bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary Behaviors and Diet Quality of Low‐Income Children in the Supplemental Nutrition Assistance Program
Authors: Leung Cindy W , Hoffnagle Elena E , Jensen Helen H , Foerster Susan B , Murphy Suzanne P , Nestle Marion , Cheung Lilian , Turrell Sophia , Mozaffarian Dariush , Willett Walter C , Blumenthal Susan J
Published: June 2021

The Supplemental Nutrition Assistance Program (SNAP) is the national nutrition safety net for low‐income families. Few studies have thoroughly examined dietary differences by children's SNAP participation status. We examined if SNAP participation was associated with weight and dietary outcomes in low‐income children. Using 1999–2008 NHANES, the study included 5,730 children aged 4–19 with household incomes <130% of federal poverty level. Diet was measured using 1–2 24‐hour recalls. No significant associations were observed between SNAP participation and weight status among all children (OR, obesity, 0.94, 95% CI 0.67, 1.32), or within age, sex and food security groups. For SNAP and non‐SNAP children, mean intakes of whole grains, fruits, vegetables, fiber, and potassium were ≤50% of the recommendations. Mean intakes of sugary beverages, saturated fat, sodium and iron were ≥150% of the recommendations. After multivariate adjustment, SNAP participants consumed 30% more vegetables (95% CI 4%, 62%), 34% more processed meats (95% CI 6%, 69%), 36% more high‐fat dairy (95% CI 13% 63%), and 63% more water (95% CI 14%, 31%) than nonparticipants. Additional dietary differences were observed among food‐insecure children only. SNAP participation was associated with a 5% lower Alternate Healthy Eating Index (95% CI 1%, 9%). Regardless of SNAP participation, the diets of low‐income children need improvement.

Grant Funding Source : Robert Wood Johnson Foundation; Aetna Foundation; National Institutes of Health
Higher levels in a continuum of processed foods are sources of both nutrients to encourage and nutrients to limit: Results from analyses of NHANES, 2003–2008, dietary data
Authors: Keast Debra R , Fulgoni Victor L
Published: June 2021

A consumer guideline describing a “Continuum of Processed Foods” available from the International Food Information Council (IFIC) classified store‐bought foods into 5 categories including (1) minimally processed foods, (2) foods processed for preservation, (3) mixtures of combined ingredients, (4) ready‐to‐eat processed foods, and (5) prepared foods/meals. This study was to quantify the nutritional contribution of the continuum's processed food levels. Using 24‐hr dietary recall data from NHANES, 2003– 2008, participants aged 2+ years whose dietary interviews were reliable (n=27,181), all consumed foods were classified by processing level, and the NCI/USDA “sources of nutrients” methodology was used to determine percentages of total nutrient intakes contributed from each “source” or processing level. After adjusting for covariates, population subgroup differences in sources of energy and 27 nutrients or dietary components (e.g., saturated fat, total and added sugars) were determined. For the total population, Level 1 provided ~300 Kcal/d and >20% dietary fiber, vitamin D, calcium, potassium and vitamin B12. Levels 2 and 5 provided only 60–70 Kcal/d and <10% of most nutrients. Level 3 provided ~360 Kcal/d and >10% dietary fiber, calcium, folate, iron with >10% saturated fat, added sugars and sodium. Level 4 provided ~740 Kcal/d and >20% dietary fiber, vitamin D, calcium, potassium, vitamin B12, folate, and iron with >20% saturated fat, added sugars and sodium. Thus, higher processed food levels provide considerable amounts of important nutrients in American diets, but greater effort is needed to choose processed foods with lower levels of nutrients to limit while preserving nutrients to encourage. Supported by the Food‐Nutrition Science Solutions Taskforce
Influence of Cultivar, Processing, and Food Form on the Glycemic Index of Barley
Authors: Aldughpassi Ahmed , Wolever Thomas MS , Abdel-Aal Elsayed M
Published: June 2021

Replacement of refined high glycemic index (GI) carbohydrates by whole grain products such as barley may prevent the progression of cardio‐metabolic disorders due to their favourable effects on glycemia. However, processing, cultivar variation, physiochemical properties and food form may alter their impact on glycemia. To examine these factors on the GI of barley, 3 experiments were performed in separate groups of 10 healthy subjects. GI was determined using standard methodology and in vitro starch digestibility was measured. Cultivars varied in starch and β‐glucan content. In Experiments 1 and 2 cultivars were processed into four fractions ranging from hull removal only to hull, bran, germ, and crease removal. Experiment 3, a wet pasta‐like product was made from 100% barley flour with wet semolina pasta as a control. In experiments 1 and 2 the GI range was (22.8 – 40.9); processing increased the GI significantly (p>0.05). Cultivars differed in their nutrient composition (p = 0.024), but without significant interaction between pearling and cultivars (p>0.05). Regardless of the cultivar, the least processed kernels had significantly lower total starch and higher total low molecular sugars, insoluble and total fiber. Resistant starch (RS) was inversely correlated with the GI (r = −0.58; p< 0.05). The barley pasta had medium‐high GI values similar to semolina pasta. Results suggest that processing affects the GI and the physiochemical properties. These results will improve knowledge in the development of low‐GI functional foods and improve future consumer guidelines. Financial support from the Ontario Ministry of Agriculture.
Effects of an inulin‐chromium complex on glycemic index in obesity
Authors: Tulp Orien L , Einstein George P
Published: June 2021

To determine the effects of an inulin fructan polymer‐chromium complex (FCC) derived from dahlia root juice on glycemic responses, groups of congenic lean and obese LA/Ntul/ /‐cp rats were administered an oral glucose tolerance (OGT) alone or in combination with measured amounts of the FCC. Glycemic responses and the glucose area under the curve (AUC) of obese rats were significantly impaired compared to similarly‐fed lean littermates. Oral FCC administration resulted in a significant 50% reduction toward normalization of the peak glycemic response during OGT in both lean and obese rats, a 75% reduction in the total glucose area of lean rats and a 50% reduction of the total glucose area of obese rats. The greatest segment of the reduced glucose area occurred during the post absorptive phase of the OGT response in both phenotypes. The results of this study are consistent with an acute FCC‐mediated improvement in glycemic control in a congenic obese animal model of impaired glucose tolerance, and suggest that the FCC complex may be a useful nutritional adjunct in the dietary treatment of obesity and other glucose‐intolerant states as they may occur in man and animals. Institutional Support.
Novel secreted maltase activity enables suckling mouse pup starch digestion
Authors: Nichols Buford Lee , Diaz-Sotomayor Maricela , Avery Stephen , Chacko Shaji , Hadsell Darrel , Baker Susan , Yan Like , Lin Amy (Hui-Mei) , Ao Zi-Hua , Quezada-Calvillo Roberto , Hamaker Bruce
Published: June 2021

Starch requires 6 enzymes for digestion to free glucose: 2 amylases (salivary and pancreatic) and 4 mucosal maltase activities; Sucrase‐isomaltase (Si) and Maltase‐glucoamylase complexes (Mgam). All 6 activities are deficient in suckling rodents.

Objective

: Test 13C‐starch digestion before weaning (at 21d) from mother's milk by measuring enrichment of blood 13C‐glucose in Mgam Null and Wild Type mice.

Methods

Mgam gene was ablated at the N‐terminal expressing the membrane binding domain. Dams were fed low 13C‐diet and litters kept on low 13C‐diet. Pups were weaned at 21d. Digestion was tested at 13d and 25d by intragastric feeding of amylase predigested UL 13C‐α‐limit dextrins (LDx). Blood 13C‐glucose enrichment was measured by GCRMS using pentaacetate derivatives.

Results

4 h after feeding, blood 13C‐glucose MPE% was enriched by 26×103 in null and 18×103 foldat 13d and 0.3×103 and 0.2×103 fold at 25d (vs. fasting p = 0.045 and p = 0.045). By jejunal enzyme assay, imunohistochemistry or Western blots there was no maltase activity or brush border staining with anti‐Si or Mgam antibodies at 13d but these were fully developed in WT by 25d. In 13d null mice lumenal contents were stained by both specific antibodies.

Conclusion

1. 13C‐LDx was rapidly digested to 13C‐glucose in 13d mice independent of age, Mgam genotype or mucosal maltase activity. 2. This novel observation demonstrates that a maltase activity is secreted which enables suckling pup starch digestion well before brush border enzyme development. 3. The origin of this secreted starch digesting activity is presently uncertain.
The metabolic effects of a high fructose versus a high glucose diet in healthy overweight men
Authors: Johnston Richard David , Stephenson Mary , Crossland Hannah , Cordon Sally , Taylor Moira , Aithal Guruprasad P , Macdonald Ian
Published: June 2021

This study assessed the effects of high glucose and fructose intakes, both at energy balance and with overfeeding, on liver and muscle lipid contents and other metabolic parameters.

32 healthy, centrally overweight men (age 34 yrs and BMI 29.4kg/m2) were randomised to 2 periods each of 2 weeks of either a high fructose or high glucose intake (25% energy). Groups were well matched at study entry. In the first 2 weeks, isoenergetic status was maintained by providing all foodstuffs, followed by a 6 week washout. The second 2 week period was an ad libitum overfeeding period.

Period
Fructose
Glucose
Sig between groups

Mean
SD
Mean
SD

Weight (kg)
Isoenergetic
−0.26
0.9
−0.12
0.7
0.62

Overfeeding
1.03*
1.4
0.57 *
1.0
0.29

Hepatic lipid (%)
Isoenergetic
0.30
2.2
−0.05
2.1
0.65

Overfeeding
1.70*
2.6
2.05*
2.9
0.73

ALT (U/L)
Isoenergetic
−4.0
7.9
−2.9
6.5
0.67

Overfeeding
*5.8
8.7
4.1
9.8
0.62

Triglyceride (mg/dL)
Isoenergetic
−6.1
35.1
11.4
61.4
0.35

Overfeeding
31.6
70.2
28.9**
35.1
0.91

Absolute changes during each 2 week period

=p<0.05,

=p<0.01 compared to that group's baseline

There were no changes during the isoenergetic period and similar changes in each group during overfeeding. There were no inter‐group differences in intramyocellular lipid, systemic or hepatic insulin resistance or haemodynamic parameters. Changes during the overfeeding period were strongly associated with weight changes. Hence these were an energy, as opposed to a nutrient, specific effect. There was no evidence for specific, potentially deleterious effects of fructose compared to glucose.

Clinical trials registry: NCT01050140

Financial support: CORE charity UK
Diet‐induced Obesity Decreases Liver Iron Stores in Mice Fed Iron Deficient, Adequate or Excessive Diets
Authors: Hintze Korry Joseph , Killilea David W , Healy Brett J
Published: June 2021

Epidemiological and observational evidence suggests that obesity is related to poor Fe status. To determine interactions between obesity, dietary Fe intake and Fe status, male weanling C57BL/6J mice were fed either standard or high fat diets (3.8 vs 4.5 kcal/g) for 16 weeks to produce lean or obese phenotypes. Fe concentrations of both the high fat or control diet were set at 5, 50 or 500 mg Fe/kg diet. Mice fed the high fat diets had a significantly higher percentage body fat (17.9%) compared to mice fed control diets (5.3%, P < 0.001). Dietary Fe levels alone did not significantly influence body composition. Dietary Fe intake did not differ significantly between mice fed high fat or control diets matched with the same Fe concentrations. Obesity and/or dietary Fe concentration did not significantly affect plasma Fe levels. ANOVA analysis showed significant effects of diet‐induced obesity, dietary Fe and an interaction between both factors on liver Fe levels (P < 0.05). Obese mice had significantly lowered liver Fe levels compared to lean cohorts fed the same amount of dietary Fe (P < 0.05 for all comparisons). Moreover, lean mice fed the 5 mg Fe/kg diet had similar liver Fe levels (127 mg Fe/kg ± 0.04) compared to obese mice fed 50 mg Fe/kg diet (132 mg Fe/kg ± 0.05). These data suggest that obesity, independent of dietary Fe intake, influences liver Fe stores.
Development and validation of a sensitive, high throughput bioassay of radiolabeled E. coli adhesion to uroepithelial cell in vitro
Authors: Mathison Bridget D , Kimble Lindsey L , Kaspar Kerrie L , Khoo Christina , Chew Boon P
Published: June 2021

Anti‐adherent properties of cranberry bioactives have been used to prevent uropathogenic E. coli adherence to uroepithelial cells (UEC). We developed and validated an assay to assess anti‐adhesion activity of cranberry extracts and urine. P‐fimbriated E. coli (CFT073) were labeled with 3H‐uridine then co‐incubated with HTB‐4 UEC at a 400:1 ratio. Cranberry extract (0–20 mg PACs/mL) was pre‐incubated with 3H‐labeled E. coli yielding a sensitive inhibition curve; LLD and IC50 were 0.5 mg and 1.85 mg PAC equivalents/mL, respectively; intra‐and inter‐assay CV were <10% and <15%, respectively. NIST cranberry extract showed identical inhibition of E. coli adhesion. Serial dilutions of urine from human subjects who consumed cranberry beverages showed a linear decrease in anti‐adhesion activity in the assay. Adding extract to UEC prior to or at the time of bacteria:UEC co‐incubation produced similar anti‐adhesion activity. Mannose had no inhibitory effects indicating little to no Type I‐fimbriation. Anti‐adhesion assays conducted with urine from a pilot human cranberry intervention study showed good agreement with results obtained using the hemagglutination assay. Therefore, we have developed a sensitive, high throughput, biologically relevant anti‐adhesion assay using 3H‐E. coli co‐incubated with UEC which can be used for studying the action of cranberry bioactives. Funded by Ocean Spray Cranberries Inc.
Preparation, Characterization, and Induction of Cell Apoptosis of Cocoa Procyanidins‐Gelatin‐Chitosan Nanoparticles
Authors: Gu Liwei , Zou Tao , Percival Susan S. , Cheng Qiong , Li Zheng , Rowe Cheryl
Published: June 2021

The objective of this study is to enhance the stability and bioactivity of cocoa procyanidins (CPs) by fabrication of CP‐sgelatin‐ chitosan nanoparticles. The CPs‐gelatin‐chitosan nanoparticles were fabricated by the self‐assembling method. Using CPs‐gelatin‐chitosan mass ratio of 0.75: 1: 0.5, the resultant nanoparticles had a particle size of 344.7 nm, zeta potential of +29.8 mV, particle yield of 51.4%, loading efficiency of 50.1% and loading capacity of 20.5%. The nanoparticles were spherical with a smooth surface as observed by scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FTIR) suggested that the primary interaction between the CPs and gelatin was hydrogen bond and hydrophobic interaction, while electrostatic interaction was the main binding force between the chitosan and CPs‐gelatin nanoparticles. Nanoencapsulation of the CPs significantly improved the stability of the CPs at 60 °C. The CPs‐gelatin‐chitosan nanoparticles showed the same apoptotic effects at lower concentrations in human acute monocytic leukemia THP‐1 cells compared with the CPs in solution.
Metabolism of [6]‐shogaol in mice and in cancer cells
Authors: Chen Huadong , Warin Renaud F , Sang Shengmin
Published: June 2021

Ginger has received extensive attention because of its antioxidant, anti‐inflammatory, and antitumor activities. However, the metabolic fate of its major components is still unclear. In the present study, the metabolism of [6]‐shogaol, one of the major active components in ginger, in mice and in cancer cells (HCT‐116, HT‐29, H‐1299, and CL‐13), was examined for the first time. Our results indicated that [6]‐shogaol is extensively metabolized in human cancer cells and in mice and the mercapturic acid pathway is one of the major biotransformation pathways of [6]‐shogaol. The major phase I and phase II metabolites of [6]‐shogaol were purified from mouse fecal samples and identified using 1D and 2D NMR spectra as well as tandem mass spectra (MSn, n=2–3). The effects of the major metabolites of [6]‐shogaol on the growth inhibition and induction of apoptosis on human lung and colon cancer cells will also be discussed. This work was supported by NIH Grant CA138277 and CA138277S1 to S. Sang.
Botanical Oil Supplementation Alters Insulin Levels in Diabetic/Metabolic Syndrome Subjects in a Genotypic‐Specific Manner
Authors: Sergeant Susan , Lee Tammy C. , Ivester Priscilla , Ainsworth Hannah C. , Case L. D. , Chilton Floyd H.
Published: June 2021

Dietary botanical seed oils may influence common diseases associated with systemic inflammation such as asthma and diabetes. We have previously determined the optimal dietary concentrations of two botanical seed oils from borage (Borago officinalis) and echium (Echium plantagineum) necessary to reduce ex vivo leukotriene generation in asthmatic subjects. The current study compared the impact of dietary supplementation with borage/echium and corn (placebo) or fish oil on insulin levels in 59 diabetes/metabolic syndrome subjects. Serum PUFA levels changed as expected based on the dietary oil (corn, botanical or fish) provided to subjects. When examined as a group, there was no statistical difference in insulin levels among the dietary groups. A variant LTC4 synthase genotype (−444 LTC4S or rs730012) has been identified that is associated with high levels of mRNA for LTC4S, leukotrienes and responsiveness to leukotriene blockers The borage/echium oil combination, but not placebo or fish oil, markedly increased insulin levels in individuals with the LTC4S variant. These data suggest that dietary supplementation with borage/echium oils alters insulin levels in diabetic/metabolic syndrome subjects in a genotypic‐specific manner. Supported by NIH PO50AT0027820.
Liver antioxidant status after hemorrhage in inbred rat strains fed diets varying in levels of methyl group donors for 9 weeks
Authors: Dubick Michael A , Rose Rajiv , Grubbs Dana L , Barr Johnny L , Klemcke Harold G
Published: June 2021

To further characterize epigenetic influences on survival time after hemorrhage (STaH) in rat strains, the present study investigated liver antioxidant status in hemorrhaged BN/Mcwi and DA rats (n=5–12/group) fed synthetic diets deficient (SD), sufficient (SC) or supplemented (SS) with methyl group donors (folic acid, choline, methionine and vitamin B12). Rats were bled 50% of their blood volume and STaH observed up to 240 min. Liver tissue was collected at death and frozen at −80°C until assayed for total antioxidant potential, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), nitric oxide (NO), NAD+ and superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase and aconitase activities. Total antioxidant potential was 60% lower and TBARS 2‐fold higher in DA SD‐fed rats compared to the other groups. All antioxidant enzyme activities and levels of GSH, NO and NAD+ were also significantly lower (p<0.05) in the SD‐fed rats compared to the SC and SS fed rats. Similar observations were noted in the BN/Mcwi SD fed rats, but changes from their SC and SS groups were of lower magnitude. In addition, STaH was lower (p<0.013) in DA than BN/Mcwi rats for all diets. Taken together these data suggest that putative changes in DNA methylation, subsequent to different methyl group donor diets, can differentially influence liver antioxidant status and STaH in inbred rat strains.
Advertising for food and dietary supplements in the print media in South Africa
Authors: Temple Norman Joseph , Mchiza Zandile , Abrahams Zulfa , Steyn Nelia P
Published: June 2021

Objective

To survey advertisements for food and beverages, slimming products, and dietary supplements in South African magazines.

Methods

We examined the 5 most popular magazines over one year. They are in 3 major languages (English, Afrikaans, and Xhosa).

Results

We recorded 959 advertisements; 776 (80.9%) were for food and beverages, 86 (9.0%) for slimming products, and 97 (10.1%) for dietary supplements. The most common advertisements for food and beverages were for restaurants (n=126), supermarkets (119), alcoholic beverages (79), cereal products (59), confectionery items (59), vegetables (40), and fruit or fruit juices (29). Thirty‐nine of them (4.1%) carried health claims; these were most common for dairy products (14), followed by non‐alcoholic beverages (7), and meat and poultry (6). The most commonly mentioned health benefits were for healthy growth (18), performance claims (13), therapeutic claims (9), and non‐serious risk reduction claims (6), and for and energy (6). The majority of health claims referred to iron (17), B vitamins (12), vitamins and minerals (11), essential fatty acids (11), and calcium (9).

Conclusion

A fraction of advertisements were for healthy products (such as vegetables, fruit, and low‐fat milk) but the large majority were for unhealthy foods. Misleading advertisements for slimming products and dietary supplements are becoming common.
The Study of Androgen Effects on LPA‐induced ROS in Different Human Prostate Cancer Cell Lines
Authors: Lin Yueh-Chien , Huang Yuan-Li , Lee Hsinyu
Published: June 2021

Treatment of prostate cancer by using androgen deprivation therapy often ensued relapse such as independency of androgen stimulation, producing androgen‐refractory tumors with increased invasion, proliferation, and malignancy. The detailed molecular mechanism of this gradual transition is not clearly understood. Recent reports found that androgen negatively regulates vascular endothelial growth factor‐C (VEGF‐C) effects in prostate cancer cell line. Additionally, androgen deprivation leads to intracellular reactive oxygen species (ROS) generation in rat prostate and also in human prostate cancer cell line LNCaP. Furthermore, our previous study indicated that lysophosphatidic acid (LPA) induces the expression of VEGF‐C through ROS generation in PC‐3 cell, a human androgen‐independent prostate cancer cell line. Therefore, we attempt to investigate whether LPA‐induced ROS generation is affected by androgen in different prostate cancer cells. The flow cytometric analysis results revealed that LPA triggers ROS generation in different prostate cancer cell lines. In addition, our results further demonstrated that androgen agonist Mibolerone decreased LPA‐induced ROS generation. In contrast, LPA‐induced ROS production was enhanced by treatment with androgen antagonist Bicalutamide. In summary, our results demonstrated that androgen might regulate LPA‐induced ROS generation in human prostate cancer cells.
Calreticulin up‐regulates VEGF‐A and VEGF‐C in SK‐N‐DZ and SH‐SY5Y neuroblastoma cell lines
Authors: Lin Kuan-Hung , Wu Pei-Yi , Lee Hsinyu
Published: June 2021

Neuroblastoma is a childhood cancer with low survival rate and great potential of metastasis. Even though there are several clinically relevant prognostic markers have been identified, the molecular mechanism underlying the tumorigenesis of NB is still unknown. Calreticulin (CRT), an endoplasmic reticulum chaperone protein, is one of the biomarker of neuroblastoma. It was suggested to play a critical role in the tumorigenesis and differentiation in neuroblastoma. In gastric cancer cells, it was also found that CRT strongly enhances angiogenesis by promoting the expression of vascular endothelial growth factors (VEGFs). In this study, we aim to investigate the correlation between CRT and VEGF‐A and VEGF‐C in neuroblastoma. Overexpression of CRT in SK‐N‐DZ and SH‐SY5Y cells upregulates VEGF‐A and VEGF‐C expression at both mRNA and protein level. Furthermore, CRT siRNA efficiently suppresses CRT expression resulted in downregulation of VEGF‐A and VEGF‐C. These results indicate that CRT plays a role in regulating VEGFs expression, which may affect angiogenesis and lymphangiogenesis in neuroblastoma. However, the underlying mechanism of how CRT regulating neuroblastoma metastasis still remain to be clarified.
The discriminative stimulus effects of nitrous oxidea
Authors: Richardson Kellianne J , Slavova-Hernandez Galina , Shelton Keith L.
Published: June 2021

Despite both widespread clinical use and a long history of abuse, there is a limited understanding of the CNS actions of the anesthetic gas nitrous oxide (N2O). In vitro data has shown that N2O oxide alters the function of NMDA and GABAA receptors, amongst others. Our overarching goal was to use drug discrimination to assess the neurotransmitter systems responsible for producing the intoxicating, subjective stimulus effects of N2O. Sixteen male B6SJLF1/J mice were trained to discriminate 10 min of exposure to 60% inhaled N2O/40% O2 versus 100% O2 in daily 5‐min operant sessions. Subsequently substitution tests with other compounds were conducted. We hypothesized that if the discriminative stimulus effects of N2O are mediated by NMDA antagonism, drugs which attenuate NMDA receptor function would substitute for N2O. Thus far the NMDA channel blockers ketamine and MK‐801 have shown partial substitution for N2O. Of 8 mice tested with ketamine, 5 fully substituted at 7 or 10 mg/kg. Of 6 mice thus far tested with MK‐801, 4 fully substituted at 0.1– 0.3 mg/kg. The NMDA competitive antagonist CGS 19755 did not substitute for N2O, producing exclusively vehicle appropriate responding in 4 of 5 mice. These results suggest the subjective stimulus properties of N2O overlap with those of noncompetitive NMDA antagonists, suggesting common underlying mechanisms of action. Supported by NIDA grant RO1‐DA020553.
Nitrous oxide (N2O) increases brain L‐arginine in production of its antinociceptive effect in mice
Authors: Zhang Yao , Sayre Casey L. , Chung Eunhee , Ohgami Yusuke , Davies Neal M. , Quock Raymond M.
Published: June 2021

C57BL/6 (C57) inbred mice respond to N2O with an increase in brain nitric oxide synthase (NOS) activity and a robust antinociceptive effect; DBA/2 (DBA) fail to exhibit these responses (Ishikawa and Quock, Brain Res. 976:262–263, 2003a). This study was conducted to determine whether increasing the availability of nitric oxide (NO) by administration of L‐arginine might increase responsiveness of DBA mice to N2O and ascertain the effect of N2O on brain levels of L‐arginine. Sensitivity to N2O was assessed using the acetic acid‐induced abdominal constriction test. Whole brain levels of L‐arginine were quantified by HPLC. Intracerebroventricular preloading of L‐arginine in subthreshold doses enhanced the N2O‐induced antinociceptive effects in both C57 and DBA mice. A 60‐min exposure to 70% N2O produced a 12‐fold increase in brain L‐arginine levels of C57 mice, compared to room air exposure. Similar treatment of DBA mice resulted in a 5‐fold increase in brain L‐arginine levels. While the cause of the differential responsiveness of inbred mice to N2O remains to be determined, it is apparent that N2O increases brain L‐arginine levels to produce its antinociceptive effect. (Supported in part by NIH Grant GM‐77153 and the Allen I. White Distinguished Professorship.)
Allergen‐derived proteinases: Isolation, characterization and signaling via proteinase‐activated receptors (PARs)
Authors: Polley Danny , Mihara Koichiro , Saifeddine Mahmoud , Renaux Bernard , Vliagoftis Harrisios , Boitano Scott , Daines Michael , Hollenberg Morley D.
Published: June 2021

Objective

To identify proteolytic enzymes in insect and mould asthma allergens and assess their ability to signal via the PARs.

Methods

Allergen‐derived trypsin‐like enzymes known to contribute to lung inflammation were visualized (western blot) with serine proteinase activity‐based probes (ABP). Enzymes were purified using ion‐exchange chromatography and analyzed for their ABP reactivity & substrate‐inhibitor kinetic profiles. Each enzyme was tested for its activation of PAR–dependent calcium signaling in PAR1 & PAR2‐expressing KNRK cells. ABP‐labeled enzymes were isolated by avidin affinity chromatography and their peptide sequences determined (mass spectrometry).

Results

Both allergens contained ABP‐labeled proteinases with MWs of 20–26 kDa. Each enzyme had distinct kinetic substrate (Km) and inhibitor (Ki) profiles. The enzymes all caused PAR2 calcium signaling, and differentially activated PAR1. Sequencing revealed that the allergen proteinases were homologous with previously described trypsin‐like enzymes.

Conclusions

Diverse allergens contain multiple biochemically distinct trypsin‐like proteinases that are able to target PARs 1 and 2. Thus, PARs and their activating allergen proteinases may be attractive therapeutic targets for the treatment of allergen‐induced asthma.

Support: Canadian Institutes of Health Research, NIH USA and Lung Association of Alberta & NWT
Ubiquitination of protease‐activated receptor‐1 reveals a new mode of signal regulation
Authors: Grimsey Neil Justin , Le Phillip , Trejo JoAnn
Published: June 2021

Protease‐activated receptor‐1 (PAR1) is irreversibly proteolytically activated by thrombin. The regulatory mechanisms that control PAR1 signaling are critical for proper cellular responses. Here, we show that PAR1 is ubiquitinated after agonist stimulation. However, ubiquitin is not essential for PAR1 internalization and lysosomal degradation. This study seeks to identify the PAR1 ubiquitin modifying enzymes and the function of receptor ubiquitination. A candidate siRNA screen revealed a NEDD4 family E3 ligase to be a critical mediator of agonist‐stimulated PAR1 ubiquitination. NEDD4 formed a stable interaction with PAR1 after agonist stimulation. To determine if PAR1 signaling regulated NEDD4 recruitment we examined the role Ca2+. Treatment with the Ca2+ chelator BAPTA blocks PAR1‐ stimulated Ca2+ mobilization, NEDD4 recruitment and receptor ubiquitination. Furthermore, agonist induced PAR1 ubiquitination mediates the recruitment and activation of MAPK signaling. This study is the first example of GPCR stimulation, in this in case PAR1, regulating the activation and recruitment of NEDD4 E3 ligase to mediate itâ€™s own modification, which is critical for activation of MAPK signaling. Supported by NIH R01 HL073328
β‐arrestin2 and ARRDC proteins have distinct roles in β2AR trafficking and signaling
Authors: Han Sang-oh , Kommaddi Reddy Peera , Venkataramanan Vidya , Shenoy Sudha K
Published: June 2021

Objective

To investigate the roles of the 7TMR adaptor β‐arrestin2 (βarr2) and the related arrestin domain containing α‐arrestins (ARRDC 2, 3, and 4) in β2 adrenergic receptor (β2AR) trafficking and signaling.

Results

βarr2 binding to activated β2ARs is rapid and detected at the plasma‐membrane, whereas GFP‐tagged ARRDC binding is delayed and detected on endosomes. Gene‐silencing of βarr2, but not of ARRDC3 blocks internalization, MAP Kinase activation, receptor ubiquitination and lysosomal trafficking of activated β2AR. βarr2 binds clathrin, AP‐2 and pERK robustly upon agonist‐activation of the β2AR whereas ARRDC3 does not. ARRDC3 has canonical poly‐proline domains, which are binding regions for WW‐domain containing E3 ubiquitin ligase Nedd4 that ubiquitinates agonist‐activated β2AR. Mutations within the poly‐proline domains in ARRDC3 not only ablate its binding with Nedd4, but also prevent its localization on endosomes and eliminate the interaction with internalized β2AR.

Conclusions

The role of βarr2 in β2AR internalization, ubiquitination, lysosomal trafficking and ERK signaling cannot be substituted by ARRDC3. The apparent association of ARRDC3 with the β2AR results from ARRDC3/Nedd4 primarily and Nedd4/β2AR association secondarily. ARRDC2 or ARRDC4 can substitute for ARRDC3 in associating with Nedd4 during β2AR post‐endocytic trafficking. (Supported by HL 080525 to SKS).
Adenylyl cyclase 6 defines a distinct cAMP compartment that increases somatostatin expression by airway smooth muscle cells
Authors: Bogard Amy S , Elam Jacob M , Ostrom Rennolds S
Published: June 2021

All adenylyl cyclases (AC) catalyze synthesis of cAMP, but the 9 isoforms differ in localization, regulation and interactions with other signaling molecules. Human bronchial smooth muscle cells (hBSMC) express AC isoforms 2, 4, and 6. Distinct pools of cAMP maintained by AKAPs/PDEs allow cells to respond differently to activation of various GPCR/ACs. Our goal was to examine AC isoform‐specific effects on gene expression. We overexpressed AC2 (non‐raft localized isoform) or AC6 (lipid raft localized isoform) in hBSMC. A PCR array showed a number of genes that were differentially regulated when AC2 or AC6 were overexpressed and activated by forskolin (Fsk). AC6 increased Fsk‐stimulated expression of somatostatin (SST), while overexpression of AC2 led to Fsk‐induced SST mRNA levels below control cells. Activation of Gs coupled GPCR induced SST expression to levels greater than Fsk, suggesting that GPCR might also invoke a non‐cAMP pathway. βAR‐induced SST expression was similar in control cells and those overexpressing AC2 or AC6, while EP2R‐induced SST expression was enhanced by overexpression of AC6 but not AC2. Overexpression of either AC increased Fsk‐stimulated cAMP, but receptor‐stimulated cAMP levels differed between AC2 and AC6. cAMP driven SST expression appears to be coupled to AC6, but not AC2, illustrating that cAMP compartments differentially regulate gene expression. Supported by HL079166.
Differential Regulation of Brown Adipose Tissue Activation by PDE3 and PDE4
Authors: Kraynik Stephen M. , Hinds Thomas R. , Beavo Joseph A.
Published: June 2021

Recent studies indicate that adult humans possess brown adipose tissue (BAT), though the impact of BAT on human energy expenditure is still unclear. The activation of key BAT processes by adrenergic stimulation, namely lipolysis and Ucp1 expression, are mediated through cAMP‐dependent signaling. Phosphodiesterases (PDEs) catalyze the breakdown of cyclic nucleotides, and therein control subsequent downstream effects. This makes PDEs attractive pharmacological targets to activate BAT in obese humans in order to potentially reduce fat stores. We have identified six different PDE subtypes in mouse interscapular BAT that can hydrolyze cAMP. We found that a combination of PDE3 and PDE4 inhibitors can highly synergistically stimulate basal cAMP, CREB‐phosphorylation and Ucp1 mRNA expression, whereas individual inhibitors were ineffective when used alone. However, a PDE3 inhibitor alone could shift the adrenergic agonist dose‐response curves for cAMP and Ucp1 mRNA to the left. Additionally, we found the same synergistic effect of PDE3 and PDE4 inhibitors on lipolysis in primary brown adipocytes, but in this case also identify PDE4 as a major regulator in the basal state. These results identify a functional synergy between PDE3 and PDE4 in the control of BAT function, and suggest that one must inhibit both in order to fully activate the tissue in the basal adrenergic state. Supported by NIH grant GM08392.
The role of phospholipase c‐epsilon in COX‐2 expression and inflammation
Authors: Dusaban Stephanie , Cho Min K. , Masliah Eliezer , Smrcka Alan V. , Brown Joan Heller
Published: June 2021

Brain injury triggers a response characterized by astrocyte proliferation, inflammatory gene expression, and migration. During injury thrombin is generated and can activate the G‐protein coupled protease‐activated receptor 1 (PAR1). Using primary mouse astrocytes, we demonstrated that PAR1 activation triggers phosphoinositide hydrolysis through the novel phospholipase C epsilon (PLCε). Here we asked whether PLCε signaling mediates inflammatory gene expression in astrocytes in response to PAR1 activation. Thrombin stimulation induces 8 and 2.5‐fold increases in COX‐2 mRNA and protein expression, respectively, and these are significantly inhibited in PLCε KO astrocytes. Protein kinase D (PKD) is also activated in response to thrombin and lost in PLCε KO astrocytes, and studies using siRNA or inhibitors show PKD activation is important for COX‐2 expression. Furthermore, we found that thrombin induces NFκB translocation, which in turn regulates COX‐2 expression and that this is dependent on PLCε and PKD. Finally, using scratch assays and a stab wound model to mimic astrogliosis in vitro and in vivo, respectively, we demonstrate PLCε dependent COX‐2 expression. Our studies show that PLCε is the link between activation of PAR1 and COX‐2 expression and this is mediated through PKD and NFκB activation. In vitro and in vivo data indicate that this pathway has important implications in astrogliosis.
Targeting phosphoinositide 3‐kinase γ in airway smooth muscle cells to suppress interleukin‐13‐induced mouse airway hyperresponsiveness
Authors: Jiang Haihong , Xie Yan , Abel Peter W. , Toews Myron L. , Townley Robert G. , Casale Thomas B. , Tu Yaping
Published: June 2021

Excessive activation of different bronchoconstrictor G‐protein coupled receptors (GPCRs) in airway smooth muscle (ASM) contributes to airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. We previously reported that phosphoinositide 3‐kinase γ (PI3Kγ), a down‐stream effector of multiple types of GPCRs, directly regulates ASM contraction by modulating Ca2+ oscillations. The aim of this study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR induced by interleukin‐13 (IL‐13) in a murine model of asthma. We found that intranasal administration of IL‐13 into mice induced AHR after 48 h, as assessed by invasive tracheostomy. Acute treatment with a PI3Kγ‐specific inhibitor attenuated IL‐13 effects in vivo and IL‐13‐augmented airway contractility of precision‐cut mouse lung slices in vitro. In addition, silencing PI3Kγ expression in isolated mouse ASM cells markedly attenuated IL‐13‐augmented Ca2+ oscillations and contractility of ASM cells induced by different bronchoconstrictor GPCRs. Our studies demonstrate that PI3Kγ in ASM cells is important for AHR and acute treatment with a PI3Kγ inhibitor can ameliorate AHR by a broader‐based suppression of unwanted GPCR signaling in a murine model of asthma. This work was supported by American Asthma Foundation Early Excellence Award and State of Nebraska Research Funds.
Effects of diacetyl vapor exposure on human cultured airway epithelial cell ion transport
Authors: Zaccone E. J. , Goldsmith W.T. , Thompson J.A. , Shimko M.J. , Fedan J.S.
Published: June 2021

In August 2000, NIOSH investigated the development of bronchiolitis obliterans following inhalation of the butter flavoring, diacetyl, in employees at a popcorn manufacturing plant. Rats exposed to diacetyl vapor for 6 h developed diacetyl dose‐dependent epithelial damage of the upper airways 18 h after exposure. Nevertheless, diacetyl inhalation did not result in hyperreactivity to methacholine (MCh) both in vivo (inhaled MCh aerosol) and in vitro (isolated, perfused trachea), despite damage to the epithelium. In order to investigate the direct effects of diacetyl on airway epithelium, we examined ion transport of human air‐interface, cultured epithelial cells following diacetyl exposure. We hypothesized that altered epithelial ion transport may be among the earliest events associated with the onset of diacetyl toxicity. Using an exposure system designed for cultured cells, cells were exposed for 6 h to diacetyl vapors (100, 200, 300, 360 ppm). Eighteen hours after exposure, cells were placed into an Ussing chamber to measure transepithelial potential difference (Vt). The results demonstrated that, whereas inhalation of diacetyl had no effect in vivo or in vitro, all concentrations of diacetyl abolished Vt in cultured cells. Our results indicate that diacetyl causes substantial damage to airway epithelium in culture at exposure concentrations that do not alter reactivity of the airways in vivo or in vitro.
Identification of Dysregulated microRNAs in the Lungs of Allergen‐challenged Mice
Authors: Sun Guizhen , Phillips Shirley , Kaimal Vivek , Tay Jia , Xu Jessica , Jackson Aimee , Karras Jim
Published: June 2021

microRNAs (miRs) are important regulators of gene expression that interact with the RNA‐induced silencing complex (RISC) in the cellular cytoplasm. miRs have been implicated in inflammatory diseases such as asthma. We utilized intranasal administration of synthetic complementary sequence anti‐miRs in mice to demonstrate interference of engagement of the let 7 miR with its target mRNAs. Bioinformatic analysis of let7 target mRNAs showed activity of the anti‐miR in lung tissue. To identify miRs involved in allergen‐induced pulmonary inflammation, we characterized house dust mite (HDM) allergen‐induced pulmonary inflammation models. Intranasal HDM administration in the absence of alum produced both an eosinophilic and neutrophilic airway inflammatory response that was suppressed by Dexamethasone. miR and mRNA profiling was assessed using nanostring and microarray in the acute HDM model and identified a number of dysregulated miRs. The expression of miRs in HDM‐challenged mice was compared to the mRNA pathway signature and suggested the potential involvement of multiple miRs in the pulmonary inflammatory response. Ongoing experiments are assessing the effects of anti‐miR inhibition of these miRs in HDM challenge models to support drug discovery activities for pulmonary disease applications.
Detecting thromboxane signaling abnormalities in experimental models of pulmonary arterial hypertension
Authors: Detweiler Neil D , Hirenallur-S Dinesh K , Haworth Steven T , Gordon John B , Rusch Nancy J
Published: June 2021

An increased ratio of thromboxane to prostacyclin metabolites has been reported in children and adults with pulmonary arterial hypertension (PAH). Thus, increased thromboxane signaling may contribute to the pathogenesis of PAH. Thromboxane A2 (TXA2) acts on vascular smooth muscle cells to trigger constriction and proliferation. Since the mechanism of enhanced thromboxane signaling has not been elucidated, we designed studies to detect abnormalities of this pathway in two animal models of hypoxia ‐ induced PAH, the neonatal piglet and the adult rat. The Western blot technique revealed a 2.8‐fold increase in the expression of thromboxane synthase in pulmonary arteries (PAs) of neonatal piglets exposed to 3 weeks of hypoxia compared to control neonates exposed to normoxia. Subsequently, we initiated studies to determine if abnormalities of the thromboxane signaling pathway extend to PAs from rat models of adult PAH. This is a preclinical model in which vasodilator therapeutics commonly are tested for efficacy. The expression of the receptor for TXA2, the TP receptor, appeared similar between PAs of adult rats exposed to 5 to 6 weeks of chronic normoxia or hypoxia. Now we are exploring if upstream mediators including an upregulation of thromboxane synthase may contribute to the increased abundance of thromboxane metabolites in adult models of PAH. Supported by UL1RR029884 (ND) and R01 HL083013 (NR) from the NIH.
ROS Sensitive Calcium Channel TRPM2 Regulates VEGF Induced Angiogenesis
Authors: Mittal Manish , Malik Asrar B
Published: June 2021

TRPM2 is a cation channel involved in calcium influx in response to cellular oxidative stress. We hypothesized that TRPM2 regulates VEGF induced vascular angiogenesis secondary to ROS production. We show that TRPM2 knockdown suppressed VEGF induced human lung endothelial cell (hLEC) hyper‐permeability, which is essential for angiogenesis, as measured by trans‐endothelial resistance and albumin influx (EBA). VEGF induced phosphorylation of VE cadherin at Tyr658 residue was inhibited by TRPM2 knockdown. Additionally, VEGF stimulation increased ROS production in hLEC, and application of apocynin (ROS scavenger) inhibited VEGF induced phosphorylation of Tyr658 of VE cadherin. TRPM2 knockdown reduced VEGF induced calcium entry in hLEC, and delayed migration of HUVEC. VEGF induced angiogenesis measured by in vivo matrigel plug assay was markedly reduced in TRPM2 KO mice compared to wild type. We conclude that calcium influx from ROS activated TRPM2 channels on VEGF stimulation is a key mechanism signaling angiogenesis.

Research support: NIH grants RO1 HL45638‐23 and P01 HL077806
Protection by a transdermal patch system for the prophylaxis against soman poisoning in rhesus monkey
Authors: Cho Young
Published: June 2021

A transdermal patch system containing procyclidine, an N‐methyl‐D‐aspartate receptor antagonist possessing anticholinergic action, and physostigmine, a reversible cholinesterase inhibitor, was developed, and its prophylactic efficacy against soman intoxication was investigated. Male rhesus monkeys were shaved on the dorsal area, attached with a matrix‐type patch with various sizes (2 × 2 to 7 × 7 cm) for 24 or 72 hours, and challenged with 2 × LD50 doses (13 μg/kg) of soman. The smallest patch size for the protection against lethality induced by soman intoxication was 3 × 3 cm, resulting in blood procyclidine concentration of 10.8 ng/ml, blood physostigmine concentration of 0.54 ng/ml, which are much lower concentrations than maximum sign‐free doses, and blood cholinesterase inhibition of 42 %. The drug concentrations and enzyme inhibition rate corresponding to a diverging point of survivability were presumably estimated to be around 7 ng/ml for procyclidine, 0.35 ng/ml for physostigmine, and 37 % of enzyme inhibition. Separately, in combination with the patch treatment, the posttreatment consisting of atropine (0.5 mg/kg) plus 1‐[([4‐(aminocarbonyl)pyridinio]methoxy)methyl]‐2‐[(hydroxyimino)methyl]pyridinium (HI‐6, 50 mg/kg) exerted protection against 5 × LD50 challenge of soman, which means the posttreatment remarkablely augmented the efficacy of the patch. Additionally, it was found that brain injuries induced by soman toxicity were effectively prevented by the patch treatment according to histopathological examinations. These results suggest that the patch system could be an effective alternative for diazepam, an anticonvulsant, and the current pyridostigmine pretreatment, and especially in combination with atropine plus HI‐6, could be a choice for quality survival from nerve‐agent poisoning.
Selective induction of CYP3A4‐dependent vitamin D catabolism via pregnane X receptor in human hepatocytes and healthy volunteers
Authors: Wang Zhican , Lin Yvonne S. , Poulton Emma-Jane , Dickmann Leslie J. , Lampe Johanna W. , Eaton David L. , Thummel Kenneth E.
Published: June 2021

Long‐term therapy with certain drugs, especially hPXR ligands, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1 and CYP3A4 can inactivate vitamin D, although their relative contributions appear to be tissue‐specific and not well characterized. In this study, formation of specific metabolites, 4β,25(OH)2D3 (CYP3A4) and 24R,25(OH)2D3 (CYP24A1) from 25OHD3 was measured, along with mRNA levels for these VDR/PXR target genes. In human hepatocytes, treatment with rifampin significantly increased the levels of CYP3A4 but not CYP24A1 mRNA. Cells treated with 25OHD3, after rifampin pretreatment, showed an 8‐fold increased formation of the major metabolite, 4β,25(OH)2D3; this effect was diminished by addition of DHB, a specific CYP3A4 inhibitor. 24R,25(OH)2D3 was a minor metabolite, and its formation occurred predominantly from induction of CYP24A1 via VDR activation. In healthy volunteers, the plasma level of 4β,25(OH)2D3 were increased 60% (p < 0.01) after rifampin administration. In contrast, a small reduction in plasma 1α,25(OH)2D3 (−10%; p = 0.03) and no change in 24R,25(OH)2D3 (−8%; p = 0.09) levels were observed. Analysis of the plasma metabolite/25OHD3 ratios indicated selective induction of the CYP3A4 pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug‐induced osteomalacia.