bluuebunny/crossref_metadata_2025 huggingface dataset:
PGE2 – EP3 Receptor Signaling Involves Rho‐kinase Pathway in Hyperoxia‐Induced Airway Hyperresponsiveness
Authors: Neziri Burim , Sopi Ramadan B , Dreshaj Ismail A , Beqiraj Qendresa , Cenaj Asllan , Kurtishi Ilir , Krasniqi Avdyl , Dreshaj Shemsedin , Jakupaj Muharrem
Published: November 2023

Hyperoxic exposure is associated with airway hyperresponsiveness and it was shown that hyperoxia upregulates synthesis of prostaglandin E2 (PGE2), which induces contraction of airway smooth muscle via EP1 and EP3 receptors. It is not clear through which signaling pathway PGE2 exerts contractile effect on airway smooth muscle under hyperoxic conditions. Therefore, we hypothesized that under hyperoxic condition PGE2 induces contraction of airway smooth muscle via EP3 receptors by activating Rho‐kinase signaling pathway. Tracheal cylinders were obtained from 12 days Wistar rat pups exposed to hyperoxia (≥ 95% O2) or room air for seven days. These cylinders were used to study contractile responses evoked by EP3 agonist (sulprostone, 10−9 – 10−5 M); methacholine (MCh, 10−8 – 10−4 M) in absence or presence of an EP3 antagonist (L‐798106, 10 μM) or Rho‐kinase inhibitors (Y‐27632, 10 μM or fasudil, 10 μM).

Both, sulprostone and MCh induced dose‐dependent contractile responses of airway smooth muscle, respectively. The contractile responses were significantly higher in preparations obtained from animals exposed to hyperoxia as compared to those obtained from room air controls. These enhanced contractile responses in hyperoxic tissues were reversed by pre‐incubating tissues with Y‐27632 or fasudil. EP3 antagonist also significantly attenuated contractile responses evoked by sulprostone. The results of this study revealed that PGE2 – EP3 receptor signaling involves Rho‐kinase pathway and we speculate that the use of Rho‐kinase inhibitors or EP3 antagonist could attenuate the contractile effects of PGE2 and this might be an effective therapeutic approach to reverse hyperoxia‐induced airway hyperresponsiveness.

Support or Funding Information

Supported by MEST
Effects of nanoparticle size and surface charge on uptake into rat alveolar epithelial cells
Authors: Sipos Arnold , Peti‐Peterdi Janos , Chow Robert H , Borok Zea , Kim Kwang‐Jin , Crandall Edward D
Published: November 2023

Physicochemical properties of nanoparticles, including size, composition and surface charge, are likely to affect nanoparticle interactions with biological cells/tissues. We reported previously that uptake of polystyrene nanoparticles (PNP) from apical fluid into primary cultured rat alveolar epithelial cell monolayers (RAECM) takes place in a manner dependent on exposure time and dose. Classical endocytosis pathways do not appear to play a role in the uptake of PNP, consistent with a ‘diffusional’ process. We also reported that autophagy is involved in cellular processing of PNP, in that accumulation of PNP in lysosomes occurs via autophagic flux. Lysosomal exocytosis of PNP (modulated by intracellular [Ca2+]), takes place in parallel with a slower ‘diffusional’ egress process. In this study, we investigated the effects of nanoparticle size and surface charge on uptake into RAECM grown on Anopore filters. RAECM were exposed apically to 80 μg/mL carboxylated or amidinated 20, 100 or 200 nm PNP (with near‐infrared fluorescent label) and intracellular [PNP] was monitored over 24 hr using live cell imaging. Results showed that intracellular [PNP] reached a plateau of ~0.6 mg/mL after ≥12 hr of apical exposure to both 20 nm carboxylated or amidinated [PNP] at 80 μg/mL. As PNP size increased from 20 to 100 to 200 nm, steady state intracellular [PNP] decreased from ~0.6 to ~0.3 to ~0.1 μg/mL, respectively, after ≥12 hr. These data indicate that, for RAECM, PNP uptake is strongly dependent on PNP size, while the role of surface charge appears to be less important. Additional factors (e.g., nanoparticle‐associated protein corona) might alter the charge effect. In general, understanding the role of physicochemical properties of nanoparticles in cellular interactions will lead to improved nanoparticle design for protection from cellular injury and approaches to therapeutic utilization in drug/gene delivery.

Support or Funding Information

Funding: NIH; Hastings and Whittier Foundations.
GPR171 in the Basolateral Amygdala Regulates Stress and Reward‐Related Behaviors
Authors: Bobeck Erin , Pena Darlene , Gomes Ivone , Fujita Wakako , Devi Lakshmi
Published: November 2023

Recently we deorphanized GPR171, a hypothalamic G protein‐coupled receptor, as the receptor for the abundant neuropeptide, Big‐LEN. By virtual screening of a library of small molecules, we identified compounds that selectively target GPR171. Using these compounds we found that the activation of GPR171 within the hypothalamus regulates feeding. Additional characterization of the distribution of GPR171 revealed high expression in the basolateral amygdala. This led us to explore a role for GPR171 in various stress and reward‐related behaviors using lentiviral shRNA‐mediated knockdown of GPR171. While decreases in expression of GPR171 in the basolateral amygdala had a minor or no effect on anxiety and depression, the decrease in expression had a major effect on fear learning and morphine conditioned place preference. In addition, we investigated these behaviors using GPR171 selective ligands and found a reduction in anxiety. Interestingly, we also find that mice subjected to repeated stress show an increase in GPR171 mRNA. Together, these results suggest a role for GPR171 in stress and reward‐related behaviors within the amygdala.

Support or Funding Information

This work was supported in part by NIH grants NS026880 and DA019521 to L.A.D and a NIDA postdoctoral training grant DA007135 to E.N.B.
Adenosine A3 Receptor Expression and Function in Mitochondria
Authors: Doyle Timothy M. , Phillps Grady , Tosh Dilip K. , Gratton Michael Anne , Jacobson Kenneth , Bennett Gary J. , Salvemini Daniela
Published: November 2023

Mitochondrial dysfunction is emerging as a common mechanism of a variety of neurodegenerative diseases, including chemotherapy‐induced neuropathic pain. We have recently found that administration of selective agonists to the G protein‐coupled, adenosine A3 receptor (A3AR) prevents the development of chemotherapy‐induced mitochondrial dysfunction in peripheral sensory neurons that result peripheral neuropathy and chronic pain. Recent evidence also indicates the presence of G proteins and G‐coupled protein receptors within mitochondria, including the adenosine A1 and A2A receptors. Bioinformatic analysis revealed potential mitochondrial‐associated sequences in published A3AR protein sequences, thus prompting us to hypothesize that A3AR is associated with mitochondria and its signaling could regulate mitochondrial function. Using Western blot analysis and transmission immune‐electron microscopy, we report for the first time that A3AR is present in normal rat mitochondria purified from liver, spinal cord and peripheral blood lymphocytes. Moreover, we also report that when normal rodent mitochondria are pretreated with the selective A3AR agonist, MRS5890, they produced more ATP following ADP‐stimulation than mitochondria treated with its vehicle (0.002% DMSO). We also find that MRS5980 counteracts normal mitochondrial membrane depolarization during calcium mobilization. Collectively, our data provide the first evidence for the presence A3AR in mitochondria and initial mechanistic insight to its functional role in mitochondria that potentially could serve as a target for the treatment of neurodegenerative disease.

Support or Funding Information

NIH‐NCI CA169519
A load of hypergravity inhibits AMPKα and causes liver injury in mice, which can be prevented by preconditioning loads
Authors: Lee Sang gil , Lee Chan gyu , Kim Sang geon
Published: November 2023

An understanding of the effects of hypergravity on energy homeostasis is necessary in managing proper physiological countermeasures to assure safe and effective aerospace missions and human survival. This study investigated whether a single or multiple load(s) of mice to hypergravity has an effect on the molecules associated with energy metabolism. In the liver, AMPKα level and its signaling pathway were repressed 6 h after a load of +9 Gz hypergravity for 1 h, and then gradually returned toward normal up to 24 h. AMPK β1 and γ1/2/3 levels were unchanged. AMPKα level was restored after 3 consecutive daily loads to +9 Gz, suggestive of adaptation of animals to repetitive preconditioning. In cDNA microarray analyses, 221 genes were differentially expressed by +9 Gz (P<0.05 and a 1.5‐fold cutoff), and the down‐regulated genes included Nrf2 targets. Nrf2 knockout abrogated the recovery of AMPKα elicited by 3 loads to +9 Gz, indicative of the role of Nrf2 in the adaptive increase of AMPKα. In addition, +9 Gz stress inhibited other transcription factors including STAT3, FOXO1/3 and CREB, and this effect was attenuated during the resting time. Similarly, apoptotic markers were enhanced with liver injury 6 h after a load of hypergravity, indicating that the liver may be vulnerable to hypergravity stress. Hepatocyte death was also abolished after multiple hypergravity loads. Our results show that a load of mice to +9 Gz hypergravity causes AMPKα repression with liver injury, which can be overcome by multiple loads to hypergravity as mediated by Nrf2.

Support or Funding Information

As supported by the NRF grant funded by the Korea government (NRF‐2014M1A3A3A02034698)
Metformin Protects against 7, 12‐Dimethylbenz[a]anthracene‐Induced Breast Cancer Initiation and Adduct Formation In Vivo Rat Model Through CYP1A1/CYP1B1 Pathway
Authors: Belali Osamah M , Attafi Ibraheem M , Moraished Bader A , Ansari Mushtaq A , Korashy Hesham M
Published: November 2023

Recent studies have established that metformin (MET), an oral anti‐diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen‐induced animal models and cell lines. In that, a study from our laboratory showed that MET protected against 7, 12‐dimethylbenz[a]anthracene (DMBA)‐induced breast carcinogenesis in vitro human non‐cancerous epithelial breast cells (MCF10A). However, whether MET can protect against breast cancer initiation in vivo rat model has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET against DMBA‐induced rat breast carcinogenesis and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1/1B1 pathway. In this regard, treatment of rat with DMBA (25 mg/kg) induced initiation of breast carcinogenesis in rat as evidenced by changes in biochemical and histopathological analysis. Importantly, the protective effect of MET against DMBA‐induced breast carcinogenesis was supported by the capability of MET (500 mg/kg) to restore the induction of the mRNA and protein expression levels of several DNA repair genes ( OGG1, XRCC1), and the level of 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG), a DNA damage marker. In addition, MET was able to restore DMBA‐mediated changes in the gene expression of apoptotic (caspase‐3 and BAX) and inflammatory (TNF‐α and IL‐6) mediators. Interestingly, these changes were associated with proportional decrease in DMBA‐induced CYP1A1 and CYP1B1 gene expression. In conclusion, the present work provides the first evidence that MET protects against DMBA‐iduced rat breast carcinogenesis by inhibiting DNA adduct formation, inducing apoptosis and inhibiting the expression of CYP1A1/CYP1B1 pathway.

Support or Funding Information

This Project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (12‐MED3131‐02)
Investigate Cigarette Smoke Induced IL‐8 Secretion in Ovarian Cancer Cells and Estrogen Mediated Modulations
Authors: Sumanasekera Wasana , Beckmann Taralein
Published: November 2023

Cigarette smoking is a global health crisis and exposure to cigarette smoke (CS) leads to variety of diseases including many cancers. Although there are few clinical reports involving CS exposure with the ovarian cancer, laboratory based reports are rare. The focus of our laboratory is to investigate whether CS contributes to the development of ovarian cancer and unveil the role of estrogen in CS induced effects. Our preliminary data shows that CS induced functional changes in ovarian cancer cells (OCCs). The objective of current work is to investigate IL‐8 release as one of the contributing mechanism for CS induced effects. The specific aims are 1). Detection of CS induced IL‐8 release 2). Investigate whether estrogen cause IL‐8 release 3) Examine estrogen's role in modulating CS induced IL‐8 release.

Methods

OCCs were cultured using standard procedure. Following 10 nM estradiol pre‐treatment, OCCs were exposed to two different concentrations of CS, which is created in‐house using a published method. Following CS exposure, released IL‐8 was detected using IL‐8 Enzyme Linked Immunosorbant Assay (ELISA). ELISA was conducted based on manufacturer's instructions (Boster Bio).

Results

CS increased IL‐8 release in OCCs. 10 nM estradiol pre‐treatment also increased IL‐8 release. However, when OCCs are exposed to both estrogen and CS, estrogen pre‐treatment attenuated CS induced IL‐8 release showing a protective effect

Support or Funding Information

Sullivan University Faculty Development Grant
Effect of gallic acid on preneoplastic liver lesions in vivo
Authors: Escutia‐Gutiérrez Rebeca , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Lourdes
Published: November 2023

One of the main interests in cancer studies is the research for natural, bioactive and nontoxic compounds that modulate or inhibit the initiation stage in the development of cancer. Gallic acid is a polyphenol with potential properties as antioxidant, anticancer, and antimutagenic. The aim of this study is to investigate the effect of gallic acid on preneoplastic liver lesions induces by administration of the diethylnitrosamine (DEN) and carbon tetrachloride (CCl4). We used thirty male Wistar rats, which were randomly divided into six groups. Control, gallic acid (AG) 50 mg/kg v.o. for 8 weeks, preneoplasia (P) a single dose of DEN 200 mg/kg i.p. and two weeks after a single dose of CCl4 2 mL/kg i.p., pre‐treated gallic acid for 5 weeks after DEN and CCl4 (AG + P), post‐treated DEN and CCl4 then gallic acid for 5 weeks (P + AG) and simultaneous (AG=P), gallic acid for 8 weeks and concurrently DEN and CCl4. Histopathological analysis of liver tissue showed three schemes of gallic acid administration decreased dysplastic cells, restored nucleo‐cytoplasmic relation and improved morphological architecture of hepatic tissue in 60% of the animals. Gallic acid significantly decreased serum levels of the enzymes ALT and FA in AG + P, P + AG and AG =P groups, with respect preneoplasia group. Expression of the biomarker PCNA was performed by immunohistochemistry on liver tissue, reduced in the P + AG and AG = P groups, with respect preneoplasia group. In summary, gallic acid was able to reduce the hepatic preneoplastic lesions, could therefore be used as a preventive and therapeutic agent in liver cancer
Pancreas Proteome of Palmatine and Purified Fraction E Extract of Coscininum Fenestratum ‐Supplementation in STZ‐Induced Diabetic Rats
Authors: Okechukwu Patrick Nwabueze , Ekeuku Sophia Ogechi , Sena Teo Swee , Akuwoah Gabriel Akyirem , Froemming Gabriele Anisah
Published: November 2023

Previous studies have reported the antidiabetic and antioxidant activity of Purified Fraction E extract from stem of Coscinium Fenestratum and Palmatine. Therefore in this study the mechanism of action of Palmatine, Fraction E and Tolbutamide were evaluated using proteomics. The proteome of pancreas harvested from STZ‐ induced diabetic rat and supplemented with Palmatine, Fraction E and tolbutamide for 90 days were evaluated using 2‐D gel electrophoresis and MALDI‐TOF MS/MS. The identified proteins which were expressed based on their functions were: chaperone/heat shock proteins (calreticulin, protein disulfide isomerase a2/a3, glucose regulatory protein 78 and heat shock protein 70); digestion protein (pancreatic amylase 2a3/1a, chymotrypsinogen, anionic trypsin 2/trypsin v‐a); lipid digesting proteins (bile salt activated lipase protein, Enoyl CoA hydratase and pancreatic lipase); channel proteins (ATP synthase B and voltage dependent anion channel2 protein); protection proteins (selenium‐binding protein1 and Lactoyl glutathione lyase) and antioxidant proteins (glutathione‐s‐transferase, peroxidoxin 4 and serum albumin). The differential protein expression of tolbutamide, Palmatine and Fraction E‐treated groups suggest that their antidiabetic effect maybe by the stimulation of chaperone/heat shock proteins, digestion protein, lipid digesting proteins, channel proteins and protection proteins. However, Palmatine and Fraction E stimulated antioxidant proteins expression, which were absent in Tolbutamide treated group.

Support or Funding Information

CERVIE UCSI University
Hypoglycemic and Hypolipidemic Effects of Acalypha Wilkesiana Leaves in Alloxan Induced Diabetic Rats
Authors: Olukunle Johnny Olufemi , Jacobs Eniope Bamidele , Oyewusi Joseph Adeoye
Published: November 2023

Hypoglycemic and hypolipidemic activities of 21day, once a day dosing of aqueous extract of Acalypha wilkesiana administered orally at a dose of 400mg/kg was studied in alloxan‐induced diabetic rats. The hypoglycemic effect of a single dose of 400mg/kg methanolic extract of Acalypha wilkesiana was also evaluated within 24 hours of dosing the rats, Glibenclamide, Polyoxyethylene sorbitan monooleate (Tween 80) and distilled water were orally administered to equal number of rats as positive and negative controls, respectively. Effect of oral administration of aqueous extract Acalypha wilkesiana (400 mg/kg) on level of blood glucose, triglycerides (TG), low density lipoprotein (LDL), high density lipoprotein (HDL) were determined.

The 400 mg/kg aqueous extracts caused 50.8% reductions in blood glucose when compared to 70.6% reduction caused by Glibenclamide, a standard antidiabetic agent. The aqueous extract also caused statistically significant (p<0.05) decrease in the values of serum cholesterol (121±0.5) mg/dl, LDL (61.70±1.4) mg/dl and statistically significant (p<0.05) increase in the value of HDL (42.95±1.0) mg/dl when compared to the untreated control. The hourly percentage glucose reductions obtained, revealed a steady decrease from 10.7% at 30mins post administration to 43.8% reduction at 24 hours, this is comparable with the 52.4% reduction obtained at 24 post administration of standard antidiabetic agent by Glibenclamide. This result establishes the hypoglycemic and hypolipidemic activities of Acalypha wilkesiana leaves.
Depletion of Dietary microRNAs from Cow's Milk Causes an Increase of Purine Metabolites in Human Body Fluids and Mouse Livers
Authors: Lozano Ana Gabriela Aguilar , Baier Scott , Adamec Jiri , Sadri Mahrou , Giraud David , Zempleni Janos
Published: November 2023

Background

MicroRNAs (miRNAs) play essential roles in gene regulation. We have recently reported the ground‐breaking discovery that miRNAs are not exclusively derived from endogenous synthesis but can also be obtained from dietary sources such as cow's milk. Evidence suggests that milk consumption is inversely correlated with serum uric acid (UA) concentrations, and 19 miRNAs regulate 11 pathways in purine metabolism.

Hypothesis

Consumption of a diet depleted of milk miRNAs causes changes in purine metabolism.

Methods

In a first screen, C57BL/6 mice were fed a diet depleted of miRNAs (encapsulated in exosomes) from age 3 to 7 weeks [denoted Exosome (E) Minus, E−], whereas controls were fed a miRNA‐sufficient diet (E+). Mouse liver metabolites were analyzed using non‐targeted LC/MS‐MS, and targeted enzymatic assays for purine metabolites. Subsequently, we assessed purine metabolites in urine and plasma from human dairy avoiders and dairy consumers. The statistical significance of differences was assessed by t‐test.

Results

Principal Components Analysis (PCA), a dendrogram performed with hierarchical clustering, Variable Important in Projection (VIP) scores, and a heat map analysis consistently suggested that concentrations of purine metabolites were higher in E− livers than in E+ livers, and that purine metabolites were affected by milk miRNA intake to a greater extent than any of the other metabolites that were identified (p<0.01; n=5). As for effect size, the concentrations of xanthine were 16.6 ± 3.3 μM and 10.1 ± 1.0 μM in E − and E+ livers, respectively (p<0.05). Likewise, the concentrations of plasma xanthine and urinary uric acid were 81.6% and 19.3% higher in human dairy avoiders compared with dairy consumers (n=6).

Conclusion

Dietary depletion of milk miRNAs causes an increase in purine metabolites in mouse tissues and human body fluids.

Future plans

A reversed‐phased HPLC was developed to quantify eight purine metabolites in a comprehensive analysis of purine metabolism; gene expression analysis and purine feeding studies will be used to identify the step(s) in purine metabolism targeted by milk miRNAs.

Support or Funding Information

NIFA/USDA 2015‐67017‐23181, NIH 1P20GM104320, Gerber Foundation, Egg Nutrition Center, USDA W‐3002, Hatch and NIH P30GM103335
A Genome‐Wide Association Study of Plasma α2‐Macroglobulin Concentrations in Young Adults
Authors: Jamnik Joseph , Borchers Christoph , El‐Sohemy Ahmed
Published: November 2023

Background

Gluten intake has been shown to be associated with circulating levels of α2‐macroglobulin in young adults without celiac disease. Gluten‐free foods have increased in popularity over the past decade despite a poor understanding of the physiological effects of gluten intake in those without celiac disease. Plasma α2‐macroglobulin represents a physiologically important molecule involved in inflammation and cytokine release, but the contribution of common genetic variation to the observed variation in circulating concentrations of α2‐macroglobulin remains unclear.

Objective

The objective was to identify genetic variants that are associated with α2‐macroglobulin in a population of young adults using a genome‐wide approach.

Methods

A genome‐wide association study (GWAS) was conducted on circulating levels of α2‐macroglobulin in a population of young Caucasians without clinically diagnosed celiac disease from the Toronto Nutrigenomics and Health Study (n=488). Concentrations of α2‐macroglobulin were measured in plasma using a multiple reaction monitoring HPLC‐MS/MS assay. Genome‐wide genotyping was conducted for 822,000 single nucleotide polymorphisms (SNPs) using the Affymetrix 6.0 chip. The association between genome‐wide genetic variants and plasma α2‐macroglobulin was explored using linear regression with an additive mode of inheritance.

Results

Circulating α2‐macroglobulin was associated with variation in ICOSGL (p = 3.9×10−7), a T‐cell stimulatory gene involved in cytokine release and various immune‐related disorders including celiac disease.

Conclusion

These results suggest that the physiological effects of gluten intake may be mediated by genes involved in T‐cell proliferation and cytokine release.

Support or Funding Information

Research support from the Advanced Foods and Materials Network.
Interaction between PhIP from meat intake and polymorphism in superoxide dismutase 2 gene increases risk of oxidative stress
Authors: Carvalho Aline , Steluti Josiane , Carioca Antonio Augusto , Loureiro Ana Paula , Gattas Gilka , Fisberg Regina , Marchioni Dirce
Published: November 2023

Background

Heterocyclic amines (HCA), especially 2‐Amino‐1‐methyl‐6‐phenylimiazo[4,5‐b]pyridine (PhIP) from meat intake have been linked to cancer due to oxidative stress generated during the metabolism. This process is mediated by detoxifying enzymes, as superoxide dismutase 2 (SOD2), which is encoded by a polymorphic gene. The presence of this polymorphism Val16Ala (rs4880) decreases the enzyme activity increasing oxidative stress and risk for chronic disease as cancer. The aim of the present study was to investigate if there is an interaction between PhIP from meat intake and SOD2 polymorphism on biomarkers of oxidative stress.

Methods

We performed a cross‐sectional study in adults and older adults from Brazil (n=239). PhIP intake was assessed using one 24‐hour dietary recall and a structured questionnaire with cooking methods and levels of meat doneness. It was used PCR‐based assays to detection SOD2 polymorphism. Malondialdehyde was measured in plasma after derivatization by thiobarbituric acid and separation on HPLC. Multiple linear regression models were performed to verify the association and interaction between PhIP intake and SOD2 on risk of oxidative stress. Sensitivity analyses were performed in a subsample (n=79) using a DNA adduct marker, 8oxodG measured by ELISA, as a dependent variable.

Results

The frequency of the minor allele (C) was 45%. PhIP mean intake was 254ng/day (95% CI: 200;308ng/day). MDA concentration was associated with PhIP intake (β=0.04 P=0.004) after adjustment for energy and fruit intake, sex, age, BMI, race, physical activity, C reactive protein, global methylation, and smoking. We also noted a significant interaction between SOD2 polymorphism and PhIP intake on MDA (P=0.035). Subjects who had one or two risk alleles presented a significant relationship between PhIP and MDA concentration (β=0.06 P=0.01) compared with who had the wild‐type genotype (β=0.01 P=0.870). Moreover, we confirmed a significant interaction between SOD2 and PhIP intake on 8oxodG (P=0.022) after adjustment.

Conclusion

PhIP was associated with oxidative stress and interacted with polymorphism in SOD2 gene increasing oxidative stress and DNA adducts in a general population.

Support or Funding Information

Researches relating to this abstract were funded by Municipal Health Secretariat of Sao Paulo, National Counsel of Technological and Scientific Development and Sao Paulo Research Foundation
Genetic variation in the sucrase‐isomaltase (SI) gene is associated with dietary intake in a young adult population
Authors: Sorkin Riva , Wolever Thomas , El‐Sohemy Ahmed
Published: November 2023

Background

Genetic variation contributes to individual differences in energy and nutrient intake and may be a contributing factor to the selection of certain foods and macronutrient preference. Sucrase‐isomaltase is a brush border enzyme encoded by the SI gene and plays an important role in starch digestion. Research on genetic variation in SI has mostly focused on rare mutations that result in congenital sucrase‐isomaltase deficiency, a severe digestive disorder. The objective of this study was to determine whether common variants in the SI gene are associated with habitual dietary intake.

Methods

Fasting blood samples were drawn from a total of 1,400 ethno‐culturally diverse participants aged 20–29 years from the Toronto Nutrigenomics and Health Study for genotyping of 5 single nucleotide polymorphisms (SNPs) in the SI gene: rs4557160 (C > T), rs7649108 (C >T), rs9756306 (G >A), rs12496714 (A >T) and rs13069478 (C >T). Dietary intake was assessed using a one‐month, 196‐item Toronto‐modified Willett food frequency questionnaire. The prevalence of the SI gene variants was compared using χ2. An analysis of covariance adjusted for age, sex, ethnicity, BMI, and physical activity was used to determine the association between SI genotypes and dietary intake.

Results

Among Caucasians rs9756306 and rs12496714 were associated with total energy intake. Carriers of the minor allele had significantly higher energy intakes 2112 vs. 1194 kcal/day, p = 0.02 and 2011 vs. 1990 kcal/day, p = 0.03 respectively). The rs7649108 SNP was associated with starch consumption, whereby carriers of the minor allele had a significantly lower starch intake (115.8 vs. 120.9 g starch/day, p = 0.02). No associations were observed between rs4557160, rs13069479 and dietary intake.

Conclusion

Polymorphisms in the SI gene are associated with dietary intake patterns. Identifying the genetic determinants of dietary intake and food preference may enhance our understanding of excessive caloric intake and obesity, a common risk factor of many chronic conditions.

Support or Funding Information

Research support from Mitacs, Prof. Ahmed El‐Sohemy
Netrin‐1 is a Novel Regulator of Endothelial Function in Diabetes
Authors: Toque Haroldo A. , Fernandez‐Flores Aracely , Mohamed Riyaz , Vilapakkam Punithavathi , Caldwell Ruth B. , Ramesh Ganesan , Caldwell R. William
Published: November 2023

Netrin‐1, a secreted laminin‐like protein and axon guidance molecule, has been shown to be critically important within the cardiovascular system. Recent studies have revealed it to be pro‐angiogenic, anti‐apoptotic and anti‐inflammatory as well as cardioprotective against myocardial injury in diabetic mice. We examined the role of netrin‐1 in diabetes‐ and high glucose (HG)‐induced vascular endothelial dysfunction using netrin‐1 transgenic (Tg3) and WT mice and bovine aortic endothelial cells (BAECs). Mice rendered diabetic with streptozotocin (STZ) and BAECs were treated with HG (25 mM). Levels of netrin‐1 and its receptor DCC, markers of inflammation, senescence, apoptosis and vascular function were assessed in aortas. Vascular netrin‐1 in WT mice was reduced under diabetic conditions. Aortas from non‐diabetic Tg3 and WT mice showed similar maximum endothelium‐dependent relaxation (MEDR) (83% and 87%, respectively). MEDR was markedly impaired in aorta from diabetic WT mice (51%). This impairment was largely prevented in Tg3 diabetic aorta (MEDR, 70%). Improved vasorelaxation in diabetic Tg3 mice were associated with increased levels of phospho‐ERK1/2 and reduced levels of reactive oxidative species (ROS), mediators of inflammation (NFkβ, COX‐2), cellular senescence (p16INK4A) and apoptosis (cleaved caspase‐3) in comparison to diabetic WT mice. In BAEC, netrin‐1 treatment prevented the HG‐induced decrease in nitric oxide (NO) production and elevation of ROS and apoptosis. In conclusion, overexpression of netrin‐1 prevents diabetes‐induced impairment of vascular endothelium‐dependent relaxation and reduces markers of oxidative stress, senescence, and apopotosis. Netrin‐1 treatment of endothelial cells attenuates HG‐induced impairment of NO synthase function, and elevation of ROS and apoptosis. Enhancement of netrin‐1 levels may be a useful therapeutic means for restoring vascular function in diabetes.

Support or Funding Information

Supported by Scientist Development Grant from AHA (13SDG17410007)
H2S‐RELEASING ASPIRIN EXERTS PROTECTIVE EFFECT IN ESOPHAGEAL AND GASTRIC MUCOSAL STRESS‐ASSOCIATED INJURY
Authors: Zayachkivska Oksana , Bula Nazar , Pavlovsky Yaroslav , Gavriluk Elena , Wallace John
Published: November 2023

Aspirin is one of the widely prescribed antiplatelet and anti‐inflammatory drugs and last data expanded its role into complex biological processes such as cancerogenesis, tissue repair, and aging, despite that well know its effects for peptic ulcer or gastrointestinal (GI) bleeding which can develop even in an achlorhydric environment. Recent extensive research of activities of hydrogen sulfide (H2S) has proven that the addition of H2S‐releasing moiety to the classical non‐steroidal anti‐inflammatory drugs (NSAID) results with GI cytoprotective activity.

The objective of this study was to evaluate the effects of novel NSAID‐H2S compound ‐ ATB 340 (Antibe Therapeutics Inc), hydrogen sulfide‐releasing derivative of aspirin (H2S‐aspirin) in esophageal and gastric mucosal defence in a rat model of stress injury.

Methods

Rats were treated with vehicle (control), classical aspirin (10 mg/kg), ATB‐340 (17,5 mg/kg) with or without being subjected to injury according to the University Ethical Committee Approval (21.05.2014, No 5). The damage of the esophageal mucosa (EM) and gastric mucosa (GM) was estimated via scoring lesion index by histomorphological analysis. Serological levels of VCAM‐1, IL‐6 by ELISA Kits (Cedarlane, Canada) were evaluated.

Results

Treatment by ATB‐340 resulted in protective effect and lower grade of EM & GM lesions in twice by damage index. The basal levels of VCAM‐1, IL‐6 from aspirin‐treated rats and suspected to stress‐injury were higher that of control animal. Pre‐treatment by ATB‐340 exerted an anti‐inflammatory effect by decreasing VCAM‐1 and IL‐6 vs classical aspirin.

Conclusion

These results indicate that inhibiting inflammation and improving endothelial functions modulate protective effect of H2S‐aspirin.
Contractile Actions of Hydrogen Sulfide Donors on the Bovine Isolated Iris: Role of Prostaglandins
Authors: Ajelabi Evelyn , Anyikwa Christina , Ly Christine , Chua Cherie , Chua Cheryl , Robinson Jenaye , Mitchell Leah , Opere Catherine , Njie‐Mbye Ya Fatou , Ohia Sunny
Published: November 2023

We have previously reported that hydrogen sulfide (H2S) donors can relax pre‐contracted, porcine isolated irides, an effect that was dependent upon endogenous production of H2S and prostaglandins, and on the activity of KATP channels (Ohia et al. Curr. Eye Res. 35: 402, 2010). In the present study, we examined the pharmacological actions of H2S donors (NaHS and L‐cysteine) on basal tone in bovine isolated irides. Furthermore, we studied the effect of inhibition of cyclo‐oxygenase (COX) on this response.

Methods

Isolated bovine iris muscle strips were set up in an organ bath containing oxygenated Krebs buffer solution maintained at 37°C and gassed with 95% O2: 5% CO2. The muscle strips were set to a resting tension of 0.3 g and longitudinal isometric tension was recorded via a Grass FT03 force‐displacement transducer and analyzed using PolyView Computer Software.

Results

NaHS (1 nM – 10 μM) and L‐cysteine (100 nM – 1 mM) caused a concentration‐dependent contraction of isolated bovine irides yielding EC50 values of 10 nM and 30 μM, respectively. Both COX inhibitors, flurbiprofen (10 μM) and indomethacin (10 μM) abolished the contractile actions of NaHS and L‐cysteine on this tissue.

Conclusions

We conclude that H2S donors can elicit contraction of isolated bovine irides, an effect that is dependent upon the production of endogenous prostaglandins. Based on data reported previously in the porcine isolated irides in which L‐cysteine had no direct contractile action on this tissue, the present finding that L‐cysteine can elicit contractions of bovine isolated irides indicates that a species difference exists in the response of mammalian irides to exogenously applied H2S donors.
NEUROPROTECTIVE ACTIONS OF L‐CYSTEINE AND METH‐ANADAMIDE IN THE BOVINE ISOLATED NEURAL RETINA
Authors: Mitchell‐Bush Leah , Robinson Jenaye , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou
Published: November 2023

Evidence from literature suggests that hydrogen sulfide (H2S) and endocannabinoids can protect the central nervous system against glutamate‐induced excitotoxicity and oxidative stress. In the present study, we compared the neuroprotective actions of H2S (using L‐cysteine as a substrate) with that elicited by a cannabinoid (meth‐anandamide) against H2O2‐induced oxidative stress in the isolated bovine retinae.

Methods

Isolated neural retinae were pretreated with the H2S substrate L‐cysteine (10 nM ‐ 1 μM) or meth‐anandamide (1 nM–100 nM) for 30 minutes prior to the application of oxidative insult with H2O2 (100 μM) for 10 minutes. Lipid peroxidation was assessed by measuring the levels of 8‐isoprostane in tissues via enzyme immunoassay.

Results

In the presence of H2O2 (100 μM), there was a 20% increase in 8‐isoprostane levels in isolated retinal slices when compared to untreated controls. Pretreatment of tissues with L‐cysteine or meth‐anandamide blocked the H2O2‐induced increase in 8‐isoprostane levels. For instance, L‐cysteine (10 nM) significantly (P<0.001) reversed H2O2 (100 μM)‐induced elevation of 8‐isoprostane levels in the neural retina. Furthermore, meth‐anandamide (1 nM) completely prevented the H2O2 (100 μM)‐induced increase in the level of the lipid peroxidation product.

Conclusions

Both H2S producing compounds and cannabinoids can protect the bovine neural retina from H2O2‐induced oxidative stress with cannabinoids displaying a higher degree of potency.
Sodium hydrosulfide alleviates cecal ligation and puncture (CLP) ‐ induced sepsis and elevates the regional blood flow in septic shock
Authors: Ahmad Akbar , Szabo Csaba
Published: November 2023

Cecal ligation and puncture (CLP) induced sepsis is a serious medical condition, caused by a severe systemic infection resulting in a systemic inflammatory response. The aim of the present study was to assess the effect of hydrogen sulfide in sepsis, with special reference to its effect on the modulation of regional blood flow. To this purpose, we infused sodium hydrosulfide (NaHS) (1 mg/kg/h, 3 mg/kg/h or 10 mg/kg/h through the carotid artery) in control rats or rats 24 hours after CLP measured blood flow using fluorescent microspheres. We have also evaluated the effect of hydrogen sulfide on survival and organ function by injecting NaSH (3 mg/kg) intraperitoneally (i.p) after 24 hrs of CLP. We found that in normal control animals, NaHS induced a characteristic redistribution of blood flow, and reduced cardiac, hepatic and renal blood flow in a dose‐dependent fashion. In contrast, we found that, in rats suffering from CLP‐induced septic shock, the regional blood flow was significantly reduced, and infusion of NaSH (1 mg/kg/h and 3 mg/kg/h) significantly increased the blood flow. In other words, the effect of H2S on regional blood flow was dependent on the status of the animals (i.e. a decrease in blood flow in normal controls, but an increase in blood flow in septic animals). After 24 h of CLP, the glomerular function was significantly impaired, as evidenced by markedly increased (over 4‐fold over sham control baseline) blood urea nitrogen and creatinine levels; this increase was also significantly reduced by treatment with NaSH (3 mg/kg i.p). Treatment of the animals with NaSH reduced myeloperoxidase levels (an index of neutrophil infiltration) in heart as well as in lung and malondialdehyde levels (an index of oxidative stress) in heart as well as in liver. Plasma levels of TNF‐alpha and IL‐1beta were increased in septic shock animals, which were attenuated by NaSH. Treatment of NaSH (3 mg/kg i.p) every 6 h after 12 h of CLP improved the survival rate in CLP animals. In summary, delayed treatment with H2S (treatment starting after the CLP‐induced organ injury, inflammation and blood flow redistribution has already ensued) dose‐dependently maintains regional blood flow and exerts protective effects in rat model of CLP induced sepsis.

Support or Funding Information

This work was supported by the National Institutes of Health (R01GM107846) to C.S.
Effect of lipopolysaccharide (LPS)‐induced endotoxemia in mice genetically deficient in either one of the three major hydrogen‐sulfide producing enzymes
Authors: Ahmad Akbar , Gero Domokos , Olah Gabor , Szabo Csaba
Published: November 2023

Various strategies, targeting the pro‐inflammatory response associated with sepsis have been proposed, without clinical success. We examined the impact of LPS‐induced changes in systemic inflammation, and sulfide expression, metabolism in mice deficient in either one of the three H2S producing enzymes. We injected LPS (10 mg/kg) intraperitoneally (i.p) to wild type (WT) mice, cystathionine‐β‐synthase heterozygous (CBS+/−) mice, cystathionine‐γ‐lyase homozygous (CSE−/−) deficient mice or 3‐mercaptopyruvate sulfurtransferase (3‐MST) mutant mice. After 6 h animals were sacrificed, blood and organs were collected and the following parameters were evaluated: blood urea nitrogen (BUN) and creatinine levels in blood samples, myeloperoxidase (MPO) and malon dialdehyde (MDA) in lung samples, cytokines levels in plasma samples and the expression of the three H2S producing enzymes (CBS, CSE and 3‐MST) in various tissue samples (spleen, lung, liver and kidney) from vehicle and LPS treated CBS+/−, CSE−/−, 3MST mutant mice. We found that LPS markedly impaired the glomerular function, as evidenced by increased (over 4‐fold over baseline) blood urea nitrogen (BUN) and creatinine levels in wild‐type LPS treated mice and CSE & 3MST LPS treated mice. However, CBS+/− LPS treated mice exhibited significantly less BUN levels. Both MPO levels (an index of neutrophil infiltration) and MDA levels (an index of oxidative stress) in lung samples were significantly increased in LPS treated wild‐type mice; these effects were similar in CBS+/−, CSE−/− and 3MST mutant mice. LPS‐induced a significant upregulation of inflammatory cytokines (TNF‐alpha, IL‐1beta, IL‐12 and IFN‐gamma) in the plasma; these effects were comparable in all four animal groups. Finally, the effect of LPS on expression of the three H2S producing enzymes (CBS, CSE and 3MST) in various tissue samples (spleen, lung, liver and kidney) from vehicle and LPS treated WT, CBS+/−, CSE−/− and 3MST mutant mice, in order to test for any compensatory upregulation. We found that CBS expression in liver and kidney samples and 3MST expression in lung samples were significantly high from LPS treated CSE−/− mice compared to wild‐type LPS treated mice. In summary, the observations suggest that deficiency of any single one hydrogen sulfide producing enzymes only modestly affects the outcome of LPS induced endotoxemia, perhaps due to the fact that the H2S production by the remaining other enzymes can compensate for the lack of any single one enzyme.

Support or Funding Information

This work was supported by the National Institutes of Health (R01GM107846) to C.S.
Targeting the sphingosine‐1‐phosphate receptor using virtual screening methods
Authors: Caliman Alisha D , Miao Yinglong , McCammon J Andrew
Published: November 2023

Sphingosine‐1‐phoshate receptor (S1PR1) is a G protein coupled receptor that regulates the egress of T cells and B cells from secondary lymphoid tissues to the periphery. Given this role, identifying modulators that inhibit the function of S1PR1 and promote retention of T cells is an active area of drug discovery for diseases that are perpetuated by an over‐active immune response. In this study, I used trajectories from conventional and accelerated molecular dynamic simulations to screen S1PR1 for allosteric sites. Afterwards, I screened the allosteric sites and the orthosteric site for potential modulators using high throughput virtual screening (HTVS) and induced fit docking (IFD) algorithms. Top ranking compounds for each site will be tested in vitro.

Support or Funding Information

NIH: T32GM007752
Do CHT Inhibitors Constitute a Possible Therapeutic Strategy for Dystonia? Studies with the Novel CHT Inhibitor ML352
Authors: Ennis Elizabeth A , Gresch Paul , Blobaum Anna L , Lindsley Craig W , Dauer William T , Blakely Randy D
Published: November 2023

Dystonias are a group of disorders characterized by abnormal involuntary contracting of musculature that causes twisting and turning of a patient's body. Though the etiology of each type of dystonia varies, many forms are treated with anticholinergic medications ranging from the use of botulinum toxin to muscarinic receptor antagonists. Although the efficacy of cholinergic treatments is limited by side effects, mechanistic evidence for cholinergic dysfunction along with efficacy of current cholinergic treatments encouraged us to pursue the development of inhibitors of the high affinity choline transporter (CHT, SLC5A7) as a potential treatment of dystonia. CHT transports choline at cholinergic presynaptic terminals, is the rate‐limiting determinate of acetylcholine synthesis, and is subject to activity‐dependent regulation. In vitro brain slice studies demonstrate efficacy of CHT inhibition by the competitive, but highly toxic agent, hemicholinium‐3, in reversing maladaptive plasticities in dystonic mice (Martella et al, 2009; Sciamanna et al, 2012). Through a high‐throughput screen and subsequent chemical diversification effort (Ennis et al, 2015), we identified ML352 as a potent and, specific noncompetitive inhibitor of CHT. Our in vivo studies with CHT heterozygous knock out and overexpressing CHT BAC mice demonstrate that ML352 lethality is dependent on CHT gene dosage, consistent with the transporter as a major site of drug action in vivo. Ongoing studies using microdialysis and behavioral experiments seek to elucidate further in vivo effects of ML352 and its analogs in wild‐type and dystonia mouse models. ML352 provides a novel opportunity to evaluate CHT as a treatment for hypercholinergic disorders.

Support or Funding Information

Supported by the Dystonia Medical Research Foundation and the National Institute of Neurological Disorders and Stroke (5R01NS077730 to W.T. Dauer).
Deletion of Endothelial SIRT3 Causes Coronary Microvascular Dysfunction and HFpEF
Authors: He Xiaochen , Zeng Heng , Chen Jian‐xiong
Published: November 2023

The morbidity of heart failure with preserved ejection fraction (HFpEF) increases with advanced age and diabetes, and is associated with endothelial dysfunction. SIRT3, a mitochondrial NAD+‐dependent deacetylase, declines progressively with aging and in diabetes. However, the role of SIRT3 reduction in HFpEF remains to be elucidated. In the present study, we analyzed the cardiac function and coronary microvascular function in endothelial specific SIRT3 knockout (ECKO) mice and global SIRT3 KO mice, as well as endothelial glycolytic metabolism and angiogenesis in vitro. We found that SIRT3 ECKO mice developed left ventricle (LV) diastolic dysfunction, as evidenced by prolonged isovolumic relaxation time (IVRT) and reduction of coronary flow reserve. Western blot analysis revealed a significant decrease in glycolytic enzyme PFKFB3 in SIRT3 KO‐ECs, accompanied by a dramatic reduction in basal glycolysis, glycolytic reserve and glycolytic capacity. Moreover, SIRT3 KO‐ECs exhibited higher oxygen consumption rate and more prominent production of reactive oxygen species (ROS) than WT‐ECs. SIRT3 KO‐ECs exhibited less potent angiogenic capabilities as indicated by decreased network formation and migration. In contrast, global knockout of SIRT3 in mice resulted in progressive deterioration of LV function, as evidenced by a decrease in ejection fraction and fraction shortening at 12 months of age. SIRT3 KO mice also exhibited reduction of ANP and ERK‐1/2, and increased ROS production, gp91(phox), caspase‐3 and Wnt7 in the heart. In conclusion, we demonstrated that ablation of SIRT3 in ECs caused an HFpEF phenotype with impaired glucose metabolism and angiogenesis. Global SIRT3 KO mice, however, exhibited an HFrEF phenotype. Our data suggest cell‐specific effects of SIRT3 deletion on cardiac function.

Support or Funding Information

This study was supported by National Institutes of Health RO1 HL102042 (Chen).