bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of a method to determine lutein and lycopene levels in frozen human plasma by reversed‐phase HPLC
Authors: Abadiano Judith , Craft Neal , Yachetti Stephen , Burgher Anita
Published: June 2021

Objective

This reversed‐phase HPLC method to measure lutein and lycopene in frozen human plasma was validated to support a study of infant plasma from three countries.

Method

Human plasma was maintained at −70 ± 10°C. Unspiked pooled infant plasma provided the Low Quality Control (QC) sample, Mid QC and High QC samples were prepared from adult plasma. Lutein and lycopene in plasma were separated by hexane/tetrahydrofuran extraction then quantified with isocratic reverse‐phase HPLC with visible detection. To validate the method, accuracy, repeatability (n=6) and intermediate precision (n=3) of QC samples were determined.

Results

Lutein
Lycopene

NIST n=6
Low QC
Mid QC
High QC
NIST n=6
Low QC
Mid QC
High QC

Mean (mcg/L)
46.4
50
142
292
352
62.3
305
458

RSD* (%)
3.4
5.3
7.2
7.5
2.8
6.4
6.0
8.3

CoA ** (mcg/L)
40 – 54

300 – 380

RSD – Relative Standard Deviation

CoA – Certificate of Analysis

Plasma stability at −70° C for 38 weeks, mean lutein= 95.4% and lycopene=89.1% for the three QC sample pools. The Limit of Quantification (LOQ) for lutein = 5.46 mcg/L and for lycopene = 5.27 mcg/L and defined for this method as the sample concentration that corresponds to the lowest calibration standard.

Conclusion

The method has the sensitivity to detect lutein levels from 5.46–292 mcg/L and lycopene levels from 5.27–458 mcg/L in plasma for clinical trials.

This is funded by Pfizer, Inc.
A weight management program, including a blend of natural compounds, facilitates weight loss in overweight men and women: a pilot study
Authors: Wood Steven M , Poole Stephen , Burke Doug , Bartlett Mark , Mastaloudis Angela
Published: June 2021

Increased rates of overweight globally highlight the need for effective weight management tools. Unfortunately, the complexity of weight management creates challenges and successful interventions backed by well‐controlled studies are lacking. The purpose of this study was to test a blend of natural compounds along with a diet and exercise program for the ability to support healthy weight loss in humans.

Sixty‐nine subjects (47 Women (W) and 22 Men (M)) average age 43.9 ± 9.8 years and an average Body Mass Index (BMI) of 30.6 ± 3.4 were instructed to follow the diet, exercise and supplement program for 2 weeks. 91% of subjects lost weight, average weight loss after two weeks was −3.8 ± 3.1 lbs (−3.0 ± 2.9 lbs W; −5.4 ± 3.0 lbs M) and average fat loss of −1.4 ± 2.3% (−1.3 ± 2.4% W; −1.6 ± 1.9% M)(all changes p < 0.05). Although men lost more weight than women, women experienced greater changes in body measurements. Women exhibited statistically significant reductions in waist, hip, arm and ankle circumferences while no changes were recorded in men. No adverse events were reported and laboratory values were within normal limits.

A weight management program made up of a unique blend of natural compounds, a diet and an exercise regime, led to successful reductions in body weight and % body fat within 2 weeks. These positive findings warrant further investigation including a longer term intervention and a randomized placebo controlled trial. Research was supported by Nu Skin Enterprises, Provo, Utah
Spinal cord dysfunction does not influence leptin in a group of adolescents
Authors: Merlino Emily , Dopler‐Nelson Mindy
Published: June 2021

Obesity has been found to be more serious in spinal cord injured (SCI) and spina bifida (SB) adolescents due to decreased mobility. In a previous study, increased levels of leptin were reported in adults with SCI based on body mass index (BMI). Since the sympathetic nervous system is disrupted in SCI, this leads to question whether individuals with SCI have higher adiposity than able‐bodied individuals (controls), resulting in higher leptin concentrations. Furthermore, does higher adiposity lead to higher leptin concentration in SCI compared to controls of comparable adiposity. The analysis of adiposity and leptin in controls and spinal cord dysfunction individuals, using DXA rather than BMI, shows that there is a significant difference between the obese and non‐obese leptin values (p<0.0001) when controlled for weight (obese individuals have higher leptin than non‐obese). When comparing SCI and SB patients with controls, there is no significant difference in leptin when controlled for by weight (p<0.9429, p<0.9308 respectively). A greater number of SCI and SB than controls moved from non‐obese to obese when categorizing by DXA versus BMI. Therefore we conclude that spinal cord dysfunction does not have significant effect on leptin secretion when controlled for by adiposity.
Hypolipidemic effect of myricetin in db/db mice
Authors: Choi Ha‐Neul , Jang Yang‐Hee , Kang Min‐Jung , Kim Jung‐In
Published: June 2021

Aggressive treatment of dyslipidemia is associated with the reduced risk for cardiovascular diseases in diabetic patients. The aim of this study was to investigate the effect of myricetin on dyslipidemia in animal model of type 2 diabetes. Four‐week‐old male C57BL/KsJ‐db/db mice (n=14) were fed AIN‐93G diet or diet containing myricetin (150 mg/kg) for 7 weeks after one week of adaptation. After the animals were sacrificed after an overnight‐fast, serum triglycerides, total cholesterol, and HDL‐cholesterol were measured by enzymatic methods. Atherogenic index (AI) was calculated as follows: [(Total cholesterol ‐ HDL‐cholesterol)/HDL‐cholesterol]. Myricetin decreased serum triglycerides and total cholesterol levels compared with the control group (p<0.05). Serum HDL‐cholesterol levels were not significantly different between the two groups. AI of the myricetin group was significantly lower than the control group. These results indicate that myricetin could be beneficial in management of type 2 diabetes by controlling dyslipidemia. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012015763).
The effectiveness of carnitine on triglyceride catabolism in fatty liver cultured cell model
Authors: Miyazaki Teruo , Honda Akira , Ikegami Tadashi , Matsuzaki Yasushi
Published: June 2021

Background/Aim

Fatty liver is closely associated with lifestyle‐related illness and liver diseases. However, the effective therapy for fatty liver has not been established yet. Carnitine is an essential factor to transport fatty acid into mitochondria. This study examined the effectiveness of carnitine on fatty acid catabolism in fatty liver culture cell model.

Methods

In human Fa2N4 and mouse AML12 cells, fatty liver induced by synthetic LXR‐ligand (To901317) exposure was treated with 0–10 mM carnitine for 3 days. The levels of triglyceride (TG) and fatty acid metabolism‐related genes were evaluated. Further, normal cell was loaded to 200 μM parmitic acid with 1 mM carnitine for up to 20h, and β‐oxidation metabolites in medium was measured.

Results

In both cells, TG level was significantly and dose‐dependently decreased by carnitine treatment. The enhanced mRNA levels of SREBP‐1, ACC, FAS, and SCD were significantly decreased by carnitine. After 20h of fatty acid load, ketone body excretion from cell was significantly increased with carnitine compared to that without carnitine. The mRNA levels of PPARα, CPT‐1α, PGC‐1α, and OCTN2 were significantly increased after 20h compared to these at 0 time.

Conclusion

In the cell model, fatty liver was improved by carnitine treatment through β‐oxidation activation. Therefore, carnitine administration would be a possible therapy for metabolic syndrome with fatty liver.
Effect of peanut consumption on satiety and energy intake
Authors: Barbour Jayne Anne , Howe Peter R.C. , Buckley Jonathan D. , Bryan Janet , Coates Alison M.
Published: June 2021

Background

Despite their high oil content there is evidence that peanuts do not contribute to weight gain. The nutrient profile of peanuts may assist with weight management by increasing satiety.

Objective

To examine effects of eating normal and Hi‐Oleic peanuts compared with potato crisps on satiety and subsequent energy intake.

Experimental Design

24 participants were randomized in a crossover trial to consume unsalted iso‐energetic serves of three test foods, viz. 60–80g of normal (oleic acid as 55% of total oil content) or Hi‐Oleic (oleic acid as 75% of total oil content) peanuts or 70–90g of potato crisps, after an overnight fast. Hunger/satiety was assessed at baseline, 30, 60, 120 and 180 minutes with visual analogue scales (VAS), after which a cold buffet meal was freely consumed. The same quantity of each food was consumed daily for a further 3 days during which food intake was recorded. Participants crossed over to the alternate foods and repeated the protocol twice at weekly intervals.

Results

Compared with potato crisps, Hi‐Oleic and normal peanuts reduced subsequent total energy intake both acutely (−21%; P<0.001, and −17%; P<0.004) and over the subsequent 4 day period (−11%; P<0.001 and −9%; P<0.008). There were no significant differences in perceived satiety estimated from the VAS scales.

Discussion

Consumption of peanuts reduced subsequent food intake and may assist with weight management.
Oat and Barley β‐Glucans Induce Satiety and Reduce Energy Intake ‐ a Study on Acute and Longer‐term Effects
Authors: Kristensen Mette , Ibrügger Sabine , Mikkelsen Mette Skau , Poulsen Malene Wibe , Ejsing Johnny , Bak Monika Judyta , Dragsted Lars Ove , Jespersen Birthe Moeller , Engelsen Soeren Balling , Bügel Susanne
Published: June 2021

Objective

We investigate the acute and longer‐term effects of three different β‐glucan preparations camouflaged in food matrices on appetite and food intake.

Methods

Fourteen participants completed this randomized crossover study with four 3‐week periods with daily consumption of 3.3g of fibers (control (no fiber), mutant barley β‐glucan, mother barley β‐glucan and oat β‐glucan of a similar molecular mass). Before and after each period (day 0 and 21) a 4‐hour meal test was completed, during which appetite ratings were measured and ad libitum energy intake at the next meal was recorded.

Results

No significant difference in ad libitum food intake was seen on day 0 (p=0.14), however AUC for satiety and fullness both tended to differ between meals on day 0 (p=0.10 and p=0.08) with higher values for oat and mutant barley meals compared to control. Interestingly, the participants consumed less energy (~10%) after all fiber‐rich meals compared to control (p<0.05) on day 21, despite no differences in AUC for satiety, fullness or hunger (p>;0.50).

Conclusions

The difference in effect on appetite and food intake could be speculated to derive from differences in the fiber molecular block‐structure seen for mutant barley, mother barley and oat and then in turn small differences in fermentation patterns.
Inhibitory effect of genistein on nonalcoholic fatty liver disease development in ApoE−/− mice fed a high‐fat diet
Authors: Kwon Young Hye , Jeon Sookyoung , Park Youn‐Jin
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) occurs in a wide variety of clinical presentations, ranging from asymptomatic hepatic steatosis to severe non‐alcoholic steatohepatitis (NASH). Recent studies reported that ApoE−/− mice fed a high‐fat diet (HFD) can be used as a model of NASH as well as arteriosclerosis. Because genistein has been shown to alleviate hepatic steatosis, we investigated the effects of genistein on the development of NASH in ApoE−/− mice. Both wildtype and ApoE−/− mice were fed either an HFD or an HFD supplemented with genistein (0.5 g/kg diet). After 24 weeks on an HFD, serum triglyceride, total cholesterol, and oxidized lipid levels were more exacerbated in ApoE−/− mice compared to wildtype mice. In addition, ApoE−/− mice exhibited higher serum monocyte chemoattractant protein 1 (MCP‐1) and alanine aminotransferase levels. Cholesterol levels in both serum and liver were alleviated by genistein in ApoE−/− mice. Furthermore, ApoE−/− mice exhibited a reduced serum MCP‐1 levels and hepatic pro‐inflammatory gene expressions (MCP‐1 and cyclooxygenase‐2) in response to genistein. In conclusion, genistein alleviated metabolic abnormalities including hypercholesterolemia and NASH in ApoE−/− mice fed an HFD. Moreover, our findings confirmed that the ApoE−/− mice fed an HFD is a promising long term NASH model. This work was supported by National Research Foundation of Korea.
Identification of the role of mangostin in adipocytes with high‐throughput biosensor platform
Authors: Shen Qiwen , Chitchumroonchokchai Chureeporn , Gushchina Liubov , Failla Mark , Ziouzenkova Ouliana
Published: June 2021

We developed a biosensor platform based on 3T3‐L1 cell lines that stably express nuclear factor κB (NF‐κB) response element/GFP, fatty acid binding protein 4 (Fabp4) promoter/CFP, and Nrf2 promoter/YFP fluorescent reporters. The fluorescence signals indicate a direct correlation to the inflammatory, adipogenic and antioxidant status in adipocytes, respectively. α‐Mangostin (MG), a polyphenolic xanthone derivative from mangosteen fruit, has been reported to have anti‐adipogenic and anti‐inflammatory activities, while the activity of γ‐MG remains largely unknown. We applied biosensor assays to identify inflammatory and adipogenic properties of γ‐ as compared to α‐MG. α‐MG suppressed the activation of NF‐κB reporter in adipocytes in a dose‐dependent manner. α‐ and γ‐MG suppressed the activation of NF‐κB reporter by 30% and 40%, respectively, as compared to control adipocytes. This was confirmed by a significant decrease in mRNA expression of adipogenic marker PPARγ and pre‐adipocyte marker pref‐1. γ‐MG was a more potent inhibitor than α‐MG of MCP1 production as quantified by ELISA. MG also regulated Fabp4 and Nrf2 reporters during adipogenesis. Our studies suggest that the high‐throughput fluorescence biosensor platform facilitated the discovery of the potential therapeutic properties of γ‐MG in reducing obesity and inflammation.

OIA Gateway Research Seed Grant OSU
Impact of a new total Western diet for rodents on metabolic health and colorectal carcinogenesis
Authors: Monsanto Stephany Perez , Ward Robert E , Hintze Korry J , Lefevre Michael , Frauen Shelby , Benninghoff Abby D
Published: June 2021

Our group developed a new total Western diet (TWD) for rodents that mimics typical human nutrition patterns with respect to macronutrient composition (amounts and types of sugar, fat, protein) and micronutrient content (vitamins and minerals) on a nutrient density basis. The goal of the present study was to determine the relative roles of macro‐ and micronutrients in dysregulating metabolism. Mice fed the TWD appeared to compensate for its high energy content by reducing food consumption, whereas animals fed a commercial DIO diet did not. However, food and energy intakes in mice fed a diet modified only for vitamin and mineral content was similar to the AIN93‐fed controls, whereas food and energy intakes by mice fed a diet modified for macronutrient composition were highly similar to DIO‐fed mice. These patterns were reflected in the average final body weight for each group, and body weight gain was entirely attributed to an increase in fat mass. Overall, based on our observations, the altered micronutrient composition of the TWD blocked the macronutrient driven increase in body weight. Significant differences in fasting glucose or oral glucose tolerance were observed only for mice fed the DIO diet. Our observations point to a complex interaction between macro‐ and micronutrients with respect to satiety and energy intake. We are currently examining the impact of these westernized rodent diets on colon carcinogenesis.
A Comparison of Quality of Life and Nutritional Status after Gastrectomy in Korean Gastric Cancer Patient
Authors: Lim Hee Sook , Kim Soon Kyung , Cho Gyu Seok , Jeong Gui Ae
Published: June 2021

The purpose of this study was to evaluate QoL (quality of life) and nutritional status after surgery in 222 patients with gastric cancer during followup duration. Malnutrition status was 67.7% at 6 months postoperatively and nutritional satus improved as time went on (p<0.001). In the survey of the nutrient intake, malnutrition group showed significantly lower intake of energy, protein, phosphorous, sodium, potassium, zinc, niacin, folic acid. The QoL mean score of malnutrition group was 22.3 ±14.6 and normal group was 17.2±11.8(p<0.001). Malnutrition group experienced worse QoL in eating restrictions (p<0.001), anxiety (p<0.001), taste problem (p<0.01), and hair loss (p<0.001). After postoperatively, also showed a trend to experience worse QoL in taste problem (p<0.001), body image (p<0.01). Factors that independently influenced QoL were follow up duration (p<0.05), extent of gastric resection (p<0.01), chemotherapy (p<0.001), and nutritional status (p<0.01). These results suggest that nutritional status improvement may be associated with QoL and early recovery.
Bioelectrical impedance phase angle: predictor of cell disturbance, metabolism and priority for nutritional intervention?
Authors: Almeida Ana Isabel , Ferreira Catarina , Grillo Isabel Monteiro , Camilo Maria , Ravasco Paula
Published: June 2021

Rationale

Cancer & undernutrition may result in disturbed electric tissue properties, translated in altered phase angle (PA). We aimed to assess the predictive value of PA in identifying cancer pts with major priority for nutritional intervention.

Methods

We included 51 ambulatory cancer pts. Nutritional status was assessed by Patient‐Generated Subjective Global Assessment (PG‐SGA). PA values were assessed by bioelectrical impedance analysis (Biodynamics 450®) & compared with age/sex reference percentiles. Comparisons were made using non‐parametric tests.

Results

Undernutrition was prevalent (39%) & 63% pts had indication for nutritional intervention. Most pts with a PA<5th percentile were undernourished (75%) & had indication for nutritional intervention (88%). A PA<5th percentile did predict a significantly worse PG‐SGA intervention score (p<0.02). PG‐SGA A score was worse in pts with a PA<5th percentile vs pts with PA>;5th percentile (p<0.01), particularly on food intake, symptoms & functional capacity (p<0.05).

Conclusion

A PA<5th percentile was associated with critical need for nutrition intervention. PA integration in clinical practice may be of great value; while simple and easy to use, it provides key information on cell disturbance & metabolism, which may be useful as a first approach to prioritize the urgent need of nutritional intervention & symptom management. No research support.
Absorption of Marine vs Non‐Marine sources of EPA and DHA
Authors: Ketz John Samuel , Rodavich Mary C , Barnes Kimberly M
Published: June 2021

DHA from algae oil has been incorporated into tissues at a lower level and been less effective than fish oil (FO) at reducing body fat. Our objective was to determine the fecal excretion of EPA and DHA from different sources. Male mice (n=100) were fed a 12% lipid diet with soy oil (SO), FO, yeast oil (YO, equal EPA to FO diet), algae oil (AO, equal DHA to FO diet), or AO+YO (equal EPA + DHA as FO diet). For 4 weeks fecal samples were collected over night once per week. There were no differences in feed intake, body weight, or body fat (P>;0.05). FO‐fed mice did have heavier livers (P<0.05). AO+YO‐fed mice had greater fecal DHA content than AO‐fed mice and FO‐fed mice for the first 3 weeks (P<0.05) and AO‐fed mice had greater (P<0.05) fecal DHA content than FO‐fed mice in weeks 2–4. FO‐fed mice had greater fecal EPA than YO‐fed mice and AO+YO‐fed mice on week one (P<0.05). FO‐fed mice and YO‐fed mice had greater fecal EPA on week 1 than weeks 2–4 (P<0.05). Thin layer chromatography was performed and verified that the oil sources were primarily composed of triglycerides. In conclusion DHA in FO appears to be more bioavailable than in AO.
How do microbes control their metabolic operation
Authors: Sauer Uwe
Published: June 2021

While our knowledge on regulation events is steadily increasing, we are much less informed about the functionality of individual regulation events and their quantitative relevance for controlling a given biological function. For metabolism this function is the flux of small molecules that can be quantified network‐wide through methods of 13C‐flux analysis (1). This ability to quantify metabolic function allows us to investigate the question which of the multiple overlapping regulation mechanisms are employed by microbial cells to manage their small molecule traffic (2, 3). By combining various omics methods with computational analysis, we delineate actively controlling regulation events from the much larger number of co‐occurring regulation events. From large‐scale 13C‐flux experiments with deletion mutants in different microbes we found, somewhat surprisingly, that only few transcription factors are actively involved in controlling the distribution of flux in the network. Here I will discuss the relevance of enzyme phosphorylation in controlling fluxes through yeast central metabolism and of allosteric metabolite‐enzyme interactions in controlling E. coli fluxes.
Role of miR‐19a in the development of abdominal aortic aneurysm
Authors: Zhang Yali , Park Ho‐Jin , Galper Jonas B
Published: June 2021

Abdominal aortic aneurysm (AAA) is a significant health problem that affects 6–9% of men over 65 years of age. Surgical repair is the only effective means of AAA treatment. The mechanistic basis for the initiation and progression of the disease which might serve as a therapeutic target for treatment of AAA has not been established. AngII treatment of ApoE−/− mice is a model for AAA formation. MicroRNAs (miRs) are endogenous small RNAs that negatively regulate gene expression in a sequence‐specific manner. MicroRNA arrays demonstrated that miR‐19a is increased in AngII‐induced AAA tissue in ApoE−/− mice. SOCS1 (Suppressor of Cytokine Signaling 1) has been shown to play a protective role in inflammatory diseases via inhibition of p‐STAT3 activity. Based on a computational analysis, we found the binding sequence of miR‐19a in 3′UTR of SOCS1 mRNA suggesting that miR‐19a might inhibit SOCS1 protein expression and decrease the inhibition of inflammation by SOCS1. We demonstrated that SOCS1 is decreased and pSTAT3 is increased in AngII‐induced aneurysmal tissue from ApoE−/− mice. Furthermore, treatment of bone marrow derived macrophages with AngII resulted in increased expression of mir‐19a and decreased SOCS1 and increased pSTAT3. These data suggest the conclusion that miR‐19a might play a role in AngII‐induced AAA formation by inhibiting SOCS1 inhibition of inflammation.
Single nucleotide polymorphisms in the 3′ untranslated and near gene regions of transthyretin may play a role in senile systemic amyloidosis
Authors: Sikora Jacquelyn L , Spencer Brian H , Prokaeva Tatiana B , Connors Lawreen H
Published: June 2021

Senile Systemic Amyloidosis (SSA) is a disease occurring mainly in elderly Caucasian males, estimated to affect approximately 25% of individuals over 80 years of age. SSA usually presents as cardiomyopathy caused by extracellular deposits of amyloid fibrils composed of the plasma protein, transthyretin (TTR). We hypothesized that genetic variation in regulatory regions of the TTR gene may contribute to SSA. We examined SNP variations in a region of ~800 bases spanning the 3′ untranslated and near gene regions of the TTR gene in a group of patients with SSA (n=88). For comparison, a group of controls (n=10) matched for age, gender, and race were analyzed. Of 24 previously reported SNPs in this region, we identified one novel and 3 common SNPs, rs62093482, rs1791228, and rs75032823 in our samples. The odds ratios (OR) calculated under allele analysis for these SNPs were >;1, indicating at least one SNP may be associated with SSA. Genotype analysis revealed OR of 7 and 5 under the recessive model for rs62093482 and rs1791228, respectively, and OR of 3 under the dominant model for rs75032823. Haplotype analysis revealed a haplotype block unique to the SSA group including the three SNPs. The common haplotype identified had an OR of 2.5, indicative of association with SSA, and included alleles with high OR for each SNP.

This research is supported by the Gruss Foundation, Boston University Amyloid Fund, the Young Family Amyloid Research Fund, and NIH grant RO1AG031804 (LHC).
Effects of dietary resistant starch on beta‐catenin in azoxymethane‐treated colonic epithelium
Authors: Cray Nicole , Pillatzki Angela , Zhao Yinsheng , Birt Diane F , Whitley Elizabeth M
Published: June 2021

Resistant starches (RS) have been suggested to reduce colon cancer through production of short chain fatty acids by microflora. Aberrant Wnt signaling is common in colorectal cancer, with mis‐regulation and over‐expression of beta‐catenin. We hypothesize that RS will modulate the Wnt pathway in colonic epithelial cells. We evaluated mRNA levels of beta‐catenin in colonic epithelial cells, comparing results from F344 rats fed for 8 weeks with diets containing Guat209 (Guat), AR16035 (AR), or a cross (ARxGuat), with 34.5% 1.2%, and 1.9% RS, respectively. Carcinogenesis was initiated by azoxymethane (AOM) before RS was fed. Populations of histologically normal epithelial cells of the mucosal surface and crypt base were collected by laser microdissection and quantative PCR performed for beta‐catenin. Preliminary results demonstrate no difference between beta‐catenin mRNA levels in colonic epithelium from non‐AOM‐treated rats fed low‐ or high‐RS diets, or associated with AOM‐treatment in rats fed low‐RS diets. Without AOM initiation, regardless of the diet, expression of beta‐catenin is similar in each niche. Dietary RS may provide a means to regulate Wnt signaling in the colon.
TRP‐ML1 channels‐Mediated Ca2+ Release Contributes to FasL‐Induced Lysosomal Trafficking and Interactions with the Sarcoplasmic Reticulum in Coronary Arterial Myocytes
Authors: Xu Ming , Li Xiaoxue , Zhang Yang , Li Pin‐Lan
Published: June 2021

Activation of death receptor, Fas has been reported to produce a local Ca2+ burst via lysosomal transient potential receptor‐mucolipin 1 (TRP‐ML1) channels followed by a large Ca2+ release response from the sarcoplasmic reticulum (SR). The present study further tested how the lysosomal Ca2+ burst is coupled with SR Ca2+ release and thereby results in cell death, a common action of Fas activation in mouse CAMs. By confocal microscopy, we found that transfection of CAMs with TRP‐ML1 siRNA substantially inhibited FasL‐induced lysosome Ca2+ bursts and consequent SR Ca2+ release. In contrast, transfection of CAMs with plasmid containing a full‐length TRP‐ML1 gene construct enhanced the FasL‐induced two‐phase Ca2+ release. We further demonstrated that FasL significantly increased the colocalization between lysosomal marker Lamp1 and ryanodine receptors 3 (RyR3) and the dynamic trafficking of lysosomes to the SR in CAMs. When CAMs were treated with TRP‐ML1 siRNA, FasL‐induced interactions between lysosomes and SR were substantially blocked. Flow cytometry analyses demonstrated that FasL‐induced apoptosis and activation of calpain and calcinurin, the Ca2+ sensitive proteins mediating apoptosis, in CAMs was significantly attenuated by silencing TRP‐ML1 gene. However, this FasL‐induced apoptosis and activation of calpain and calcinurin were enhanced in CAMs when these cells were overexpressed with TRP‐ML1. It is concluded that TRP‐ML1 channel‐mediated lysosomal Ca2+ bursts upon FasL stimulation promotes lysosomal trafficking and interactions with the SR, increasing intracellular global [Ca2+] and apoptosis of CAMs (supported by NIH grants HL057244, HL091464 and HL075316).
Role of Endothelial TRPC3 Channels in Endoplasmic Reticulum Stress Induced Apoptosis in Human Coronary Endothelial Cells
Authors: Smedlund Kathryn , Ingels Marcel , Vazquez Guillermo
Published: June 2021

Atherosclerosis is a chronic inflammatory disease of the arterial wall and represents the major cause of coronary artery disease. The balance between apoptosis and survival signaling pathways is crucial to maintaining endothelial integrity during vascular injury, and alteration of these processes has been associated to atherogenesis. Endoplasmic reticulum (ER) stress has emerged as a critical player in development of atherosclerotic lesions. Despite its beneficial and essential corrective functions, chronic ER stress, through persistent activation of the unfolded protein response (UPR) can alter the balance between apoptosis and survival favoring lesion progression. In recent studies, we found that in human coronary artery endothelial cells (HCAECs) Transient Receptor Potential Canonical 3 (TRPC3), a member of the larger TRP superfamily of channel forming proteins, plays a role in cell survival and apoptosis. In HCAECs knockdown of TRPC3 results in an increase in the number of apoptotic cells in response to the ER stressor thapsigargin. This correlated positively with increased expression of typical ER stress markers such as CHOP and BiP. Altogether, this data suggests that TRPC3 expression and function may have a protective role in HCAECs and that TRPC3 deficiency and/or deregulation may sensitize HCAECs to the pro‐apoptotic actions of sustained ER stress. Research support: AHA 12POST11910042 to KS, R01HL111877–01 to GV.
Gα12‐dependent constitutive EC‐specific vWF secretion
Authors: Rusu Luiza , Liu Guoquan , Visintine David , Kim Kyungho , Malik Asrar B , Du Xiaoping , Cho Jaehyung , Meigs Thomas E , Minshall Richard D
Published: June 2021

A poorly understood component of hemostasis and thrombosis is von Willebrand factor (vWF) secretion by endothelial cells (EC). Here, we used genetic approaches in vitro and in vivo to determine the role of G proteins, RhoA, and αSNAP in constitutive vs. evoked vWF secretion. As expected, αSNAP siRNA completely abolished constitutive and evoked vWF secretion by HUVECs. In addition, siRNA‐mediated depletion of G protein 12 α subunit (GNA12) inhibited both constitutive and thrombin‐induced vWF secretion, while over‐expression of activated GNA12 promoted vWF secretion. In GNAq, p115 RhoGEF and RhoA‐depleted HUVECs, thrombin‐induced vWF secretion was reduced by 40% whereas basal vWF levels were unchanged. GNA12−/− mice showed increased tail‐snip clotting time, reduced laser‐injury arterial clot formation, and reduced vWF levels in blood and in thrombin‐stimulated blood‐free mouse lung perfusate. Finally, in vitro binding assays revealed a novel role of the GNA12 N‐terminal domain in binding and activation of αSNAP. Taken together, these studies uncover obligatory and complementary roles of GNA12 and GNAq/11 in constitutive vs. evoked EC vWF secretion. Supported by:NIH P01 HL60678, R01 HL109435, and P30 HL101302
Macrophages Polarize during Acute Lung Injury and Resolve Lung Vascular Dysfunction through Sphingosine 1 Phosphate
Authors: Sukriti Sukriti , Chava Koteswara Rao , Tauseef Mohammad , Sadikot Ruxana
Published: June 2021

Macrophages (Mϕ) induce neutrophils sequestration during acute lung injury (ALI) but their influence on lung vascular barrier function is unclear. We exposed CD11b‐diptheria toxin receptor (DTR) transgenic mice to endotoxin, LPS, followed by i.p. DT to deplete Mϕ to test the hypothesis that Mϕ polarizes during ALI and regulate lung vascular permeability (LVP). In WT mice, LPS increased LVP, determined by measuring lung wet‐dry ratio and albumin accumulation into lung parenchyma, at 4 hrs that resolved within 24 hrs. However, LPS induced persistent increase in LVP in Mϕ depleted mice which resolved following adaptive transfer of WT‐bone marrow derived macrophages (BMDM). M1Mϕ were increased during onset of ALI while M2Mϕ dominated during resolution phase. Sphingosine kinase (SPHK)‐induced sphingosine 1 phosphate (S1P) stabilizes endothelial cell (EC) barrier function. Thus, we applied conditioned media (CM) from WT, SPHK1−/− or SPHK2−/− BMDM on sub‐confluent EC monolayer to address if Mϕ strengthens barrier function by secreting S1P. CM from WT and SPHK1−/− BMDM enhanced basal barrier function and facilitated EC barrier recovery following the increase in EC permeability by edemagenic agents. However, CM from SPHK2−/− BMDM failed to induce this response. Thus, Mϕ polarizes during ALI and secretes S1P to resolve lung vascular barrier dysfunction thereby mitigating ALI.
Structure‐Function Relationship and Local Subcellular Signaling using Heart Failure Relevant Molecular Defined Muscle Specific mAKAP Mutants
Authors: Rababah Abeer , Craft John , Singh Sonal , Atrooz Fatin , Wijaya Cori , McConnell Bradley
Published: June 2021

Beta adrenergic stimulation signals through phosphorylation of downstream proteins such as protein kinase A (PKA). PKA substrate phosphorylation is facilitated through its co localization with its signaling partner by A kinase anchoring proteins (AKAPs). Particularly, mAKAP (muscle selective AKAP) localizes PKA and its target substrates such as phosphodiesterase 4D3 (PDE4D3), ryanodine receptor and protein phosphatase (PP2A). We have recently identified potentially important human mAKAP mutations located within or near key protein binding sites critical to beta adrenergic receptor signaling. Three mutations (P1400S, S2195F and L717V) were cloned for the purpose of comparing whether these substitutions disrupted mAKAP binding to the PKA regulatory subunit RIIα or PDE4D3 binding domain and to understand whether these mutations mediated altered signaling. Interestingly, S2195F, a mutation located in PP2A binding site, showed similar protein expression (p=0.3) to wild type mAKAP, but with a significant increase in binding propensity to RIIα after cell stimulation. This result was corroborated by immunopreciptation (p=0.0003). Consequently, PKA activity showed a significant increase with mAKAP S2195F mutation, as compared to wild type. In conclusion, mAKAP S2195F displayed modified activity and binding propensities and may participate in hypertrophy. This work was supported by NIH (HL085487).
Characterizing the Structural Elements Involved in β1‐ Adrenergic Receptor Interaction with SAP97
Authors: Nooh Mohammed M. , Bahouth Suleiman W
Published: June 2021

In this study we characterized the domains in SAP97 that are involved in binding to the β1‐AR and to AKAP79. Among the three PDZs of SAP97, PDZ2 displayed the highest binding affinity to the β1‐AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology‐3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3‐SAP97) did not affect the binding of SAP97 to the β1‐AR. However, ΔI3‐SAP97 could not rescue the recycling of the β1‐AR because it failed to incorporate AKAP79 into the SAP97‐ β1‐AR complex. On the other hand, we demonstrated that mutations to alanine at positions 0, −2, and −3 of β1‐AR inhibited recycling of the agonist‐internalized β1‐AR and coimmunoprecipitation of β1‐AR with SAP97 in HEK‐293 cells. Also, we determined that mutating β1‐AR V477 to other small hydrophobic residues (I or L) did not affect recycling of the agonist‐internalized β1‐AR or co‐immunoprecipitation of full β1‐ AR with SAP97. These and other data to be presented define the key elements required for organizing β1‐ adrenergic receptosome involved in connecting the β1‐AR to trafficking and signaling networks.

Funded by NIH HL‐085848
Function of the first intracellular loop for human D1 and D5 dopaminergic receptors
Authors: Zhang Boyang , Tiberi Mario
Published: June 2021

The human D1‐class dopamine receptors (hD1R and hD5R) are positively linked to adenylyl cyclase (AC) activation and play critical physiological roles in the brain. To study the function of their first intracellular loop (IL1), we have designed human D1‐ class mutants (hD1‐PD1 and hD5‐PD1) whereby all serines and threonines of IL1 have been replaced by alanines and valines, respectively. Both PD1 mutants exhibited a loss of dopamine affinity but unaltered binding properties for inverse agonists. Furthermore, constitutive activity of PD1 mutants was abolished, implicating that serines and threonines of IL1 are critical for the transition of D1‐class receptors from inactive (R) to active (R*) states in the absence of activating ligands. Dopamine potencies for hD1‐PD1 and hD5‐PD1 were drastically reduced compared to those of parent receptors. Interestingly, despite such warranted uncoupling from the G protein, both PD1 mutants displayed a higher dopamine‐mediated maximal activation of AC in relation to wild‐types. We speculate that this observation is attributed to an impairment in dopamine‐mediated internalization by PD1 mutants. Taken together, our study has demonstrated that IL1 can modulate ligand binding, G protein‐coupling, and possibly receptor trafficking, poising this region as an important structural determinant for D1‐class receptors. This research was funded by NSERC (grant #203694–2010).
Targeted delivery of Kv1.3 siRNAs with functionalized lipid nanoparticles
Authors: Hajdu Peter , Thompson Tyler , Yun Yeoheung , Conforti Laura
Published: June 2021

Hyperactive effector memory T cells (TEM) play a key role in cardiovascular complications of the autoimmune disease systemic lupus erythematosus (SLE). SLE TEM's have higher intracellular Ca2+ level, which contributes to their hyperactivity. Kv1.3 channels facilitate Ca2+ influx in T cells. Therefore silencing the Kv1.3 gene selectively in TEM's can be used as novel immunosuppressive therapy. Here we demonstrate that lipid nanoparticles (NPs) can be used to selectively deliver Kv1.3 siRNAs into TEM cells. Specificity and efficacy of Kv1.3 siRNAs was determined by RT‐qPCR and electrophysiology. NPs were made from a mixture of phosphatidylcholine, phosphoethanolamine‐PEG2000‐biotin and cholesterol. NPs were functionalized with inactive biotin‐CD45RO (TEM marker) antibodies via fluorophore‐conjugated streptavidin. Human T and MOLT‐4 (TEM cell line) cells were used to test the specific binding of NPs to TEM cells. NPs (ca. 100 nm) were characterized by dynamic light scattering and fluorescent/electron microscopy. Incubation of T/MOLT‐4 cells with CD45RO‐coated NPs resulted into the selective attachment and endocytosis of the NPs into TEM's via the internalization of the membrane CD45RO tyrosine kinase. Consequently, the Cy3‐labeled siRNA encapsulated into the NPs was released into the cytosol. These finding set the foundation for a novel therapeutic approach in autoimmunity. (NIH R21AR060966)
Amphetamine and methamphetamine differentially regulate biophysical properties of dopamine transporter
Authors: Saha Kaustuv , Villarroel Laura , Davari Paran , Mehr Shahram Ejtemaei , Zarindast M R , Richardson Ben , Khoshbouei Habibeh
Published: June 2021

Dopamine transporter (DAT) substrates, amphetamine (AMPH) & methamphetamine (METH), compete with dopamine (DA) uptake & induce DA reverse transport via DAT albeit by poorly understood mechanism. Whole‐cell recordings of cells overexpressing DAT indicate that small differences in chemical structure or chirality of substrates influence the nature of DAT‐mediated currents. D‐AMPH elicits a DAT‐mediated persistent depolarizing current larger than that of the racemic mixture of METH (S(+), R(‐) isomers) or R(‐)‐METH alone. Extracellular S(+)‐METH elicits larger DAT currents than R(‐)‐METH or the racemic mixture. Thus, R(‐)‐METH may be a DAT partial agonist which decrease DAT current elicited by S(+)‐METH, the more effective stereoisomer (or full agonist). Ongoing experiments examine whether drug access to the internal DAT binding site is required to elicit a response or whether simultaneous or stepwise drug access to the internally accessible binding site, in addition to an externally accessible DAT binding site, is required for maximum response. Via recording pipette, mouse DA neurons will be directly dialyzed with these compounds or their stereoisomers while measuring DAT current & firing activity. Funding was provided by DA026947/DA/NIDA and NS071122/NS/NINDS
Putative pain‐like states alter the discriminative stimulus effects of morphine in mice dependent on sex
Authors: Neelakantan Harshini , Ward Sara Jane , Walker Ellen A
Published: June 2021

Sex‐differences in pain sensitivity, opioid analgesia, and reinforcing effects of opioids are observed in rodents. The purpose of our study is to test the hypothesis that the presence of an acute noxious stimulus and/or chemotherapy‐induced chronic neuropathy will reduce the potency of morphine to produce discriminative stimulus effects in mice. Saline and morphine (3.2 mg/kg) were established as discriminative stimuli for food‐reinforced responses using a two‐choice operant training procedure in male and female C57Bl6 mice. After obtaining morphine dose‐response curves, mice were tested with acetic acid (0.4% conc.)+ morphine combinations to determine the effects of noxious stimulus on morphine potency. Next, mice were administered with saline or the chemotherapeutic agent paclitaxel (8 mg/kg) on days 1, 3, 5, and 7 while training was suspended for a week. Morphine dose‐curves were re‐established between days 8–14 post‐paclitaxel treatment (period of peak allodynia). The presence of an acute noxious stimulus and chemotherapy‐induced chronic neuropathy significantly decreased the potency of morphine to function as a discriminative cue in male but not in female mice suggesting a differential modulation of the stimulus effects of morphine in the presence of pain between sexes. Overall, these results may have implications for the understanding of potential sex differences in the clinical management of pain and abuse liability of prescription opioids in humans. (Supported by R01 CA129092).
Antinociceptive effects of α7 nicotinic acetylcholine receptor positive allosteric modulators type I and II in models of acute and chronic pain in mice
Authors: Freitas Kelen , Negus S Stevens , Carroll F Ivy , Damaj M Imad
Published: June 2021

Positive allosteric modulators (PAMs) of the α7 nicotinic acetylcholine receptor (nAChR) have been divided into Type I PAMs (little or no effect on agonist‐induced desensitization) and Type II PAMs (delayed desensitization). This study compared antinociception produced by intraperitoneal delivery of the Type I PAM NS‐1738 and the Type II PAM PNU‐120596 in adult male ICR mice using assays of (1) paw licking induced in two phases by intraplantar formalin, (2) thermal hyperalgesia induced by intraplantar carrageenan, and (3) mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve. Antinociceptive interactions between PNU‐120596 and intrathecal delivery of the endogenous α7 agonist choline in the formalin test were also examined with isobolographic analysis.

Results

NS‐1738 reduced formalin‐induced licking only in the first phase, whereas PNU‐120596 and choline decreased licking in both phases. Furthermore, PNU‐120596 potentiated choline's effect synergistically. NS‐1738 was partially antihyperalgesic in the carrageenan model but had no effect in the CCI model, whereas PNU‐120596 was efficacious in both models.

Conclusion

These results suggest that Type II α7 nAChR PAMs may produce antinociception across a broader range of conditions than Type I PAMs. Type II PAM antinociception may be mediated by a synergistic interaction with endogenous spinal choline. Funded by NIDA.
Selective effects of a morphine conjugate vaccine on heroin and metabolite distribution and heroin‐induced behaviors in rats
Authors: Raleigh Michael D , Pravetoni Marco , Harris Andrew C , Birnbaum Angela K , Pentel Paul R
Published: June 2021

Morphine conjugate vaccines attenuate the behavioral effects of heroin in rodents and primates. To better understand how these effects are mediated, heroin and metabolite distribution studies were performed in rats vaccinated with a morphine conjugate vaccine. In non‐vaccinated control rats 6‐monoacetylmorphine (6‐ MAM) was the predominant opioid in plasma and brain as early as 1 minute after i.v. administration of heroin and for up to 14 minutes. Four minutes after heroin administration vaccinated rats showed substantial retention of all three opioids in plasma compared to controls, and reduced 6‐MAM and morphine, but not heroin, distribution to brain. Vaccination reduced heroin‐induced analgesia and blocked heroin‐induced locomotor activity throughout 2 weeks of repeated testing. Higher serum opioid‐specific antibody concentrations were associated with higher plasma opioid concentrations, lower brain 6‐MAM and morphine concentrations, and lower heroin‐induced locomotor activity. Serum antibody concentrations over 0.2 mg/ml were associated with substantial effects on these measures. These data support a critical role for 6‐MAM in mediating the early effects of i.v. heroin and suggest that reducing 6‐MAM concentration in brain is essential to the efficacy of morphine conjugate vaccines. Funded by NIDA [Grants DA026300, DA030715 and T32‐DA07097].
Perivascular macrophages mediate endothelium dysfunction in the middle cerebral artery of hypertensive rats
Authors: Pires Paulo Wagner , Girgla Saavia Singh , Lee McClain Jonathon , VanRooijen Nico , Dorrance Anne McLaren
Published: June 2021

Hypertension increases macrophages (MΦ) around the cerebral arteries. These MΦ may release mediators that could modulate endothelial function. We hypothesized that MΦ depletion would improve middle cerebral artery (MCA) endothelial function in stroke‐prone spontaneously hypertensive rats (SHRSP). Liposome‐encapsulated clodronate (CLOD) was used to deplete MΦ, control rats received PBS containing liposomes; rats were treated from 6–12 weeks of age. Data are mean±SEM, PBS vs CLOD. Dilation data is shown for the highest concentration of the dilator. SHRSP+CLOD had less perivascular MΦ around the cerebral arteries (0.7±0.1 vs 0.3±0.1, n=5, p<0.01). Pressurized MCAs from SHRSP+CLOD showed improved dilation to ADP (change from baseline: 32.8±3.4 vs 50.8±9.7μm, n=6, p<0.05). Endothelial function was further assessed with acetylcholine (Ach)±N‐nitro‐L‐arginine methyl ester (L‐NAME) by wire myography (n=7). MCAs from SHRSP+CLOD had improved dilation to Ach (%KCl constriction: 64.8±5.0 vs 51.8±4.4, p<0.05), which was blunted by L‐NAME (%KCl constriction: 80.8±7.6 vs 79.8±5.0). Endothelium‐independent dilation to sodium nitroprusside was unchanged (%KCl constriction: 39.4±3.2 vs 43.1±2.1). These data suggest that perivascular MΦ might reduce endothelial nitric oxide generation in MCAs from SHRSP.
Absence of Mrp2 leads to differences in severity and pattern of mercury nephrotoxicity in mice
Authors: Zalups Rudolfs K , Joshee Lucy , Bridges Christy C
Published: June 2021

The multidrug‐resistance associated protein 2 (Mrp2) is located on the luminal membrane of proximal tubular epithelial cells and has been shown to mediate the export of numerous endobiotics and xenobiotics such as mercury. Although Mrp2 is present in all proximal tubular cells, there is axial heterogeneity in the magnitude of expression along the three segments of the proximal tubule. The purpose of the current study was to assess differences in renal injury and disposition of mercury between Mrp2−/− and FVB mice following a toxic dose of mercuric chloride (HgCl2). Mrp2−/− and FVB mice were injected intraperitoneally with doses of HgCl2 ranging from 18 – 30 μmol/kg. Mice were sacrificed after 24 h and organs were harvested. The renal burden of mercury was significantly greater in Mrp2−/− mice than in FVB mice. Significant differences in pattern and severity of mercury‐induced injury were observed between Mrp2−/− and FVB mice, with the most severe changes observed in Mrp2−/−mice. Histological analyses showed that degenerative change and necrosis occurred as low as 18.5 μmol/kg HgCl2 and the severity of these changes increased as the dose of HgCl2 increased. Our results suggest that the activity of Mrp2 is an important factor in the disposition and nephrotoxicity of HgCl2. This work was supported by NIH ES019991–01 (NIEHS) awarded to CC Bridges.