bluuebunny/crossref_metadata_2025 huggingface dataset:
ExoY from Pseudomonas aeruginosa, a preferential guanylyl and uridylyl cyclase (610.2)
Authors: Seifert Roland , Beckert Ulrike , Frank Dara
Published: June 2021

ExoY from Pseudomonas aeruginosa is injected into host cells via the type III secretion system (Yahr TL et al., Proc Natl Acad Sci USA 95:13899 (1998)). ExoY is structurally similar to CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis. CyaA and EF are nucleotidyl cyclases with a broad substrate specificity, producing cyclic AMP, and, to a much lesser extent, cyclic GMP, cyclic UMP and cyclic CMP (Beste KY et al., PLoS ONE 8:e70223 (2013)). These data prompted us to test the hypothesis whether ExoY is a nucleotidyl cyclase, too. For analysis of cyclic nucleotides, we used a highly specific and sensitive high‐performance liquid chromatography/tandem mass spectrometry (HPLC‐MS/MS) method. As model system, we studied rat B103 neuroblastoma cells. Transfection of B103 cells with ExoY, but not the catalytically inactive mutant ExoY‐K81M, resulted in massive increases in cyclic GMP and cyclic UMP. Similar data were obtained when B103 cells were infected with ExoY‐expressing Pseudomonas aeruginosa. ExoY induced necrosis of B103 cells. The effects of ExoY were mimicked by membrane‐permeable acetoxymetylesters of cyclic GMP and cyclic UMP. In marked contrast to ExoY, CyaA and EF preferentially increased cyclic AMP in B103 cells. In conclusion, ExoY is a nucleotidyl cyclase with a unique substrate‐specificity, and cyclic UMP may serve a novel role as second messenger.
Escherichia coli’s aerobic growth is diminished by small C‐terminal deletions in the regulatory ε subunit of ATP synthase (615.1)
Authors: Shah Naman , Duncan Thomas
Published: June 2021

FOF1 ATP synthase is a rotary nanomotor involved in energy metabolism. It is also a target of antimicrobial drugs. In bacteria, the ε subunit can inhibit the enzyme with its C‐terminal domain (CTD), which partially enters the rotor cavity to block functional rotation. However, the in vivo role of ε inhibition is unclear since, with the entire εCTD absent, E. coli can grow to nearly normal levels on a non‐fermentable carbon source such as succinate. In the inhibited enzyme, the terminal segment of εCTD is deeply buried in the rotor cavity and interacts with one catalytic subunit. To test the importance of this final segment of εCTD, we genetically truncated the last 5 (εdel5) or last 10 (εdel10) amino acids of εCTD. Growth on succinate was reduced 蠅50% with εdel5 and the effect was similar with εdel10. Thus, perturbing interactions of εCTD with the enzyme is more detrimental to in vivo function than removing the entire εCTD. Initial assays with isolated mutant membranes show i) ATPase activity similar to wild‐type membranes and ii) no dramatic effects on the coupling between the catalytic (F1) and H+‐transporting (FO) complexes. Other in vitro tests are in progress to determine how these truncations diminish the in vivo capacity for respiratory synthesis of ATP. This finding indicates compounds that perturb ε’s regulatory interactions could provide a means to prohibit aerobic growth.

Grant Funding Source: Supported by R01GM083088
Fostering remembering: the use of outside of class activities to link learning to prior knowledge (618.14)
Authors: Taylor Ann , Novak Walter
Published: June 2021

The constructivist model of learning is that concepts are built in the mind of the learner. Biochemistry is a cumulative field, and builds on concepts from general chemistry, organic chemistry, and general biology. The hypothesis of this study is that by correcting errors in these underlying concepts and connecting biochemistry learning to prior concepts will cause better understanding of both the prerequisite knowledge and biochemistry. Students completed activities either targeted at key prerequisite concepts or control activities on a weekly basis. Targeted concepts included equilibrium, bond energy, acid/base chemistry, hydrogen bonding, free energy, stereoisomers, protein function, and alpha helices. Performance was evaluated on a pre/post test of the targeted concepts and on specific questions of a biochemistry final exam.

Grant Funding Source: Supported by NSF‐TUES grant #1140711
On‐line lectures: a strategy to liberate class time for active learning (618.25)
Authors: Muth Gregory
Published: June 2021

Increasing class size, demands on student time and the desire to implement “high impact practices” in the classroom have all brought about recent changes in the approach to teaching 300‐level biochemistry classes at St. Olaf College. One innovation that satisfies both student expectations for lecture and the instructor’s desire to present material quickly is the “On‐line Lecture”, a video podcast that allows students to receive a digital lecture outside of class, focus their textbook reading and review as frequently as they wish. By moving lecture outside of class time, alternative activities can be implemented in‐class with the goal of improving student’s problem solving skills, fostering independence and promoting confidence in a supportive environment. Analysis of in‐class exams both pre and post “flip” showed no change in the overall grades earned but the exams themselves were more difficult with respect to the number of questions asked at the higher Bloom levels. Details on preparing and using the On‐Line Lectures and examples of in‐class activities will also be presented.
Evaluating sodium and potassium measuring technologies for human milk (622.1)
Authors: Hartmann Peter , Geddes Donna , Lai Ching Tat
Published: June 2021

Sodium (Na) and potassium (K) in human milk are associated with the permeability of the lactating breast. We hypothesized that the measurement using an ion selective electrode is similar to inductively coupled plasma atomic emission spectroscopy (ICP‐AES) that requires very low sample volumes.

Four groups (low to high fat) of milk samples (n= 30 per group, analyzed in duplicate) were warmed and homogenized. An aliquot (0.3 mL) was used to determine [Na] and [K] by the ion selective electrodes (HORIBA, Japan). Another aliquot (0.01 mL) was digested with nitric acid/hydrogen peroxide and used to determine [Na] and [K] by ICP‐AES (Thermo Scientific, USA). Linear mixed models were used to test whether differences between the two techniques were significantly different from zero for the full data set.

Although the measurements using both technologies were within published values, measurement by ICP‐AES for Na and K were (mean mM±SD) 9.8±5.0 and 11.0±4.8,, respectively, which were higher (p<0.05) than by ion selective electrode (5.8±1.5 and 9.1±3.1, respectively). This might due to the complete release of Na and K ions of human milk by the nitric acid/hydrogen peroxide digestion. Therefore, a conversion factor can be applied to the data to allow comparison of values obtained using these two techniques.

Grant Funding Source: Medela, AG
Examining participation in school food and nutrition programs among students in grades 6‐8 in Vancouver, Canada (624.23)
Authors: Stephens Teya , Black Jennifer , Chapman Gwen , Velazquez Cayley
Published: June 2021

Objective: In Canada, both diet‐related health conditions and the environmental sustainability of the food system are growing concerns being addressed by school food and nutrition programs (SFNPs). The objective of this study was to quantify participation in SFNPs among grade 6‐8 students in Vancouver, and whether participation differed by gender, school‐level, or among schools.

Methods: A cross‐sectional survey in 26 diverse Vancouver schools assessed student participation in SFNPs (n=937). Rao‐Scott corrected chi‐square tests were used to evaluate associations between participation, gender, and school variables (p < 0.05).

Results: SFNP participation varied significantly between schools, yet < 50% of students reported engaging in most SFNPs examined, including: food preparation (36%), choosing or tasting healthy foods (27%), learning about Canada’s Food Guide (CFG) (45%), learning about foods grown in British Columbia (BC) (35%), growing food in a garden (21%), composting (32%), and recycling (51%). Girls were more likely to report learning about CFG or BC foods, and secondary students were more likely to participate in food‐specific activities compared to elementary students.

Conclusions: Despite recent efforts to engage Vancouver students in SFNPs, participation in most SFNPs remains low in most schools. Consequently, few students in grades 6‐8 are being exposed to the whole food cycle.

Grant Funding Source: Canadian Institutes of Health Research, the UBC Food Nutrition and Health Vitamin Research Fund
Barriers and facilitators to New Mexico Farmers’ Markets’ acceptance of SNAP and WIC benefits (632.21)
Authors: Keane Patricia
Published: June 2021

Objective: Federal nutrition assistance program benefits allow for purchase of fresh produce at farmers' markets, but the administrative burden on markets is challenging, particularly in small, rural communities. This study evaluates the barriers and facilitators faced by NM farmers' markets in accepting these benefits and suggests policy recommendations and technical assistance approaches to highlight what farmers' markets require in order to fully realize the benefit of participating in these programs, and to ensure low‐income families’ access to fresh fruits and vegetables at farmers’ markets.

Methods: Mixed methods Results: Data collection and analysis will be complete at time of presentation.

Grant Funding Source: Supported by the University of New Mexico Prevention Research Center
The challenge of classifying foods and beverages marketed to children (632.3)
Authors: Murray Chelsea , Mansuri Sudaba , L'Abbé Mary
Published: June 2021

Banning the marketing of “unhealthy” foods to children has been proposed as a strategy to address the rising rate of childhood obesity. Criteria have been developed to define what constitutes marketing to children; however, we hypothesize that application of these criteria may yield variable results due to the inherent subjectivity of such criteria. The objective of this study was to evaluate inter‐rater variability by nutrition experts in the classification of products as marketed to children. Eight experts independently evaluated the complete package label of 100 foods from a database of Canadian prepackaged products using the following criteria: 1) allusions to fun/play; 2) child‐oriented lettering/graphics; 3) cartoon/children's characters; 4) toys/prizes/contests/coupons; 5) games; 6) unusual flavour/shape/colour of product; and 7) children's product lines. Classification was inconsistent for 55% of products, with the highest levels of disagreement found for criteria 1) allusions to fun/play and 2) child‐oriented lettering/graphics. The highest agreement among raters was for criteria 4) toys/prizes/contests/coupons and 3) cartoon/children's characters; nevertheless, for these types of product marketing, there was unanimous agreement only 25% and 20% of the time, respectively. This subjectivity illustrates potential challenges in implementing recommendations to ban the marketing of “unhealthy” products to children.

Grant Funding Source: Supported by McHenry Chair grant (ML), OGS (CM), CIHR Master's (CM), CIHR PICDP (CM)
Using a lipidomic approach to reveal omega‐3 response phenotypes (635.1)
Authors: Zivkovic Angela
Published: June 2021

The potential benefits of ω3 fatty acids for reducing disease risk have recently been called into question. Could this be due to variations in individual responses to ω3 intervention? We set out to determine whether comprehensive measurement of lipidomic profiles in response to 6 weeks supplementation with ω3 fatty acids (1.9 g/d eicosapentaenoic acid (EPA) and 1.5 g/d docosahexaenoic acid (DHA)) would reveal ω3 response phenotypes. Fatty acids, lipid classes, lipoprotein distribution, and oxylipins were examined. Multivariate analyses showed that lipidomic and oxylipin profiles changed significantly after supplementation across all subjects (p=0.00007 and p=0.00002 respectively). Oxylipin profiles were positively correlated with EPA and DHA in different lipid classes (r2=0.93). Individuals responded differently in both direction and magnitude across several key ω3 and ω6 fatty acid metabolites including prostaglandin E2 (PGE2) and thromboxane B2 (TXB2). Even among those metabolites that increased in response to supplementation in all subjects (e.g. EPA and DHA), there was a high degree of variability in magnitude of response. Lipoprotein particle distributions were driven mainly by gender. Individuals who were hyperresponders had distinct profiles from those who were hyporesponders. We used a lipidomic based phenotyping approach to determine whether differences in individual responsiveness to ω3 fatty acid supplementation could be used as a means to personalize ω3 interventions to modify disease risk.
Effect of obesity in children on the response to influenza virus vaccination (639.6)
Authors: Rebeles Jennifer , Paulson Justin , Paich Heather , Milner Justin , Xiao Ping‐Jie , Hudgens Michael , Noah Terry , Beck Melinda
Published: June 2021

Obesity is a global health concern and more than one‐third of the adult population in the United States is obese. Childhood obesity is also on the rise, with obesity rates doubling in children over the past 30 years and tripling in adolescents. Obese adults are at greater risk for increased severity of infections with influenza virus and obesity has recently been found to be an independent risk factor for increased morbidity and mortality from influenza infection. Vaccination with influenza virus vaccine is the best method to protect from or lessen infection with influenza. However, studies in our laboratory have demonstrated that obese adults, when compared with healthy weight adults, have a suboptimal response to influenza vaccination. To expand our findings to children, we enrolled children, ages 2‐17 with a range of BMI percentiles to study the effect of weight status on the response to influenza vaccination. We collected a pre‐vaccination blood sample, and 30 days later, a post‐vaccination blood sample. Peripheral blood mononuclear cells were isolated and stimulated ex vivo with pH1N1 influenza virus, a strain present in the vaccine, and markers of activation and inflammation were measured by flow cytometry. We found that obese children displayed a significant decrease in the CD4+ T cell activation marker CD69, which may suggest that obese children may have a poorer response to influenza infection compared with healthy weight children. In addition, we found that unstimulated T cells from obese children had increased markers of inflammation, including IFNγ and granzyme B, when compared to healthy weight children, suggesting that PBMCs from obese children may not respond appropriately when challenged with antigen. Therefore, much like the obese adult population, obese children also have immune impairments that may put them at greater risk for infections, even when vaccinated.

Grant Funding Source: NC TraCS Institute
The effects of a beef‐based meal compared to a calorie matched bean‐based meal on appetite and food intake (640.2)
Authors: Bonnema Angela , Thomas William , Slavin Joanne
Published: June 2021

Protein and fiber have strong satiety‐inducing potential. Beef is a high quality, protein‐rich food. Beans contain moderate levels of protein as well as fiber. The objective of this study was to determine the effects of a high protein meal (beef) compared to a moderate protein, high fiber meal (beans) on subjective appetite and energy intake at a subsequent meal. Twenty‐eight adults, 14 men (ages 24±5y, BMI 23±2 kg/m2) and 14 women (ages 25±5y, BMI 22±2 kg/m2) consumed two test lunches including a “meatloaf” made from either beef or beans. The beef meal provided 22 grams of protein and 2 grams of fiber while the bean meal provided 14 grams of protein and 10 grams of fiber. An ad libitum snack was given 3 hours after the test meal. Visual analogue scales were used to assess hunger, satiety, fullness, and prospective food intake. Gastrointestinal (GI) tolerance was assessed over 24 hours. No difference between the beef and bean was observed for appetite ratings over 3 hours, food intake at the subsequent meal (632±75 kcal vs 611±75 kcal, respectively), or sum of GI score (2.2±0.5 vs 2.9±0.5, respectively). Gas and bloating were reported more often after the bean meal than the beef meal (2.0±0.4 vs 1.3±0.4, p‐value 0.057). A beef‐based meal with high protein and a bean‐based meal with moderate protein and high fiber produced similar satiety, while the bean‐based meal resulting in higher, yet moderate, gas and bloating.

Grant Funding Source: MN Beef Council
Metabolic partitioning of oxidative metabolism in ketotic rat brain (643.2)
Authors: Zhang Yifan , Zhang Shenghui , Xu Kui , Puchowicz Michelle
Published: June 2021

We have previously reported decreased cerebral metabolic rate of glucose in brain of diet‐induced ketotic rats which inversely correlated with blood ketone levels. These data suggest a “switch” in substrate utilization from glucose to ketone bodies. The metabolic effect of ketosis on brain intermediary metabolism remains to be explored. In this study we investigated the effects of chronic ketosis on partitioning of substrate oxidation. We characterized the 13C fractional contribution of ketones and glucose into citric acid cycle (CAC), GABA and neuro‐amino acids in ketotic rat brain using infusions 13C‐stable isotope tracers. Isoptopomer analysis, gas and liquid chromatography mass spectrometry were applied. Results: 13C percent label contribution from ketone bodies increased 100 fold towards GABA, and 7.9 and 4 fold for glutamate and glutamine with ketosis compared to non‐ketotic rats. The data show chronic ketosis increases ketone body carbon entry towards GABA which is supplied via CAC, suggesting a possible mechanism of neuroprotection.

Grant Funding Source: NIH R01 HL092933‐01, R21 NS062048‐01
Tocotrienols suppress non‐small lung cancer cells via downregulation of the Notch‐1 signaling pathway (644.1)
Authors: Rajasinghe Lichchavi , Gupta Smiti
Published: June 2021

Non‐small cell lung cancer (NSCLC), accounting for 87% of all lung cancers with a 5‐year survival rate of 16%, is the leading cause for global lung cancer deaths. Tocotrienols, isomers of Vitamin E have been shown to exhibit anti‐tumor activity via inhibition of different signaling pathways in tumors. The Notch ‐1 pathway has been reported to be up regulated in lung cancer patients in vivo and in vitro studies. Therefore, the objective of this study was to investigate the interactions and effects of commercially available tocorienol isomers on the Notch‐1 pathway in adenocarcinoma (A549) and squamous cell carcinoma NSCLC (H520) cell lines. Treatment with Tocotrinols resulted in a dose dependent significant decrease in cell growth, cell migration, tumor invasiveness and induction of apoptosis. Molecular mechanisms behind these changes were explored by testing the expression of Notch‐1 and its downstream stream genes by RT‐PCR and western blot analysis. A dose dependent decrease in expression was observed in Notch‐1 and its downstream genes related to proliferation, apoptosis and invasion (Hes‐1, Survivin, PARP, MMP‐9, VEGF, Bcl 2 and Bcl‐XL). In addition, we found a mechanistic link between the Notch‐1 pathway and. NF‐kB. Thus, our data suggests that commercially available Tocotrienols inhibits cell line growth, cell migration, and tumor cell invasiveness via down regulation of Notch 1and NF‐kB pathway while inducing apoptosis, and could therefore be a potential therapeutic approach.
Cranberry extract initiates apoptosis in HL‐60 cell line through intrinsic apoptotic pathway (644.9)
Authors: Mansouri Rasha , Khoo Christina , Percival Susan
Published: June 2021

The potential anticancer activity of cranberry was reported for the first time in 1996. Proanthocyandins (PACs), the most abundant and complex flavonoid in cranberry, have been described as the major contributors of the anticancer activity reported in the literature. One of the proposed chemopreventative mechanisms is the induction of apoptosis by cranberry PACs. However, limited studies have been done to investigate the role of cranberry in initiating apoptosis in cancer cells and its underlying mechanism of action remains undetermined. Therefore, the overall aim of this study was to investigate the apoptotic pathway that cranberry affects, intrinsic vs. extrinsic pathway. In our study, HL‐60 cells were incubated with 25 µg/ml cranberry‐(60‐65%)PAC extract for 24 hours. Cranberry treatment showed an 85 % increase in caspase 3/7 activity. The data also showed a great enhancement in the activation of caspase‐9 (379 %), but it did not exhibit any change in caspase‐8 activity. Further tests revealed that cranberry treatment caused a loss of mitochondrial transmembrane potential leading to an increase in the mitochondrial release of cytochrome C (87.5%) and Smac (41%). These findings indicate that this cranberry‐PAC extract can induce apoptosis in HL‐60 cells through triggering the intrinsic (mitochondrial) apoptotic pathway.

Grant Funding Source: Funded by Agricultural Experiment Station and Ocean Spray Cranberries, Inc.
Consuming iron‐biofortified pearl millet increased hemoglobin concentrations and prevented a decline in energy efficiency in Indian girls (646.7)
Authors: Beer Hannah , Luna Sarah , Pompano Laura , Przybyszewski Eric , Udipi Shobba , Ghugre Padmini , Haas Jere
Published: June 2021

Objective: To determine if the change in iron status from an iron biofortified efficacy trial results in a change in energy efficiency for Indian girls.

Methods: 56 Indian girls (12‐15 years) were randomly assigned to receive iron biofortified pearl millet or traditional pearl millet for 6 months. Blood and energy efficiency measurements were taken before and after the intervention. Energy efficiency was calculated by recording O2 and CO2 consumption during sub‐maximal exercise tests on a Monark 874E ergometer. General linear models were used to evaluate the effect of the iron biofortification intervention on energy efficiency of the girls.

Results: Consuming iron biofortified pearl millet resulted in an increase in hemoglobin after 3 months (p=0.05). Energy efficiency decreased in both treatment and control groups, but was significantly higher in the treatment group (p=0.02).

Conclusion: Consuming biofortified pearl millet improved hemoglobin status in Indian girls and prevented a decline in energy efficiency over 6 months.

Grant Funding Source: Supported by HarvestPlus
Chemical analysis and anti‐tumor activity test of the volatile oil from Arisaema amurense Maxim tubers (647.13)
Authors: Yang Xiao‐Hong , Li Gui‐Ying , Wang Xing‐Yu , Dong Lei
Published: June 2021

Arisaema amurense Maxim (AAM) is perennial plant native to East Asia and its tubers have been used as Chinese herbal medicine. The extracts from fresh AAM have been suggested to have anti‐tumor property based on the in vitro suppressive effect on the growth of tumor cell lines. While the previous studies have focused on the non‐volatile fractions in AAM extracts for chemical and functional analyses, little is known about volatile fractions in AAM. Studies have shown that for many herbal medicines the effective components are present in the volatile oil. In this study, using GC‐MS method we isolated and identified 46 chemical constituents in the volatile oil from AAM tubers. We further determined effect of the volatile oil from AAM tubers on growth of tumor cell lines Hep2 and Hct‐116 using MTT method. We found that AAM volatile oil inhibited tumor cell growth in a dose‐dependent manner, with IC50 at 54.83 and 15.36 μg/mL for Hep2 and Hct‐116 cells, respectively. These results suggest that the volatile oil from AAM tubers has anti‐tumor activity. Further investigation is needed to validate this effect in the in vivo study and to further identify the effective components and working mechanism.
Effect of MonaVie Balanceœ on stress levels and mood state in moderately stressed subjects (647.22)
Authors: Perkins Logan , Young Devin , Talbott Shawn
Published: June 2021

BACKGROUND: A variety of medicinal plants are used traditionally to reduce anxiety and induce relaxation. Modern herbal supplements, including Magnolia bark (Magnolia officinalis), Tongkat ali (Eurycoma longifolia), and Ashwagandha (Withania somnifera) have been shown to reduce both cortisol exposure and the perception of stress/anxiety, while improving weight loss in subjects with stress‐related eating. OBJECTIVE: We conducted a pilot study in 20 moderately stressed subjects to assess changes in psychological mood state following dietary supplementation for 4 weeks with a combination product (MonaVie BalanceTM) intended to reduce stress and improve well‐being. The product contains a blend of Magnolia bark, Phellodendron bark, Tongkat ali root, Ashwagandha leaf, Amla fruit, and New Zealand pine bark. METHODS: The 65‐question Profile of Mood States (POMS) survey and an abbreviated stress/vigor index were administered before and after 4 weeks of supplementation. RESULTS: Following 4 weeks of supplementation, improved scores were found for chronic stress index (‐23%), well‐being index (+23%), and psychological vigor (+70%). CONCLUSION: These preliminary results suggest that supplementation with MonaVie Balance reduces perceived stress while improving a variety of mood state parameters, suggesting an effective natural approach to the detrimental health effects of chronic stress.
Techological valorization of anthocyanin‐rich Brazilian berries (647.36)
Authors: Alves Andressa , Lacerda Ellen , Inada Kim , Silva Laís , Silva Tamirys , Castelo‐Branco Vanessa , Finotelli Priscilla , Torres Alexandre , Monteiro Mariana , Perrone Daniel
Published: June 2021

This study aimed to add value to two anthocyanin‐rich Brazilian berries by technological processes. Jabuticaba (Myrciaria cauliflora) and Jussara (Euterpe edulis) juices were pressurized at 200 MPa for 5 and 10 min. Jussara juice was microencapsulated by spray drying using Capsul® and Capsul® and inulin (1:1) as encapsulating agents. Jussara oils were obtained by cold extraction with either petroleum ether (JPO) or ethanol (JEO). Anthocyanin contents in products were determined by HPLC‐UV and tocopherols contents in oils were determined by HPLC with fluorescence detection. Jussara products contained both cyanidin‐3‐O‐glucoside and cyanidin‐3‐O‐rutinoside, whereas Jabuticaba juices contained only the former anthocyanin. Although pressurization of Jussara juice for 5 min led to a decrease of 19% in anthocyanins in comparison to control (3.87 g/L), the content was restored when the juice was pressurized for 10 min. In contrast, pressurization of Jabuticaba juice for 5 min led to an increase of 29% in anthocyanins in comparison to control (1.11 mg/L), but this increase was not maintained after pressurization for 10 min. Capsul®:inulin microcapsules showed higher contents of both cyanidin‐3‐O‐glucoside (7.14 g/kg) and cyanidin‐3‐O‐rutinoside (8.98 g/kg) than those prepared with Capsul® alone (5.00 and 5.73 g/kg, respectively). Although JPO extraction yield was higher (48.9%) than that of JEO (37.7%), the latter showed a 24‐fold higher anthocyanins content (21.1 and 22.1 mg/100 g of cyanidin‐3‐O‐glucoside and cyanidin‐3‐O‐rutinoside, respectively). Tocopherols contents in JEO were 1.6‐fold higher (150.9mg/100g) than in JPO, with alpha‐ and delta‐tocopherols corresponding to 56% and 40% of total tocols, respectively. As a whole, our results suggest that pressurization, microencapsulation and oil extraction are suitable technologies for Brazilian berries’ valorization.

Grant Funding Source: FAPERJ, CNPq, CAPES, UFRJ
Activation of mitochondrial‐u‐calpain sensitizes opening of the mitochondrial permeability transition pore during ischemia‐reperfusion (648.11)
Authors: Chen Qun , Hu Ying , Lesnefsky Edward
Published: June 2021

BACKGROUND: Opening of the mitochondrial permeability transition pore (MPTP) increases cardiac injury during ischemia‐reperfusion (IR). IR activates both cytosolic and mitochondrial calpains. Administration of MDL‐28170 (MDL, a calpain inhibitor) decreases injury in isolated hearts during IR. We asked if inhibition of calpains during IR decreases MPTP opening and if activation of mitochondrial u‐calpain (m‐u‐calpain) predisposes to MPTP.

METHODS: Buffer‐perfused mouse hearts (C57BL/6) were subjected to 25 min ischemia and 30 min reperfusion with or without MDL (10 uM) treatment. Opening of MPTP was evaluated by calcium retention capacity (CRC, nmol calcium/mg protein) in mitochondria isolated from IR hearts. In control mitochondria (protease‐purified to remove cytosolic contamination), exogenous calcium was used to activate mito‐u‐calpain. The ADP‐stimulated change in inner membrane potential (ΔΨ) during the transition from state 4 to state 3 respiration (reflected by alteration in TMRM fluorescence arbitrary units, A.U.) was used to assess inner membrane integrity.

RESULTS: Inhibition of calpains improved CRC in mitochondria following IR, supporting that calpain activation enhanced MPTP opening. In control mitochondria, exogenous calcium progressively depolarized ΔΨ, whereas inhibition of m‐u‐calpain attenuated the extent of depolarization.

CONCLUSION: Inhibition of calpain in purified mitochondria reduces the calcium‐induced permeation of the inner membrane, indicating that activation of m‐u‐calpain likely contributes to MPTP opening and cardiac injury during IR.

Isolated hearts
LDH release
LVDP
CRC

IR
615±47
13±2
424±50

MDL + IR
383±55*
29±5*
560±16*

Isolated mito
Î”Î¨, TMRM, A.U.

Calcium uM
0
50
100

DMSO + mito
0.088±0.009
0.050±0.003â€
0.013±0.003â€

MDL+ mito
0.087±0.006
0.066±0.004â€¡
0.036±0.013â€¡

Mean ± SEM; LVDP‐developed pressure mmHg; LDH‐mU/g/min; *p<0.05 vs. IR; †p<0.05 vs. calcium (0 uM); ‡ p<0.05 vs. untreated mitochondria (mito).

Grant Funding Source: Supported by Scientist Development Grant from AHA
Activation of HIF‐1 pathway by cholesterol in hepatocytes under normoxia (649.1)
Authors: Anavi Sarit , Hahn Obercyger Michal , Madar Zecharia , Tirosh Oren
Published: June 2021

Hypoxia inducible factor‐1 (HIF‐1) and cholesterol have been separately implicated in the development of several liver pathologies. The aim of the present study was to investigate possible activation of HIF‐1 pathway by cholesterol in AML12‐hepatocytes.

Increased HIF‐1α protein accumulation was evident in hepatocytes after 2h of incubation with cholesterol while enhanced HIF‐1 transcriptional activity was observed after 6h. Investigating the molecular mechanism revealed that cholesterol inhibited HIF‐1α proteasomal degradation. Hepatocytes incubated with cholesterol exhibited enhanced Nitric oxide (NO) levels, which corresponded with increased iNOS induction. Inhibition of iNOS had no effect on HIF‐1α stabilization at 2h but effectively attenuated cholesterol‐induced HIF‐1 accumulation and transcriptional activity at 6h. Mitochondrial dysfunction and enhanced mitochondrial ROS generation were observed in 2h cholesterol‐treated cells. In order to establish a role for mitochondria‐derived ROS in HIF‐1α protein stabilization, mitochondrial DNA‐depleted cells were utilized. In these cells, the ability of cholesterol to induce HIF‐1 pathway was significantly abolished.

These results demonstrate that cholesterol promotes HIF‐1 activation in hepatocytes in a ROS‐dependant manner. Therefore, alterations of HIF‐1 may contribute to the deleterious effects of cholesterol in the liver.
Garcinol attenuates TGF‐β1/Smad signaling in dimethylnitrosamine‐induced liver fibrosis (649.8)
Authors: Cheng An‐Chin , Lee Ming‐Fen , Tsai Chen‐Yu , Li Chun‐I , Pan Min‐Hsiung
Published: June 2021

Garcinol is a polyisoprenylated benzophenone derivative from Garcinia indica and other related species. Many studies have shown that garcinol is a pleiotropic agent against inflammation. Our previous studies indicated that Garcinol supplementation alleviated the dimethylnitrosamine (DMN)‐induced liver damage, including the expression levels of alanine transaminase and aspartate transaminase and the histopathological status. In this study, we further investigate the effect of garcinol on the transforming growth factor‐β1 (TGF‐β1)/Smad signaling. Sprague‐Dawley rats were intraperitoneally given DMN (10 mg/kg) three days per week for four weeks. Garcinol (10 or 20 mg/kg) was administered by oral gavage daily. Hepatic tissue sections were stained for α‐smooth muscle actin (α‐SMA) and collagen I, and the hepatic TGF‐β1 expression was examined. Our results indicated that garcinol suppressed collagen deposition and α‐SMA formation in the DMN‐treated animals. These data demonstrated that garcinol attenuated TGF‐β1/Smad signaling in liver fibrosis.

Grant Funding Source: Supported by Chang Jung Christian University Grant
Serine protease modulation of epithelial barrier function through the tight junction (650.8)
Authors: Ronaghan Natalie , MacNaughton Wallace
Published: June 2021

Inflammatory mediator‐induced disruption of the tight junction may contribute to pathogenesis of IBD through increased intestinal permeability. We have previously found apical addition of the serine proteases trypsin and matriptase to colonic epithelial cell lines increase transepithelial electrical resistance (TER). We aimed to elucidate the role of tight junction (TJ) modulation in this phenomenon. Confluent monolayers of SCBN colonic epithelial cells were mounted on Ussing chambers after being transfected with siRNA against occludin and treated with 45 BAU/mL trypsin. Trypsin‐induced changes in TER and permeability to 4 kDa FITC‐dextran were monitored. siRNA reduced occludin levels by 90% (Western blot) and reduced the trypsin‐induced increase in TER to 29.8 ± 6.0% (n=4 p<0.05) of control response. However, no changes in trafficking of any TJ protein were observed by Triton X‐100 fractionation and Western blotting after trypsin treatment. In a calcium‐switch assay performed in Ussing chambers, trypsin did not induce faster recovery or have an affect on TER until 30 minutes post‐recovery of the monolayer. In conclusion, we have shown that occludin is essential in the serine protease‐mediated increase in TER but this is not through trafficking to the TJ, and that trypsin does not induce the formation of the tight junction.

Grant Funding Source: Supported by Canadian Institutes of Health Research
Oxidative stress induces apoptosis of MCF7 cells treated by HDAC inhibitors (655.13)
Authors: Hirata Aiko , Senanayake Thulani , Woster Patrick , Hirata Fusao
Published: June 2021

When MCF7 human breast adenocarcinoma cells were treated by HDAC inhibitors (HDACi), SAHA or THS79‐12, considerable portions of MCF7 cells became floated and underwent apoptosis. While annexin A1 was induced in both floated and attached cells, this protein in floated cells was mono‐ubiquitinated as determined by Western blots, suggesting that DNA damage took place. PML, a protein that plays an important role in DNA damage induced apoptosis was acetylated, and Bclxs was induced, and caspase 3 was cleaved, demonstrating features of apoptosis.

8‐Oxoguanosine contents of cells treated with HDAC inhibitors increased by 8 folds in floating cells as compared with those in attached cells. These apoptotic changes did not occur in attached cells treated with HDACi, but their growth was suppressed as seen with annexin A1 transfected MCF7 cells. These observations suggest that apoptosis of MCF7 cells treated with HDACi is attributed to oxidative stress induced by HDACi rather than to annexin A1 induction by HDACi.

Grant Funding Source: Supported in parts by a grant from The Susan G. Komen Breast Cancer Foundation.
A novel nociceptin receptor antagonist inhibits feeding behavior in rodents (656.1)
Authors: Statnick Michael , Toledo Miguel , Witkin Jeffrey , Song Min , Hu Chalie , Ansonoff Michael , Pintar John , Lafuente Celia , Alma Jiménez Alma , Ana Benito Ana , Martínez‐Grau Mariangles , Pedregal Conception , Chen Yanyun
Published: June 2021

Background: Nociceptin is a 17 amino acid peptide that produces physiological effects through activation of the ORL1 (NOP) receptor. We have identified a novel NOP antagonist (Cpd 1) and found it to have high binding affinity (Ki = 0.11 nM), antagonist potency (Kb = 0.17 nM) and selectivity for NOP. Cpd 1 is orally active, CNS penetrant and potently occupies NOP receptors in vivo. Herein, the effects of Cpd 1 on feeding behavior and metabolism were studied.

Methods: Male, lean and dietary‐induced obese (DIO) rats, DIO mice and NOP knockout (KO) mice were used in feeding and indirect calorimetry studies.

Results: Consistent with the literature, Cpd 1 reduced food intake in rodents. In lean rats, Cpd 1 inhibited consumption of a highly palatable (high fat/sucrose) diet, reducing the calorie intake to control chow levels. In DIO rats, Cpd 1 inhibited feeding induced by a mild caloric restriction. In DIO mice, Cpd 1 decreased 24 hr free access food intake and decreased the respiratory quotient indicating that metabolism was driven by fat utilization. Fasting‐induced feeding was inhibited by Cpd 1 in 129S6 mice, an effect that was absent in NOP KO mice. Moreover, under vehicle conditions fasting‐induced feeding was reduced in NOP KO mice.

Conclusions: These data support that Cpd 1 potently inhibits feeding behavior and stimulates fat utilization in rodents. Moreover, the hypophagic effect of Cpd 1 is dependent on NOP receptors.

Grant Funding Source: Eli Lilly and Company
Critical interaction of the dopamine D1 receptor with ergolines: the roles of conserved aromatic residues in transmembrane six segment (656.5)
Authors: Lee Sang‐Min , Mailman Richard
Published: June 2021

We have investigated the interaction of the dopamine D1 receptor with ergoline‐based ligands to understand how non‐catechols activate this receptor. Here, we examined the roles of conserved aromatic residues (F6.51 and F6.52) in transmembrane six segment that are reported to be conserved sites for receptor activation. Mutations of F6.51 and F6.52 to alanine (F6.51A and F6.52A) dramatically decreased the binding affinity of ergolines. Consistent with the affinity data, the functional potency of these ligands at D1‐mediated cAMP synthesis was eliminated at the F6.51A receptor. Whereas F6.52A significantly reduced the potency of these ergolines (consistent with their decreased affinity), it did not decrease the maximal cAMP production. We also studied F6.51Y and F6.51W that were more likely to conserve aromatic/hydrophobic interactions of F6.51. Although F6.51Y did not restore the affinity decrease by F6.51A, it partially restored the potency and fully restored cAMP production. F6.51W was essentially identical to the wild‐type receptor in terms of both affinity and activity of the ergolines. Using a D1 receptor homology model, we hypothesize that the B and D ring of the ergoline backbone has a docking pose close to F6.52 and F6.51, respectively. The F6.51W mutation mimics the hydrophobic interaction that ergolines have with the wild‐type receptor. These data can be useful for designing non‐catechol agonists for the D1 receptor.

Grant Funding Source: Supported by Grant PDF‐SFW‐1208 from the Parkinson's Disease Foundation
Probing partial/biased agonism at the G protein‐coupled A2B adenosine receptor (662.11)
Authors: Gao Zhan‐Guo , Balasubramanian Ramachandran , Kiselev Evgeny , Jacobson Kenneth
Published: June 2021

The A2B adenosine receptor (AR) is involved in many important functions and couples to several G proteins, but the endogenous effects have not been well documented partly due to the lack of selective agonists at this subtype until recently. In the present study, we probed the potential partial and/or biased agonism at the A2B AR in several types of cells, including HEK293 cells and T24 bladder cancer cells, using a series of A2B agonists of adenosine analogs substituted at 5’, N6 and C2 positions, including NECA, MRS5911 and MRS3997, and the non‐nucleoside A2B agonist, BAY60‐6583. All A2B agonists tested induced accumulation of cyclic AMP fully or partially in all cells tested, with NECA being the most efficacious. However, only 5’‐substituted analogs are full agonists in calcium mobilization and ERK1/2 phosphorylation. Over‐expression of the A2B AR in HEK293 cells increased the agonist potency and efficacy in inducing the Gs‐mediated accumulation of cyclic AMP, but not the ability to induce calcium mobilization by the C2‐substituted adenosine derivatives or non‐adenosine A2B agonists. Results from an arrestin translocation assay showed that the 5’‐substituted NECA is more efficacious than adenosine derivatives substituted at C2 and N6 positions and non‐adenosine agonists. BAY actually did not induce arrestin translocation in this assay. The data suggest that 5’‐substituted adenosine analogs are balanced agonists of different pathways, whereas the C2‐ and N6‐substituted adenosine analogs and the class of non‐nucleoside A2B agonists are Gs‐biased agonists. The data also suggest that BAY and some N6‐ and C2‐substituted adenosine analogs may be full agonist, partial agonist or antagonist under various conditions. Indeed, BAY behaves as an A2B antagonist to induce insulin release in MIN6 pancreatic β cells. Thus, differences in efficacy and effector pathways might explain some of the contradictory results reported for A2B AR activation.

Grant Funding Source: Supported by NIDDK Intramural Research, NIH
The G protein‐coupled estrogen receptor (GPER)/GPR30 C‐terminal PDZ motif regulates receptor plasma membrane retention and constitutive Gαi/o‐independent inhibition of cAMP production (662.13)
Authors: Broselid Stefan , Berg Kelly , Clarke William , Olde Björn , Leeb‐Lundberg Fredrik
Published: June 2021

G protein‐coupled estrogen receptor (GPER), or GPR30, is a G protein‐coupled receptor reported to bind 17β‐estradiol (E2), couple Gαs, and mediate non‐genomic estrogen responses. However, there remains a debate about the receptor pharmacological profile, effector coupling, and membrane trafficking. We addressed receptor membrane trafficking and effects on cAMP production in well‐defined model systems, HEK293 cells and CHO cells, and MDCK cells containing a Cre reporter, transfected to express GPER. We also addressed the role of the receptor C‐terminal type‐I PDZ motif (‐SSAV) by deleting the motif (GPERΔSSAV). E2 and G‐1, a reported GPER agonist, did not stimulate or inhibit cAMP production either in the absence or in the presence of wild‐type (WT) GPER or GPERΔSSAV. However, WT GPER constitutively inhibited forskolin (FSK)‐ and agonist‐stimulated cAMP production in a PDZ‐dependent and pertussis toxin‐insensitive manner. Consistent with this, attenuation of native receptor expression with siRNA increased FSK‐stimulated cAMP production in MDCK cells. Confocal microscopy and cell sorting of HEK293 cells stained with antibody live revealed that both WT GPER and GPERΔSSAV matured to the plasma membrane, but that GPERΔSSAV was subject to much higher constitutive endocytosis. Thus, GPER depends on a PDZ motif for plasma membrane retention and constitutive Gαi/o‐independent inhibition of cAMP production.
Identification of a novel, highly potent D3 dopamine receptor‐selective agonist (662.8)
Authors: Moritz Amy , Free R. , Conroy Jennie , Weiner Warren , Doyle Trevor , Southall Noel , Ferrer Marc , Javitch Jonathan , Aubé Jeffrey , Frankowski Kevin , Sibley David
Published: June 2021

Dopamine receptors (DARs) are involved in the development and/or treatment of many neuropsychiatric disorders including schizophrenia and Parkinson’s disease. Many currently available dopaminergic drugs modulate both D2 and D3 DARs due to high homology in their orthosteric binding sites, leading not only to potential unwanted side effects, but also uncertainty as to the roles each DAR subtype plays in normal and pathological processes. In order to discover compounds that target unique, less conserved allosteric sites of these DARs, our lab employed a high throughput screening approach. Through the NIH Molecular Libraries Program, compound 3843 was originally identified as a D2 antagonist in a screen of a 380,000+ small molecule library. Counter‐screening assays of beta‐arrestin recruitment revealed that this compound selectively activates the D3 DAR, yet also acts as an antagonist at the D2 DAR. Over 270 analogs were synthesized and tested to explore the structure‐activity relationship of 3843 for the D2 and D3 DARs, and several compounds with a 1,000‐fold or greater increase in D3 DAR agonist potency were found. Initial competition binding assays suggest these compounds act in an allosteric manner at the D3 DAR, and initial ADME studies performed on 3843 were favorable. We ultimately hope these probes will be useful in vitro and in vivo pharmacological tools to elucidate D3 DAR specific physiology and pathology.

Grant Funding Source: Supported by NIH IRP
Mitochondrial telomerase regulates flow mediated dilation by suppressing mitochondrial derived free radical production (664.1)
Authors: Santos Janine , Gutterman David , Beyer Andreas
Published: June 2021

Mitochondrial (mt) reactive oxygen species (ROS) are key modulators of vascular reactivity. Coronary artery disease (CAD) changes the mechanism of flow‐mediated dilation (FMD) from NO to mtH2O2. Decreased telomerase activity, prominent in cellular senescence and tissue aging, correlates with mtROS, and pharmacological inhibition of telomerase mirrors this change. We hypothesize that telomerase regulates the mechanism of FMD by mitigating mtROS.

FMD, analyzed by video microscopy, was reduced in mesenteric arteries (~200 µm) of mice lacking the catalytic subunit of telomerase (TERT‐/‐). (% Max dilation at 100 cmH2O WT 79.8 ±5.2; TERT‐/‐ 42.5 ±9.6; n蠅6 P< 0.05 ANOVA). L‐NAME inhibited FMD in controls whereas in TERT‐/‐ catalase blocked dilation indicating NO and H2O2 as mediators respectively.
TERT is present both in nucleus and mitochondria. To differentiate the mtROS‐decreasing activity as requiring the nuclear vs. mt activity of TERT, we used cultured human fibroblast expressing WT or a mutant TERT unable to localize to mitochondria (nucTERT). nucTERT cells had higher mtROS, decreased MnSOD protein levels and produced less ATP, which paralleled mitochondrial ultrastructure abnormalities. These functional changes were also observed in endothelial cells mitochondria from TERT‐/‐. Taken together, these data indicate that mitochondrial TERT is a key regulator of mtROS in the microvasular endothelium.
Transformed human brain pericytes enhance endothelial wound healing and angiogenic sprout formation (670.5)
Authors: Mayo Jamie , Bearden Shawn
Published: June 2021

We have recently discovered that density‐stressed human brain pericytes transform to a stem cell‐like phenotype; they form spheres that self‐renew and generate daughter cells with varying morphologies. Co‐culture of transformed pericytes with endothelial cells in a scrape wound assay increased the rate of wound healing as compared with non‐transformed co‐cultures. The goal of this study was to identify mechanisms by which transformed pericytes increase the rate of wound healing and to test the hypothesis that transformed pericytes increase the rate of angiogenic sprout formation. To stimulate sprout formation, primary human brain endothelial cells and pericytes were co‐cultured in a collagen matrix with vascular endothelial growth factor. In a scrape wound assay, lipophilic cell membrane dyes (DiO, DiD; Molecular Probes) were used to visualize and track respective cell types. Transformed pericytes increased sprout formation by 2.13 ± 0.08 fold (p<0.05) compared to endothelial cells co‐cultured with non‐transformed pericytes. In the scrape wound assay, both endothelial cells and transformed pericytes were present at the wound edge. In contrast to the non‐transformed co‐cultures, many of the transformed pericytes fused with endothelial cells during wound repair. Cell fusion was positively correlated with a higher rate of wound healing. Transformed pericytes may be a source of stem cell‐like cells with pro‐angiogenic potential.

Grant Funding Source: NIH HL10654801