bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of protein kinase C impairs endothelium‐dependent vasorelaxation in isolated human omental resistance arteries
Authors: Grunewald Zachary Ian , Ghiarone Thaysa , Quinones‐Morales Mariana , Ramirez‐Perez Francisco , Martinez‐Lemus Luis , Padilla Jaume
Published: June 2021

Chronic diseases such as obesity and diabetes are associated with endothelial dysfunction and increased cardiovascular disease risk. Notably, protein kinase C (PKC) has been identified as a major molecular contributor to chronic disease‐associated microvascular impairments. However, the molecular mechanisms by which PKC activation causes microvascular endothelial dysfunction are not fully understood. Herein, we tested the hypothesis that PKC activation impairs microvascular vasorelaxation through an endothelial nitric oxide synthase (eNOS)‐dependent mechanism in human isolated resistance arteries and cultured endothelial cells. Accordingly, resistance arteries were isolated from the omentum of 32 patients (52±3 years of age, 43.3±1.7 BMI, 76% women) that underwent abdominal surgery and analyzed for vasomotor function ex vivo using wire myography. We found that pharmacological inhibition of PKC (ruboxistaurin, 1μM) for 30 minutes enhanced endothelium‐dependent vasorelaxation in response to bradykinin relative to untreated arteries (p<0.05). This effect of ruboxistaurin was abrogated by NOS inhibition (L‐NAME, 300μM), suggesting that improved endothelium‐dependent relaxation with PKC antagonism is nitric oxide‐dependent (p<0.05). Next, we found that treatment with the PKC activator, phorbol 12‐myristate 13‐acetate (PMA, 0.5μM), for 30 minutes impaired endothelium‐dependent and ‐independent vasorelaxation, which was abolished with concomitant incubation of ruboxistaurin (p<0.05). Furthermore, we found that PMA treatment reduced Akt and eNOS activation in cultured endothelial cells (HUVEC; p<0.05). In conclusion, these findings suggest that in isolated human abdominal adipose tissue resistance arteries, PKC activation impairs microvascular endothelial function likely through an eNOS‐dependent mechanism. Follow‐up experiments are ongoing to determine the involvement of specific PKC isoforms and upstream molecular regulators contributing to PKC‐mediated impairments in endothelial function.

Support or Funding Information

Research support: National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Secreted Klotho Protects Against Aging‐associated Coronary Endothelial Dysfunction via SIRT1 Activation
Authors: Chen Kai , Sun Zhongjie
Published: June 2021

Background and Objective

Coronary heart disease (CHD) is the leading cause of death in the world. Endothelial dysfunction is an initial step in the development of CHD and may be a critical early target for the prevention of CHD. Klotho, originally identified as an aging‐suppressor gene, is predominately expressed in kidneys. Klotho protein is secreted into the circulation. The objective of this study is to investigate whether secreted Klotho protein protects against aging‐associated coronary endothelial dysfunction and CHD.

Methods and Results

We found that aging‐associated coronary endothelial dysfunction assessed by endothelium‐dependent vasodilation and coronary blood flow was accompanied by a reduction in serum level of secreted Klotho (sKL) protein. Using klotho‐hypomorphic (KL (−/−)) mice, we confirmed that Klotho deficiency caused coronary endothelial dysfunction. Interestingly, the impaired endothelium‐dependent vasorelaxation and coronary flow were remarkably improved by in vivo delivery of exogenous sKL protein in aged mice and KL (−/−) mice, suggesting that sKL rescued aging‐induced coronary artery endothelial dysfunction. In addition, sKL treatment protected against aging‐associated and Klotho deficiency‐induced cardiac dysfunction, as evidenced by restoration of stroke volume and cardiac output in aged and KL (−/−) mice. sKL treatment also enhanced coronary endothelial nitric oxide bioavailability and attenuated oxidative stress and apoptosis. Mechanistically, aging via Klotho deficiency suppressed SIRT1‐AMPKα‐eNOS pathway in the coronary endothelial cell through activation of transcription repressor HIC‐1. Furthermore, pharmacological activation of SIRT1 by SRT 1720 protected against Klotho deficiency‐induced coronary artery endothelial dysfunction and cardiac dysfunction.

Conclusion

Klotho regulates coronary artery endothelial function via SIRT1 activation. Supplement of exogenous Klotho protein represents a promising therapeutic strategy for the treatment of aging‐associated coronary heart disease.

Support or Funding Information

AG049780, HL118558

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Building a Data Collection Architecture for Exercise Training and Health Monitoring
Authors: Lee Hsiu An , Hsu Chien‐Yeh
Published: June 2021

The selection of athletes for a sports competition is based on his/her achievements and experience. According to research, in the professional sports program, the selection criteria should be based on the characteristics of the competition, the size, physiology, test value, psychological and physiological development and characteristics of the athletes. However, the qualifications of athletes on cross‐disciplinary and skill have not been fully implemented.

This study is to build a data collection architecture for exercise training and health status monitoring for athletes. The data items which will be stored including sports training data, biochemical indicators, dietary intake, psychological and physiological data, fatigue index etc. There is a data analysis platform build for future data processing.

The design of the collection architecture is divided into five phases, including definition, capture, contribution, share, and analysis. The definition phase and the capture phase are happened in each data source unit and data are transferred and stored in a central data repository. Each data source plays as a contribution role which will follow a prearranged research protocol of the central repository. The information acquisition and storage mechanism are also designed for the repository. The sharing stage is to design a transferring and sharing mechanism for athlete training and competition data to build an exercise research database which is used as a base in the analysis platform in the repository.

The system includes five core modules: Data Contributor, Data Dictionary, Data Access, User Management, and Repository Management. The Data Contributor module provides users with a research case on the web page so that participants can upload data. A screening process is also designed to verify the correctness of the sports data and research database. The Data Dictionary module is to provide the definitions of the Basic Data Elements (BDE) which is the most commonly used fields defined by defined by the project, and Extended Data Elements (EDE) which is the user defined data fields and can be added to the BDE.

Data Access module is the control module for data access permission. The owner of the research case can share the information, once shared, all members of the team will have the right to view and obtain all data, and can query the data in the database in the system. The User Management module is the research team member and its data usage rights. The research owner can set the usage rights of each member. The Repository Management module manages the database structure and provides views of the contents of the database. Using the data collection architecture, the researcher can manage, share, distribute, and process the information and training schedules of participating athletes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endurance Exercise Capacity and Substrate Metabolism in Male and Female Mice
Authors: Rowe Patrick , O'Neill Caitlin , DeWitt Elizabeth , Kolwicz Stephen C
Published: June 2021

The investigation of sex‐specific differences in physiological and pathophysiological processes is of increased importance in scientific research. Previous studies, in both rodents and humans, observed that females generally have a higher endurance exercise capacity (EEC) than males. Additional research suggests that the increased EEC in females is mediated, in part, by differences in substrate metabolism during exercise. The objective of this study was to determine whether changes in exercise substrate metabolism contributed to sex‐specific differences in EEC in mice. To evaluate EEC, male and female, C57BL/6‐NCrl mice (n=9 each group) were subjected to a graded exercise test on a motorized treadmill. The treadmill was set at a 10% incline and the speed gradually increased from 10.2 m/min to 22.2 m/min at fixed intervals for up to 2.5 hrs. At exhaustion, blood glucose (BG) was measured from a tail clip using a hand‐held glucometer. Blood and tissues (heart, liver, skeletal muscle, and adipose) were harvested from all mice. Serum levels of non‐esterified fatty acids (NEFA), triglycerides (TG), cholesterol (CHOL), lactate (LAC), and ketone bodies (KET) were measured using commercially available kits. Endurance exercise times were ~25% higher in females compared to males (121.2 ± 4.0 min vs. 97.3 ± 4.3 min, P<0.05). At exhaustion, BG was significantly lower in females (66.7 ± 3.0 mg/dl) vs. males (75.8 ± 2.4 mg/dl); however, there was no significant difference in LAC. Although post‐exercise serum TG levels were similar, CHOL was significantly lower in females (40.3 ± 2.5 mg/dl vs. 50.6 ± 3.0 mg/dl). Interestingly, serum levels of both NEFA and KET were ~50% higher in females at exhaustion. Although body weight was significantly greater in males (25.6 ± 0.8g vs. 20.1 ± 0.6g), the quadriceps to body weight ratio was similar, suggesting that body or muscle mass was not a factor in the differences. In conclusion, female mice have higher EEC than male mice, which may be due to the supply of exogenous substrates. In particular, female mice have elevated serum levels of fatty acids and ketone bodies at exhaustion, which may reflect an increased ability to mobilize these substrates to prolong endurance exercise. Additional studies will investigate whether endogenous cellular metabolism, especially glycogen and triglycerides in skeletal muscle, contribute to the enhanced endurance capacity phenotype.

Support or Funding Information

This work was supported by funding from the American Heart Association and Ursinus College.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of temperature on athletic performance in the pelagic Mahi‐mahi (Coryphaena hippurus)
Authors: Heuer Rachael M , Stieglitz John D , Enochs Ian C , Pasparakis Christina M , Benetti Daniel D , Grosell Martin
Published: June 2021

Temperature is an environmental factor that can have profound impacts on physiological processes, behavior, and performance in poikilotherms. Temperatures that deviate too far from thermal tolerance are thought to reduce organismal performance by constraining aerobic scope. The objective of the present study was to examine how temperature influences metabolic rate, aerobic scope, and swimming performance in the circum‐global pelagic teleost Mahi‐mahi (Coryphaena hippurus). Mahi were acclimated to a range of temperatures experienced in their natural habitat (20, 24, 28, and 32°C) for 1–2 weeks and then aerobic performance was assessed using swim tunnel respirometry. Using this technique, maximal sustained swimming speed (Ucrit), maximal and minimal metabolic rate, and aerobic scope were measured. Mahi exposed to the lowest (20°C) and highest temperatures (32°C) experienced a reduction in aerobic scope compared to intermediate temperature treatments. A reduction in maximal metabolic rate accounted for the reduction in cold‐acclimated animals, while an increase in minimal metabolic rate led to the reduction in warm‐acclimated animals. Maximal sustained swimming speed showed similar but, non‐significant trends. These results indicate that performance is impaired in mahi at temperatures at the extreme ends of their natural range. These results are particularly relevant in the context of future climate change predictions for mahi residing in areas like the Gulf of Mexico, where 32°C is the mean temperature expected during summer months throughout surface waters by year 2100. Pairing data from the present study to environmental parameters may help assess habitat utilization by many pelagic species as oceans continue to warm.

Support or Funding Information

This research was made possible in part by a grant from The Gulf of Mexico Research Initiative. Data are/will be publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearchinitiative.org

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Simvastatin Inhibits Allergen‐induced IL‐33 Secretion in Human Bronchial Epithelial Cells by Blocking ATP Release and Ca2+ Uptake
Authors: Srisomboon Yotsawee , Maniak Peter , Squillace Diane , Kita Hirohito , O'Grady Scott
Published: June 2021

Previous studies have demonstrated that simvastatin possesses immunomodulatory effects in airways, but the precise mechanisms and therapeutic potential are poorly understood. The objective of the present study was to investigate the inhibitory effects of simvastatin and b‐methyl‐cyclodextrin (β‐MCD) on the innate immune response of the airway epithelium induced by Alternaria alternata (100 μg/ml) and house dust mite (200 μg/ml, HDM) allergens. Experiments were performed with cultured human bronchial epithelial cells transfected with the human IL‐33 gene to produce a robust and reproducible cell model for the study of IL‐33 secretion. Cells were pretreated with varying concentrations of simvastatin ranging from 0.1 to 10 μM for 24 hours prior to allergen exposure. IL‐33 release was quantified by ELISA (Quantikine®) following the procedure provided by the manufacturer. Simvastatin produced a concentration‐dependent inhibition of IL‐33 release that was nearly abolished at 10 μM (IC50; 1.0 μM). In contrast, simvastatin was ineffective at blocking the effects of HDM on IL‐33 release (IC50; >10 μM). Similarly, pretreatment with β‐MCD (5 mM) for 30 minutes also inhibited the Alternaria response, suggesting that depletion of cholesterol from the plasma membrane was responsible for the effects of simvastatin and β‐MCD. Subsequent experiments explored the effects of simvastatin and β‐MCD on Alternaria‐evoked Ca2+ uptake, which is necessary for activating IL‐33 release. Simvastatin (10 μM, 24 hrs) and β‐MCD (5 mM; 30 minutes) both abolished the increase in [Ca2+]i induced by Alternaria. We next examined the possibility that abolition of Ca2+ uptake resulted from inhibition of ATP release, since ATP activates P2X7 receptors that mediate Ca2+ influx in response to Alternaria exposure. ATP release was measured in real time using a luciferin/luciferase‐based photon counting assay. Both simvastatin and β‐MCD blocked ATP release. Moreover, addition of 100 μM cholesterol to the extracellular media rescued the effects of simvastatin. These results demonstrated that inhibition of IL‐33 secretion by simvastatin and β‐MCD following Alternaria exposure involved suppression of ATP release that was dependent on membrane cholesterol content. Thus, membrane cholesterol likely plays a pivotal role in innate immune function of airway epithelial cells.

Support or Funding Information

This study was supported by NIH R01 AI128729 to HK and SMO.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Estrogen on Alveolar Macrophage Polarization in Response to Particulate Matter Exposure
Authors: Fuentes Nathalie , Pesantez Jonathan , Nicoleau Marvin , Wal Randy Vander , Chroneos Zissis , Silveyra Patricia
Published: June 2021

Background

Lung diseases affect more than 500 million people worldwide. Exposure to air pollutants such as particulate matter (PM) contributes significantly to lung disease, especially in women, indicating an intersection of air pollution and sex hormones in lung disease outcomes. Studies have been performed on the effects of estrogens on lung immunity, particularly in alveolar macrophages (AM). AMs provide one of the first lines of defense in the airways and are important mediators of inflammation. However, the mechanisms by which estrogens control the immune response to PM exposure remain unclear. Here, we hypothesized that PM exposure triggers AM polarization to the pro‐inflammatory (M1) macrophage phenotype in a sex‐dependent matter, and that this process is mediated by estrogens in female cells.

Methods

We tested this hypothesis by exposing male (RAW264.7, BALB/c) and female mouse AMs (J774A.1, BALB/c) to a synthetic PM preparation in the presence or absence of estrogen agonists/antagonists. Cell viability and cytotoxicity were measured at 8h to determine PM dose‐responses. Cells were then stimulated with LPS or IL‐4, which have been described as mediators of AM polarization. The polarization phenotype, determined by expression of specific markers (iNOS, Arg1), was evaluated by quantitative real‐time PCR.

Results

Our results demonstrate that estrogen treatment decreases iNOS expression and increases Arg1 expression, impairing the LPS‐induced pro‐inflammatory phase and triggering resolution of inflammation in RAW 264.7 cells. In addition, induction of AM polarization towards different phenotypes not only was altered by PM and estrogen treatment, but also depended on the sex of the cell. Interestingly, PM treatment led to decreased expression of estrogen receptors when compared to the non‐PM treated cells.

Conclusions

We conclude that estrogens regulate the immune response to PM in a sex‐dependent manner. These findings may be relevant to elucidate the transcriptional mechanisms involved in the sex‐specific inflammatory response and particularly for the identification of novel hormone receptor modulators efficacious in the prevention or mitigation of chronic inflammation in men and women.

Support or Funding Information

This research was supported by funding from NIH K01HL133520, the Center for Research on Women and Newborn Health and the American Physiological Society Porter Physiology Development Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cloning, purification, and enzymatic activity of the quorum sensing signal synthase RhlI
Authors: Wetherald Noah , Ricciardi Meghan , Ulrich Scott Michael
Published: June 2021

Quorum sensing is a bacterial cell‐cell communication system that functions through the synthesis, secretion, and detection of signaling molecules called autoinducers. Quorum sensing enables bacteria to assess their cell density and coordinate group behaviors that are advantageous at high cell density. Many bacteria that cause acute and chronic infections in humans use quorum sensing to control expression of virulence factors and formation of biofilms, so inhibitors of quorum sensing hold significant promise as novel antibacterial agents. By targeting bacterial virulence as opposed to growth and survival, quorum sensing inhibitors avoid the selective pressure for drug resistance that is inherent to traditional antibiotics.

The main quorum sensing system in Gram‐negative bacteria is based on the synthesis and detection of acyl homoserine lactone (AHL) autoinducers. Efforts to inhibit AHL‐mediated quorum sensing have focused on developing synthetic AHL analogs that act as antagonists of AHL receptors. These compounds have been shown to block virulence controlled by quorum sensing, validating quorum sensing as an antibacterial drug target.

The overall goal of the project is to develop inhibitors of AHL synthase enzymes, an alternative quorum sensing drug target that remains under‐explored. Towards this goal, we have cloned and purified RhlI from Pseudomonas aeruginosa and established its enzymatic activity as an assay platform for candidate synthetic inhibitors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crossing Blood‐Brain Barrier with Carbon Quantum Dots
Authors: Seven Elif S. , Zhou Yiqun , Seven Yasin B. , Mitchell Gordon S. , Leblanc Roger M.
Published: June 2021

The blood‐brain barrier (BBB) is a semi‐permeable structure, which is essential for the protection of the central nervous system (CNS). BBB selectively allows the passage of the necessary small molecules/nutrients from the blood to the brain while blocking pathogens or toxins. However, BBB becomes a major obstacle during disease since it significantly hinders the drug delivery to the CNS. Therefore, developing a drug delivery system to cross BBB is highly desired. Recently, nanoparticles (NPs) have become emergent tools to assist drugs to cross BBB. Since most NPs cannot cross BBB by themselves, the nanoparticle‐mediated drug delivery heavily depends on conjugating the NPs to the ligands such as transferrin to pass through the BBB via receptor‐mediated endocytosis.

Here, we designed Carbon quantum dots (CDs) that can cross BBB without the need of a ligand. We hypothesized that the saccharide‐based CDs will have an amphiphilic structure with surface groups that resemble the saccharide precursor. Therefore, these CDs will be able to cross BBB either with the aid of glucose transporter proteins or by passive diffusion.

CDs were prepared via a bottom‐up approach using hydrothermal carbonization of saccharides and characterized using various spectroscopic, microscopic and surface chemistry methods such as UV/vis, fluorescence, FTIR/ATR, TEM, AFM and Langmuir monolayers. To test our hypothesis, CDs were injected into the heart of wild‐type zebrafish, Danio rerio. The confocal images showed the accumulation of CDs in the CNS of zebrafish confirming the ability to cross BBB. CDs were also shown to pass through rat BBB 45 min after intravenous tail injection (2.5 mg/kg). Following transcardial perfusion, rat spinal cord is post‐fixed in paraformaldehyde overnight and cryoprotected in sucrose. Presence of CDs in the CNS was confirmed in 40 μm tissue sections via epifluorescence microscopy. These proof‐of‐principle studies suggest that saccharide‐based CDs can cross BBB in different vertebrate species. Further studies should address if the amount of BBB penetration is sufficient to cause a biologically meaningful effect in the CNS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineered High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation
Authors: Woods Robert J. , Wu Shengcheng , Meng Lu , Gerner‐Smidt Christian , Sanders Matthew J. , Ben‐Ayre Shani Leviatan , Padler‐Karavani Vered , Yang Loretta
Published: June 2021

As alternatives to established carbohydrate‐detection reagents (antibodies and lectins), Lectenz® Bio is developing lectin‐like, enzyme‐derived proteins (known as Lectenz®), that target glycan sequences for which existing reagents either do not exist or are sub‐optimal. Lectenz® are engineered from catalytically inactivated glycan‐processing enzymes that have been optimized for high affinity towards specific glycan sequences. The conversion of such enzymes into affinity reagents is facilitated by computationally‐guided directed evolution. Lectenz® are being developed for a variety of glycan detection and enrichment applications including affinity chromatography, Western blot, and immunohistochemistry. Here we demonstrate the performance and specificity of two novel sialic acid recognizing Lectenz® engineered from a sialidase enzyme.

Support or Funding Information

NIH R44 GM113351, R44 OD024964

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polyphenols and other Phytochemicals in Cancer Prevention and Management
Authors: Asuzu Peace C , Besong Samuel Ayuk , Aryee Alberta NA
Published: June 2021

Phenolic compounds and other phytochemicals are significant resources for drug discovery and development. Plant‐derived natural products have contributed in preventing and managing oxidative stress, and in cancer therapeutics. Recent reports indicate that 83% of new anti‐cancer agents are of natural product origin. The aim of this study was to assess the content and properties of bioactive phytochemicals in water, ethanol, methanol, acetone, and dichloromethane extracts of Prunus africana, Pausinystalia yohimbe, Moringa oleifera, Momordica charantia and Orthero spp, and test their therapeutic effect on a wide range of diseases. In the first part of the study, the content of total polyphenols (TPC), flavonoids (TFC), carotenoid, anthocyanin, and antioxidant properties of the extracts of the different plant parts of these five plants were measured using the Folin‐Ciocalteu, 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity (DPPH), ferric reducing antioxidant power (FRAP), and 2,2′‐Azino‐bis(3‐ ethylbenzthiazoline‐6‐sulfonic acid) radical scavenging (ABTS) assays. For most samples, extraction yields were highest when either ethanol or methanol were used as extraction solvent. The FRAP values were in the range of 6.04 – 112.00 μM Fe2+/g, while the highest TPC and TFC were seen in the methanol extract of P. africana bark from Cameroon (1397.33 mg GAE/g) and acetone extract of M. charantia leaf (217.33 mg RU/g), respectively. The concentration required to obtain a 50% antioxidant effect (EC50) for the acetone and methanol extracts of P. africana bark (Cameroon), methanol and ethanol extracts of P. yohimbe leaf and the methanol extract of P. yohimbe root were comparable to that of ascorbic acid. Seven phenolic acids were identified in the extracts of P. africana and P. yohimbe using high performance liquid chromatography (HPLC). The most abundant phenolic acids were; vanillic acid (116.41 mg/g dry extract in P. yohimbe leaf methanol extract) and trans‐sinapic acid (102.22 mg/g dry extract in P. africana bark water extract). This study shows that these plants parts are valuable sources of natural polyphenols and antioxidants and their individual and/ synergistic effect may modulate cellular and signaling events involved in growth and invasion of cancer cells.

Support or Funding Information

USDA‐NIFADelaware State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the Insulin Staining of LEW.1WR1 and LEW/SsNHsd Rats in Response to Polyinosinic‐Cytidylic Acid
Authors: Wade Amber , Wimalarathne Madushika , Cantrell Kayleigh , Gibson Helen , Love‐Rutledge Sharifa
Published: June 2021

The experimental model LEW.1WR1(1WR1) rats show increased type 1 diabetes (T1D) onset with the administration polyinosinic‐polycytidylic acid (PIC). The susceptibility window of inducible T1D in the LEW rat occurs between 21–35 days (3–5 weeks) of life, which is a similar life stage of the onset of human T1D (peak age of onset is 14 years old). The susceptibility of this model is related to FAT10 overexpression, a ubiquitin‐like protein that plays an unclear role in the regulation of lipid metabolism. The objective of this study was to compare the size and number of insulin stained pancreatic islets in 1WR1 rats treated with PIC with control rats during disease induction. Using immunohistochemistry we were able to visualize pancreatic islet infiltration by CD3+ and CD68+ stained cells. We observed increased immune cells in the PIC treated pancreata. There were also no significant differences in blood insulin level which appeared to be trending up. Using blinded trained scorers we observed islet size varied as well as intensity of staining. We believe that the beta cells may be responding to the inflammatory stimulus which could play a role in increasing the susceptibility to T1D.

Support or Funding Information

The University of Alabama in Huntsville

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Different Antihypertensive Classes on the Complexity of Heart Rate and Blood Pressure Variability
Authors: Salgado Hélio Cesar , Silva Luiz Eduardo Virgilio , Silva Carlos Alberto Aguiar , Nobre Fernando , Fazan Rubens
Published: June 2021

Hypertension is associated with several physiological dysfunctions, including changes in the neurohumoral control of heart and vessels. Different classes of antihypertensive drugs may have distinct effects on the autonomic system and body homeostasis, but their individual effects on the complexity of cardiovascular regulation are still not established. We investigated the effect of five different classes of antihypertensive on the complexity of pulse interval (PI) and arterial pressure (AP) variability in an experimental model of renal hypertension (2 kidney 1 clip, 2K1C) in the rat. Hypertensive rats were treated (by gavage) during 15 days with: water (N=12), ramipril (2 mg/kg/day; N=7), losartan (10 mg/kg/day; N=8), atenolol (90 mg/kg/day; N=9), amlodipine (10 mg/kg/day; N=7) or hydrochlorothiazide (20 mg/kg/day; N=8). A normotensive (sham‐operated) control group (N=10) received water for the same period. After the treatment, rats had their raw AP recorded and beat‐by‐beat series of systolic AP and PI were created. The complexity of AP and PI was assessed by multiscale entropy (MSE) and detrended fluctuation analysis (DFA). For PI series, hypertensive rats treated with water showed decreased MSE and increased DFA in the middle range of scales. For AP series, hypertensive rats untreated or treated with amlodipine have lower MSE at short and middle time scales. Hypertensive rats treated with amlodipine also showed altered DFA in comparison to the other groups. All antihypertensive drugs were efficient to restore AP to normal levels. Moreover, 2K1C untreated (water) rats presented the lowest cardiovascular complexity and all antihypertensive drugs, except for amlodipine, were capable to restore the cardiovascular complexity to normal levels. Therefore, amlodipine seems to have a deleterious effect on the cardiovascular integrative control, probably due to the increased sympathetic activity induced by amlodipine.

Support or Funding Information

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance Code 001, São Paulo Research Foundation (FAPESP, Proc. 2013/20549‐7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 402076/2016‐8).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Peripherally Administered Proinflammatory Cytokines Increase Afferent Renal Nerve Activity in Rat
Authors: Cao Yiling , Yu Yang , Bell Balyssa , Felder Robert B. , Wei Shun‐Guang
Published: June 2021

Inflammation plays an important role in sympathetic activation in cardiovascular diseases. Systemic administration of the proinflammatory cytokines (PICs) tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) increases mean blood pressure (MBP), heart rate (HR) and efferent renal sympathetic nerve activity. However, the renal nerve contains both afferent and efferent fibers. The afferent renal nerve is composed of chemoreceptor and mechanoreceptor fibers, and delivers peripheral signals to the brain that have been reported to increase sympathetic outflow in hypertension and heart failure. The effect of circulating PICs on afferent renal nerve activity (ARNA) has not previously been investigated. The present study sought to determine whether peripherally administered PICs TNF‐α and IL‐1β increase ARNA. Urethane anesthetized male Sprague Dawley rats (300–350 g) underwent an intravenous (IV) or a renal artery injection of TNF‐α or IL‐1β. MBP (mmHg), HR (beats/min) and ARNA (% change from baseline) were continuously recorded for 4–5 hours. IV injection of TNF‐α (500 ng/kg) or IL‐1β (500 ng/kg) significantly (* p<0.01 vs. baseline) increased MBP (20.1 ± 2.2* and 23.4 ± 2.1*, respectively), HR (51.5 ± 6.7* and 58.8 ± 7.1*, respectively) and ARNA (37.2 ± 3.5* and 42.1 ± 3.5*, respectively) with peak responses 20–30 mins after injection. Similarly, renal artery injection of the same doses of TNF‐α or IL‐1β induced increases (*p<0.01 vs. baseline) in MBP (17.0 ± 2.1* and 19.9 ± 2.2*, respectively), HR (45.5 ± 5.6* and 49.3 ± 4.6*, respectively) and ARNA (30.7 ± 3.5* and 38.4 ± 4.5*, respectively) that began within 5–10 mins and peaked at 20–30 mins after the injection. Quantitative real‐time PCR showed high mRNA levels of TNF‐α receptor 1 and IL‐1β receptor in renal cortex and medulla. These data indicate that circulating TNF‐α and IL‐1β can act at the kidney to augment afferent renal nerve activity, suggesting that circulating PICs may activate ARN fibers to elicit a sympathetically mediated pressor response. PIC activation of renal afferent nerves may be an important mechanism contributing to inflammation‐driven sympathetic activation in heart failure and hypertension. These findings provide support for the potential therapeutic benefit of renal afferent denervation.

Support or Funding Information

Supported by NIH grant R01 HL139521 (S.G. Wei)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The apical membrane localization and trafficking of uromodulin (UMOD) is critically dependent on nonmuscle myosin II isoforms Myh9 and Myh10 in mouse thick ascending limb epithelial cells
Authors: Otterpohl Karla , Busselman Brook , Hart Ryan , Shan Kevin , Lin Brendon , Surendran Kameswaran , Chandrasekar Indra
Published: June 2021

Mutations that alter the trafficking and function of uromodulin, a membrane associated GPI anchored protein expressed in the TAL segment, result in autosomal dominant tubulointerstitial kidney disease (ADTKD)1. Proper localization and function of GPI‐anchored proteins requires intact ER to Golgi to apical membrane transport. Recently Nonmuscle myosin II (NM2) have been linked to vesicular transport2, and we have identified a novel function for NM2 isoforms Myh9 (NM2A) and Myh10 (NM2B) in regulating membrane remodeling in mammalian cells3. Due to the differential expression and unique localization of NM2 isoforms in the murine renal tubules4, we hypothesized that NM2 isoforms are involved in the specialized transport pathways of membrane associated proteins in the TAL epithelial cells. Conditional genetic knockout of Myh9 & Myh10 within the renal tubules of mice results in initial mis‐localization and gradual loss of the TAL‐specific protein: Uromodulin. These mice develop rapidly progressing tubulointerstitial kidney disease and are moribund by 5–6 months of age. In order to understand the mechanisms underlying the mis‐localization of the Uromodulin, we generated an immortalized TAL cell line from the adult kidneys of Pax8‐>rtTA; Tet‐o‐cre; Myh9F/F; Myh10F/F mice crossed to mice harboring Immorto +/Tg transgene. These cells can be maintained at 33C indefinitely due to the expression of the temperature sensitive SV40 T‐antigen transgene. To promote polarization and endogenous expression of uromodulin and other membrane‐associated proteins in these cells, we have modified a recently established culture protocol with cells grown on polyester membranes at 37C on an orbital shaker5. We observe robust expression of endogenous uromodulin detected by immunohistochemistry and western blot analysis. Furthermore, induction of Myh9 and Myh10 inactivation by doxycycline treatment of these cells results in altered levels of uromodulin in the cells possibly indicating changes in N‐glycosylation of uromodulin along the ER‐Golgi transport pathway. Loss of NM2 also decreases the intracellular sodium levels in the knockout cells, observed using sodium green™ indicator. Our studies indicate a novel relationship between NM2 isoforms and uromodulin transport along the TAL segment and are suggestive of a role for MYH9 and MYH10 in tubulointerstitial kidney disease associated with abnormal renal epithelial transport.

Support or Funding Information

Funding support from NIH Grant P20GM103620 and Sanford Research Program Funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Accurate segmentation of prostate cancer histomorphometric features using a weakly supervised convolutional neural network
Authors: Bukowy John David , Foss Halle , McGarry Sean D. , Lowman Allison , Hurrell Sarah , Ickzkowski Kenneth A , Banerjee Anjishnu , Barrington Alexander , Dayton Alex , Unteriner Jackson , Jacobsohn Kenneth , See William , Nevalanian Marja , Nencka Andrew , Ethridge Tyler , Jarrard David , LaViolette Peter
Published: June 2021

Prostate cancer (PCa) arises from the glandular epithelium. To confirm the presence of cancer, a pathologist uses stained tissue samples taken either from biopsy or prostatectomy. Due to the relationship between epithelium and cancer, special consideration is given to regions identified as epithelium. Histomophometric techniques have long been used to identify areas of epithelium within the tissue for automated detection and classification pipelines; however, they are often rigid in their implementation and their performance suffers on large datasets where variation in staining, imaging, and preparation is difficult to control. The recent development and popularity of deep learning methods for image processing and segmentation offer great promise for developing robust classification pipelines for such ends; however, they require large labeled datasets for training.

The goal of this study was to combine weakly labeled datasets generated using histomophometric techniques, and high‐quality labeled datasets from human observers to train a convolutional neural network. In doing so we developed a pixel‐wise segmentation algorithm for classification of stromal, epithelium, and lumen (SEL) regions for use on both biopsy core and whole‐mount bright‐field H&E stained tissue. We provide evidence that by simply training a deep learning algorithm on weakly labeled data, we can improve the robustness of the classification. Finally, we show that not only does this method carry primary improvement on SEL labeling within tissue, but that the information provided by the deep learning generated labels improves cancer classification in a higher‐order algorithm over the histomorphometric labels that it was trained on.

Support or Funding Information

Funding was provided by the State of Wisconsin Tax Check‐off Program for Prostate Cancer Research (RO1CA218144 and R01CA113580) and the National Center for Advancing Translational Sciences (NIH UL1TR001436 and TL1TR001437).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A three‐dimensional model of the pterygopalatine fossa significantly enhances the learning experience
Authors: Tanner Jordan A , Sakaguchi Alan , Bartanuszova Maria , Ruparel Nikita , Jackson Jeffrey , Johnson Linda , Nation Haley , Jethwa Beeran , King Thomas S , Bhattacharya Arunabh , Sharma Ramaswamy
Published: June 2021

The pterygopalatine fossa (PPF) is a small bilateral space deep within the skull that acts as a major neurovascular junction between the oral cavity, nasopharynx, orbit, infratemporal fossa, and middle cranial fossa. Because of its important crossroads of afferent sympathetic, and parasympathetic fibers, students, radiologists, ophthalmologists, neurosurgeons, and otorhinolaryngologists need to have a firm understanding of the PPF. However, its small volume, poor accessibility and numerous neurovascular connections make it a difficult space to comprehend using two‐dimensional illustrations and a single cadaveric dissection. We hypothesized that a 3D model of the PPF significantly improves the student's understanding of the PPF's boundaries, its communicating channels, and neurovascular structures as compared to traditional forms of pedagogy by promoting visual and kinesthetic learning experiences. We developed a 3D model of the PPF and an instructional manual describing the model. We evaluated our model by analyzing student performance on pre‐ and post‐quizzes and a student user satisfaction survey based on 5‐point Likert scale. A total of 43 undergraduate students enrolled in pre‐medical and dental schools completed the study; 21 students used a human half‐skull (control group) and 22 students used the 3D model (intervention group). The intervention group performed significantly better on the post‐quiz (p=0.01) when compared to the pre‐quiz; however, the control group did not improve their quiz scores (p=0.17). Surveys also indicated that the model enhanced the learning experience. We conclude that anatomy educational tools such as 3D models that would be easily accessible outside of the laboratory promote student learning ability.

Support or Funding Information

UT System Shine Academy of Health Science Education Small Grants Program to Drs. Ramaswamy Sharma and Arunabh Bhattacharya

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation and characterization of human iPSCs from human fibroblasts in respect to osteochondral regeneration
Authors: Danisovic Lubos , Csobonyeiova Maria , Nicodemou Andreas , Novakova Zuzana Varchulova , Miko Michal , Zamborsky Radoslav , Varga Ivan
Published: June 2021

Cartilage and bone disorders are a group of varied acute and chronic traumatic, degenerative, malignant or congenital conditions affecting the joints and bones. Recently, cell‐based therapy appears to be a very promising tool of regenerative medicine and it has attracted the attention of many researchers and physicians. The huge attention is given to induced pluripotent stem cells (iPSCs). In the experiment, the human fibroblasts originally derived from subcutaneous tissue of infant due to cytogenetical analysis were used. Fibroblasts from 2nd passage were thawed and were sub‐passaged two more times in D‐MEM low glucose with 10% fetal bovine serum and antibiotics (Sigma Aldrich). Then, fibroblasts were reprogrammed using the StemRNA‐SR Reprogramming Kit (Stemgent) following the manufacturer's protocol with slight modifications. Briefly, 7 × 104 fibroblasts were plated into one well of a 6‐well plate coated with Geltrex (Thermo Fisher) on day 0, and cultured at 37 °C and 5% CO2 overnight in D‐MEM with 10% fetal bovine serum. Then, a medium was replaced by StemMACS™ iPS‐Brew XF medium (Miltenyi Biotec) and 500 μL NM‐RNA transfection complex solution was added to wells. This step was repeated during the next 3 days. Then, cells were cultivated in StemMACS™ iPS‐Brew XF medium. After 13 to 15 days, when the colonies emerged they were identified, mechanically picked and sub‐passaged. Obtained iPSCs were characterized by flow cytometry, real‐time PCR, and by histochemistry. Moreover, their chondrogenic and osteogenic differentiation potential was assessed according to the expression of specific genes. Normal fibroblasts were used as negative control. Obtained results showed that fibroblasts are easily accessible and they are suitable for generation of iPSCs, which have the potential to differentiate into chondroblasts and osteoblasts. It may be emphasized that after performing further studies focused on their bio‐safety they will represent unique tool for osteochondral regenerative medicine.

Support or Funding Information

APVV No. 14‐0032.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Aldh1l1 Knockout On Metabolic Phenotype in Mouse Liver
Authors: Sharma Jaspreet , Krupenko Natalia I. , Krupenko Sergey A.
Published: June 2021

ALDH1L1 (10‐formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in liver. This enzyme metabolizes 10‐formyltetrahydrofolate to produce tetrahydrofolate (THF) and is involved in the regulation of reduced folate pools, de novo purine biosynthesis, and the flux of folate‐bound methyl groups. The goal of this study was to survey the metabolic impact of the Aldh1l1 gene loss in mouse liver. Aldh1l1−/− (KO) mice were generated using gene‐targeted ES cell clone (C57BL/6N background) from KOMP repository. KO mice were viable and did not show obvious phenotypic differences from the wild type animals. Flash frozen hepatic tissues from 12–14 weeks old Aldh1l1+/+and Aldh1l1−/− mice were profiled to identify metabolite changes and define associated pathways. In this study, mice were kept on standard AIN‐93 diet with 2.2 ppm of folic acid. Global metabolomic analysis was performed by Metabolon Inc. (Durham, NC) using Ultrahigh Performance Liquid Chromatography‐Tandem Mass Spectroscopy (UPLC‐MS/MS) for five samples from each group. This analysis has identified 628 known metabolites and principal component analysis demonstrated good segregation between the two genotypes. The difference for 91 metabolites was statistically significant between the two groups, with 31 compounds being decreased and 60 being elevated in Aldh1l1−/− compared to the Aldh1l1+/+ mice. This analysis indicated that KO mice might experience functional folate deficiency. Specifically, the intermediate of the histidine degradation pathway serving as a marker of folate deficiency, formiminoglutamate, was increased more than 15‐fold in KO mice. Furthermore, tissue folic acid and dihydrofolate were strongly depleted (more than 2‐fold and 5‐fold, respectively) in these animals, which would be in agreement with folate deficiency. In addition, a 2‐fold decrease in glycine was detected in KO mice. Strong drop in several glycine conjugates was also observed in the KO genotype, which is apparently associated with the deficiency of glycine. The depletion of glycine was likely caused by the decrease of the THF‐dependent synthesis of glycine from serine, with the underlying basis for this phenomenon being the decrease of the THF pool observed in Aldh1l1−/− mice. The depletion of glycine and/or folate could be the cause of several secondary effects on metabolism including the decrease in glutathione, lower availability of ascorbate, impaired mitochondrial lipid metabolism, oxidative stress, and less efficient remediation of toxic products. Our data also imply a compromised liver function in the KO mice and associated changes in gut microbiome activity. Overall, our study indicated significant metabolic perturbations caused by the Aldh1l1 gene knockout.

Support or Funding Information

NIH R01DK 54388

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.