bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimating the cost of scaling up direct nutrition interventions at national and subnational levels: insights from India (632.15)
Authors: McDonald Christine , Menon Purnima , Chakrabarti Suman
Published: June 2021

Background: Maternal and child undernutrition in India is high. National programs are designed to provide recommended direct nutrition interventions, but current coverage is low and little is known about costs of closing gaps. Costs of scaling up nutrition are calculated at the global level, but they do not capture national policy guidelines, delivery platforms or local unit costs.

Objective: To estimate national and state‐level costs of delivering direct nutrition interventions in India and identify local costing needs.

Methods: Interventions included for implementation by government through local delivery platforms were costed. Size of target populations (TP) for each intervention were estimated using national data. Unit costs (UC) were derived or obtained from programmatic data & other sources. Cost of delivering an intervention at 100% coverage = (UC*projected TP).

Results: Over US$ 5 billion/year is required to deliver chosen interventions in India. Cost estimates are constrained by limited local data on UC rather than data on TP. Differences between government budget & expenditures (B&E) and intervention‐based cost estimates are difficult to calculate because B&E are often not reported by intervention.

Conclusion: There is an urgent need for local cost studies on delivery of high‐impact interventions to ensure accurate national and sub‐national nutrition budgeting in India and in other countries.

Grant Funding Source: Supported by: Bill & Melinda Gates Foundation, through POSHAN, led by IFPRI.
Average infant formula and breastmilk intake among WIC infants reflects food package changes (632.9)
Authors: Reat Amanda , Crixell Sylvia , Von Bank Julia , Thornton Hannah , Friedman B.J.
Published: June 2021

The Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) provides food vouchers in “packages” to low‐income, at risk participants. Prior to 2009, food packages for all infants included vouchers for 806 ounces/month of infant formula, regardless of feeding mode, a potential barrier to breastfeeding. In 2009, the WIC packages were modified to improve nutrient intake and to address the historically low rates of breastfeeding among WIC mothers. Formula is now allocated based on feeding mode, with more formula provided to fully formula fed (FF), less to partially formula fed (PF), and none to fully breastfed (BF) infants. The intent of this study was to determine whether formula and breastmilk intake of infants participating in a central Texas WIC clinic reflected this new allocation of formula. Using ANOVA and Tukey’s post hoc test, we analyzed 24‐hour recall data collected from 82 WIC caregivers of infants 4‐12 months using Nutrition Data System for Research. Among infants 4‐6 months, PF and BF infants consumed significantly less formula and more breastmilk than their FF counterparts (P<0.05); BF infants also consumed significantly more breastmilk than PF infants (P<0.05). Among infants 6‐12 months, fully BF consumed significantly less formula than FF infants (P<0.001); the difference in formula intake between PF and BF infants approached significance (P=0.054). Intake of breastmilk was significantly different among all groups; BF infants consumed more breastmilk than PF (P<0.001) and FF infants (P<0.001); PF infants consumed more than FF infants (P=0.001). These results suggest that infants receiving less formula from WIC packages actually consumed less formula and more breastmilk, in accordance with the intent of the policy change.

Grant Funding Source: Texas State University Research Enhancement Program
Enhanced aerobic exercise performance in women by a combination of three mineral chelates plus two conditionally essential nutrients (634.7)
Authors: DiSilvestro Robert
Published: June 2021

Active, young adult women often eat less than optimal levels of iron, zinc and copper, a situation not always corrected by use of multi‐vitamin‐mineral supplements (which often omit iron and often only include zinc and copper in non‐ideal forms). Similarly, intake of carnitine and phosphatidylserine, two conditionally essential nutrients (CENs), may fall below what maximally enhances aerobic energy metabolism. Therefore, a study was done where aerobically fit, young adult, non‐anemic women consumed one of the following for about a month: placebo, glycinate complexes of the 3 minerals + the CENs, or generic versions of the 3 minerals + the CENs. Before and after the treatment, subjects were tested for time in a 3 mile run, followed shortly by distance covered in 25 minutes of stationary biking, followed shortly by performance in a 90 second step test. The mineral glycinates + CENs produced substantial improvements in all three tests (ie mean run time decrease of 54 seconds). The placebo had a significant effect only on the biking distance, though it was less than the mineral glycinates + CENs. The generic minerals + CENs only significantly affected the step test. In summary, a combination of certain mineral complexes plus two non‐essential organic nutrients improved aerobic exercise performance in fit young adult women.

Grant Funding Source: Gatorade Institutue
Iodized salt benefits children school readiness: preliminary results from a cluster randomized trial in Ethiopia (635.2)
Authors: Bougma Karim , Marquis Grace , Aboud Frances , Melka Tizita , Samuel Aregash
Published: June 2021

Iodine deficiency is thought to contribute to delayed mental development. The effectiveness of iodized salt programs in reducing this delay has not been studied in children 6 years and under. Using a randomized cluster design, 1835 children 54 to 60 mo old were sampled from 60 villages in the Amhara region of Ethiopia. The iodized salt and control groups were comparable on all baseline variables, including maternal schooling (89.6% had none) and child dietary diversity (mean: 2.2 out of 7 food categories). Once national production of iodized salt began, the project facilitated early delivery to intervention village marketplaces; iodized salt arrived to control village marketplaces approximately 6 months later. The same children (92% of the original sample) were re‐assessed at 69 to 78 mo of age. Intervention group children had a significantly higher score on the 25‐item school readiness test (mean: 8.3 vs 7.8, respectively, p<0.05). No significant differences were found on the two Wechsler tests of reasoning, analyzing raw or standardized scores (0‐19); standardized mean values were very low: Matrix Reasoning (4.0) and Similarities (5.5). In conclusion, along with the exposure to iodized salt, the school readiness skills of 5‐ and 6‐year‐old children benefited possibly because they had exposure to older siblings' literacy and math skills but not to cognitive and verbal reasoning.

Grant Funding Source: Supported by Micronutrient Initiative, Canada
In vivo and in vitro evidence that leptin stimulates the hexosamine biosynthetic pathway (640.9)
Authors: Zimmerman Arthur , Harris Ruth
Published: June 2021

We previously reported that glucosamine administration, which activates the hexosamine biosynthetic pathway (HBP), causes leptin resistance in rats. Here we tested whether nutrient activation of the HBP results in leptin resistance. Male Sprague‐Dawley rats received intraperitoneal infusions of saline or leptin (40 μg/day) for 12 days and were fed chow or a choice diet of chow, 30% sucrose solution, and lard. The choice diet caused leptin resistance and increased hepatic glutamine fructose 6‐phosphate amidotransferase (GFAT), a rate limiting enzyme in the HBP, and O‐linked N‐acetylglucosamine (O‐GlcNAc) protein modification. Leptin infusion increased GFAT and O‐GlcNAcylation in chow‐fed rats, whereas it had no additive effect in choice rats. To test for direct effects of leptin and glucose, HepG2 cells were incubated with high concentrations of glucose (18.5 mM) or with 5.5 mM glucose and leptin (0, 5, 10, 50, 100 or 500 ng/mL). Both leptin and glucose stimulated cellular GFAT and protein O‐GlcNAcylation. Thus, leptin stimulates the HBP both in vivo and in vitro, and activation of the HBP due to increased nutrient flux is associated with leptin resistance.

Grant Funding Source: Supported by NIH grant DK 53903
Endoplasmic reticulum stress signaling through C/ebp homologous protein is required for adaptation to a high‐fat diet (641.15)
Authors: Grant Ryan , Youm Yun Hee , Ravussin Anthony , Albarado Diana , Dixit Vishwa
Published: June 2021

Endoplasmic reticulum (ER) stress leads to activation of the NLRP3 inflammasome and cleavage of pro‐caspase‐1 to its active form, resulting in the secretion of IL‐1B and IL‐18. C/ebp homologous protein (CHOP) is an ER stress signaling protein that amplifies IL‐1B secretion. We hypothesized that CHOP‐mediated ER stress signaling links adipose tissue leukocytosis with metabolic dysfunction during obesity. Male C57BL/6 and CHOP‐/‐ mice were fed a 60% high‐fat for 5 months. CHOP‐deficient mice had increased body weight and body fat, which was explained by a small reduction in energy expenditure. Contrary to our hypothesis, CHOP‐/‐ mice displayed a large increase (P<0.05) of adipose tissue B cells, T cells, and macrophages. In accordance with increased adipose tissue inflammation CHOP‐/‐ mice had decreased glucose and insulin tolerance, which was accompanied by an increase in hepatic steatosis. These results indicate that during obesity CHOP reduces recruitment of leukocytes to adipose tissue, which may be related to its role in promoting apoptosis in response to cell stressors. This work was funded by the Pennington Foundation and RWG is supported by NIH T32 DK064584.
d‐δ‐Tocotrienol suppresses the differentiation of murine 3T3‐F442A preadipocytes (641.8)
Authors: Mo Huanbiao , Yeganehjoo Hoda , Torabi Sheida
Published: June 2021

Suppression of adipogenesis is one of the potential approaches in obesity prevention. The statins, competitive inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, suppress the differentiation of adipocytes via mevalonate deprivation. We hypothesize that d‐δ‐tocotrienol, a vitamin E molecule shown to accelerate the degradation of HMG CoA reductase, suppresses adipocyte differentiation and adipogenic gene expression. Adipo‐Red assay and oil Red O staining showed that an 8‐d incubation with 2.5 ‐ 10 μmol/L d‐δ‐tocotrienol dose‐dependently reduced the intracellular triglyceride content of murine 3T3‐F442A adipocytes, reminiscent of the lovastatin (1.25 μmol/L) effect. Concomitantly, d‐δ‐tocotrienol dose‐dependently inhibited glucose uptake by 3T3‐F442A cells and the expression of GluT4, pAKT, and HMG CoA reductase proteins. Rosiglitazone (1 µmol/L), an agonist of peroxisome proliferator activator receptor‐activated receptor γ ‐ a key mediator of adipocyte differentiation ‐ reversed the effects of d‐δ‐tocotrienol on cellular lipid content and glucose uptake but not those of lovastatin. In contrast, mevalonate (100 µmol/L) reversed the effects of lovastatin. d‐α‐Tocopherol at 10 µmol/L did not have any impact on 3T3‐F442A differentiation. Trypan blue staining showed no changes in cell viability following 48‐h incubation of 3T3‐F442A cells with d‐δ‐tocotrienol (0‐80 µmol/L), suggesting that the adipogenesis‐suppressive activity of d‐δ‐tocotrienol was independent of cytotoxicity. Dietary d‐δ‐tocotrienol may have potential as anti‐adipogenesis compounds.

Grant Funding Source: Supported by Texas Department of Agriculture and TWU Research Enhancement Program
Mechanistic study for non‐oxidative anticancer activity of epigallocatechin‐3‐gallate in MCF‐7 human breast cancer cells (644.6)
Authors: Ono Misaki , Takeshima Mikako , Higuchi Takako , Chen Chen , Nakano Shuji
Published: June 2021

Despite its strong antioxidant activity, epigallocatechin‐3‐gallate (EGCG), a green tea polyphenol, induces growth inhibition and apoptosis in many human tumor cells through oxidative stress by producing H2O2. Because these cellular events were reversed by exogenous catalase, H2O2 has been considered as the main cause of the cytotoxicity of EGCG. However, its cytotoxicity was not fully reversed by catalase. We investigated such non‐oxidative anticancer mechanisms of EGCG using MCF‐7 human breast cancer cells. EGCG showed dose‐dependent cytotoxicity, with pronounced membrane alterations characterized by bleb. Co‐treatment with catalase drastically reduced the cytotoxicity from 95% to 48%. To determine non‐oxidative anticancer activity of EGCG, cells were treated with 50 µM EGCG in the presence of catalase, and cell cycle progression and signaling proteins for growth and apoptosis were assessed. EGCG arrested cells in G1/S phase and increased apoptotic cell population. Upon treatment with EGCG, Erk and Akt were inactivated with subsequent reductions of activities of downstream mTOR and S6K1. Anti‐apoptotic Bcl‐2 was reduced while pro‐apoptotic Bax increased. These results suggest that non‐oxidative anticancer activities of EGCG were mediated by G1/S arrest and apoptosis through ablation of signal transduction involving Erk, Akt, mTOR pathways as well as suppression of Bcl‐2 and enhancement of Bax.
Separation of α‐retinyl palmitate and quantification of vitamin A after consumption of carrots containing α‐carotene (645.24)
Authors: Goetz Hilary , Kopec Rachel , Riedl Ken , Cooperstone Jessica , Narayanasamy Sureshbabu , Curley Robert , Harrison Earl , Schwartz Steven
Published: June 2021

Vitamin A (VA) is vital for proper growth, vision, and immune function. Provitamin A carotenoids are an important source of VA for humans, yet the bioequivalence of VA from nonsymmetrical provitamin A carotenoids is poorly characterized. In humans, α‐carotene (αC) is cleaved to VA and inactive α‐retinol (αR) ‐ both of which are esterified with palmitate (P). Analytical methods are needed to separate VA esters from αRP to avoid overestimation of the VA bioequivalence of αC containing foods. To accurately assess the VA obtained from an αC‐rich meal, an αRP standard was synthesized from α‐ionone following adapted methods based on the Wittig‐Horner reaction. A high performance liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed to resolve αRP from VA esters. Separation was confirmed by MS, where parent ester m/z and differing intensities of daughter ions allowed for distinction of αRP from VA esters. The developed LC‐MS/MS method was applied to the analysis of post‐prandial triglyceride rich lipoprotein fraction of plasma from healthy humans after consumption of carrots and αRP and VA esters were individually quantified. These results provide important insights about the fate of αR in humans consuming αC‐rich foods and the bioequivalence of VA from commonly consumed nonsymmetrical provitamin A carotenoids.

Grant Funding Source: Supported by The Ohio State University Food Innovation Center
A role for BCMO1 beyond carotenoid metabolism: regulation of androgen status and signaling (645.4)
Authors: Smith Joshua , Ford NIkki , Clinton Steven , Erdman John
Published: June 2021

In human studies, higher serum lycopene (Lyc) levels are inversely associated with risk of prostate cancer (PCa), which is driven by androgens. Genomic alterations in carotene‐15,15’‐monooxygenase (BCMO1), which cleaves provitamin A carotenoids to yield retinal, have been shown to alter Lyc accumulation. We hypothesized that Lyc and tomato powder (TP) feeding may impact the prostate by altering testosterone (T) action and that BCMO1 may modulate this process. Nine‐ to fourteen‐week‐old male WT and Bcmo1‐/‐ mice were fed AIN‐93G‐based control or diets containing TP or Lyc for four days. Surprisingly, we found that independent of Lyc or TP feeding, loss of Bcmo1 decreased testicular esterified cholesterol (p<0.05) and testicular mRNA expression of 17β‐hydroxysteroid dehydrogenase 3 (p<0.001), which converts androstenedione to T. In parallel, Bcmo1‐/‐ mice show reduced serum T and reduced androgen receptor‐mediated gene expression in the prostate. Regardless of diet, the weight of the prostate and the seminal vesicles was correlated (R2=0.12, p=0.0015, and R2=0.33, p<0.0001, respectively) with logarithmic concentrations of serum T, in agreement with published androgen‐driven growth kinetics. In summary, BCMO1 seems to play a significant role in androgen and prostate physiology independent of its function in carotenoid metabolism, highlighting a potential role in PCa.

Grant Funding Source: Supported by NIH grant PHS‐1‐RO1 CA125384
Flavonoids from rooibos tea promote cell death in an osteosarcoma cell line (647.10)
Authors: Nash Leslie , Ward Wendy
Published: June 2021

The Saos2 cell culture system is a well characterized osteoblast‐like cell line used to assess the effects of dietary components such as flavonoids on the production of bone mineral. Saos2 cells are derived from human osteosarcoma and possess desirable characteristics for assessing molecular interactions and mechanistic routes in which nutrients may influence mineral production. While screening rooibos tea flavonoids for potential positive effects on bone mineralization, several flavonoids reduced mineralization, while exhibiting cellular death. Mineral, measured using an Alizarin red assay, was reduced in cells treated with isoquercetin (蠅10uM, p<0.001) vitexin (蠅5uM, p<0.05) and luteolin (蠅15uM, p<0.001) in comparison to controls. Metabolic activity of the mitochondrial reductase was analyzed over 72 hours as a marker for cell viability by measuring the reduction of a tetrazolium dye to a formazan salt. The addition of isoquercetin (p<0.001), vitexin (p<0.001) or luteolin (p<0.001) was shown to attenuate mitochondrial activity in a concentration dependent manner, and was in agreement with the mineralization data. Human osteosarcoma is known for its ability to resist chemotherapy. Thus, further investigation of these compounds may help develop natural strategies that enhance chemotherapeutic agents in addition to reducing toxic side effects.

Grant Funding Source: Supported by a NSERC Discovery Grant to W. Ward
Synergy of bioactives from camelina seed meal for health promotion (647.16)
Authors: Das Nilanjan , Berhow Mark , Jeffery Elizabeth
Published: June 2021

Camelina sativa L. Crantz is under development as a novel oil seed crop yet there are few studies about the bioefficacy of camelina. Camelina seed meal contains several potentially bioactive compounds: two major aliphatic GSLs, glucoarabin (9‐(methylsulfinyl)nonylglucosinolate; GSL9), glucocamelinin (10‐(methylsulfinyl)decylglucosinolate; GSL10), with trace amount of a third, 11(methylsulfinyl)undecylglucosinolate (GSL11) and several flavonoids, mostly as quercetin glycosides. We tested hydrolyzed glucosinolates (hGSL) and flavonoids quercetin and rutin for their ability to upregulate detoxification enzymes, as biomarkers of anti‐cancer activity. None of these compounds either caused induction of CYP1A1 activity, or inhibition of β‐naphthoflavone‐induced CYP1A1 activity. In contrast, hGSL9 and hGSL10, quercetin and rutin alone and in combination all caused induction of quinone reductase (NQO1) activity. We found synergistic induction of NQO1 activity when Hepa1c1c7 cells were treated with a combination of all the four major bioactives in the proportions present in camelina seed extract. Although potency of hGSL9 was somewhat less than that of sulforaphane, we found that GSL9 and quercetin appear responsible for this synergy. As Camelina seed extract possesses NQO1 inducing activity, it should be evaluated for anticancer activity, similar to other Brassicaceae family members, such as Broccoli.

Grant Funding Source: Supported by USDA/NIFA #AG 2010‐65200
Cytotoxic activity of gypenosides and gynogenin from Gynostemma pentaphyllum against A549 cells (647.25)
Authors: Piao Xiang‐Lan , Chen Dao‐Jin , Xing Shao‐Fang , Kim Sung Woo , Piao Xiangshu
Published: June 2021

Gynostemma pentaphyllum (Thunb.) Makino is one of the well‐known traditional Chinese herbal medicines, which has been demonstrated to be effective against chronic diseases, such as lung cancer. In the present study, seven dammarane‐type compounds were isolated from the raw, heat processed and alkaline hydrolyzed G. Pentaphyllum and compared for their cytotoxic activities against non‐small cell lung carcinoma A549 cells using MTT cytotoxicity assay. By means of chemical and spectroscopic methods, their structures were established as gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin A, damulin B and 20S‐dammar‐24‐en‐2α, 3β, 12β, 20‐tetrol. Gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin A, damulin B and 20S‐dammar‐24‐en‐2α,3β,12β,20‐tetrol all showed strong cytotoxic activity against A549 cells, with the IC50 values of 105.89±2.48, 52.63±8.31, 34.94±4.23, 50.96±9.55, 26.98±0.51, 4.56±0.58, 12.54±0.53 μg/ml respectively, whereas the IC50 value of 20S‐Rg3 (positive control) was 55.36±3.20 μg/ml. Comparing their structures with cytotoxic activity, these compounds might possess some kind of structure‐activity relationship. Based on the structure and cytotoxic activity relationships of these seven constituents, the OH group in C‐2, the double bond in C20(21) and C20(22) positions, the connected sugar number and the configuration in C‐20 were important for cytotoxic activity against A549 cells.
Hepatocyte‐specific γ‐catenin deletion lacks an overt phenotype (649.6)
Authors: Zhou Lili , Shou Weinian , Monga Satdarshan (Paul) Singh
Published: June 2021

We have previously shown that β‐catenin loss in hepatocytes led to an increase in γ‐catenin at the adherens junctions (AJ) where γ‐catenin complexed with E‐cadherin in the absence of β‐catenin. γ‐Catenin (also known as Plakoglobin), is normally a major component of the desmosomes and its role in liver biology remains unexplored. Intriguingly both tumor suppressive as well as tumor promoter roles of γ‐catenin have been suggested in hepatocellular cancer (HCC). In the current study we conditionally deleted γ‐catenin in hepatocytes by interbreeding albumin‐cre and γ‐catenin floxed mice. We successfully deleted γ‐catenin genetically and at the protein level in mouse hepatocytes using this strategy in the γ‐catenin knockout mice (KO). These mice lacked any overt phenotype up to 3 months of age so far. However, male KO displayed larger size of liver that appeared softer in texture, although no change in liver weight/body weight ratio was evident. No change in basal cell proliferation, apoptosis or fibrosis was evident in KO mice. Additional histological analysis of KO was unremarkable. There was also no obvious difference in serum biochemistry between the WT and KO mice. Molecular analysis using whole cell lysates reveals comparable levels of E‐cadherin but showed marginally increased β‐Catenin. There were some changes in levels of key desmosomal proteins. Thus γ‐catenin loss in hepatocytes appears to be well‐tolerated in an unchallenged mice.
Primary human cardiomyocyte culture: a model for assessment of cardiovascular disorders (652.12)
Authors: Collins Atijah , Harper Emily , Braimah Ayemoba , Sharma Avadhesh
Published: June 2021

The purpose of this research is to establish an in vitro cardiac system using primary human cardiomyocyte cultures (PHCC). We hypothesized that norepinephrine (NE) and endothelin‐1 (ET‐1) will contribute to human cardiomyocyte growth. PHCC were obtained from Celprogen, San Pedro, CA and grown with varied concentrations of NE or ET‐1 for 72h. Protein expressions of contractile proteins were analyzed via Immunoblot and immunocytochemistry. Analysis of confocal images showed a significant decrease in nuclear area in all NE treatment groups. NE (10 µmol) stimulated significant increases in fluorescent intensities of the contractile proteins, f‐actin, troponin and tropomyosin. The protein expression of myosin light chain was significantly lower with increasing concentrations of NE. In addition, the average cell area increased in all groups of ET‐1. ET‐1 (1, 10, 100 nmol) exhibited a significant decrease of fluorescent intensities of f‐actin, troponin and tropomyosin. PHCC were also grown in the presence of LPS (1, 10, 100µg/ml) in a 6‐well plate for 72h. Viability data showed the cells recovered from the endotoxin treatment and maintain steady growth in each concentration of LPS. In contrast to NE, ET‐1 exhibited a decrease on the expression of contractile proteins in PHCC.

Grant Funding Source: Office of Chief Science Officer, PCOM
Role of autophagy related protein 5 induction by globular adiponectin in the suppression of ethanol‐induced apoptosis in liver cells (653.16)
Authors: Nepal Saroj , Kim Mijin , Shrestha Anup , TiliJa pun Nirmala , Park Pil‐Hoon
Published: June 2021

Hepatocellular apoptosis, an early response of liver cells to hepatic injury on chronic alcohol consumption, has been an area of active research for the treatment of ALD. Adiponectin has been shown to protect liver against chronic ethanol consumption. We have previously demonstrated that ethanol and gAcrp affect expression of genes related with autophagy in liver cells, and further reported the potential utility of autophagy activation by adiponectin in the suppression of ethanol‐induced apoptosis. Herein, we aim to find out whether adiponectin induces autophagy related protein 5 (Atg5), an essential Atg for the elongation of autophagosomal membrane, and if so, explore the role of Atg5 in the suppression of ethanol‐induced apoptosis in liver cells.

In the present study, ethanol treatment suppressed Atg5 expression in both primary rat hepatocytes and HepG2 cells while globular adiponectin significantly increased Atg5 expression. Furthermore, pretreatment with globular adiponectin significantly up regulated ethanol‐suppressed Atg5 expression in both primary rat hepatocytes and HepG2 cells.

Our initial findings suggest that gAcrp induces Atg5 expression in liver cells that might play a role in the suppression of ethanol‐induced apoptosis.
Experiment‐guided virtual screening provides new high affinity ligands for the benzodiazepine site on GABA‐A receptors (654.4)
Authors: Sigel Erwin , Middendorp Simon , Baur Roland , Puthenkalam Roshan , Ernst Margot
Published: June 2021

Often novel high affinity ligands are required for a well‐established target protein. Reasons include side effects, resistance development to or sub‐optimal pharmacokinetic or pharmacodynamic properties of an established drug. Here we describe the identification of 19 compounds (14 different scaffolds) that are high affinity ligands for the benzodiazepine‐binding site on mammalian GABA‐A receptors, hitherto un‐crystallized proteins. The procedure includes biochemical techniques that may be generally applied to other proteins. Prerequisites are a ligand that can be chemically modified with cysteine‐reactive groups, knowledge on amino acid residues contributing to the binding pocket on the protein and either crystal structures of proteins homologous to the target protein or even better a crystal structure of the target itself. The procedure includes virtual screening that when used without additional techniques often only results in low affinity ligands, even when a crystal structure of the target protein is available. Combination of crystallization with a functional approach may replace the use of crystallization alone in efforts towards finding new high affinity ligands of an established target.

Grant Funding Source: Supported by Swiss National Science Foundation grant 31003A‐132806/1 to E.S.
Functional screening system for GPCR‐agonists using peptide‐secreting yeasts (654.5)
Authors: Shigemori Tomohiro , Kuroda Kouichi , Ueda Mitsuyoshi
Published: June 2021

G‐protein coupled receptors (GPCRs) which feature a seven‐transmembrane structure are involved in various diseases as well as in regulating physiological functions. Phage display has been used for discovering bioactive peptides, but the methodology is never effective for screening GPCR‐agonists because phage‐displayed peptides are screened based only on binding ability and are different from their final soluble form. In this study, we established a functional screening system for GPCR‐activating peptides by combining peptide‐secreting yeasts with GPCRs‐producing mammalian cells. Glucagon‐like peptide‐1 receptor (GLP1R) and its peptide agonist exendin‐4 (Ex4) were used as model molecules. Ex4‐secreting yeast (Ex4‐yeast) was constructed by introducing the Ex4‐encoding gene linked to secretion signal genes of glucoamylase or yeast α‐factor into Saccharomyces cerevisiae. Detection of yeast‐secreted Ex4 was performed by evaluating cAMP production in GLP1R‐producing CHO cells exposed to yeast culture medium incubated in glass tube and microplate. Model screening of Ex4‐yeast from mixture containing Ex4‐ and control‐yeasts (ratio of each cell number, 1:9) was carried out on microplate. The results indicated that the functional screening system with peptide‐secreting yeasts would be promising for efficient discovery of novel peptide‐based GPCR agonists [1]. [1] Shigemori et al. submitted.
Synthesis, characterization and toxicity studies of [Ru2(Aap)4cl]: a diruthenium complex (655.12)
Authors: Naidu Naga , Deng Yuanjian , Wei Xin , Yakubu Momoh
Published: June 2021

Cisplatin has been identified as one of the leading transition metal‐based drug and widely used as anticancer agent. But tumor resistance and development of serious, to overcome these problems we have synthesized a diruthenium complex [Ru2(Aap)4Cl], instrumentally confirm synthesis and evaluate its anti‐proliferative and toxicity effects on A549 cell line and SMC. Aap ligand was synthesized by reaction between 2‐Chloro‐4‐aminopyridine and excess amount of aniline. The Aap was reacted with Ru2(CH3COO)4Cl to yield the Ru2(Aap)4Cl complex. The ligand and the diruthenium complex were analyzed for their thermal, elemental, spectroscopic (NMR (1H & 13C NMR), IR, and MS) and structural properties. Cell proliferation, viability and toxicity were performed using the MTT assay. Spectroscopic and thermal data were consistent with the proposed structures, single X‐ray crystallography of the ligand and the complex further revealed that: I) the 4‐amino group on the pyridine ring was not involved in complex formation; ii) the diruthenium complex was a (4, 0) isomer; and iii) interesting conformational changes were also observed in the Aap ligand after the complex formation. The anti‐proliferative property of the new diruthenium complex was evaluated at 24, 48, and 72 hrs following treatment and showed that the [Ru2(Aap)4Cl] complex reduced cell proliferation by 50% with an IC50 value of 5 ±1 μM for A549 cell line, and 75 ± 5 μM for SMC. These results suggests that the Ru2(Aap)4Cl complex is a promising anticancer agent and needs further studies to fully evaluate the anticancer properties by evaluating the molecular mechanism by which it reduces cellular proliferation.

Grant Funding Source: supported by grants from NHLBI: HL03674 and HL070669.
Epigallocatechin‐3‐gallate sensitizes therapeutic efficacy of doxorubicin involving autophagy inhibition in hepatoma Hep3B cells (655.2)
Authors: Ye Hui‐Lan , Chen Li , Zhang Guo , Chen Xing‐Zhou , Yao Wen‐Min , Zhang Fa‐Can
Published: June 2021

The green tea component (‐)‐Epigallocatechin‐3‐O‐gallate(EGCG), has promisingly been found to enhance the efficacy of several chemotherapy agents in hepatocellular carcinoma (HCC) treatment and its detailed mechanisms remain unclear. Herein, this study aimed to confirm the synergistic interaction of EGCG and DOX on Hep3B cells and to define whether the autophagic flux is involved in this cooperated fashion. Firstly, by MTT and trypan blue assay, both EGCG and DOX exhibited cellular growth inhibition and cell death induction effects. However, by western blotting and real time RT‐PCR, DOX dramatically induced the expression of Atg5 and beclin1, the autophagic signaling molecules, whereas EGCG presented a markedly and dose‐dependently inhibitory role in autophagy signaling. Secondly, EGCG and DOX combined treatment amazingly caused a synergistic effect of about 50% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. Interestingly, the DOX‐induced autophagy was abolished by this combination therapy. Thirdly, the anticancer effect of either DOX or combination with EGCG treatment was substantially impaired by either rapamycin, an autophagic agonist, or Atg5 as well as beclin1 siRNAs. Consistently, significantly tumor growth reduction and apoptosis increment were found in combination therapy compared with DOX alone in vitro study. And immunohistochemisty analysis indicated that the suppressed tendency of autophagic hallmark LC3 expression was consistent with thus combined usage in vitro. Taken together, the current study suggested that EGCG emerges as a chemotherapeutic augmenter and synergistically enhances DOX anticancer effects involving autophagy inhibition in HCC.

Grant Funding Source: Supported by NSFC Grants 881172260, 30630145, 81360077 to G.Z. & 81102495 to L.C.
Nrf2‐Keap1 pathway does not have an important role in cisplatin resistance in neuroblastoma (655.8)
Authors: Tanoshima Reo , Yang MingDong , Dalvi Pooja , Wu Alex , Matsuda Kensuke , Zhang Libo , Fujii Hisaki , Baruchel Sylvain , Harper Patricia , Ito Shinya
Published: June 2021

Drug resistance is one of the major causes of treatment failure in neuroblastoma (NB). Cisplatin is one of the key chemotherapeutic agents for NB. Nuclear factor erythroid 2‐like‐2 (Nrf2) is a transcription factor that regulates anti‐oxidative stress enzymes and drug transporters, negatively regulated by Keap1 (kelch‐like ECH‐associated protein 1). Loss‐of‐function mutations of Keap1 gene and subsequent Nrf2 activation have been shown to be a mechanism of cisplatin resistance in adult cancers. We conducted this study to clarify the role of Nrf2‐Keap1 pathway in cisplatin resistance in NB. We investigated the basal activity of Nrf2 in nine NB cell lines (IMR‐32, LAN5, CHLA‐15, SH‐SY5Y, CHLA‐20, SKN‐FI, SK‐N‐BE(2), BE(2)C, CHLA‐90) using Antioxidant Response Element luciferase reporter assay. No cell lines had high reporter activities. In order to evaluate whether cisplatin activates Nrf2, we analyzed the mRNA expression of Nrf2 and its target genes (Glutamate‐Cysteine Ligase, Modifier Subunit: GCLM and NAD(P)H Dehydrogenase, Quinone 1: NQO1) after 24h treatment with cisplatin. In this experiment, we identified CHLA‐90 as one of the 3 cell lines, which showed high induction of Nrf2 target genes upon cisplatin exposure. However, knockdown of Nrf2 expression by small interfering RNA did not improve MTT assay‐measured dose‐response survival rate of CHLA‐90 with cisplatin treatment.

We conclude that the Nrf2‐Keap1 pathway does not necessarily play an important role in cisplatin resistance in NB cells.

Grant Funding Source: Supporter by CIHR, TCCSG, and Joseph M. West Family Memorial Fund
The apoptotic effect of ramalin on human breast cancer cells (657.11)
Authors: Park Bongkyun , Lee Chunggi , Kim Jiyun , Pyo Suhkneung
Published: June 2021

Ramalin is the compound isolated from Antarctic lichen, Ramalina terebrata. Previous study showed that ramalin has antioxidant and anti‐inflammatory activity. However, the effect of ramalin on MCF‐7 human cancer cells has not been addressed. We investigated the effect of ramalin on cell proliferation, apoptosis, and its mechanism in MCF‐7 cells. MTT assay and western blotting showed that ramalin decreased concentration‐dependently cell proliferation, induced apoptosis related proteins (Bax, Bid, AIF), and activated caspase‐3 and 8, respectively. In addition, Annexin V/PI analysis revealed that apoptotic cells were remarkably increased in ramalin‐treated MCF‐7. Our data also showed that ramalin increased phosphorylation of ERK1/2, p38 and JNK in a concentration‐ and time‐dependent manner. Taken together, ramalin induces apoptosis through Mitogen‐activated protein kinases (MAPKs) and caspase dependent signaling pathways in MCF‐7 cells and may be a potential anticancer agent for the treatment of human breast cancer.
Effects of a heroin vaccine on heroin self‐administration and its pharmacokinetic correlates (658.5)
Authors: Raleigh Michael , Pentel Paul , LeSage Mark
Published: June 2021

Vaccination with a heroin vaccine (M‐KLH) produced a compensatory increase in heroin self‐administration (HSA) at high heroin unit doses, but a decrease in HSA at low unit doses. Vaccination also blocked heroin‐primed reinstatement of HSA responding during extinction. To study the PK correlate of vaccine‐induced increases in HSA at high unit doses, repeated i.v. heroin injections were administered to vaccinated and control rats that simulated heroin exposure during HSA (0.5 mg/kg/hr heroin in control rats and 1.0 mg/kg/hr heroin in vaccinated rats). 6‐monoacetylmorphine (6‐MAM) and morphine, but not heroin, distribution to brain were reduced in vaccinated rats compared to controls. 6‐MAM concentrations in brain were similar between control rats given 0.5 mg/kg and vaccinated rats given 1.0 mg/kg, suggesting that 6‐MAM was the main heroin metabolite mediating compensation of HSA. These data show that vaccination with M‐KLH altered HSA and reinstatement, that 6‐MAM concentrations in brain mediate these effects, and that high antibody to drug ratios are most effective at reducing heroin’s reinforcing effects.

Grant Funding Source: Supported by DA026300, DA030715, and DA07097
Characterization of the kappa opioid receptor‐mediated G protein signaling in mouse striatum using a [35S]GTPγS binding assay (659.6)
Authors: Zhou Lei , Stahl Edward , Lovell Kimberly , Slauson Stephen , Frankowski Kevin , Aube Jeffrey , Bohn Laura
Published: June 2021

The kappa opioid receptor/dynorphin system is an endogenous system involve in modulating stress responses. Disruption of the ability to respond appropriately to stress can lead to elevated risks of developing mood disorders including depression, anxiety, and drug addiction. The dynorphin peptides are products cleaved from prodynorphin of varying amino acid length and are considered the cognate high affinity ligands for the KORs. However, they also promiscuously bind to and activate other opioid receptors in the brain. Here we seek to determine what component of dynorphin signaling can be attributed specifically to KOR by investigating the pharmacological profiles of dynorphin peptides in wild‐type (WT), KOR knockout (KOR‐KO), and mu opioid receptor knockout (MOR‐KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that dynorphin peptides are efficacious in stimulating [35S]GTPγS binding in WT striatum in a manner that resists concentration‐induced plateau. While KOR activation contributes significantly to dynorphin‐induced [35S]GTPγS binding in striatum, our studies identify a significant contribution of MOR‐induced coupling as well. Moreover, we find that KOR displays a constitutive activity in striatum that can be inhibited by antagonist pretreatment; the constitutive activity is not present in KOR‐KO striatum. Recently developed, functionally selective small molecule KOR agonists as well as new selective KOR antagonists were also tested in striata. Our studies in characterization of dynorphin’s effects at the KOR in brain will be essential for developing ex vivo assays that assess the selective contributions of biased KOR ligands that have been recently developed.

Grant Funding Source: Supported by NIDA grant R01 DA031927(LMB, JA)
Tolerance and cross‐tolerance to behavioral effects of phenethylamine‐ and tryptamine‐derived hallucinogens in mice (661.2)
Authors: Smith Douglas , Williams Diarra , Fantegrossi William
Published: June 2021

Tolerance refers to a decrease in effect(s) of a drug due to repeated administration, and develops rapidly to psychoactive effects of some hallucinogens in man. Tolerance to various effects of hallucinogenic drugs can also be readily induced in laboratory rodents, and is presumably mediated by downregulation of serotonin 5‐HT2A receptors. The hallucinogen‐induced head twitch response (HTR) in mice is a reliable rodent model for psychedelic effects because it is mediated by agonist activity at 5‐HT2A receptors, but the role of tolerance in HTR remains somewhat controversial. In these studies, two phenethylamine hallucinogens (2,5‐dimethoxy‐4‐iodoamphetamine [DOI] and 2,5‐dimethoxy‐4‐n‐propylthiophenethylamine [2C‐T‐7]) and two tryptamine hallucinogens (dipropyltryptamine [DPT] and diisopropyltryptamine [DIPT]) were administered to male NIH Swiss mice to determine dose‐effect functions for HTR. The most effective dose of each compound was then selected for repeated administration to assess the development of tolerance to the HTR. Finally, mice received daily treatment with DOI for 3 days, then were substituted with different doses of DOI, 2C‐T‐7, DPT or DIPT the next day to assess surmountability of tolerance and cross‐tolerance among drugs. Repeated administration of the phenethylamines ‐ but not the tryptamines ‐ resulted in rapid tolerance to HTR in mice which was not surmountable with increased dose. Although the tryptamines did not induce tolerance to HTR with repeated administration, cross‐tolerance to HTR elicited by DPT was observed in mice previously treated with DOI, and this cross‐tolerance was not surmountable with increased DPT dose. Explanations for these distinct effects of phenethyltamine‐ and tryptamine‐based hallucinogens may include the involvement of 5‐HT2C receptors (for the phenethylamines) or 5‐HT1A receptors (for the tryptamines.)

Grant Funding Source: Supported by RR020146, RR029884, and by an ASPET SURF award to DS
miR‐125b transcriptionally induced by Nrf2 inhibits AhR repressor for AhR activation (663.12)
Authors: Joo Min Sung , Lee Chan Gyu , Koo Ja Hyun , Kim Sang Geon
Published: June 2021

MicroRNAs (miRNAs) play a role in the cellular defense mechanism. Nrf2 may control cell survival against toxicant challenge. miRNA transcriptionally controlled by Nrf2 had not been characterized. In this study, we explored the miRNAs regulated by Nrf2 and the responsible biological function. Bioinformatic approaches enabled us to extract 6 candidate miRNAs. Of them, only miR‐125b was increased in the kidney of mice treated with oltipraz. Nrf2 overexpression enhanced primary, precursor and mature miR‐125b levels. Functional assays revealed MIR125B1 is a bona fide target gene of Nrf2. Oltipraz treatment protected the kidney from cisplatin toxicity with increase of miR‐125b. Consistently, Nrf2 knockout abrogated an adaptive increase of miR‐125b by cisplatin, augmenting kidney injury. An integrative network of miRNA and mRNA changes enabled us to predict miR‐125b as an inhibitor of AhRR and cell survival. In the experiments using 3′‐UTR reporter, miR‐125b mimic and its inhibitor, miR‐125b inhibited AhRR, activating AhR, which caused the induction of mdm2. Consequently, p53 activation by cisplatin was diminished by either miR‐125b transfection or oltipraz treatment. In summary, miR‐125b is transcriptionally activated by Nrf2 and serves as an inhibitor of AhRR, which contributes to the protection of cell from injury.
Ubiquitination of glycogen phosphorylase by the malin‐laforin complex regulates glycogen catabolism (663.17)
Authors: Dukhande Vikas , Paasch Bradley , Shaikh Nazeer , Gentry Matthew
Published: June 2021

Lafora disease (LD) is fatal progressive myoclonic epilepsy characterized by pathological aberrant glycogen accumulations in brain and various other organs. Autosomal recessive mutations in genes encoding malin or laforin cause LD. Our group identified laforin as a novel glycogen phosphatase essential for maintaining glycogen in a soluble form. Malin, a RING‐type E3 ubiquitin ligase, is known to ubiquitinate proteins involved in glycogen metabolism such as laforin, glycogen synthase, protein targeting to glycogen and glycogen debranching enzyme. However, malin’s role in LD pathogenesis is not clear. In this study, we aimed to discover substrate/s of malin and its function in LD pathogenesis. We used malin‐ and laforin knockout mice, affinity pull‐down and mass spectrometry, immunoprecipitation, western analysis, fluorescence microscopy, glucan‐binding assay, and enzyme activity measurements. We discovered glycogen phosphorylase (GP) as a novel substrate of malin. GP is vital in catabolism of cellular glycogen. The expression and phosphorylation of GP was decreased in tissues from malin knockout mice. Similarly, overexpression of LD mutations of malin in HEK‐293 cells decreased GP expression. Overexpression of malin in HEK‐293 cells increased the endogenous expression of GP. Malin interacted with and ubiquitinated GP, and presence of malin increased nuclear localization of GP. Malin also altered glucan‐binding of GP. Thus, we have identified a novel control mechanism of glycogen catabolism. Further exploration into the mechanisms of this regulation will unravel insights in glycogen metabolism, physiological functions of the malin‐laforin complex and will aid in betterment of LD pharmacotherapy.

Grant Funding Source: Supported by NIH grants R00NS061803, R01NS070899 and University of Kentucky College of Medicine star
Fullerene C60 chemically linked for biocompatible polymers for in vivo radical trapping (663.2)
Authors: Tegegn Cheregn , Megos Welay , Simpson Duane , Volkis Victoria
Published: June 2021

Free radicals can be formed in living tissues under UV irradiation and/or when oxygen interacts with certain bio‐molecules. Once formed these highly reactive particles are capable to start number of chain reactions damaging living tissues and are believed to be a reason for some kinds of cancer. It is thus important to capture and study free radicals in live biological tissues and samples, which is complicated due to the short‐lived life span of free radicals. Mostly indirect methods focus on studying tissues affected by free radicals instead of direct observation and trapping of the latter. Here we present a method for the synthesis of biocompatible polymers based on polycyclic esters, in which fullerene C60, is chemically linked to the polymeric matrix. Fullerene is biocompatible and also known as a good radical scavenger capable to trap multiple small radicals from solutions. In such a way, radical trapping from living tissues and blood can happen on the surface of a biocompatible polymer which is in contact with physiological sample (for example as a filter built‐in into infusion device) allowing in‐vivo studying of free radical effects. In this project we first use the Diels‐Alder reaction to connect C60 to amino‐anthracene. Next we use the Schiff‐base or reductive amination reactions of carbonyl groups of poly‐cyclic esters with primary amines in order to link the fullerene radical trap to the polymeric matrix. Like pure fullerene C60 this biocompatible polymeric hook for free radicals may be used in tissues and physiological liquids in in‐vivo experiments. The synthesis, product isolation and characterization and preliminary trapping experiments will be presented.

Grant Funding Source: Supported by Grant T34GM008411, from the National Institute of General Medical Sciences