bluuebunny/crossref_metadata_2025 huggingface dataset:
Aplysinopsins Inhibit Growth of Crypotococcus neoformans by Interfering with Inositol Metabolism
Authors: Moss Jackson , Clouse Alexandria , McCullough Donna , Wloch‐Salamon Dominika , Johnson Paul , Bialonska Dobrusia
Published: June 2021

Cryptococcus neoformans is a widespread microscopic fungus (yeast), that humans are frequently exposed to via inhalation. Such exposure is usually harmless, resulting in no adverse reactions. However, for those with a weakened immune system, particularly those with advanced HIV/AIDS, C. neoformans may present as an opportunistic, facultative, intracellular, pathogen causing the disease cryptococcosis, defined by meningitis and/or encephalitis. While the advent of antiretroviral drugs has reduced the risk of cryptococcal meningitis for patients in the US and other developed countries, ~250,000 cases of cryptococcal meningitis are reported each year with a resulting ~200,000 deaths, it remains an acute concern for immunocompromised patients in developing nations with limited access to modern healthcare.

In this work, we screened three molecules of the tryptophan derived aplysinopsin family of compounds (aplysinopsin, 5‐bromoaplysinopsin, and 6‐bromoaplysinopsin) for their ability to inhibit the growth of the pathogenic yeast C. neoformans. These compounds were originally isolated from marine invertebrates, but have since been synthesized in the lab for this work. All of the studied compounds exhibited antifungal activity with a potency similar to the current front line treatment for cryptococcosis, Amphotercin B, as the identified IC50 for these compounds was between 9–20 μg/mL. In order to further characterize these compounds activity, we also investigated the underlying mechanisms involved in their antifungal activity via genetic approaches.

In order to characterize the underlying mechanisms of this antifungal activity, we used Saccharomyces cerevisiae as a model organism. We identified two mutants lacking genes for metabolism of inositol that showed increased susceptibility to these compounds. These mutants were also more susceptible to aplysinopsins, compared to the wild type, indicating that our compounds may be targeting synthesis of inositol, an important precursor for several secondary messengers in fungi. While the mechanisms of Cryptococcus neurotropism remain poorly understood, recent studies have revealed that C. neoformans has evolved sophisticated inositol acquisition systems and inositol is abundant in the human central nervous system. Thus, inositol utilization in C. neoformans, and its likely contribution to Cryptococcus virulence, may represent one example of a common trait for the emergence of pathogens from environmental reservoirs as well as a target for novel antifungal compounds.

Support or Funding Information

UNG FUSE grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How Entamoeba histolytica goes vampire: the hunt for new genes in the conserved process of trogocytosis
Authors: Feeney Shea E. , Ralston Katherine S.
Published: June 2021

The eukaryotic pathogen Entamoeba histolytica (histo‐: tissue; lytic‐: dissolving) causes amoebiasis in humans. The profound tissue damage during infection is likely driven by E. histolytica's ability to kill human cells through trogocytosis (trogo‐: nibble). During trogocytosis, amoebae pinch off and internalize bites of membrane and intracellular contents from human cells, which eventually results in cell death. In contrast, amoebae ingest pre‐killed human cells whole via phagocytosis. Beyond E. histolytica, trogocytosis is part of an underappreciated, emerging theme in eukaryotic biology. While there are some shared features between trogocytosis and phagocytosis, we hypothesize that there are differences. Since E. histolytica is capable of both trogocytosis and phagocytosis, it represents an excellent model to dissect the trogocytosis‐specific mechanism. Both phagocytosis and trogocytosis are under feed‐forward gene regulation in E. histolytica. To identify genes differentially involved in trogocytosis versus phagocytosis, E. histolytica were fed live or pre‐killed human cells, respectively, to induce upregulation of genes involved in these processes. RNA‐seq analysis was used to identify genes that changed substantially in comparison to amoebae that had not been fed human cells. Over 1000 genes were found to be significantly differentially expressed between trogocytosis and phagocytosis and, were involved in various processes including cytoskeletal rearrangement. To begin to explore the roles of select genes that were significantly upregulated in ingestion, we are currently generating RNAi knockdown mutants and evaluating their phenotypes. This work should have broad impact since trogocytosis is directly relevant to amoebiasis and also appears to be a fundamental form of eukaryotic cell‐cell interaction.

Support or Funding Information

NIH MCB T32 GM007377 (to SEF); Pew Charitable Trusts (to KSR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Early Alterations in the Expressions of MCTS and HIF‐1α in Experimental Diabetes Mellitus
Authors: Encinas Jéssica Freitas Araujo , Perez Matheus Moreira , Peiró Carlos Henrique Foncesca , Petri Giuliana , Simões Diogo Pimenta , Mathia Vanessa Lopes , Mendonça Maria Isabel Sacchi , da Costa Aguiar Alves Beatriz , Fonseca Fernando Luiz Affonso , da Veiga Glaucia Luciano
Published: June 2021

Aim

To evaluate alterations due to the establishment of experimental diabetes mellitus (DM) alloxan‐induced in biochemical parameters and the expression of genes MCT1, MCT4, CD147 and HIF‐1α in different tissues and in the blood profile.

Methods

Wistar rats (120–180g) were conditioned at the FMABC bioterium in conditions of 12 hours light/dark, climate temperature (21 ± 2 °C) and ad libitum supply of water and food. Animals of the DM group were treated with Alloxan monohydrate (120 mg/kg) intraperitoneal (ip); the control group sham (NDS) received injections of sodium chloride 0,9% (ip). The glycemic values were measured weekly during the period of treatment through a commercial glycosimeter by puncturing of the caudal vein of the animal; animals with glycemia above ≥200 mg / dL were determined as diabetic. Evaluated the biochemical parameters: glycemia, blood creatinine and urea, and the expression of molecular biomarkers in the heart, brain, kidney and blood by the RT‐qPCR technique. Data was analyzed by GraphPad Prism® 6.0, statistical differences were observed by the Mann‐Whitney test and Student T test with an established significance level in 5% (descriptive value of p <0.05).

Results

The biochemical parameters found were: glycemia (DM7 619.1 ± 140.7 * p <0.005, n = 10 vs NDS 192.5 ± 43.64, n = 4), blood creatinine (DM7 0.46 ± 0.05 * p <0.005, n = 10 vs NDS 0.40 ± 0.07, n = 4) and urea (DM7 96.7 ± 26.6 * p <0.005, n = 10 vs. NDS 46.5 ± 7.0, n = 4) these values follow the established standards for the diabetic profile in the present study. In the molecular analyzes an increase of CD147 in the brain was observed (DM7 26.72 ± 27.45, * p <0.005, n = 9 vs NDS 1,489 ± 1,648, n = 4). In the blood profile an enhancement in the expressions of all the genes studied in the animals with DM were found; MCT1 (DM7 3,739 ± 3,057 * p <0.005, n = 10 vs NDS 0.009745 ± 0.007489, n = 4); MCT4 (DM7 7.364 ± 7.3, * p <0.005 n = 10 vs NDS 0.06399 ± 0.04844, n = 3); CD147 (DM7 4,295 ± 4,424 * p <0.005, n = 10 vs NDS 0.2212 ± 0.01653, n = 4) and HIF‐1α (DM7 1,413 ± 1,087 * p <0.005, n = 10 vs NDS 0.003833 ± 0.003741, n = 4). A positive correlation between glucose and MCT1 in the blood profile (r = 0.8061, * p <0.05, n = 10) was also noted, Spearman test.

Conclusions

Preliminary data demonstrate that even early DM promotes serious biochemical changes accompanied by modifications in the expression of genes involved with glycolytic metabolism and in the cellular hypoxia mechanism. Apparently, the brain is the first tissue affected by these changes.

Support or Funding Information

CAPES and CNPq

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generating highly potent and efficacious antibodies to the cholecystokinin B (CCK‐B) receptor by ribosome display for the treatment of neuropathic pain
Authors: Kunamneni Adinarayana , Montera Marena , Gott Katherine , Durvasula Ravi , Westlund Karin
Published: June 2021

Development of effective non‐opioid therapeutics tailored to specific chronic pain syndromes is sorely needed. Our recent microchip gene expression profile identified upregulated cholecystokinin B (CCK‐B) receptor in trigeminal ganglia (TG) as one suitable target among many others. Thus, the CCK‐B receptor is an ideal candidate to impact both nociceptive and limbic components of chronic pain. More importantly, its inhibition can reverse opiate tolerance. Based on the literature and the gene expression changes over time, the CCK‐B receptor is implicated as one of the persisting functional targets suitable for development of therapeutics to inhibit chronic pain‐ and anxiety‐related behaviors. Here we describe the isolation and characterization of a panel of CCK‐B receptor specific antibodies using ribosome display technology. Furthermore, we demonstrate in vivo efficacy of our antibodies in a relevant model of chronic neuropathic pain. This is the first example, to date, of scFv antibodies that can block the CCK‐B receptor directly with high potency and specificity. This highlights the exciting potential therapeutic opportunity of an antibody for blocking this type of receptor.

Support or Funding Information

This work was supported by Startup dollars from Loyola University Medical Center (RD) and University of New Mexico (KW).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes in VEGF and HIF 1α gene expression levels as potential diagnostic/prognostic markers in liquid biopsies on breast cancer patients
Authors: Peiró Carlos Henrique Foncesca , Encinas Jéssica Freitas Araújo , Aquino Glauco Sérgio Avelino , Veiga Glaucia Luciano , Perez Matheus Moreira , Fonseca Fernando Luiz Affonso , Alves Beatriz Costa Aguiar
Published: June 2021

Aim

This study aimed to evaluate the potential diagnostic and/or prognostic value of VEGF and HIF 1α gene expressions in peripheral blood of patients with breast cancer under chemotherapy treatment.

Methods

Peripheral blood samples and tumor biopsies were collected from 125 patients with breast cancer and from 25 healthy women (control). Blood samples were collected at diagnosis and after 3 and 6 months of the beginning of chemotherapy. Total RNA isolation was performed using TRIZol reagent. RNA was then converted to cDNA and the gene expression analysis was made by RT‐qPCR. Results were expressed as mean ± standard deviation and analysed by GraphPad Prism® 6.0 using Mann‐Withney, Friedman and Spearman tests, with a significancy level established in 5% (value of p< 0,05).

Results

Gene expression analysis showed an increase of HIF 1α gene expression in patients at diagnosis when compared to that of healthy women (Patients: 0.3362 ± 0.7729, n=34 vs Donors: 0.00212 ± 0.001509, n=25, *p<0.0001). Analysis showed that in patients HIF 1α and VEGF expression is higher in peripheral blood than tumor samples (blood: 0.1927 ± 0.4754, n=108 vs Tumor: 0.09498 ± 0.2141, n=91, *p<0.0001 for HIF 1α and blood: 0.001436 ± 0.002469, n=117, Tumor: 0.002364 ± 0.007736, n=91 and *p=0.0041 for VEGF). HIF 1α also showed a differential expression along treatment (at diagnosis: 0.1432 ± 0.2161, n=102; 3 months after chemotherapy: 0.2010 ± 0.3456, n=102 and 6 months after chemotherapy: 0.1657 ± 0.2150, n=102, *p=0.0021). This differential expression along treatment was also observed for VEGF expression (at diagnosis: 0.001314 ± 0.001780, n=112; 3 months after chemotherapy: 0.001891 ± 0.002446, n=112 and 6 months after chemotherapy: 0.001674 ± 0.002260, n=112, *p<0.0001). It was found a correlation between the gene expression levels of HIF 1α and VEGF in peripheral blood (r=05881, *p<0.0001) as well as in the tumor samples (r=0.2804, *p=0.0123).

Conclusions

Data suggest that HIF 1α could be a diagnosis marker since there's a significant increase in its expression in blood samples from breast cancer patients when compared to healthy women. It's also observed that there's a positive correlation between VEGF and HIF 1α gene expression in peripheral blood samples.

Support or Funding Information

Financing: CNPq (process number: 133505/2018‐9) and FAPESP (Process number: 2017/03558‐3). Associated author: Carlos Henrique Foncesca Peiró Association: ASBMB (Number: 64413).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ate1 Controls Cellular Warburg Effects by Modifying Hif1a with Arginylation
Authors: Moorthy Balaji T. , Jiang Chuanhua , Patel Devang M. , Kumar Akhilesh , Birnbaum Micheal D. , Alfonso Belkis , Huang Jingyu , Barrientos Antoni , Lampidis Theordore J. , Fontanesi Flavia , Zhang Fangliang
Published: June 2021

The hypoxia‐inducible factor 1a (HIF1a) is a key regulator of stress response and metabolism. Its expression level is known to be controlled by oxygen sensing with two critical proline residues followed by ubiquitination via the pVHL pathway. Here, we report that the degradation of HIF1a is also controlled by N‐terminal arginylation mediated by Arginyltransferase 1 (Ate1) according to the N‐end rule pathway. We found that this mechanism is separate from the classical pVHL‐pathway and it affects cellular Warburg effect, which is the preferred usage of glycolysis that are commonly seen in cancer cells. Furthermore, we found that Ate1, the only arginylation enzyme in animals, appears to have originated from mitochondrial gene transfer. At least a subset of Ate1 is located inside mitochondria and appears to be essential for the composition and functions of respiration complex II. Our study therefore suggest Ate1 as a master regulator of metabolism in cancer cells through arginylation modification of HIF1a.

Support or Funding Information

This study is supported by NIGMS R01 GM 107333, and a start‐up funding from the University of Miami.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ganoderma Lucidum polysaccharide inhibits lipid accumulation in vivo and in vitro by activating HIF‐1a through ERK1/2 signaling pathway
Authors: Wang Ying , Guo Chenjie , Pan Haitao , Sang Tingting , Wang Yujie , Guo Dandan , Wang Xingya
Published: June 2021

Ganoderma lucidum polysaccharide (GLP) has been proposed to have beneficial effects against obesity. However, how GLP affects lipid accumulation has not been examined. In this study, we used 3T3‐L1 preadipocytes and a high‐fat diet‐induced obese mice model to investigate the effects and mechanism of GLP on lipid accumulation. Our results revealed that GLP significantly inhibited adipogenic differentiation in a dose‐dependent manner in 3T3‐L1 preadipocytes determined by Oil Red O staining and Triglyceride measurement. In order to explore the mechanism, we conducted a dynamic transcriptome study during 3T3‐L1 preadipocytes differentiation (0 h, 9 h, 18 h, 48 h, 120 h) with or without GLP treatment. Transcriptome analyses showed that GLP significantly caused cellular hypoxia by blocking respiration chain, and inducing the expression of HIF‐1a which is a major regulator of cell cycle arrest during hypoxia by activating the ERK1/2 signaling pathway. And then delayed the mitotic clonal expansion(MCE) which is required for differentiation of 3T3‐L1 cells, eventually inhibiting a cascade of adipogenesis pathway. These has been verified by qRT‐PCR or Western blot. Moreover, flow cytometry data revealed that GLP does arrest cell cycle at G0/G1 phase at 9 h of differentiation. Furthermore, knocking‐down of HIF‐1a by siRNA can abolish the GLP‐induced inhibition of adipogenesis in 3T3‐L1 cells, indicating that HIF‐1a was directly required for GLP‐induced inhibition of aidopogenesis. In addition, we demonstrated that GLP induces the phosphorylation of ERK1/2. Interestingly, pretreatment with ERK inhibitor, significantly rescued GLP‐induced inhibition of adipogenesis. We further demonstrated that HIF‐1a is the downstream target of ERK1/2. Furthermore, GLP treatment significantly suppressed hepatic lipid accumulation by activating ERK1/2/HIF‐1a signaling pathway to improve non‐alcoholic fatty liver disease(NAFLD) and insulin resistance in high‐fat diet‐induced obese mice. Taken together, GLP inhibits lipid accumulation by activating HIF‐1a through ERK1/2 signaling pathway in vivo and in vitro. These findings also indicate that GLP possesses protective effects in NAFLD and IR‐related metabolic disorders.

Support or Funding Information

National Natural Science Foundation of China (Grant No. 81473397).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct Missense Mutations of the Parkinson's Disease‐Related Ubiquitin Kinase PINK1 Alter Auto‐ or Substrate‐Phosphorylation
Authors: Springer Wolfdieter , Truban Dominika , Caulfield Thomas R , Hudec Roman , Hou Xu , Lewis‐Tuffin Laura J , McCarty Arthur M , Soto Alexandra I , Walton Ronald L , Puschmann Andreas , Wszolek Zbigniew K , Ross Owen A , Fiesel Fabienne C
Published: June 2021

The mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PRKN (Parkin) together mediate the selective degradation of damaged mitochondria via the autophagy‐lysosome system (mitophagy). Complete loss of either gene function results in failure of this mitochondrial quality control and thus accumulation of damaged organelles. While homozygous or compound heterozygous mutations in PINK1 or PRKN are the most common causes of early‐onset Parkinson's disease (PD), the contribution of heterozygous variants to PD is debated. However, recently we identified a partial dominant‐negative effect of a heterozygous PINK1 missense mutations that is associated with a significant increased risk and an earlier onset compared to late‐onset, sporadic PD. This underscores that not all missense mutations are alike and suggests critical threshold functions of this stress‐activated, neuroprotective pathway.

Here, we analyzed the underlying mechanisms in greater detail and found that the pathogenic PINK1 mutant, which can dimerize with wild‐type PINK1 protein, is aberrantly auto‐phosphorylated, impacting its enzymatic activity towards the substrates Ub and PRKN. Phosphorylation of both PINK1 targets however, is critical for PRKN recruitment as well as efficient labeling of mitochondria with phosphorylated poly‐Ub chains that serve as the mitophagy tag. In addition to cells from PD patients, we confirmed the altered phosphorylation pattern of mutant PINK1 under endogenous conditions in isogenic CRISPR/Cas9 genome engineered cells. Excitingly, while studying the pathogenic mechanisms in detail with a structure‐function approach, we identified a novel PINK1 variant that exerts even greater kinase activity than wild type PINK1. Our results further suggest that this hyperactive form of PINK1 likely modulates substrate receptivity thereby resulting in enhanced kinase activity and greater mitophagy rates. Going forward, it will be interesting to study if enhanced PINK1 signaling translates into greater neuroprotection. An in‐depth understanding of the regulation and enzyme functions of PINK1 may provide a rationale for a structure‐function based drug design in the future.

Support or Funding Information

This work was partially supported by funding from the Mayo Clinic Foundation, the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) [R01#NS085070] and the Michael J. Fox Foundation for Parkinson's Research to W.S.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Designing and Synthesis of a siRNA against Angiopoietin‐2 for the Treatment of Neovascularization Associated to Diabetic Retinopathy
Authors: Becerra Sandra Edith Cabrera , Villafaña Santiago
Published: June 2021

Diabetic retinopathy (RD) is a pathology that causes changes in the retina, for example exudates, microhemorrhages, neovascularizations, etc., which leads to loss of sight and is the third cause of blindness in the world. Among the mechanisms involved in neovascularization are angiogenic factors like angiopoietins (Angpt), especially angiopoietin‐2 (Angpt2), which has been reported that increase the formation of new vessels. For this reason, the aim of this study is to design and synthesize an siRNA against to Angpt2. Male Wistar rats were administered with streptozotocin (STZ) to induce diabetes, after 4 weeks the biochemical parameters, the neovascularization (junctions and lacunarity) and the expression of the Angpt2 were determined in the retina. The siRNA was designed with the siRNA Wizard v3.1 and RNA fold webserver software programs and was synthesized by MERMADE 8 equipment. The results showed that the number of junctions and the levels of Angpt2 of the diabetic rats were higher than the controls significantly, while the lacunarity was significantly lower than controls, on the other hand, the region susceptible to silencing could be from 1416 to 1436. Thus, we can conclude that early diabetes produces an increase in Angpt2 and neovascularization, which could be reduced by the siRNA to Angpt2.

Support or Funding Information

Grants: Instituto Politécnico Nacional (SIP‐20194983), Programa Institucional de Formación de Investigadores del IPN (BEIFI No. 2177), Scholarship by Consejo Nacional de Ciencia y Tecnología (CONACYT No.418913) and Comisión de Operación y Fomento de Actividades del IPN (COFAA).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oleocanthal as a Promising Natural Therapeutic Agent: the Experimental Leishmaniasis Paradigm
Authors: DOTSIKA ELENI , KARAMPETSOU KALLIOPI , KOUTSONI OLGA S. , ANGELIS APOSTOLOS , CHALAMBALAKI MARIA , SKALTSOUNIS LEANDROS‐ALEXIOS
Published: June 2021

Oleocanthal (OC) is a phenolic compound of Extra Virgin Olive Oil (EVOO), the main source of fatty acids in the Mediterranean diet that is associated with beneficial health effects such as reduced incidence of cardiovascular mortality and cancer. OC is responsible for the localized irritation in the oropharangeal region, is homologous with the non‐steroidal anti‐inflammatory drug ibuprofen and several studies evidenced relevant pharmacological properties of OC in different pathogenic processes. Our group has previously elucidated the anti‐leishmanial effect of Total Phenolic Fraction derived from EVOO and of the phenolic compound Oleuropein. Current study aimed to evaluate the effect of OC in murine experimental models of cutaneous and visceral leishmaniasis since there is no effective vaccine and the chemotherapeutic treatments have serious drawbacks such as toxicity, parasite resistance and high cost. BALB/c mice were infected either subcutaneously in the footpad with 106 L. major promastigotes (Cutaneous Model ‐ CM) or intravenously in the tail vein with 107 L. infantum promastigotes (Visceral Model ‐ VM), according to EC regulation conformed by FASEB Statement of Principles for the use of Experimental Animals. At 7 days post infection, mice were intraperitonealy treated with 5 and 10 mg/kg body weight of OC, 3 days per week for 5 weeks. Mice of the positive control group received orally 15 mg/kg b.w of the reference drug miltefosine (HePC), for 25 days. In the CM, results demonstrate significant reduction of the footpad swelling in OC‐treated mice along with the reduction of parasite load in the popliteal lymph nodes, 1 month post‐treatment termination. Correspondingly, in the VM, OC promoted a severe reduction of parasite burden in target organs, liver and spleen at 10 days post‐treatment termination. Moreover, in order to validate the profile of cytokines induced by OC treatment, intracellular cytokine production in splenocytes of all experimental groups was determined for IL‐4 (Th2‐type cytokine) and IL‐12 (Th1‐type cytokine) at 48h post Soluble Leishmania Antigen in vitro re‐stimulation and revealed that OC was able to stimulate important reduction in IL‐4 production by CD4+ T cells in both CM and VM, while the IL‐12 production was equal in all experimental groups. Furthermore, specific antibodies of both IgG1 and IgG2α isotypes, were detected in the plasma of OC‐treated mice. These data indicate that OC treatment resulted in the induction of a mixed Th1/Th2 immune response moderately polarized towards Th1 type that effectively mediated the elimination of parasitic spread in both disease models. Therefore, OC appears as a potential therapeutic agent into the leishmaniasis drug discovery pipeline.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Airway Caliber is Linked to Wall Thickness in Mice: Implications for Morphometry
Authors: Meyerholz David K , Beck Amanda P
Published: June 2021

Morphometry of lung tissues often involves evaluation for changes in airway wall components such as collagen and/or smooth muscle. Alterations of these tissue parameters may be markers of diseases like asthma, chronic obstructive pulmonary disease or cystic fibrosis. The mouse lung is composed of airways that exhibit monopodial branching during development and these intrapulmonary airways are exclusively bronchioles (i.e. no intra‐pulmonary bronchi). As such, many airways can look alike for analysis by inexperienced observers. We tested if the airway caliber in wild type B6 mice could influence the detection and scoring of airway collagen or smooth muscle composition. We examined 15 lungs from archival blocks that came from studies with appropriate institutional approvals. Airway lumen diameter as well as wall thickness (sum of epithelium, smooth muscle and collagen) measurements of the 2 largest and 2 smallest airways were taken. These were then cumulatively analyzed for wall thickness (y axis) and airway lumen diameter (x axis). There was a significant positive slope (P=0.0017). We then grouped the data into large (lumen >130 microns) and small (lumen <130 microns) airways and compared the wall thickness, which was significantly thicker in the larger airways (P<0.0001). These data indicate that morphometry of murine airways requires analysis of similar sized airways for accurate assessment of changes and we speculate this finding will be relevant across other species as well.

Support or Funding Information

NA

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 Acute Cadmium Exposure on the Vascular Endothelial Barrier Antigen and Astrocyte Morphology in the Developing Rat Central Nervous System
Authors: Ibiwoye Michael O , Matthews Qiana , McElroy Pamela , Foster James D
Published: June 2021

Cadmium is a non‐biodegradable heavy metal and a major worldwide environmental pollutant. Clinical and experimental studies have documented the neurotoxic and behavioral effects of cadmium. However, the exact pathophysiologic mechanism(s) of the toxicity of cadmium for the human central nervous system is not completely understood. A literature review indicates that disruption of the blood‐brain barrier (BBB) structures is a key event in the pathogenesis of central nervous system disorders including cadmium neurotoxicity and infectious diseases. Several BBB‐associated proteins, including occludins, junctional adhesion molecules and claudins, among others, have been identified and used for evaluating BBB disruption under pathological conditions. A developmentally regulated rat blood‐brain barrier (BBB)‐specific capillary endothelial marker, the endothelial barrier antigen (EBA), has been previously isolated and classified by Sternberger and others. In previous studies, we have demonstrated that anti‐EBA exclusively recognizes barrier‐competent microvessels in the rat central and peripheral nervous system (CNS and PNS) and is negative in endothelial cells of peripheral tissues such as the heart and kidney. The antigen is also absent in brain microvessels possessing fenestrated endothelia such as the median eminence, organum vasculosum of lamina terminalis (ovalt) and pineal gland. In our current study, we applied a double, sequential indirect immunofluorescent protocol with anti‐EBA and an antibody directed against glial fibrillary acidic protein (GFAP), to examine the immunoreactivity patterns and morphological alterations in BBB microvessels and astrocytes, following acute exposure to a single, high dose of cadmium in young rats. We detected a moderate reduction in immunoreactivity and number of microvessels labelled by the anti‐EBA in the forebrain, cerebellum and midbrain in cadmium‐exposed rats compared to normal controls. Within the white and gray matter of cadmium‐exposed brains, we observed weakly GFAP‐reactive astrocytes displaying cell bodies with ill‐defined borders and blurry cytoplasm. The astrocyte nuclei appeared markedly enlarged, intensely hyperchromatic and exhibited marked chromatin condensation with varying degree of nuclear fragmentation. The weakly GFAP‐positive cell processes were hypertrophic and showed less branching compared to the strongly GFAP‐positive, highly branched astrocytes. Thus, indicating that EBA is involved in the pathogenesis of cadmium neurotoxicity in the rat model system.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐inflammatory activity of Sageretia thea in LPS‐stimulated RAW264.7 cells
Authors: Park Gwang Hun , Eo Hyun Ji , Jeong Jin Boo
Published: June 2021

Sageretia thea (S. thea) has been reported to exert various pharmacological activities such as anti‐oxidation, anti‐cancer and anti‐human immunodeficiency virus activity. However, there is little study on the anti‐inflammatory activity of S. thea. Thus, we evaluated the anti‐inflammatory effect of extracts of leaves (ST‐L) and branches (ST‐B) from S. thea in LPS‐stimulated RAW264.7 cells. ST‐L and ST‐B significantly inhibited the production of the pro‐inflammatory mediators such as NO, iNOS, COX‐2, IL‐1β and IL‐6 in LPS‐stimulated RAW264.7 cells. ST‐L and ST‐B blocked LPS‐induced degradation of IκB‐α and nuclear accumulation of p65, which resulted to the inhibition of NF‐κB activation in RAW264.7 cells. ST‐L and ST‐B also attenuated the phosphorylation of ERK1/2, p38 and JNK in LPS‐stimulated RAW264.7 cells. In addition, ST‐L and ST‐B increased HO‐1 expression in RAW264.7 cells, and the inhibition of HO‐1 by ZnPP reduced the inhibitory effect of ST‐L and ST‐B against LPS‐induced NO production in RAW264.7 cells. Inhibition of p38 activation and ROS elimination attenuated HO‐1 expression by ST‐L and ST‐B, and ROS elimination inhibited p38 activation induced by ST‐L and ST‐B. ST‐L and ST‐B dramatically induced nuclear accumulation of Nrf2, but this was significantly reversed by the inhibition of p38 activation and ROS elimination. Collectively, our results suggest that ST‐L and ST‐B exerts potential anti‐inflammatory activity by suppressing NF‐κB and MAPK signaling activation, and activating HO‐1 expression through the nuclear accumulation of Nrf2 via ROS‐dependent p38 activation. These findings suggest that ST‐L and ST‐B may have great potential for the development of anti‐inflammatory drug to treat acute and chronic inflammatory disorders.

Support or Funding Information

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF‐2016R1D1A3B03931713 and NRF‐2018R1A6A1A03024862), and by a grant from National Institute of Forest Science in 2019.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Activator of G‐protein Signaling 3 (AGS3) and the signaling protein Dishevelled: Implications for the regulated distribution AGS3 within the cell and its functional diversity
Authors: VURAL ALI , LANIER STEPHEN
Published: June 2021

Activator of G‐protein Signaling 3 (AGS3), a receptor independent activator of G‐protein signaling, consists of 7 tetratricopeptide repeats (TPR) upstream from 4 G‐protein regulatory motifs (GPR), each of which interact with GαGDP. AGS3 moves among multiple cellular compartments including cytosol, the cell cortex, the Golgi apparatus, the aggresomes and cellular puncta, with suggested oscillation between hydrophilic and hydrophobic domains. Such a regulated subcellular distribution is intimately related to the functional diversity of the protein, which remains mechanistically elusive. We recently reported that a mutation of a single threonine residue in the AGS3‐GPR domain alters the apparent phosphorylation status of the protein and results in the localization of AGS3‐T602A to punctate subcellular structures in contrast to the more diffuse, cytosolic distribution observed with WT AGS3. Moreover, these puncta are distinct from defined intracellular organelles or vesicles and their localization is regulated by interaction with AGS3 binding partners and potentially by the phosphorylation status of AGS3. In a search for proteins that exhibited a similar punctate distribution, we determined that the Dishevelled protein 2 (Dvl‐2) exhibits a punctate cellular distribution that is actually a “mirror” image of that observed with the AGS3‐T602A. However, co‐expression of AGS3‐T602A with Dvl‐2 indicated that the two proteins were localized to distinct punctate populations in the cell. In contrast AGS3‐WT, which typically has a non‐homogeneous, diffuse distribution in the cytosol, was actually redistributed to Dvl‐2 puncta upon co‐expression of the two proteins. The Dvl‐2 induced localization of WT AGS3 to puncta was reversed by coexpression of Giα3, which likely reflects the interaction of Giα3 with the AGS3 GPR domains. This series of observations suggest that the positioning of AGS3 as part of the Dvl‐2 puncta may be phosphorylation‐dependent and regulated by G‐proteins. In addition, these observations taken together, suggests a potential role for AGS3 in canonical and noncanonical WNT signaling pathways involving Dvl‐2.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aging influences cerebrovascular function via BK channel in a sex‐specific manner
Authors: Pabbidi Mallikarjuna , Pareek Tanya , Reed Joseph , Sontakke Sumit
Published: June 2021

The myogenic response of middle cerebral arteries (MCA) protects the brain by altering the diameter in response to lumen pressure to maintain constant cerebral blood flow. Large conductance potassium (BK) channels are known to regulate the myogenic response. However, it is not clear how aging will influence the myogenic response via altering BK channel function in males and females. Here, using vascular reactivity, patch‐clamp and biochemical methods, we found that age‐associated changes in BK channel subunit expression and function alter myogenic response in a sex‐specific manner. Aging exaggerated the myogenic response of MCA of females but did not change the myogenic response in males. Aging abolished differences in myogenic tone of MCA (MT as determined from active and passive diameters) in males and females. Aging, while decreased wall to lumen ratio in females, it increased in males revealing a sex‐specific difference in MCA remodeling. Aging while decreased total K channel and BK channel currents in vascular smooth muscle cells (VSMCs) isolated from female vessels, it did not affect males. Spontaneous transient outward currents (STOC) that represent BK function are also decreased with aging in both males and females. However, the magnitude of the decrease in STOC amplitude and frequency is much greater in aged females than aged males. While aging increased BKα subunit, it decreased β1 subunit protein in cerebral vessels of both males and females. Age‐associated increase in BKα subunit is similar between males and females. However, there is a greater and significant reduction of BKβ1 subunit protein in aged females than aged males. Activation of BKα and β1 subunits either individually or both failed to decrease MT of MCA isolated from aged males and females at physiological lumen pressures. Besides, aging diminished acute estradiol (17βE)‐mediated decrease in MT either alone or combined with BKβ1 agonist in females. However, this combination significantly decreased MT in ovariectomy females that are chronically supplemented with 17βE via increasing BKβ1 protein content. Overall our results suggest that age‐associated changes in cerebral vascular structure and function and that of BK channel subunit expression and function are sex‐specific. Chronic supplementation of 17βE increases BKβ1 protein content, and its activation by agonist combined with acute 17βE attenuates MT in aged females.

Support or Funding Information

Supported by AHA SDG (13SDG14000006) and IRSP from UMMC to Mallikarjuna R. Pabbidi.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nitric Oxide‐Mediated Nanomolar Vasodilator Potencies of Glycosylated Anthocyanins do not Correlate with Redox Potential
Authors: Garcia‐Huidobro J Pablo , Calfio Camila , Donoso M Veronica , Donoso Francisca , Silva Camila
Published: June 2021

Calafate is an endemic Patagonian shrub (Berberis Microphylia); its berries are rich in polyphenols, including 3‐glycosylated anthocyanins (GA, delphinidin, petunidin or malvidin) or mono or disaccharide conjugated quercetin (Q). The chemical profile of Calafate berries is well characterized and indicates its high redox potential; pharmacological properties are virtually unknown. From ethno‐pharmaceutical reports we hypothesized that Calafate polyphenols are vasodilators and ascertained whether this effect is related to their cell antioxidant activity (CAA).

Vascular responses of GA and Q were assessed perfusing the intact rat arterial mesenteric bed or following endothelium denudation. The role of NO was assessed in mesenteries perfused with an eNOS inhibitor (L‐NNA). Luminally accessible NO was determined by chemiluminescence; intracellular NO was quantified by DAF fluorescence in isolated mesentery endothelial cells. Antioxidant capacity was quantified either by 1,1‐Diphenyl‐2‐picryl‐hydrazyl antioxidant assay (DPPH) or by CAA assessment. Extracellular ATP was assessed by chloroacetaldehyde derivatization to yield fluorescent purines; the nucleotide is related to NO production in endothelial cells.

GA proved potent vasodilators with EC50s of 84.1 ± 0.3, 65.8 ± 0.4 and 17.7 ± 0.1 nM, respectively; aglycones exhibited similar EC50s. The maximal effect of GA did not exceed 56 ± 3.7% efficacy compared to acetylcholine (EC50 was 43.4 ± 0.1 nM with 100% efficacy) while the Q EC50 was 12.8 ± 0.1 μM, with 99 ± 4.1%. The GA‐induced vasodilatation was totally endothelium‐dependent; while the Q response showed partial endothelium‐dependence. The degree of GA vasodilatation correlated with luminal NO (r2 = 0.83), confirming NO as the final intracellular signal. 30 μM Q increased both extracellular ATP secretion from endothelial cells plus intracellular DAF‐fluorescence (P<0.05), an indication that part of the Q response involves NO as the final effector. While Q exhibited the highest antioxidant effect in both the DPPH or CAA (p<0.01), the GA showed a 60‐fold lower potential than Q in the CAA test, indicating that the CAA is not related to its vasodilator profile, which apparently relies on eNOS allosteric modulation.

In sum, GA are novel and potent endothelium‐dependent vasodilators, linked to modulation of NO production.

Support or Funding Information

Funded by FONDECYT 117‐0842 and 21141226 to CC and CEDENNA FB0807

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of T1R3 reduces atherosclerosis and liver triglyceride accumulation in ApoE−/− mice fed a high‐fat diet
Authors: Shojaat Shayla , Engman Samuel , Hofferber Jason , Keomanivong Faithe , Wauson Eric M
Published: June 2021

To maintain homeostasis, cells continuously evaluate and adjust to their nutrient availability and current energy state. A cell's response to the fed state requires the presence of amino acids and other nutrients, which signal to the mammalian target of rapamycin complex 1 (mTORC1) coordinating cell growth, protein translation, and autophagy. The umami taste receptor, which is a heterodimer between the G protein‐coupled receptors T1R1 and T1R3, is a direct sensor of amino acid availability and signals to mTORC1. Reducing the expression of T1R1/T1R3 in mice and pancreatic beta and cardiomyocyte cell lines diminishes the activation of mTORC1 by amino acids, thereby inducing autophagy. Previous research suggests that elevated autophagic flux reduces the development and progression of atherosclerosis by protecting cells from oxidative stress, reducing apoptosis within atherosclerotic lesions, and promoting lesion stabilization by degrading intracellular components that are harmful to the cell. Other studies suggest that increased autophagic flux decreases the progression of Non‐Alcoholic Fatty Liver Disease (NAFLD) by reducing hepatocyte lipid accumulation and liver inflammation. We hypothesized that the deletion of T1R3 in ApoE−/− mice would decrease aortic atherosclerotic plaque buildup and decrease hepatosteatosis by increasing autophagic flux. Our data suggest that T1R3−/− ApoE−/− mice have less liver lipid accumulation and reduced atherosclerotic plaque area when compared to T1R3+/+ApoE−/− mice. Taken together, these data suggest that modulation of nutrient and amino acid‐sensing GPCRs like T1R1/T1R3 could be used as a potential therapeutic strategy for cardiovascular disease.

Support or Funding Information

American Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (E.M.W.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐adipogenic Activity of Terminalia pallida
Authors: BADRI KAMESWARA RAO
Published: June 2021

Obesity rates are rising globally, with 1.5 billion overweight and 500 million obese worldwide. According to The Centers for Disease Control and Prevention, almost 2/3rds of the United States population is either overweight or obese. Growing obesity rates in children and adolescents makes the obesity pandemic more alarming and solutions more critical. Further, non‐Hispanic Black and Hispanic Americans are more prone to this disease/condition, making it also a health disparity. Obesity increases the risk for insulin resistance, diabetes mellitus, dyslipidemia, hypertension, sleep apnea, cardiovascular disease and certain types of cancer (4). Only limited obesity management options are available, including behavioral interventions, lifestyle modification and invasive surgeries.

New drug development remain the cornerstone of anti‐obesity research due to the limited availability of drug treatments. Further, due to reduced costs, fewer side effects and cultural significance, many are opting for complementary treatments. Terminalia pallida (TP) is one such plant that is been used ethno‐botanically to treat metabolic diseases. Hence, here we investigated the anti‐adipogenic activity of TP. We used 3T3‐L1 embryonic fibroblast‐preadipocyte cells treated with or without adipogenic cocktail (1 μM dexamethasone, 10 μg/mL insulin and 0.5 mM 3‐isobutyl 1 methylxanthine) to induce adipogenesity. We treated the 3T3‐L1 cells with various concentrations of ethanolic extract of Terminalia pallida fruit (TP‐50, 25 and 10 μg/ml) or Simvastatin (1 μM), a known anti‐lipidemic drug. Ten days post‐adipogenic induction, the adipogenic cocktail‐treated 3T3‐L1 preadipocytes differentiated into adipocytes. However, TP‐treated groups showed significant inhibition of adipocyte formation. The Simvastatin‐treated group also showed anti‐adipogenic activity, as evidenced by the reduced size and number of adipocytes. Expression of the key adipogenic markers PPARγ2 and C/EBPα was decreased in the TP‐treated group compared to untreated control. We further investigated the cellular mechanisms by which TP elicit the antiadipogenic function. Our studies conclude that TP significantly decreased proliferation and elevated apoptosis in 3T3‐L1 adipocytes, indicating that partially TP shows its anti‐adipogenic function by regulating preadipocyte and adipocyte cell cycle.

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