bluuebunny/crossref_metadata_2025 huggingface dataset:
Konjac glucomannan and inulin oligosaccharide attenuated the progression of colitic‐associated colon carcinogenesis and modulated immune response in mice
Authors: Wu Wen‐Tzu , Tsai Yi‐Ting , Chen Hsiao‐Ling
Published: November 2023

The aim of this study was to determine effects of konjac glucomnanan and inulin oligosaccharide on tumor development and immune function using a colitis‐associated colon carcinogenesis mouse model. Male C57BL/6J (n = 36, six‐week‐old) mice were fed AIN‐93G diet and initially divided into vehicle control and AOM‐DSS groups. Mice were given an intraperitoneal injection of azoxymethane (AOM, 10 mg/kg BW) followed by three cycles of 1–1.5% (w/v) dextran sodium sulfate (DSS) in drinking water to induce colon cancer. The vehicle group was received 0.9% saline and distilled water instead. Three days after the last DSS cycle, the AOM‐DSS treated mice were randomly assigned to the following group: control (AIN‐93G diet), KGM (2%, w/w), or inulin (2%) for 9 weeks. The severity of adenocarcinoma, leukocyte phagocytic capacity, and spleen proinflammatory cytokines were determined. Results indicated that colonic adenocarcinoma occurred in all AOM‐DSS treated mice. Supplementation of KGM and inulin effectively reduced the extent of tumor invasion. AOM‐DSS treatment reduced the leukocyte phagocytic ability and increased the spleen interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) levels, which was partially reversed with KGM and inulin supplementation. This study suggested that supplementation of KGM or inulin (2%, w/w) during the cancer promotion period could reduce the progression of colitis‐associated colon carcinogenesis. The mechanisms mediating effects of these fibers may involve in the improvement of leukocyte phagocytic capacity and attenuation inflammatory cytokine in the spleen.

Support or Funding Information

This study was supported by the National Science Council Grant MOST‐103‐2320‐B‐040‐013‐MY3, Taipei, Taiwan.
α‐Asarone augments autophagophore elongation in oxysterol‐challenged macrophages
Authors: Park Sin‐Hye , Kang Young‐Hee
Published: November 2023

Macrophage apoptosis plays a major role in the advanced atherosclerotic lesions, which is induced by several stimuli including endoplasmic reticulum (ER) stress and oxidative stress. In the recent studies, autophagic responses make cells escape from cellular apoptosis through promoting phagocytosis of misfolded proteins. α‐Asarone is isolated from Perilla frutescens, a traditional herb in the East‐Asia, and this compound possesses several effects on allergic inflammation. This study investigated whether α‐asarone alleviated macrophage apoptosis by promoting autophagic responses. To induce lipotoxicity in macrophages, this study treated 7β‐hydroxycholesterol in richly present in oxidized LDL. 7β‐Hydroxycholesterol induced cellular apoptosis by decreasing cell viability by ≈70% within 18 h, which were recovered by treating 10 μM α‐asarone. To induce ER stress by this oxysterol, the activation and induction of pancreatic ER kinase (PERK), translation initiation factor (eIF) 2α, activating transcription factor (ATF) 4 were determined. α‐Asarone down‐regulated the activation and induction of these ER stress‐related proteins. In addition, the oxysterol induced C/EBP homologous‐protein (CHOP) and DNA damage‐inducible protein (GADD34), which was dose‐dependently ameliorated by supplementing α‐asarone. Furthermore, to reveal a relationship between cellular apoptosis and autophagy, autophagy protein (ATG) 5 siRNA was employed in oxysterol‐challenged macrophages. The CHOP induction was up‐regulated in ATG5‐deleted macrophages free of autophagy, indicating that cellular apoptosis and autophagy was reciprocal in oxysterol‐challenged macrophages. The oxysterol enhanced the formation of ATG5/12/16L complex by instigating autophagy and increased LC3 induction for the autophagosome formation. α‐Asarone attenuated the formation autophagosomes through disturbing the formation of ATG5/12/16L complex and the LC3 conversion to LC3II for mature. Therefore, α‐asarone is a protective agent inhibiting atherosclerosis by ameliorating oxysterol‐mediated macrophage apoptosis and by augmenting autophagosome formation.

Support or Funding Information

This study was supported by Food, Agriculture, Forestry and Fisheries (112085‐03‐1‐SB010) and Brain Korea 21 plus program by Ministry of Education of Korea (22A20130012421).
Effects of Moderate Ethanol Consumption on Lipid Metabolism and Inflammation through Regulation of Gene Expression in Rats
Authors: Justice Meegan , Penprase Jerrold , Cintora Patricia , Erwin Danielle , Medrano Octavio , Brasser Susan M , Hong Mee Young
Published: November 2023

Evidence supports a J or U‐shaped correlation with consumption of alcohol and cardiovascular disease (CVD) risk and suggests that light to moderate drinking as opposed to excessive consumption may provide some cardiovascular benefits. The present study examined the underlying mechanisms of how moderate alcohol consumption provides a protective effect against cardiovascular disease. Twenty‐four 109 day old, male Wistar rats were divided into a control group and a treatment group (n=12/group). The treatment group was administered a 20% v/v ethanol solution on alternate days for 12 weeks. There was no difference in body weight gain between the two groups, however, epididymal fat weight was lower in ethanol‐fed rats (P=0.030). Blood glucose was lower in the ethanol group compared to control (P=0.030). Total cholesterol levels (P=0.036) and LDL‐cholesterol levels (P=0.031) were also lower in the ethanol group. There was a significant reduction seen in the expression of hydroxymethylglutaryl‐coenzyme A reductase (Hmgcr) and sterol regulatory element‐binding protein‐2 (Srebp‐2) in the ethanol group (P=0.026 and P=0.034, respectively), suggesting that ethanol lowers cholesterol levels via downregulation of genes involved in cholesterol synthesis. Paraoxonase‐1 (Pon‐1), which is associated with inhibition of LDL‐cholesterol oxidation, was upregulated in the ethanol group (P=0.029). Cyclooxygenase‐2 (Cox‐2) and nuclear factor NF‐kappa‐B p65 subunit (Rela) gene expression was significantly decreased in ethanol‐treated rats (P=0.016 and P=0.047, respectively), indicating possible anti‐inflammatory effects. These findings suggest that moderate ethanol consumption may reduce the risk of CVD by reducing body fat, improving blood cholesterol and blood glucose, and modulation of gene expression involved in inflammation.

Support or Funding Information

This study was funded by NIH AA02329 and supported by San Diego State University N302L Advanced Nutrition Laboratory.
Daily consumption of a dark chocolate containing flavanols prevents genotoxicity in buccal epithelial cells and improves biochemical parameters related to cardiovascular risk factors in young adults
Authors: Leyva‐Soto Aldo , Chavez‐Santoscoy Rocio Alejandra , Lara‐Jacobo Linda Ramona , Re‐Araujo Denisse , Leal‐Orozco Alma Elia
Published: November 2023

Epidemiological and medical anthropological investigations suggest that flavanol‐rich foods exert cardiovascular health benefits. Chocolate is a food that is known to be rich in flavonoids, the main flavonoids in chocolate is flavan‐3‐ol epicatechin. However, the bioavailability and the biological effects of the chocolate flavonoids are poorly understood. The objective was to quantify the effect of epicatechin ingested via dark chocolate on changes in biochemical parameters. Also to determine if the ingestion of dark chocolate rich in epicatechin exert anti‐gentoxic effect in buccal epithelial cells of young adults. Thirty apparently healthy men (20 to 30 years old) participated in a randomized, double‐blind, placebo‐controlled crossover trial during tree months. Consumption of dark chocolate or low‐flavanol chocolate (2 g) was daily ingested. Genotoxicity in buccal epithelial cells was determined by identify and quantify micronuclei in the exfoliated cells of oral mucosa in individuals before the intervention with chocolate, during the intervention (every mounth) and after the intervention. The concentration of glucose, cholesterol, HDL and LDL in plasma was determined before and after the ingestion of dark chocolate or low‐flavanol chocolate. The presence and amount of genotoxic damage was correlated with the main genotoxic factors (alcohol intake, smoking and obesity) and the ingestion of dark chocolate or low‐flavanol chocolate.

Support or Funding Information

This research was supported by the Research Chair Funds for Full Time Professors (2014) of Autonomous University of Baja California (UABC), program Number 3888. The authors would like to acknowledge Consejo Nacional de Ciencia y Tecnología (CONACYT) in Mexico.
Consumption of Selected Cruciferous Vegetables and Soy Phytochemical Dietary Supplements Can Alter Gut Microbiome Composition
Authors: Huang Haiqiu , Roman Arocho Gloricelly M , Davis Cindy , Yu Liangli , Wang Thomas T. Y.
Published: November 2023

The human gut microbiota is in direct contact with ingested dietary phytochemicals. Phytochemicals from cruciferous vegetables (indole‐3‐carbinol (I3C) and 3,3′‐diindolylmethane (DIM)) and soy (genistein (GEN), daidzein (DAI), and glycetin (GLY)) are widely used in dietary supplements for their presumed safety and claimed health benefits. Previous studies have shown that the gut microbiota can influence the metabolism of indoles from cruciferous vegetable and daidzein from soy and the specific metabolic products in the circulation. However, the effect of these phytochemicals on the composition of the gut microbiota is not well understood. Consumption of available dietary supplements can result in a concentration between 0.5–5 mM of these phytochemicals in the gastrointestinal tract. This study aimed to evaluate the potential impact of these phytochemicals, at supplement levels, on gut microbiota composition. In‐vitro culture of mouse fecal bacteria under anaerobic conditions was used as the model of gut microbiota, and turbidity was measured to evaluate total bacteria growth. DIM, but not I3C, at 100 μM significantly inhibited total bacteria growth by 9% (p<0.01). GEN, but not DAI, at 100 μM inhibited total bacteria growth by 12% (p<0.01). In contrast, 100 μM GLY was able to increase total bacteria growth by 17% (p<0.01). Relative abundance of Akkermansia, Bacteroides, Bifidobacteria, Enterobacter, Firmicutes, Lactobacillus, Prevotella, and Ruminococcus were analyzed by qRT‐PCR. GEN (100 μM) induced an 85% (p<0.01) increase in relative abundance of Lactobacillus. I3C (50 μM) treatment resulted in a 35% decrease in Bacteroides (p=0.03) and DIM (25 μM) reduced Ruminococcus by 25% (p=0.03). In summary, the current study showed that selected phytochemical dietary supplements may potentially impact the composition of gut microbiome. Future work is needed to determine whether this contributes to the health benefits of these phytochemicals.
Phytochemical Analysis and Antioxidant Capacity in Tortillas produced from Blue Maize Processed by Nixtamalization or Extrusion Cooking
Authors: Gaxiola Nallely , Mora Saraid , Cuevas Edith , Reyes Cuauhtemoc , Milan Jorge
Published: November 2023

Tortillas from blue maize elaborated from traditional nixtamalization and lime cooking extrusion process were screened for their free and bound total phenolic concentrations, profile of phenolic acids, anthocyanins and antioxidant capacity. Tortillas prepared from extruded maize flours had higher values (217.5 to 209.9 mg GAE/100 g DW) of total phenolic compared to by the nixtamalization process (140.4 to 119.9 mg GAE/100 g DW), and around 80% of total phenolic content and antioxidant capacity were found in the bound form. Analysis of phenolic acids in the free and bound fraction confirmed that ferulic acid was the most abundant (86.7%) and is mainly found in the bound fraction in tortillas made by both processes; this phenolic acid provides more than 90% of the antioxidant capacity in the maize. Both processes caused significant losses of anthocyanins (53.1 – 74.5%) when blue maize was transformed to tortilla. Tortillas prepared by the extrusion cooking process indicated higher retention of total antioxidant capacity analyzed by the two methods DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) and ORAC (oxygen radical absorbance capacity). The results clearly show that the alternative process, lime cooking extrusion, demonstrated higher retention of phytochemicals and antioxidant capacity in these products.
Probiotic Supplements: Are They Alive?
Authors: Robbins Carrie Ann , Rogatzki Matt
Published: November 2023

Over the past twenty years the industry of probiotics has grown as consumers have become more interested in the health benefits of “good” bacteria. These good bacteria have been shown clinically that they can positively affect mental health, irritable bowel syndrome, serum cholesterol and many other aspects of human health conditions. Due to the challenges of drying probiotic cultures, their viability has been brought into question. Under current legislation, dietary supplements are not required to be approved by the Food and Drug Administration (FDA) before they are marketed. The purpose of this study was to determine whether or not commercially available probiotics contain live microorganisms. Since Lactobacillus acidophilus are commonly sold in probiotic supplements, it was chosen to be the focus species of this experiment. Florajen Acidophilus, Renew Life's Vegetable Capsule Ultimate Flora, Bayer's TruBiotics, and Spring Valley's Acidophilus were tested for live bacteria. All samples used varied in price ranging from five dollars to nineteen dollars and contained varying amounts of bacterial cultures. A standard concentration of five hundred million cells per twenty mL was obtained and streaked on to plates of Tomato Juice Agar, as Lactobacillus acidophilus has been found to have enhanced growth on such media. Negative control plates were left unstreaked, while positive control plates were streaked by a stock culture of Lactobacillus acidophilus. Plates were incubated twelve to fifteen hours and observed for growth. Following the incubation periods, all positive control and experimental groups showed growth while negative control plates did not display growth. Techniques of drying probiotics can be quite challenging and lethal to bacterial cultures; however, the results of this study show promise that adequate numbers of bacteria can be delivered in the final product to consumers.

Support or Funding Information

Office of Undergraduate Research and Creative Endeavors: University of Wisconsin‐Platteville
The Stenotic Kidney in 2‐kidney 1‐clip Renovascular Hypertension is not Protected from Chronic Damage in CX3CL1 Deficient Mice
Authors: Grande Joseph Peter , Boyilla Rajendra , Osman Mazen , Hu Zeng , Kashyap Sonu
Published: November 2023

CX3CR1 is a chemokine receptor widely expressed on inflammatory cells which recognizes CX3CL1 (fractalkine) a membrane bound chemokine that can function as an adhesion receptor. Blockade of CX3CR1 has been shown to reduce inflammation and chronic renal injury in a variety of models. We sought to determine whether CX3CR1 deficient mice are protected from chronic renal injury in experimental renal artery stenosis (RAS). RAS was established in CX3CR1 knockout (KO) and wild‐type C57BLK/6 mice (WT) through placement of a cuff on the right renal artery. Sham mice underwent manipulation of the right renal artery without placement of a cuff. As assessed by MRI, mean flow reduction was 65.3% in the WT mice and 66.5% in the KO mice (p=ns) subjected to RAS. Both KO and WT mice developed hypertension, which persisted throughout the 4 week study. KO and WT mice developed a similar degree of cardiac hypertrophy (166 mg WT, 172 mg KO, p=ns; 117 mg sham). Stenotic kidney weights were reduced to a similar extent in KO and WT mice (sham 148 mg, WT 83 mg, KO 53 mg) with compensatory hypertrophy of the contralateral kidney (sham 136 mg, WT 170 mg, KO 206 mg, p=0.0016 KO vs WT). The stenotic kidney of both KO and WT mice developed severe atrophy, characterized by flattening and simplification of tubular epithelium and influx of interstitial T cells and macrophages (32.8 % cortical surface area for WT and 76.4% cortical surface area for KO, p=0.01). The contralateral kidney was without significant histopathologic abnormality. We conclude that development of severe atrophy is not prevented in CX3CR1 KO mice, indicating that alternative pathways contribute to chronic renal damage in this model.

Support or Funding Information

This work is supported by NIH Grant R01 AI100911.
Ryanodine receptor‐dependent modulation of experimental autoimmune encephalomyelitis in mice
Authors: Osipchuk Natalia C. , Soulika Athena , Cruz‐Orengo Lillian , Allen Paul D. , Fomina Alla F.
Published: November 2023

Experimental autoimmune encephalomyelitis (EAE) in mice is an established animal model of T cell‐mediated autoimmune disease. Pathological changes seen in EAE represent a stereotypic response of the CNS tissues to immunological priming following damage. EAE induced by immunizing wild‐type (WT) C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) results in CNS inflammation (macrophages and CD3+ T lymphocytes), demyelination, and axonal loss, which causes progressive development of neurological symptoms and impairment of motor functions.

The strength and duration of cytosolic calcium signals defines T cell activation, differentiation, and immune response. Our previous studies performed in T cell cultures clearly demonstrated physiological significance of ryanodine receptors (RyRs), the endoplasmic reticulum calcium release channels, in T cell calcium signaling in vitro (Thakur et al, 2012, J Biol Chem, 287: 37233–44). Here we report that treatment of activated human T cells with RyR blockers suppresses T cell proliferation and reduces frequencies of T cell subtypes secreting proinflammatory cytokines. Furthermore, manipulation of RyR activity in vivo significantly affects the course of the EAE in mice. EAE was induced by immunizing adult WT C57BL/6 mice and mice carrying gain‐of‐function RyR type 1 R163C mutation on C57BL/6 background strain (RyR1 R163C mice) with MOG35–55. We found that RyR1 R163C heterozygosity is associated with the earlier disease onset and augmentation of neurological symptoms compared with WT mice. These data indicate that a “leaky” RyR1 amplifies autoimmune response in mice. We also found that mice injected daily with RyR1 inhibitor dantrolene beginning at the time of EAE induction, displayed significantly fewer neurological symptoms, reduced CNS inflammatory infiltrate and less axon damage compared to control EAE mice injected with vehicle alone. Withdrawing the dantrolene on day 30 post‐immunization was immediately followed by the development of neurological symptoms. No pro‐inflammatory infiltrate and/or axonal damage were detected in EAE mice treated with dantrolene. These data underscoring the key role of RyR1 in mounting the normal and pathological immune responses and indicate that RyR1 represents a powerful new tool for modulation of autoimmune responses.
The Asymmetric Interaction of Cas9 with Target DNA can Promote High Efficiency Homology‐Directed Genome Editing
Authors: Corn Jacob E , Richardson Christopher D , Ray Jordan , Curie Gemma , DeWitt Mark
Published: November 2023

Cas9 is an RNA‐guided endonuclease that has been adapted from its natural role in CRISPR‐mediated bacterial immunity to enable many applications demanding site‐specific DNA recognition. Wild type Cas9 has been rapidly embraced for genome engineering, either generating knockout cells and organisms via error‐prone non‐homologous end joining (NHEJ) or introducing precise edits via homology directed repair (HDR), while catalytically dead Cas9 (dCas9) fused to various effectors has been used for transcriptional regulation and genomic imaging. However, while Cas9's interaction with target DNA is central to its biological function and use as a genome targeting tool, the molecular nature of this complex is still poorly understood. We have found that the in vitro dissociation of wild type (wt) and dCas9 from double stranded DNA (dsDNA) are both remarkably slow, with a half‐life greater than six hours. However, this stable association is highly asymmetric, and wtCas9 releases only the PAM‐distal end of the non‐target DNA strand post‐cleavage while maintaining contact with the other three strands. dCas9 introduces a bubble into the target DNA that releases the non‐target strand and leaves it accessible for annealing by a complementary single stranded DNA (ssDNA). These activities are present during genome targeting in human cells and can be used to rationally design ssDNA templates that mediate highly efficient gene editing via annealing to the released strand. Optimized templates yield strand‐specific HDR rates over 50% and can furthermore mediate HDR from a single Cas9 nickase or dCas9. Our results highlight Cas9's unusually asymmetric interaction with target DNA, which could be functionally important for CRISPR spacer acquisition or viral interference. Our work also suggests that guiding the formation of specific DNA repair intermediates can dramatically increase the efficiency of genome editing. Because dCas9 is presumably incapable of introducing off‐target breaks in a genome, its use to stimulate HDR may be valuable for precision gene editing in certain therapeutic contexts.
Acute Tolerance to the Discriminative Stimulus Effects of Nicotine in Monkeys
Authors: Moerke Megan Jo , McMahon Lance
Published: November 2023

Tolerance to the effects of nicotine, a major psychoactive component of tobacco, contributes to escalation in the frequency and quantity of tobacco use. However, mechanisms of tolerance to the effects of nicotine are not well understood. Here, drug discrimination was used to examine the rapid decrease in sensitivity to nicotine (i.e., acute tolerance), including its underlying receptor pharmacology and its relationship to nicotine pharmacokinetics. Rhesus monkeys (n=6) discriminated 0.032 mg/kg of nicotine base administered i.v. under an FR5 schedule of stimulus‐shock termination. Nicotine dose‐dependently increased drug‐lever responding; the ED50 value (95% confidence limits) of nicotine to produce discriminative stimulus effects was 0.012 (0.0077–0.020) mg/kg. The β2‐selective nicotinic acetylcholine receptor (nAChR) antagonist dihydro‐β‐erythroidine produced a 4.8‐fold rightward shift in the nicotine dose‐response function. Methyllycaconitine, an α7‐selective nAChR antagonist, up to 10 mg/kg did not antagonize the nicotine discriminative stimulus. The duration of the nicotine discriminative stimulus was less than 30 min; however, concentrations of nicotine measured in saliva were maximal (45–65 ng/ml) 10–30 min post‐injection. Pretreatment with nicotine (0.032 mg/kg i.v.) 30 min before re‐determination of sensitivity to the effects of nicotine resulted in a 3‐fold rightward shift in the dose‐response function. Firstly, these data suggest that the i.v. nicotine discriminative stimulus is mediated by β2‐containing, not α7‐containing, nAChRs. Secondly, decreased sensitivity to the discriminative stimulus effects of nicotine when preceded by an additional dose of nicotine 30 min earlier provides evidence of acute tolerance. That the peak concentrations of nicotine in saliva persist beyond the offset of its discriminative stimulus effects is also consistent with acute pharmacodynamic tolerance. Collectively, these results demonstrate that tolerance to the discriminative stimulus effects of nicotine can be produced by acute exposure to nicotine and suggest that the mechanism underlying acute tolerance may be related to rapid desensitization of β2‐containing nAChRs.

Support or Funding Information

Supported by United States Public Health Service Grant DA 25267.
Effects of Chronic Treatment with Bupropion on Nicotine, Cocaine, and Nicotine + Cocaine Polydrug Self‐Administration
Authors: Kohut Stephen , Barkin Claire , Blough Bruce , Carroll F Ivy , Bergman Jack
Published: November 2023

Clinical reports suggest that cocaine and nicotine are often used concurrently; however, medications to manage this form of polydrug addiction are lacking. Bupropion (Zyban), a dual monoamine (dopamine/norepinephrine) transport inhibitor, is a non‐nicotine‐based pharmacotherapy for smoking cessation that has been approved by the FDA and is currently used medicinally. The present study was conducted to investigate the potential utility of bupropion in managing nicotine + cocaine polydrug addiction by comparing its effectiveness in decreasing intravenous (IV) nicotine, cocaine, and nicotine + cocaine polydrug self‐administration. Adult rhesus monkeys responded under a second order (FR2 (VR16:S)) schedule of reinforcement for food or IV nicotine (0.001–0.0032 mg/kg), cocaine (0.0032–0.01 mg/kg), or nicotine (0.001–0.0032 mg/kg) + cocaine (0.0032–0.01 mg/kg) mixtures during four 1‐hr daily sessions. The effects of chronic treatment (5–10 days) with bupropion (1.0 – 1.8 mg/kg/hr, IV) on food‐ and drug‐maintained responding were then assessed. Consistent with our previous findings, combinations of nicotine (0.001 or 0.0032 mg/kg/inj) + a low dose of cocaine (0.0032 mg/kg/inj) were self‐administered to a greater extent than either drug alone. Results further indicate that consistent or significant alterations in responding for nicotine alone or cocaine alone were not observed during chronic treatment with bupropion; however, averaged data revealed dose‐dependent reductions in responding maintained by each combination of nicotine + cocaine studied. No consistent effects on food‐maintained responding were observed during bupropion treatment regimens. These results suggest that bupropion deserves further consideration for its potential use in the management of nicotine + cocaine polydrug addiction.

Support or Funding Information

Supported by NIH DA026892, DA12970, DA019377, and DA039306
Comparing Cigarette Smoke Extract‐ and Nicotine‐Seeking Behavior after Exposure to Stress in Adolescent Rats
Authors: Gellner Candice
Published: November 2023

The nearly 90% of people who begin smoking in their teens have a harder time quitting and are more likely to relapse. Therefore, it is important to understand relapse to smoking during this developmental period. To investigate the role of non‐nicotine constituents in relapse to smoking, we have established a model of intravenous self‐administration comparing cigarette smoke extract (CSE) to nicotine alone. Previous research in our lab has shown that adult male rats that self‐administer CSE extinguished more slowly and were more sensitive to stress‐induced reinstatement. We now test the hypothesis that adolescent rats will exhibit similar patterns of behavior. Adolescent rats, aged postnatal day (P) 25, were trained to lever press on a FR5TO20 schedule (1 food pellet per 5 lever presses with a time out period of 20 seconds). After rats reached the reinforced lever press threshold (R=35), they underwent surgery to implant a catheter in their right jugular vein. After three days recovery, rats (P35) began 1‐hour drug self‐administration sessions with nicotine or CSE (15 μg/kg/infusion nicotine content). Once rats reached stable responding for drug, drug was removed and drug‐seeking behavior was extinguished. Extinguished drug‐seeking behavior was reinstated with stress, cues, or a combination of stress and cues. Preliminary results suggest that baseline responding for CSE and nicotine groups is similar. When drug is removed, CSE and nicotine seeking rats display similar extinction rates. Adolescents also exhibit similar levels of stress‐ and stress+cue‐induced reinstatement of CSE and nicotine seeking behavior. Therefore, these preliminary findings suggest that the non‐nicotine constituents in cigarette smoke do not influence adolescent responding for drug, extinction or reinstatement of drug‐seeking behavior.

Support or Funding Information

TRDRP Award #21RT‐0136; NIH Award #1R01DA040440‐01
A SUMO‐Acetyl Switch in PXR Biology
Authors: Cui Wenqi , Sun Mengxi , Shen Xunan , Staudinger Jeff
Published: November 2023

Pregnane x Receptor (PXR, NR1I2) was initially described as a master‐regulator of drug‐inducible xenobiotic detoxification pathways in the enterohepatic system. However, there is increasing recognition that PXR activation has a multiplicity of ‘non‐canonical’ roles including anti‐inflammatory effects, alterations in glucose and lipid homeostasis, development of cancer therapy resistance, and in driving tumor progression. There is a clear recognition that alterations in post‐translational modifications (PTMs) of PXR play a pivotal role in regulating its biological function. Precisely how these PTMs influence each other is a current area of interest in our laboratory. We have investigated the interaction of two distinct PTMs at the level of PXR, namely, SUMOylation and acetylation. We detect acetylation of PXR in mouse hepatocytes using an immunoprecipitation approach. The enzyme that modulates PXR de‐acetylation is likely HDAC3 in a complex with a well‐known co‐repressor protein‐SMRT. PXR and HDAC3 co‐localize in mouse hepatocytes, and the acetylation alters the sub‐cellular localization of PXR. Treatment of hepatocytes with the class I histone deacetylase (HDAC) inhibitor trichostatin A (TSA) produces synergistic transactivation of a PXR‐reporter gene when combined with rifampicin, a prototypical PXR agonist. Enzymatic de‐acetylation with HDAC3/SMRT inhibits SUMO‐modification of PXR, while pharmacological promotion of acetylation with TSA promotes high levels of SUMO‐modification of PXR. Treatment with TSA produces increased acetylation of PXR in hepatocytes. The acetylation and SUMOylation of PXR also differentially modulated its ability to associate with the canonical co‐repressor proteins HDAC3/SMRT. We show that SUMOylated PXR has an intrinsic repressive function that is mediated by a currently unknown mechanism. Taken together, our data support a model in which a SUMO‐acetyl ‘switch’ occurs at the level of PXR, such that acetylation is prerequisite for SUMO‐modification of PXR to promote active repression of target gene expression.

Support or Funding Information

National Institute of Digestive Diabetic and Kidney Diseases (R01DK090558) National Institute of Health National Center for Research Resources COBRE (P20 GM103549) University of Kansas J.R. & Inez Jay Fund
STAT3 as a Multi‐organ Target for Tissue Fibrosis
Authors: Haak Andrew , Choi Kyoung , Oh Raymond , Smith Karry M. J. , Tschumperlin Daniel
Published: November 2023

Despite recent therapeutic developments, pulmonary fibrosis remains a debilitating disease with poor prognosis. In order to create effective treatments, promising molecular targets need to be identified and validated. Across multiple organs, mesenchymal derived myofibroblasts play an essential role in matrix deposition and tissue contraction. We previously carried out a high‐content, RNAi screen to characterize myofibroblast cytoskeletal phenotypes (α‐SMA and organized F‐actin) in cultured fibroblasts. Hits from this screen, including STAT3 (Signal transducer and activator of transcription 3), were validated in primary lung fibroblasts from patients with IPF (idiopathic pulmonary fibrosis) on compliant hydrogels. To further evaluate STAT3 as a therapeutic target, we have focused on LLL12, a small molecule inhibitor of STAT3. LLL12 dose‐dependently (0.3, 1.0μM) reduced RNA expression of profibrotic genes (ACTA2, CTGF, COL1A1, and FN1) as well as protein deposition of collagen III and fibronectin, in TGFβ stimulated cultured fibroblasts. LLL12 also reduced contractile force, as measured by traction force microscopy, to levels comparable to that seen with inhibition of Rho kinase (Y27632, 20μM). In an effort to support the role of STAT3 as a multi‐organ regulator of tissue fibrosis we confirmed similar effects of LLL12 in primary human hepatic stellate cells and cardiac fibroblasts. Using the bleomycin model of pulmonary fibrosis we assessed the in vivo efficacy of STAT3 inhibition. The lungs of the mice treated therapeutically (day 14–28) with LLL12 (5 mg/kg i.p. daily) showed reduced collagen and fibrotic matrix content measured by hydroxyproline and trichrome histology. RNA expression of COL1A1, FN1, CTGF, and ACTA2 also decreased (~50%) in the mice treated with LLL12. Importantly, in the mice which did not receive bleomycin, LLL12 had no effect on RNA expression of these genes. These data not only validate the RNAi screening approach, but also point towards STAT3 as an important molecular target for regulation of myofibroblast activation in pulmonary fibrosis and other fibrotic diseases.

Support or Funding Information

Supported by NIH RO1 HL092961 and HL113796, and Boehringer Ingelheim.
Lepidium meyenii inhibits MAGL and increases the expression of LRP1 in neuroblastoma SH‐SY5Y cells
Authors: Rondon‐Ortiz Alejandro , Su Pei Ying , Khan Rimmal , Böhlke Mark , Maher Timothy , Pino‐Figueroa Alejandro
Published: November 2023

Lepidium meyenii (Maca) is a Peruvian plant that has previously been implicated to have effects on the endocannabinoid system. N‐Benzylamides, also known as macamides, are compounds present in the Maca pentane extract (MPE) with a demonstrated inhibitory effect on fatty acid amide hydrolase (FAAH), one of the enzymes that terminates endocannabinoid signaling. The purpose of this project was to evaluate the effect of MPE on monoacyl glycerol lipase (MAGL), another enzyme involved in the endocannabinoid system, and additionally to demonstrate the effect of MPE on the expression of the low density lipoprotein receptor‐related protein 1 (LRP1) in neuroblastoma SH‐SY5Y cells. LRP1 is a receptor involved in the transcellular transport of peptides and proteins, including β‐amyloid.

Macamides were quantified by LC‐MS/MS, demonstrating that N‐benzylpalmitamide, N‐benzyloctadeca‐9Z, 12Z‐dienamide and N‐benzyloctadeca‐9Z, 12Z, 15Z‐trienamide were the most predominant compounds in the MPE with concentrations of 27.0, 8.4 and 5.5 mg/g, respectively. The FAAH and MAGL inhibitory activity of MPE was demonstrated with IC50 values of 9.15 and 7.12 μg/mL, respectively, supporting an action via the endocannabinoid system.

SH‐SY5Y neuroblastoma cells were treated with MPE at three different concentrations between 0.1 and 10 μg/mL. Cells were lysed and LRP1 levels were analyzed by immunoblotting, demonstrating that Lepidium meyenii was able to significantly increase LRP1 expression.

While MPE was previously demonstrated to have neuroprotective effects associated with the inhibition of FAAH, this investigation reports that MPE additionally inhibits MAGL. This dual inhibitory activity on FAAH and MAGL could be responsible for the upregulation of LRP1. It is well known that LRP1 reduction is associated with neurological disorders such as Alzheimer's disease. The increase of LRP1 levels by MPE supports a novel mechanism which might be responsible for some of the biological activity of this plant. Further studies are necessary to better elucidate the activation of transcription factors by Maca constituents.
Isolation of an anti‐tumor compound from the leaves of Annnona muricata and pharmacological characterization in PC‐3 cells
Authors: Hall James , Bedregal Steven , Kelley Charles , Pino‐Figueroa Alejandro
Published: November 2023

Annona muricata L (Annonaceae), commonly known as soursop, has a long history in herbal medicine with a lengthy recorded indigenous use. It has also been found to be a promising new anti‐tumor agent in numerous in vitro studies. The present investigation concerns the antiproliferative effects of one isolated active compound from the leaves of Annona muricata in human PC‐3 cancer cells. One of the active compounds was isolated from the crude extract of Annona muricata leaves by using a cell viability‐guided chromatography technique. The Isolated compound was characterized by C‐NMR and H‐NMR and was found to be “Rolledecin C’ which was previously reported by Zheng‐Ming Gu et al. PC‐3 cells seeded in 96 well plates were treated with different concentrations from 1 to 100 μg/ml of the isolated compound. Cell viability was measured after 24 hours of incubation showing that the isolated compound reduced cell viability in a dose‐dependent manner with an IC50 lower than 10 μg/ml. A caspase activity assay was preformed to demonstrate the possible mechanism of action. PC‐3 cells seeded in white 96‐well plates were treated with 10 μg/ml of the isolated compound for periods from 3 to 24 hours of incubation. A caspase reagent (Promega) was added to the wells followed by 30 min. incubation. The reagent produces a luminescent signal proportional to the caspase 3/7 activity in cells. The reagent contains the tetrapeptide sequence DEVD which is cleaved by caspases to release aminoluciferin, a substrate for the enzyme luciferase used in the production of light. The isolated compound produced a significant 1.8‐fold increase of caspase 3/7 activity after 6h incubation. The maximum 2.1‐fold increase was produced at 12h incubation with the isolated compound. The results thus suggest that this isolated compound is an active constituent of Annona muricata. Further studies must be conducted to better demonstrate its anti‐cancer activity.
Cytotoxic and chemo‐sensitizing effects of ferulic acid, fromTamarix aucheriana, on colorectal cancer cells: insights into mechanisms of therapeutic efficacy
Authors: Abaza Mohamed Salah I. , Afzal Mohammmad , Al‐Attiyah Rajaa , Guleri Radhika
Published: November 2023

Natural products are valuable source for anticancer agents. The present study focuses on the isolation and identification of potential chemo‐therapeutic/preventive constituents from Tamarix aucheriana using bioactivity guided fractionation. Ferulic acid (FA) was identified as an active agent. The potential of FA to control the growth of human colorectal cancer cells, enhance their sensitivity to conventional chemotherapeutic drugs and the potential biological targets and molecular mechanisms of FA action were investigated. FA showed time‐ and dose‐dependent anti‐mitogenic effect on colorectal cancer cells with little cytotoxicity for normal human fibroblasts. FA inhibited the colony formation, induced morphological changes, induced apoptosis, and inhibited DNA‐binding activity of NFkB, the various proteolytic activities of proteasome and invasion in colorectal cancer cells. FA up‐regulated p15, p18, p19, p21, p27, p53, and p57, pro‐apoptotic gene expression and down‐regulated Cdk1, Cdk2, Cdk4, and Cdk6, anti‐apoptotic gene expression. FA showed differential potential to enhance the sensitivity of colorectal cancer cells to conventional chemotherapeutic drugs. These results indicate the chemo‐therapeutic potential of FA.

Support or Funding Information

Kuwait University, Research Project No. SL05‐04
Investigation of the Lipoxygenase Pathway in Cisplatine Induced Nephrotoxicity
Authors: Al‐khamees Osama A
Published: November 2023

Nephrotoxicity is a major side effect for the widely used antineoplastic agent, cisplatin. Several studies have investigated the possible mechanisms for such toxicity. HETEs and leukotrienes were found in several studies to mediate many physiological functions in the kidney including regulation of glomerular filtration or renal vasoconstriction. This study was designed, therefore, mainly to investigate the possible involvement of lipoxygenase (LOX) pathway in cisplatin induced‐nephrotoxicity in Wistar rats. Nephrotoxicity was induced by IP injection of cisplatin (6 mg/kg/day). Genes and protein expression of the major lipoxygenases pathway biomarkers including 5‐LOX, 12‐LOX and 15‐LOX in animals’ kidneys were quantified using real time PCR and western plot, respectively. Cisplatin challenge to the animals markedly altered renal LOX pathway genes expression. There was a marked up regulation in 5‐LOX, 12‐LOX and 15‐LOX genes in the kidneys of cisplatin treated animals. Moreover, protein expressions of 5‐LOX, 12‐LOX, 15‐LOX were also significantly increased in the kidneys of cisplatin treated animals. To the best of our knowledge, the linkage between LOX pathway and cisplatine nephrotoxicity has not been investigated. Combination of several agents with the anticancer drug is frequently used to alleviate the associated side effect. Therefore, the current project is a preclinical study that might provide a therapeutic benefit LOX inhibitors combination with cispaltin.

Support or Funding Information

This research was supported by the National Science, Technology and Innovation Plan (MAARIFAH) under grant (12‐MED2780‐08).
Effects of LRH‐1 Antagonism on Pancreatic Adenocarcinoma Cells
Authors: Kothari Devraj , Rondon‐Ortiz Alejandro , Mendigure Ana , Pino‐Figueroa Alejandro
Published: November 2023

Pancreatic cancer accounts for nearly 3% of all cancers in the U.S. and about 7% of deaths associated with cancer. Liver receptor homolog‐1 (LRH‐1) is an orphan nuclear receptor previously linked to pancreas development and pancreatic adenocarcinoma. It is predominantly present in exocrine pancreas, liver, breast and gastrointestinal tissues. LRH‐1 also regulates the transcriptional targets responsible for controlling cell growth, differentiation, and proliferation. Upregulation of LRH‐1 may be linked to a more aggressive, metastatic form of cancer. The objective of this study was to determine and characterize the in vitro inhibitory effect(s) of 1‐(3′‐(1‐(2‐Morpholinoethyl)‐1H‐pyrazol‐3‐yl)biphenyl‐3‐yl)ethanone (LRH‐1 receptor antagonist) on pancreatic adenocarcinoma (AsPC‐1 cells). The drug antiproliferative effect was tested using a cell viability screening assay method. AsPC‐1 cells were cultured in a 96‐well plate using RPMI‐1640 media and 10% FBS. Cells were exposed to antagonist from 0.1 to 100 μM. Cell viability was determined 24 hours later by trypan blue exclusion and/or the dimethylthiazolyl‐carboxymethoxyphenyl‐sulfophenyl‐tetrazolium (MTS) viability assays. The effect on Caspase activity was determined after exposing the cells to 1.5 and 10 μM of LRH‐1 antagonist. The results demonstrated that LRH‐1 antagonist possessed significant (p<0.05) concentration‐dependent antitumoral activity with a 31% maximum reduction in cell viability and 38% maximum increase of Caspase‐3/7 activity. Biomarkers, such as cyclin D1, E1, and Bcl‐xl, were also measured pre‐ and post‐treatment using western blotting assay methods. Gemcitabine was compared to LRH‐1 antagonist alone and in combination to assess for possible synergistic effects. The results suggest a potential application of LRH‐1 antagonist to better understand the involvement of LRH‐1receptor in the development and progression of cancer. This drug was shown to influence proliferation and apoptotic activity in pancreatic adenocarcinoma cells. LRH‐1 antagonist could be used as a primary compound to design selective and potent LRH‐1 blockers that could be used clinically as therapeutic agents towards pancreatic or other cancers.
Acute Exposure to High Level of Hydrogen Sulfide Induces Neurodegeneration via Activation of Inflammasomes in Brain
Authors: Kim Dongsuk , Anantharam Poojya V , Whitley Elizabeth M , Gilbreath Ebony , Mahama Belinda Y , Kanthasamy Arthi , Rumbeiha Wilson K
Published: November 2023

Hydrogen sulfide (H2S) is gaseous molecule and can be produced endogenously playing important roles in cell signaling in mammalians including human. However, acute exposure to high concentrations of H2S induces brain damage, including long‐term neurological dysfunction. To better understand the exact cellular and molecular mechanisms of H2S‐induced neurological disorders, we investigated neuroinflammation aspect of H2S‐induced toxicity using the Qiagen mouse Inflammation Response & Autoimmunity RT2 Profiler™ PCR array system. C57 black mice were exposed to 310 ppm H2S by whole body inhalation for 1, 3 or 7 days for 40 minutes on day 1 and 15 minutes on subsequent days. Hydrogen sulfide‐exposed mice showed significant impairment in motor activity compared to the control. Histopathology revealed neurodegenerative lesions in the central nucleus of the inferior colliculus (IC), and IC was dissected at each terminal endpoint. Quantitative real‐time PCR based array system was performed, and 84 inflammation related genes expressions were analyzed. IL‐18, Myd88, and TLR9 were upregulated, while C3, CCL12, and CXCR4 were downregulated in H2S exposed group. Interestingly, all three genes upregulated in H2S‐treated group play important roles in activation of inflammasomes. We further investigated genes relevant to the activation of inflammasomes using quantitative RT‐PCR. IL‐1β, CASP‐1 and AIM2 were upregulated in H2S‐treated groups. Expression of NLRP3 was downregulated, whereas NLRC4 was not greatly altered in H2S‐treated groups. We also found that NLRP1b, not NLRP1a, was upregulated in H2S treated group, while we are characterizing further the activation of inflammasomes and its significance in H2S‐induced neurodegeneration. Taken together, our results demonstrated that inhalation exposure to H2S induced motor behavioral deficit and neurodegeneration and that inflammasomes may play a significant role in H2S‐associated neurological disorders (Iowa State Internal Grant).
Epoxyeicosatrienoic Acid Analogs Reduce Renal Fibrosis By Reducing Epithelial‐To Mesenchymal‐Transition
Authors: Khan Md. Abdul Hye , Sharma Amit , Falck John R. , Imig John D.
Published: November 2023

Epoxyeicosatrienoic acids (EETs) are CYP epoxygenase metabolites of arachidonic acid that demonstrate diverse biological actions including kidney protection. Chronic kidney disease (CKD) progresses to end‐stage renal disease and common pathways leading to renal fibrosis are major contributors to CKD. Renal epithelial‐to‐mesenchymal transition (EMT) plays a critical role in renal fibrosis and CKD. We investigated the ability of EET analogs (EET‐A and EET‐B) to reduce EMT and protect from kidney fibrosis in a mouse unilateral ureteral obstruction (UUO) model. C57/BL6 male mice underwent sham or UUO surgical procedure and were grouped (n=6–8) as Sham‐Vehicle, UUO‐Vehicle, UUO‐EET‐A, and UUO‐EET‐B. EET analogs (10mg/kg/d) or vehicle administrated in drinking water for 10 days following UUO surgery. Kidneys were collected for mRNA, protein, and biochemical analysis and histopathological evaluation. Kidney histopathology in UUO mice revealed a marked renal fibrosis with 80% higher kidney collagen positive area compared to Sham group. EET analogs significantly reduced kidney collagen positive area by 50–60%. Accordingly, kidney hydroxyproline content was also higher in UUO (6.2±0.4μg/10mg) compared to Sham group (2.5±0.1μg/10mg), and EET analogs reduced hydroxyproline levels by ~20–40%. Moreover, immunopositive kidney area and mRNA expression of an important renal fibrosis and myofibroblast marker, α‐smooth muscle actin (SMA), was higher in UUO compared to Sham group, and EET‐A and EET‐B reduced SMA levels. Interestingly, in UUO group renal mRNA expression of the epithelial marker E‐cadherin was reduced by 30% compared to Sham group, and EET analogs increased E‐cadherin expression. Renal mRNA expression of mesenchymal markers α‐SMA, collagen IV, and fibronectin were higher in UUO vehicle mice compared to Sham group, and EET analogs reduced expression of mesenchymal markers. Overall, our results demonstrate that EET analogs reduce renal fibrosis in UUO by inhibiting renal EMT.