bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage mitochondrial energy status regulates cholesterol efflux and is enhanced by anti‐miR33 in atherosclerosis
Authors: Karunakaran D , Thrush B , Geoffrion M , Ramphos E , Nguygen M , Shangari P , Harper M , Moore K , Rayner K
Published: June 2021

Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis and miR‐33 has emerged as novel regulator of cholesterol homeostasis. Cellular energy status can significantly influence macrophage function, and bioinformatic analysis predicts that miR‐33 represses a cluster of genes controlling energy metabolism that may contribute to macrophage cholesterol efflux. We hypothesize that miR‐33 represses mitochondria metabolic pathways to reduce cholesterol efflux. Here, we show that macrophage cholesterol efflux is regulated by mitochondrial ATP production and that miR‐33 controls a network of genes that synchronize mitochondrial function. Macrophage cholesterol efflux capacity was markedly reduced by ATP synthase inhibition, confirming the importance of mitochondria in the efflux of excess cholesterol. Specifically, anti‐miR33 derepressed the novel target genes PGC‐1α, PDK4 and SLC25A25 and boosted mitochondrial respiration and ATP production, and mitochondrial respiration was key to the pro‐efflux effects of anti‐miR33. Anti‐miR33 therapy in atherosclerotic Apoe‐/‐ mice reduced aortic sinus lesion area, despite no changes in HDL‐C. Also, this therapy increased expression of mitochondrial target genes in vivo, suggesting that the regulation of mitochondrial genetic networks occur in atherosclerotic lesions.

Conclusion

We showed that anti‐miR33 therapy de‐represses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, promotes macrophage cholesterol efflux and reduces atherosclerosis.
Regulation of TGF‐Beta Receptor Signaling and Cell Migration by Ceramide Metabolism
Authors: Gencer Salih , Senkal Can , Ponnusamy Suriyan , Oleinik Natalia , Selvam Shanmugam , Dany Mohammed , Ogretmen Besim
Published: June 2021

Recent studies indicate that ceramide species play diverse biological functions including, skin barrier function, liver homeostasis, cell death and cancer pathogenesis, highlighting the importance of ceramide synthases (CerS) in these processes. Migration, a part of these processes, also is effected by ceramide metabolism. However, the molecular mechanism of CerS/ceramide involved is unknown. Here, we investigated the effect of CerS on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4 is related to cell migration. Here, we also have generated CerS4‐/‐ mice, and these mice were viable with no lethal tissue. Interestingly, we observed that loss of CerS4 resulted in irreversible alopecia, which was associated with hyper‐proliferation and migration of keratinocytes. Mechanistically, we show that knockout/knockdown of CerS4 enhances cell migration by which ligand‐independent signaling of TGFβ receptors RI and RII in various cell types, including keratinocytes, MEFs, and cancer cells. Additionally, low level of TGFβR1‐Smad7 interaction was found in knockdown of CerS4 cells. Moreover, we found that ceramide interact with Smad7 and interaction was decreased by knockdown of CerS4. Thus, ceramide‐Smad7 binding modulates plasma membrane association of TGFβR1, and inhibits its signaling through Sonic‐Hedgehog (Shh) signaling for migration. In fact, inhibition of TGFβR/Shh signaling using molecular or pharmacologic inhibitors almost completely prevented cell migration in response to CerS4 knockdown. These data suggest that CerS4/ceramide signaling plays key roles in the regulation of cell migration via controlling the TGFβR/Shh axis.
S‐Glutathionylation promotes HSP90 degradation under oxidative stress
Authors: Shih Yu‐Yin , Lin Chun‐Hung
Published: June 2021

S‐Glutathionylation, a redox‐dependent post‐translational modification, alters protein activity and stability in response to various stimuli. Heat shock protein 90 (HSP90), a ubiquitous chaperone, is known to interact with numerous proteins to regulate multiple cellular processes, especially in cell proliferation and cell cycle progression. The increased level of HSP90 is known as a hallmark in tumor cells and tissues, revealing the importance of HSP90 in tumorigenesis. Accumulating evidences indicated that the protein degradation of HSP90 is controlled by ubiquitin proteasome system. We herein report the site‐specific S‐glutathionylation of HSP90 in vivo and in vitro. The level of HSP90 was obviously decreased after the treatment with hydrogen peroxide or oxidized glutathione (GSSG), owing to the proteasome‐mediated degradation. Further gel filtration and CD spectrometric analyses indicated that S‐glutathionylation is the trigger to alter the quaternary and secondary structure of HSP90, respectively. Taken together, these results support the idea that S‐glutathionylation causes structure alterations in HSP90 and promotes the subsequent degradation. Since high level of HSP90 is associated with drug resistance of tumors and cancer reoccurrence, the findings on the S‐glutathionylation and subsequent protein degradation of HSP90 would shed light on the therapeutic potential in cancer therapy.
Assembly and Application of Phospholipid Nanodiscs for the Study of P‐Glycoprotein
Authors: McClean Mindy , Brewer Frances , Wise John , Vogel Pia
Published: June 2021

As many as 40% of recurring cancers often develop multidrug resistance (MDR) to chemotherapeutic treatment due to over‐expression of P‐glycoprotein (P‐gp), a member of the ABC transporter protein family. Our research has focused on finding inhibitors of P‐gp that are capable of re‐sensitizing MDR cancers to therapy. In order to investigate the mechanism of action of the discovered inhibitors, we employ electron spin resonance spectroscopy and biochemical assays. Due to the inherent instability of these mammalian membrane proteins, many of these analyses are challenging. We therefore are establishing nanodisc technology to incorporate and stabilize purified P‐gp for our drug finding and mechanistic studies. Nanodiscs are phospholipid bilayers contained within a membrane scaffold protein and mimic native membrane conditions. Initial studies indicated that a cysteine‐less mutant of the mouse MDR3 P‐gp could be incorporated into nanodiscs and showed enhanced catalytic activity and stability. We are currently establishing nanodiscs as a uniform platform from which different mutants of P‐pg can be closely studied and characterized. Nanodiscs may also enable us to utilize human MDR1 P‐gp in drug finding studies. This particular protein has been shown to be too unstable in micelle solution to be successfully used in these experiments.

This work is supported by NIH NIGMS [Grant 1R15‐GM094771‐01A1] to PDV and JGW, SMU Research Council, Dean's Research Council, and CD4, and the Communities Foundation of Texas, Dallas. Undergraduate support was received from the Hamilton Family, Dallas, SMU Undergraduate Research Assistantship, and Engaged Learning Fellowship.
Biodiesel Production via Methanol Tolerant Lipase Transesterification from Proteus mirabilis in Methyl Acetate
Authors: Altier Jake , McDonnell Alaina , Carey Demetrius , Witherow Scott
Published: June 2021

Recombinant lipase catalyzed biodiesel production is potentially more economically favorable and convenient than traditional acid/base methods. Possible advantages include less time spent, less energy required due to lower temperatures, less starting material and more efficiency if enzymes can be immobilized. A lipase from Proteus mirabilis (P. mirabilis) has been previously genetically engineered for methanol resistance and shown to be an effective catalyst for the transesterification reaction leading to the production of biodiesel in the form of fatty acid methyl esters. However, the enzyme has not previously characterized using the alternate acyl acceptor methyl acetate, which produces the fuel stabilizer triacetin as a byproduct of the reaction. These enzymes have been expressed in E. coli using a pET28 bacterial expression vector and purified using metal affinity chromatography. Subsequently, the enzymes have been kinetically characterized for transesterification activity in reactions using methyl acetate, using p‐nitrophenyl palmitate and synthetic triacylglycerides as substrates, at varying pHs and temperatures.

Supported by a University of Tampa David Delo Research Professor Grant (DSW), a University of Tampa Dana Foundation Grant (DSW), and The University of Tampa Department of Chemistry, Biochemistry, and Physics (DSW, JA, AM)
Protein Disulfide Stress: Proposed Radical‐Free Bridges to Neurodegeneration
Authors: Foley Timothy , Cantarella Kristen , Gillespie Paul , Wierbowski Shayne
Published: June 2021

Oxidative crosslinking of protein thiols via disulfide bonds, promoted by non‐radical peroxides, offers alternatives to traditional, oxygen radical‐centered, perspectives with which to consider the impacts of oxidative stress on brain functions. Protein disulfides, involving the bridging of closely‐spaced (vicinal) thiol pairs, as well as protein‐glutathione mixed disulfides occur readily in cells exposed to hydrogen peroxide but the scope, pathways, and relationships to health and disease of protein thiol oxidations in the brain remain to be fully established. We demonstrated recently that (i) brains of healthy adolescent rats, from different sources, could be distinguished by ratios of oxidized to reduced glutathione and (ii) moderate increases in these ratios were associated with marked elevations in the levels of total protein disulfides but not mixed disulfides. We report here that the more oxidized brains were characterized also by increases in the protein levels of thioredoxin (Trx)‐dependent peroxiredoxin (Prx) peroxidases as well as the extents of oxidations of the catalytic vicinal thiols of these enzymes. Moreover, among the non‐Prx proteins that were oxidized most prominently were glyceraldehyde‐3‐phosphate dehydrogenase and the catalytic subunit(s) of the Na+, K+‐ATPase, critical enzymatic determinants of glucose utilization and neuronal excitability, respectively. We hypothesize that radical‐free oxidations of protein vicinal thiol motifs, controlled by the Trx/Prx system, may facilitate short‐term adaptation to oxidative stress but that prolonged oxidations, i.e., protein disulfide stress, may derail healthy brain aging by increasing the vulnerability of neurons to excitotoxic insults.
MIEN1 drives breast cancer invasion by regulating cytoskeletal dynamics
Authors: Kpetemey Marilyne , Vishwanatha Jamboor
Published: June 2021

Introduction

Previously known as C35, C17orf37, RDX12 and MGC14832, MIEN1 is a novel gene located in the chromosomal region 17q12‐21. While absent or low in normal tissues, MIEN1 is abundantly expressed in multiple cancers including breast carcinomas. A membrane‐bound signaling adaptor, MIEN1 localizes to the leading edge of migrating cells and promotes migration and invasion by increasing filopodium formation. While MIEN1 is shown to promote motility through filopodia formation, little is known on the mechanisms by which MIEN1 influence the actin cytoskeleton. Exploring underlying mechanisms, we show that MIEN1 associates with the actin cytokeleton during motility and directly influence actin polymerization.

Methods

We performed immunofluorescence, migration and invasion assays using breast cancer cell lines, MDA‐MB 231 and MCF10CA1a to investigate the mechanisms by which MIEN1 potentiates cell motility.

Results

Ablation of MIEN1 inhibited cell migration, cell adhesion and altered the phosphorylation status of motility markers. Immunofluorescence staining with FITC‐ conjugated‐phalladoin confirmed that alteration of MIEN1 expression leads to reconfiguration of the actin cytoskeleton in breast cancer cells. Furthermore we found that MIEN1 associates with cytoskeletal proteins involved in motility processes.

Conclusion

Our results show that MIEN1 is a critical regulator of cell migration and invasion. The importance of MIEN1 in tumor cell dissemination is well established; understanding the molecular mechanisms aiding the processes involved will enable the design of novel and effective treatments for metastatic tumors.
GGF2 Activation of PI3 kinase Pathway Mediates Cardiomyocyte Protection via Effects on Mitochondria
Authors: Srinivas Maya , Cao Jing , Sarmiere Patrick , Caggiano Anthony , Button Donald
Published: June 2021

Glial growth is a neuregulin‐1 isoform that mediates ERBB receptor signaling. It has been shown to improve left ventricular function in several models of heart failure, though the mechanisms are poorly understood. GGF2 regulates PI3 kinase (PI3K) and MAP kinase (MAPK) as measured by pAKT and pERK1/2 formation in numerous cardiomyocyte models, including HL‐1, neonatal rat ventricular myocytes and human iPSC‐derived cardiomyocytes. To better understand GGF2 action in the stressed heart, we have developed in vitro systems to study GGF2 protection of cardiomyocytes from doxorubicin induced toxicity associated with mitochondrial metabolic status, membrane potential and apoptosis. MTT labeling index was used as an endpoint measurement of viability. Doxorubicin decreases the MTT labeling index to 20% while GGF2 reverses this toxicity to nearly 50% of the labeling in untreated cells with an EC50 of ~80 pM. This effect appears to be more dependent upon AKT signaling than MAPK as assessed using signaling pathway inhibitors. Doxorubicin's cytotoxicity involves alteration of mitochondrial membrane potential (Δψm) and can be monitored using the voltage‐dependent dye, JC‐10. We used JC‐10 to reveal that GGF2 pre‐treatment improves Δψm in stressed cells. Mitochondrial dysfunction is closely associated with caspase activation. While caspase3/7 activity increases with doxorubicin, GGF2 prevents or delays onset of caspase activation. Our results demonstrate GGF2 has cytoprotective actions in HL‐1 cells that require PI3K activation and involves maintenance of Δψm. Current efforts are aimed at elucidating the roles for PI3K‐dependent GGF2 regulation of mitochondrial function, and identifying the genes that may be responsible for this effect.
Chemical Composition, Probiotic Survivability and Shelf Life Studies of Symbiotic Buttermilk
Authors: Zhang Dong , Wang Xibo , Guo Mingruo
Published: June 2021

Cultured buttermilk is getting popular as an ingredient for bakery and for direct consumption in the U.S.. The objective of this study was to develop a symbiotic cultured buttermilk, containing inulin as a prebiotic and probiotics Lactobacillus acidophilus and Bifidobacterium spp. The cultured buttermilk was prepared using a commercial mesophilic starter CHN22 (Lactococcus lactis sbusp. cremoris, Lactococcus lactis subsp. lactis, Leuconstoc mesenteorides subsp. cermories, Lactococcus lactis subsp. lactis biovar diacetylactis) and the probiotics. The control buttermilk prepared using CHN22 and the symbiotic buttermilk were analyzed for chemical composition, probiotics survivability, mold, yeast and coliform counts. Changes in pH and titratable acid were also determined during storage at 4° C for 12 weeks. The chemical composition of the control and symbiotic buttermilk were: protein 3.29±0.05 and 3.30±0.02%; fat 3.28±0.04 and 3.26±0.06%; carbohydrate 4.55±0.05 and 5.16±0.06%; total solids 12.81±0.05 and 12.42±0.03%; ash 0.692±0.03 and 0.701±0.01%, respectively. The populations of both Lactobacillus acidophilus and Bifidobacterium spp were initially above 107 cfu/g and remained 106 cfu/g during the 12‐week and there was no mold and yeast were detected. There were significant differences in pH and titratable acid between the control and symbiotic buttermilk (p<0.05) due to addition of the probiotics.
How Consumer Perceptions about Stevia Impact Purchasing Decisions in the United Kingdom and France
Authors: Cheatham Rachel
Published: June 2021

Worldwide obesity rates have nearly doubled since the 1980s. Non‐nutritive sweeteners can play an important role in controlling calories. To determine parents' perceptions about nutritive and non‐nutritive sweeteners, and the impact these perceptions have on purchasing behavior, a representative sample of 434 subjects from the United Kingdom (UK) and 425 subjects from France (FR) ages 18‐64 with children living in their household (i.e. “Parents”) were surveyed online. Results showed that 74% of UK and 69% of FR Parents are either “very” or “somewhat” concerned about the amount of sugar their children consume, and 88% of UK and 83% of FR Parents say they would prefer the foods and beverages they give their children to have “less” or “no sugar.” In addition, 70% of UK and 74% of FR Parents are “very” or “somewhat” concerned about the amount of “artificial” sweeteners their children consume, and 36% of UK and 55% of FR Parents say they would “probably” or “definitely not” give their children products with “artificial” sweeteners. Sixty‐three percent of UK and 65% of FR Parents agree stevia is a “natural” sweetener. Fifty‐two percent of UK and 55% of FR Parents say they would purchase beverages sweetened with stevia, while 53% of UK and 56% of FR Parents would purchase foods sweetened with stevia. Stevia is a plant‐based sweetener that can help parents reduce calories from nutritive sweeteners in the family diet.
A Magnetic Separation Method to Improve Signal‐to‐Noise Ratio in the Measurement of Iron Deficiency Biomarkers Using a Photonic Crystal Biosensor Immunoassay
Authors: Peterson Ross , Cunningham Brian , Andrade Juan
Published: June 2021

Point‐of‐care diagnostics are critically needed to identify populations that are at risk or suffering from iron deficiency anemia (IDA). Current biosensing immunoassays capable of measuring IDA biomarkers, e.g. soluble transferrin receptor (sTfR), require extensive sample preparation to reduce non‐specific binding. In this study, the authors harnessed the magnetic properties of iron‐oxide nanoparticles (IONS) conjugated with antibodies (Ab) to reduce non‐specific binding and to increase signal‐to‐noise ratio. The objectives were to 1) establish a magnetic separation protocol to evaluate binding of sTfR (85 kDa) in a dose‐response fashion using anti‐sTfR functionalized with 30‐nm IONs (Fab‐IONs), and 2) compare the accuracy of this method in quantifying WHO's sTfR reference standard with an FDA‐certified ELISA. Fab‐IONs (0.25 mg/ml) were used as immuno‐probes to separate sTfR (0.005‐0.5 µg/ml) from different media. Binding occurred while shaking samples for 2 h at 450 rpm. A neodymium magnet separated the Fab‐IONs and sTfR complexes. Supernatants and complexes were loaded onto a 384‐well photonic crystal (PC) biosensor microplate functionalized with capture Ab. The PC biosensor showed linear (R2=0.99) response [PWV=8.3 (µg/ml) ‐ 0.1253]. The analytical bias when measuring the WHO reference standard either with the ELISA (‐3.40 µg/ml, 95% CI [‐6.59 ― 0.21]) or the PC biosensor (‐2.54 µg/ml; 95% CI [‐5.16 ― 0.7]) was similar (p>0.05). Magnetic separation using Fab‐IONs is a promising method to measure key nutrition biomarkers at the point of care.
Calcium‐dependent Chemotherapeutic Mechanisms of Resveratrol
Authors: Kenealey Jason
Published: June 2021

In 2013 breast cancer killed approximately 40,000 Americans due in part to the ineffectiveness of chemotherapy. Current chemotherapeutic options are limited by their cytotoxicity to normal tissue and variable efficacy. Resveratrol is a non‐toxic natural product that is selectively deleterious to tumor cells. However, what is not understood is how resveratrol targets tumor cells. We use live cell microscopy to show that an apoptotic rise in cytoplasmic calcium concentration ([Ca2+]I) is induced by resveratrol in cancer cells, but not in normal breast cells. Further, we demonstrate that the change in fura‐2 fluorescence is due to a change in [Ca2+]I and not an increase due to overlapping fluorescent spectra. Further, we have evidence that the mobilization of ([Ca2+]I) by resveratrol activates additional apoptotic mechanisms. These data demonstrate that resveratrol stimulates a tumor cell specific apoptotic flux of calcium into the cytosol that stimulates apoptotic signaling
Testing Novel Depsipeptides for Histone Deacetylases Inhibitor Activity
Authors: Mitchell Caitlyn , Carle Sigrid , Enos Stephen , Miller Justin , Zang Xiaoyu , Vallabhaneni Deepak , Peraro Leila
Published: June 2021

Gene expression is partly controlled by posttranscriptional modifications to histones. These modifications either loosen the binding between histones and DNA, allowing for transcription, or tighten the binding, which prevents transcription. Histone acetyltransferases (HATs) are responsible for histone acetylation, while histone deacetylases (HDACs) remove acetyl groups and are involved in gene silencing. HDAC activity is often upregulated in cancer cells, leading to the repression of several genes that normally prevent tumor formation. Due to the role of HDACs in cancer development and progression, HDAC inhibitors (HDACi) are promising treatments for several cancers. Furthermore, unlike many traditional chemotherapeutics, HDACi appear to have limited effect on normal cell growth and function.

Cyclic, cysteine‐containing, depsipeptides are a promising class of HDACi. These HDACi require three major characteristics: a Lewis basic group that associates with the Zn2+ ion at the heart of the active site, a cyclic region that binds to the nonpolar amino acid residues at the rim of the active site channel, and a linker of four to seven atoms that connects the nonpolar region to the Lewis basic group, presumably mimicking the structure of lysine. We tested four analogs with altered cyclic regions.

An MTT assay was used to measure cytotoxicity induced by each depsipetide on the histoiocytic lymphoma cell line, U937. Depsipeptides that were cytotoxic were investigated for their ability to induce apoptosis using a CaspACETM Assay. Most effective novel depsipeptides were those with smaller, nonpolar cyclic regions.
The Gut Microbiota for Health Experts Exchange: An Online Network for Sharing Knowledge
Authors: Enck Paul , Campbell Kristina , Tap Julien , Arnoux Pierre‐Yves , Antoine Jean‐Michel
Published: June 2021

The gut microbiota is a topic with rising importance in the scientific community where communicating the science presents huge challenges. With studies emerging from all over the world, researchers may have difficulty knowing the context of their own research in this rapidly‐evolving field.

One way toward more accurate communication is to create a network of experts on gut microbiota who collaboratively curate and critique recent work in the field.

Gut Microbiota for Health is an initiative with 900 existing online community members, which include scientists, clinicians, journalists, institutional representatives, and members of the public. The growing worldwide network includes 1800 Twitter followers, 1800 LinkedIn members, and 2500 newsletter subscribers with a common interest in gut microbiota.

Through social media, community members engage with the content and with each other. Clinicians can also integrate the latest scientific progress on gut microbiota into their practice with the help of the Gut Microbiota for Health e‐learning materials.

Gut Microbiota for Health is driven by the Gut Microbiota and Health section of the European Society for Neurogastroenterology and Motility (ESNM), with the institutional support of Danone. It was created as the digital continuum of the Gut Microbiota for Health World Summit, whose 2014 program was organized in partnership with the American Gastroenterological Association.

You are more than welcome to join this scientific community on www.gutmicrobiotaforhealth.com
Physical Activity (PA) Guide Development for Parents of Preschoolers: Qualitative Analysis
Authors: Delaney C , Martin‐Biggers J , Byrd‐Bredbenner C
Published: June 2021

PA levels continue to fall short of recommendations. Thus, a guide promoting PA in the home was developed using social cognitive theory concepts and motivational interviewing techniques. Parents of preschoolers (n=216) were surveyed to determine current PA practices; 15 participated in the 1st round of cognitive test interviews conducted by trained researchers and 20 participated in the 2nd round to determine guide comprehension, aspects perceived as useful, and needed improvements. About half of the parents surveyed reported that they were physically active enough to either sweat or have difficulty breathing at least 4 days/week. Qualitative content analysis of round 1 revealed that Spanish (n=7) and English speakers (n=8) understood guide content, felt the guide's information was useful, especially sections that provided ideas and reminders for parents. English‐speakers suggested adding information on types of play recommended for different age groups. Spanish speakers were satisfied with the guides and had no suggestions for improvement. The guide was revised using Round 1 results, formatted as a mini‐magazine, and subjected to a 2nd round of cognitive testing. Spanish (n=10) and English (n=10) parents expressed great satisfaction with the guide. Spanish speakers wanted to have more information on the benefits of PA vs. TV watching. English speakers were most interested in information describing benefits of PA, particularly those that promote family relationships. The guide was well received, parents expressed great interest in the content, felt the guide was easily understood and beneficial, and had very minor suggestions for improvement. The refined PA guide has the potential to increase PA in families with preschoolers. USDA NIFA #2011‐68001‐30170
Cystathionine Gamma‐Lyase Inhibition in Primary Human Hepatocytes Affects the Production of Hydrogen Sulfide Biomarkers through the Transsulfuration Pathway
Authors: DeRatt Barbara , da Silva Robin , Ralat Maria , Jacobs René , Gregory Jesse
Published: June 2021

Hydrogen sulfide (H2S) has been identified as a neurotransmitter involved in smooth muscle dilation and thus, may have cardioprotective effects in‐vivo. The transsulfuration enzymes, cystathionine beta‐synthase (CBS) and cystathionine gamma‐lyase (CSE), produce the majority of endogenous H2S concurrent with the formation of lanthionine (lan) and homolanthionine (hlan) biomarkers. We compared transsulfuration pathway kinetics and H2S biomarkers in primary human hepatocytes with and without functionality of enzyme CSE. CSE was inhibited by 1000 µM propargylglycine (PAG) and metabolic fluxes were determined using [U‐13C5] L‐methionine and (3,3 D2) L‐cysteine precursors. Methionine cycle kinetics and rate of homocysteine production were not affected by the addition of PAG. In the presence of PAG, cystathionine was accumulated in cells and media (P<0.001). Intracellular hlan concentration and fractional synthesis rates (FSR) were higher in untreated cells compared to cells with CSE inhibited (P<0.041), while in media hlan concentration was 50% less due to PAG inhibition (P<0.025). In the presence of PAG, lan concentration in cells and media exceeded those of untreated cells (P<0.012). The inhibition of CSE in primary hepatocytes greatly decreased the production of H2S biomarker hlan, while lan production was not affected. These results support in‐vitro studies indicating that CSE is the primary enzyme in hlan production while CBS produces the majority of lan. (Supported by NIH DK072398 and CIHR MOP133505).
Anti‐obesity effect of Chungkugjang in 3T3‐L1 Adipocytes
Authors: Kang Soon Ah , Kim Mi‐Ja , Kim Jong‐Ae , Kim Hyun‐Sook , Jung Kwang‐Gyun , Kim Seong‐Hee , Kim Jin‐Kyung , Hwang Boyeon
Published: June 2021

Cheonggukjang (CGJ), a traditional Korean fermented soybean food, exerts immunomodulatory effects.Cells were fixed and stained with Oil Red O to assess lipid accumulation. Total lipid and triglyceride contents increased during the cell differentiation.In this study, the antioxidantactivities of Chungkugjangwere evaluated using in vitro system including DPPH, ABTS, and ORAC methods.Total phenolic contents of Chungkugjang extract were 13.87 μg trolox equivalent/mg extract and EC50 of Chungkugjang r extract for ABTS radicalswere 13.8 μmole ascorbic acid equivalent/g raw sample. Triacylglycerolcontent in 3T3‐L1 preadipocyte cells treated with 25, 50, and 100 μg/mL Chungkugjangextract decreased significantly (p<0.05) compared tothose in untreated cells. Leptincontent in 3T3‐L1 preadipocyte cells treated with Chungkugjangextract decreased significantly (p<0.05) and adiponectin content increased compared tothose in untreated cells. Also, Chungkugjang extract induced the down‐regulation of adipogenesis‐related genes such as PPARγ, GDP1 and FAS. The Chungkugjang extract showedphenolic compounds with antioxidant ability and had in vitro decrement of triglyceride accumulation on 3T3‐L1 adipocyte cells.
Modulatory effects of Acanthopanax koreanum Nakai on oxidative stress induced by combination of alcohol and high‐fat diet in C57BL/6j mice.
Authors: Kim Hae‐in , Jeon Hye‐jin , Park Min young , Kwon Oran
Published: June 2021

Excess alcohol consumption is liked to a high risk of liver problem, which can develop into steatohepatitis and liver cirrhosis in modern society. A high‐fat (HF) diet can also lead to the progression of fatty liver. Therefore the role of alcohol as a risk factor for liver damage may be clearly demonstrated by a combination of both alcohol and HF diet. We tested this hypothesis by using Acanthopanax koreanum Nakai (AK) as a model compound. C57BL/6j mice (8‐week‐old; n=56) were randomly divided into seven groups for four weeks; normal control (NC), ethanol control (EC), EC + high fat diet (EF), EC + 1% AK (ECL), EC + 3% AK (ECH), EF + 1% AK (EFL), and EF + 3% AK (EFH). AK was provided in the diet and 50% (v/v) ethanol was provided by gavage. ADH had no significant difference among all groups. Hepatic ALDH activities were significantly increased in the EH compared with the NC, whereas the ECH showed a tendency of increase in ALDH activity compared with the EC. CYP2E1 was significantly increased in both the EC and the EF compared with the NC. ECH tended to decrease CYP2E1 protein expression compared with the EC and the EFL and EFH showed a tendency to decrease compared with the EF. Peroxisomal catalase (CAT) and glutathione reductase (GR) had a tendency to decrease in the EC; CAT and GR decreased significantly in the HF than in the NC. The AK treatment groups (ECH and EFH) showed a tendency to increase in CAT and GR. Taken together, the results support the hypothesis that combination of alcohol and high‐fat diet provide better animal model for testing the alcohol‐induced oxidative stress. Based on this animal model, the results suggest that AK administration in particularly at a high dose may have protective effects against hepatic injury via anti‐oxidative function.
Green Tea Consumption Reduces Oxidative DNA Damage and Lipid Peroxidation in Smokers and Non‐Smokers
Authors: Liu Yibin , Wang Zemin , Fly Alyce , Klaunig James
Published: June 2021

Oxidative stress is associated with the pathogenesis of human diseases. Green tea reduces oxidative stress in animal models but effects in humans are equivocal. This study was conducted to examine the effects of green tea on biomarkers of oxidative stress and antioxidants in smokers and non‐smokers, using a placebo‐controlled, double‐blind crossover design. Fifteen non‐smokers and 12 smokers consumed 32 oz daily beverage of 2.75% green tea and placebo, each for 1 week, in random order. Blood and urine samples collected pre and post treatment were analyzed for 8‐hydroxydeoxyguanosine (8‐OHdG), malondialdehyde (MDA), glutathione (GSH), α‐tocopherol and ascorbic acid by HPLC. Differences between group means were examined by 3‐way ANOVA (α=0.05). Urinary and plasma MDA of smokers were significantly higher than non‐smokers (P=0.044, P=0.016). Urinary and lymphocyte 8‐OHdG were also higher in smokers, though not significantly (P=0.206, P=0.784). Urinary MDA, urinary and lymphocyte 8‐OHdG were reduced after green tea by 65.7%, 43.2% and 42.8%, respectively (P=0.0006, P=0.037, P=0.001). No changes were seen in plasma MDA, GSH, α‐tocopherol or ascorbic acid (P=0.931, P=0.611, P=0.323, P=0.963). Two groups of subjects (high versus low basal oxidative stress) were identified based on the median of each oxidative biomarker. Urinary MDA and lymphocyte 8‐OHdG in subjects with high basal oxidative stress were significantly reduced by 53.9% and 53.4% after green tea versus placebo (P=0.026, P=0.013); and urinary and plasma MDA were lowered by 47.2% and 13.7%, though not significantly (P=0.070, P=0.183). In conclusion, green tea consumption reduced oxidative DNA damage and lipid peroxidation in smokers and non‐smokers.

Supported in part by internal IU funds.
Antioxidant Properties of Native Mexican Foods : Chocolate, Vanilla and Nopal
Authors: Hernandez Monroy Blanca Mar , Tovar Armando , Torres Nimbe
Published: June 2021

The antioxidant properties of Mexican traditional foods may contribute to reduce diseases caused by an increase in free radicals and reactive species such as diabetes and obesity in Mexican population.The purpose of this study was to investigate the properties of chocolate, vanilla and nopal. Antioxidant activity, reactive species and polyphenolic content were measured by fluorometric and spectrophotometric assays. Content of vitamin C was measured by titration and antioxidant activity in vivo of vanilla was measured in plasma of healthy subjects. The results demonstrated that vanilla, nopal and handmade Mexican chocolate have higher antioxidant activity than German, Swiss chocolate and blackberry. Mexican chocolate scavenges with higher affinity OH. > O2.‐> ONOO‐ > HOCl, whereas, vanilla is a better scavenger of H2O2> OH. > HOCl. Nopal contains approximately 280 mg of vitamin C/100 g of serving, higher than guavas and orange juice, however the content of Vitamin C as well as polyphenolic content decreases as the cooking time increases. Interestingly steamed nopal contains more polyphenols than raw nopal. Finally antioxidant activity in vanilla is transferred to the human plasma after 1 hour of consumption. Thus, these results suggest that the consumption of Mexican traditional foods can help to maintain the equilibrium between reactive species formation and the antioxidant capacity produced by the consumption of foods rich in antioxidants.
Resistant Starch and Lactobacillus Feeding Improve Metabolic Functions in Diet‐Induced Obese Mice
Authors: Barouei Javad , Mishchuk Darya , Kieffer Dorothy , Martinic Alice , Martin Roy , Slupsky Carolyn , Marco Maria
Published: June 2021

Intestinal microbes have important roles in the regulation of energy homeostasis, lipid and glucose metabolism, and immune system responses. Dysbiotic gut microbiota is associated with obesity, type 2 diabetes mellitus, and metabolic syndrome. Therefore, gut microbiota modulations by fermentable carbohydrates and/or probiotics might protect against these chronic diseases. In this study, male C57BL/6J mice received a high‐fat (HF) diet (45.9% energy from fat) for 9 weeks. Mice were then provided with either a HF diet modified to contain 20% (w/w) type 2 high‐amylose maize resistant starch (HF‐HAMRS) or remained on the control HF diet for another 6 weeks. During that time, half of the mice fed either HF‐HAMRS or HF received supplements of 10^9 cells of Lactobacillus plantarum WCFS1 (Lp) every other day (n=10/grp). HAMRS‐fed mice exhibited enhanced indicators of intestinal fermentation (cecum tissue and content weights and cecal proglucagon gene expression), increased serum adiponectin levels, and sensitized cecal and ileal responses to microbiota changes as indicated by increased expression of TLR genes and ileal antimicrobial Reg3g gene. Lp‐fed mice displayed significantly increased hepatic Cpt1a gene expression relative to HF controls, indicating increased fatty acid β‐oxidation. Combining HAMRS and Lp significantly enhanced gene expression of cecal tight junction protein occludin relative to HF control. These outcomes provide a rationale for developing nutritional strategies that prevent or treat obesity and its associated metabolic disorders. Funding: American Diabetes Association.
Integrin Crosstalk Regulation of Human Neutrophils Adhered to Fibronectin and Beta‐glucan
Authors: Johnson Courtney , Byrd Angel , O'Brien Xian , Parisi Valentina , Reichner Jonathan
Published: June 2021

Complement Receptor 3 (CR3), an integrin found on neutrophils, plays an important role in fungal recognition. Co‐ligation of CR3 with fibronectin (Fn) at the I‐domain and β‐glucan (B) at the lectin‐like domain is possible due to spatially distinct locations. Dual ligation of CR3 with Fn+B induces homotypic aggregation of primed neutrophils, a response representative of the immune cells encounter with fungi within tissues, and can be blocked by anti‐CR3 antibody. Previous findings have shown that CR3‐mediated DNA Neutrophillic Extracellular Traps (NETS) coincide with PMN aggregates. To understand the molecular mechanisms of how CR3 coordinates these neutrophil responses, we explored the role of the integrins CR3, VLA3, and VLA5. Using antibody blockade and receptor‐FRET, we unexpectedly discovered a required CR3‐mediated role for VLA3, the canonical laminin receptor, in aggregate formation. The role of VLA3 was specific as blockade of other β1 integrin family members failed to prevent aggregation including VLA5, the canonical Fn receptor. Exploring the role of VLA5 using an activating anti‐VLA5 antibody, we showed active inhibition of VLA5 is necessary for aggregate formation. In assessing NETs formation, we discovered that unlike aggregate formation, VLA5 and CR3 are necessary for NET formation, where VLA3 is not. These results have identified a novel integrin crosstalk pathway in which CR3 serves as a master regulator of VLA3 and VLA5 in response to fungal infections and increased our understanding of the complex regulation of integrins. NIH GM‐066194
Scenario Based Learning for Cardiovascular Pharmacology: Explicit Development of Strategies to Solve Complex Clinical and Scientific Scenarios
Authors: White Paul , Naidu Somaiya
Published: June 2021

In order to solve complex patient scenarios in hypertension and heart failure pharmacology, students must have i) a deep understanding of complex pathophysiology principles, “knowledge”; developed critical thinking skills to deconstruct the scenario, “critical thinking”; and iii) creative thinking skills to generate and test an hypothesis that fits the scenario well “creative thinking”. Many students fail to cope with novel scenarios as they lack skills ii and iii. A staged teaching design was implemented in which students discovered principles in early classes, then tackled scenarios with increasing complexity and resemblance to real life, and then reflected on their approach to developing critical thinking and hypothesis testing skills. Two self‐selecting sub‐cohorts were compared in two units of study: those who prepared and engaged (PE, n=94) for classes based on online analytics and attendance measures, and those who were not (NPE, n=60). The two sub‐cohorts were equivalent in that was no difference between PE and NPE cohorts in average scores from the previous semester across all units of study (mean 70 vs 67, t‐test, P>0.05). PE students improved in knowledge scores from first scenario to last by 18%, their critical thinking scores improved by 24% and creative thinking by 49%. PE students performed significantly better than NPE students on all three measures (ANOVA, p<0.001), and survey responses indicated that PE students developed effective strategies to solve clinical scenarios. This study indicates that a staged, scenario‐based learning approach can help students develop skills that will allow them to deal with novel complex scenarios in cardiovascular pharmacology.
Phosphatidylethanolamine‐Binding Protein Regulates Mu Opioid Receptor Induced βarrestin2 Recruitment and Downstream Signaling
Authors: Edwards Katie , LaVigne Justin , Pangilinan Kathlene , Streicher John
Published: June 2021

The signaling regulators and pathways of the mu‐opioid receptor (MOR) are being studied to develop functionally selective MOR ligands. Such novel analgesic drugs may alleviate pain without the side effects and drawbacks of typical opioids. We have analyzed proteomics databases available in the literature to find candidate proteins that are regulated in the brain by chronic opioid treatment. One such candidate, phosphatidylethanolamine‐binding protein (PEBP), is a cytoplasmic protein with a highly conserved structure that has been shown to regulate GPCR signaling through both the Raf/MEK/ERK pathway and G protein‐coupled receptor kinase 2 (GRK2), a major feedback inhibitor of GPCRs which has been implicated in a number of neurological disorders including chronic pain. The expression of PEBP was also found to be regulated by chronic morphine treatment in a proteomic study of rodent brain, suggesting that PEBP may regulate MOR signaling, however PEBP has not yet been associated with MOR signaling. We have knocked down PEBP in MOR‐ expressing HEK, U2OS, N2a and SH‐SY5Y cells using specific siRNA to determine changes in either MOR‐induced ERK signaling or βarrestin2 (βarr2) recruitment and receptor internalization, with and without GRK2 overexpression. PEBP knockdown does not change MOR agonist induced ERK signaling efficacy or potency, but βarr2 recruitment, MOR internalization, and chronic morphine desensitization potency is increased. This suggests a role for PEBP in regulating MOR induced βarr2 recruitment and attendant opioid induced side effects. We will continue to study the role of PEBP in pain and opioid response in vivo, and are developing in vitro drug discovery screens with PEBP as a target.
Superoxide Dismutase 1 Regulation of CXCR4‐Mediated Signaling in Prostate Cancer Cells is Dependent on Cellular Oxidative State
Authors: Dupre Denis , Wertman Jaime , Purcell Chad , Young Brent , Kuang Yi‐Qun , Charette Nicholle
Published: June 2021

CXCL12, acting via its G protein‐coupled receptor CXCR4, is a chemoattractant for a broad range of cell types, including several types of cancer cells. Indeed, elevated expression of CXCR4, and its ligand CXCL12 play important roles in promoting cancer metastasis. Cancer cells have the potential for rapid and unlimited growth in a restricted blood supply; oxidative stress is a common feature of several tumors. Superoxide dismutase (SOD) is a critical enzyme responsible for regulation of superoxide radicals. Recent studies have reported enhanced expression of SOD may increase the aggressive and invasive potential of malignant cells in some cancers. Here, we report a direct interaction between SOD1 and CXCR4. Given the important roles of these two proteins in cancer progression and metastasis, we characterized the effects of SOD1 on CXCR4 signal transduction. We showed that SOD1 interacts directly with the first intracellular loop of CXCR4. We demonstrated that CXCL12/CXCR4‐mediated modulation of ERK, AKT, apoptosis and cell migration in prostate cancer cells is different under normal versus hypoxic conditions when SOD1 is present. Finally, we identified a potential regulatory mechanism by which SOD1 directly interferes with CXCR4 function, regulating G protein coupling to CXCR4, and promoting a relative switch in G protein coupling from Gi to Gq depending on environmental conditions. This study highlights a potential new regulatory mechanism by which a sensor of the oxidative environment can directly regulate signal transduction of a receptor involved in cancer cell survival and migration. Supported the Dalhousie Medical Research Foundation and Natural Sciences and Engineering Research Council of Canada.
Comparison of Real‐Time Impedance Profiles of HepaRG Cells and Primary Human Hepatocytes to Identify Biliary Toxicants
Authors: Ciurlionis Rita , Liguori M , Blomme E
Published: June 2021

Drug‐induced biliary toxicity is difficult to monitor in vivo and detect in vitro and thus remains a confounding issue in drug development. HepaRG cells are an immortalized cell line derived from progenitor cells that differentiate into a mixed population of hepatocyte‐like and cholangiocyte‐like (biliary) cells. Leveraging the unique dual biliary/hepatocellular nature of HepaRG cells, we evaluated changes in cellular impedance as an endpoint for distinguishing compounds associated with biliary toxicity. Cryopreserved HepaRG cells (Biopredic) were plated at 30K/well on electrode plates, allowed to mature over 6 days, and treated with various concentrations of prototypical biliary toxicants representing diverse mechanisms (ANIT, cyclosporine, chlorpromazine, carmustine, Bosentan, MDA), known hepatocellular toxicants (acetaminophen (APAP), cadmium chloride) and a benign control compound (penicillin). Cells were monitored for 72 hours on the ACEA xCELLigence real‐time cellular impedance instrument. In parallel, the same treatments were conducted with cryopreserved human hepatocytes (KalyCell). A comparison of profiles showed a greater dose‐dependent decrease in impedance in primary human hepatocytes vs HepaRG regardless of the toxicant class with all but one compound, APAP. Data suggest a reduced basal GSH level in HepaRG cells compared with primary hepatocytes may make these cells more susceptible to APAP toxicity. Based on the conditions and endpoints employed in this study, HepaRG cells provide no clear advantage for either the identification or the mechanistic differentiation of compounds inducing biliary toxicity, but may be more useful for monitoring certain types of hepatotoxicity, particularly those associated with reactive metabolites.
CTD Code: a Combinatorial Code for Eukaryotic Transcription
Authors: Zhang Yan , Mayfiled Joshua , Zhang Mengmeng , Etzkorn Felicia
Published: June 2021

In eukaryotes, the C‐terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as transcription template through various post‐translational modifications on its heptad repeat sequences. Recently, novel modifications on new regulatory sites of CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on two well‐characterized modification of CTD, namely serine phosphorylation and prolyl isomerization, and discuss the interplay between the enzymes regulating these modification states. To address this question, we developed chemical compounds that can be used to mimic the cis and trans isomer states of proline residues in the CTD and therefore probe the selectivity of CTD binding proteins that are sensitive for this subtle variation. Our results established that the selectivity of prolyl isomerization state of CTD by phosphatases results in different regulatory effects and can lead to differentiated CTD phosphorylation states and eventually, transcription outcome. In particular, the phosphatases that specially recognize the minor species of cis proline will display enhanced activity upon prolyl isomerase regulation which is essential for accurate transcription termination. The application of such chemical probes can help us understand the molecular mechanism of the interplay between different post‐translational modifications with prolyl isomerization state and how that affects the conformational status of CTD in transcription.