2020

Melanoma is the most serious form of skin cancer that arises from melanocytes. It is developed due to ultra‐violet (UV) radiation inducing DNA damage when left unrepaired lead to mutations, which lead to genomic instability. Unrepaired DNA, or its faulty repair, can lead to mutagenesis, which can propagate further through rounds of replication, eventually resulting in uncontrolled cell growth leading to cancer. However, cells have various kinds of DNA repair pathways, which includes specialized enzymes that can repair DNA. One of the most important DNA repair enzyme is DNA polymerase theta (Pol θ or POLQ). POLQ has been shown to be involved in double strand break repair through a non‐canonical DNA end‐joining pathway known as the micro‐homology mediated end‐joining (MMEJ). POLQ is a low fidelity DNA polymerase and it introduces various kinds of mutations during DNA repair. Previous reports show that breast cancer patients that overexpress POLQ tend to have poorer survival rate indicating a potential role for POLQ in cancer. In collaboration with the Yale SPORE in Skin Cancer and Rhode Island College, we have identified several variants of POLQ in melanoma patients. Here we demonstrate that the variants of POLQ induce genomic instability and have accumulations of DNA double strand breaks (DSBs). We show that POLQ variants are sensitive to DNA damaging agents like etoposide. In addition, biochemical studies indicate that some of these variants display a slower polymerization rate compared to wild‐type (WT) suggesting a different mechanism for nucleotide incorporation. Together, this suggests that the cancer‐associated variants lead to genomic instability. Our studies suggest that variants of POLQ alter the DNA repair landscape and has potential to impact cancer treatment.

Support or Funding Information

This research was supported by Linfield College Start‐Up Funds and Linfield College Faculty Student Research Collaborative Grant (FSRCG) to SR.
The Effects of Low Volume Versus High Volume Sled‐Push Training on Muscular Adaptation
Authors: Bernard Jeffrey , Liao Yi-Hung , Madrigal Christian , Levesque Joshua , Lee Sukho , Fraze Matthew , Del Toro Isaac
Published: April 2020

Purpose

To compared the effects of low and high volume sled‐push resistance training on muscle strength, power, and body composition.

Methods

Twenty‐four college students were recruited and matched based on baseline one‐repetition max (1‐RM) into 1 of 3 groups: 1) low volume (LV), 2) high volume (HV) resistance training groups, or 3) control (CON) (n = 8 per group). The LV training consisted of 5‐single repetitions pushing a weighted sled for 10 yards. The HV training consisted of a multi‐set protocol of 3‐sets of 5‐pushing a weighted sled for 10 yards. Training consisted of 3‐weekly workouts performed on nonconsecutive days for 6‐weeks. This study utilized a pre‐ and post‐test study design consisting of 1‐RM, Wingate power test, standing long jump, vertical jump, and body composition. The CON group only performed the pre‐ and post‐tests.

Results

Following 6‐weeks of training there was a similar, but significant, increase in 1‐RM for both training groups (pre‐test; LV= 500 ± 32.6 lbs vs HV 480 ± 42.9 lbs; post‐test; LV= 658.1 ± 33 lbs vs HV= 632.5 ± 35.3 lbs, P < .001). However, there were no improvements in the Wingate power test, standing long jump, vertical jump, or body composition between pre‐ and post‐tests for both training groups ( P > .05). There were no improvements for CON (P > .05).

Conclusion

This study suggests that low volume resistance training is as effective as a high volume protocol for improving muscle strength. However, its effectiveness on muscle power and body composition are unclear.
Structure Activity Relationship Optimization of a 1,3‐Disubstituted Urea‐based Inhibitor of Zika Virus Infection
Authors: Khachatoorian Ronik , Garcia Gustavo , Aguilera Kristina Y. , Riahi Rana , Micewicz Ewa D. , Micewicz Alina , Dawson David W. , Dasgupta Asim , Arumugaswami Vaithilingaraja , Ruchala Piotr P. , French Samuel W.
Published: April 2020

Zika Virus (ZIKV) has recently led to major epidemics in South American countries and has been reported in several other countries as well. ZIKV is the cause of microcephaly and other developmental neuroimmunopathologies in fetuses of pregnant women and the Guillain‐Barre Syndrome in adults. To date, no therapeutics or vaccines are approved for ZIKV infection, and new inexpensive treatments are needed. Here, we conducted a second round of rigorous in vitro structure activity relationship (SAR) studies on our previously reported 1,3‐disubstituted urea‐based thiadiazole compound (ASN07115927) and conducted a preliminary in vivo test of three compounds with the highest IC50 values. We synthesized and tested 50 new derivatives of the parental compound. We were able to identify a few derivatives with improved antiviral activity as well as structural features that will guide further SAR studies. The in vivo assay of three compounds in our IFNAR knockout mouse model demonstrated significant improvement in the life span of infected mice and prevented ZIKV‐induced paralysis of the limbs. These results identify compounds with improved antiviral activity which may be utilized/further optimized for the development of inexpensive and orally available anti‐ZIKV drugs. These compounds may also be beneficial for treatment of other viruses in the same family including Dengue virus.
Exploring Drug Resistance in HIV‐1 Protease using 3D Printed Models
Authors: Villeda Adan , Chivington Austin , Cobos Maximilian , Gastineau Thomas , Mares Cesar , Que Javier , Porter Lon A. , Novak Walter R.P.
Published: April 2020

HIV roughly affects 38 million people around the world. The enzyme HIV‐1 protease is essential for replication and maturation of the virus. HIV‐1 protease activates a variety of important proteins including reverse transcriptase and RNase H. Darunavir, an HIV‐1 protease inhibitor, is the most potent antiretroviral drug to combat HIV to date. Despite the potency of Darunavir, HIV‐1 protease can develop mutations both inside and outside of its active site to render Darunavir and other inhibitors ineffective. Here we develop 3D printed models to illustrate the structural basis of drug resistance. Specifically, we model the mutations V32I and L76V in HIV‐1 protease. Hands‐on models that illustrate resistance mutations near the active site of HIV‐1 protease provide students with additional visual references to better understand drug resistance mechanisms and the design of novel inhibitors to effectively overcome these mutations.

Support or Funding Information

This work was funded in part by NSF‐IUSE 1725940 for the CREST Project.