bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of BMP6 in the Regulation of Hepcidin By Iron
Authors: Ramos Emilio
Published: June 2021

Normally, hepcidin synthesis is increased in response to iron loading to limit further absorption of dietary iron and its release from stores. Mutations in HFE, TfR2 or HJV cause hepcidin insufficiency which results in the inability to downregulate the absorption of dietary iron, and eventually leads to iron overload. In order to understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin responsiveness to short and long‐term iron challenge in HFE, TfR2 and HJV mutant mouse models. While HFE mutants partially increased hepcidin expression after acute iron challenge, both TfR2 and HJV mutants showed no response. After chronic iron challenge, both HFE and TfR2 mutants increased hepcidin expression to near WT levels, but the hepcidin expression in HJV mutants remained severely impaired. We also analyzed BMP6 expression and show that BMP6 upregulation by iron is independent of HFE, TfR2 and HJV. Because of the different roles HFE, TfR2 and HJV play in the hepcidin response to short and long term iron challenges, we propose that there are two independent signals by which iron regulates hepcidin expression. The first is most likely mediated by iron‐transferrin and acts through HFE, TfR2 and HJV, while the long term signal may depend on intracellular iron concentration and requires HJV.
Effect of Cold Water Extract from Luffa cylindrical on Intracellular Triglyceride and Free Glycerol Release Level in Lipolysis of Differentiated 3T3‐L1 Adipocytes
Authors: Yang Jiwon , Jang Ja‐Young , Kim Ok‐Kyung , Lee Jeongmin , Lee Jeongmin , Jun Woo‐Jin , Hwang Kwon‐Tack
Published: June 2021

The purpose of this study was to investigate the inhibitory effect of Luffa cylindrical cold water freaction(LCC) on intracellular triglyceride and free glycerol release level in lipolysis of differentiated 3T3‐L1 adipocyte. MTT (Methylthiazolyldiphenyl‐tetrazolium bromide) was used to detect the cytotoxic effects of Luffa cylindrical on the viability of 3T3‐L1 cells. Differentiated 3T3‐L1 cells were monitored 80% ethanol, 100% methanol, hot water and cold water fraction from Luffa cylindrical by Oil Red O staining and observed by microscope. Fraction of cold water among them was the most effective in reduction lipid accumulation among four fractions. The treatment of LCC at concentration of 200~1000 ug/mL inhibited the lipid accumulation. These were induced free glycerol release and intracellular triglyceride. These results showed that following treatment LCC was elevated by stimulating lipolysis of differentiated 3T3‐L1 adipocyte. In conclusion, the use of LCC was the most effective in the induction of lipolysis and inhibition of lipid accumulation in 3T3‐L1 cells.
Generation of an intestinal‐specific hephaestin knockout mouse
Authors: Fuqua Brie Katherine , Wolkow Natalie , Bell Austin G. , Hsu JoAnn , Nguyen Michelle P. , Yu Catherine C. , Dunaief Joshua L. , Vulpe Chris D.
Published: June 2021

Hephaestin (Heph) is a multicopper ferroxidase (MCF) important for the export of inorganic dietary iron from vertebrate intestinal cells to the blood. The gene is mutated in the sex‐linked anemia (sla) mouse and results in severe iron deficiency. However, sla mice still retain some hephaestin ferroxidase activity, and they survive, breed, and their anemia improves with age. In order to clarify the role of Heph in intestinal iron absorption, we have used the cre‐lox system to generate a knockout mouse model with intestinal ablation of Heph. The knockout mice are small and anemic compared to wild‐type littermates, and mice born to knockout mothers exhibit truncal hair loss that improves after weaning. The viability of these mice supports the hypothesis that other mechanisms exist, MCF‐dependent or not, for iron acquisition. These mice will serve as a valuable tool to study the role of hephaestin and other proteins involved in intestinal iron transport. Support: NIH (CDV, JLD), NSF (BKF)
Production and nutritional characteristics of extract from fermented brown rice
Authors: Matsuo Tatsuhiro , Shiomi Saho , Higaki Shunsuke , Kumazaki Takahito , Inai Reiko
Published: June 2021

The extract from brown rice (ER) fermented by Aspergillus Oryzae is recognized as a functional food in Japan. Dried ER is expected to be usefull as a dietary supplement because it contains the following nutritional ingredients, in grams per 100 grams of ER: protein, 39; carbohydrate, 19; fiber, 20; free amino acids, 3; nucleic acids, 4; and other trace elements. We studied the effects of dietary ER on the maturity and digestive function of the small intestine in growing rats. Twenty‐seven male Wistar rats (3 weeks old) were equally divided into three groups: control (C), nucleic acid (NA) and ER groups. The NA and ER groups were both fed diets containing 5g of nucleic acids per kilogram diet, but the NA diet contained yeast RNA and salmon sperm DNA. The three groups of rats had the same body weight gain and food intake. The liver weight was significantly lower in the NA group than in the C group. The lengths of the jejunum and ileum were significantly greater in the ER group than in the NA group. The mucosal weight of the small intestine was significantly greater in the ER group than the other groups. However, the villous height, crypt depth and wall thickness in the ileum did not differ among the three groups. These results suggest that ER in the diet increases the growth and maturity of the rat digestive tract. The functional elements in ER may not be only nucleic acids but may also include other macro and micronutrients.
Effects of a high vs. low carbohydrate diet on fat and carbohydrate oxidation in cats
Authors: Shoveller Anna Kate , Flickinger Elizabeth A. , Atkinson Jim L. , Duncan Ian J.H. , Gooding Margaret A.
Published: June 2021

Low or high carbohydrate (CHO) diets (18.9 and 46.3%, LC and HC, respectively) and length of adaptation were investigated in cats (Felis catus; 10 ± 1 mo, 3 ± 1 kg). Cats received each treatment for 14 d in a crossover design and 20 h indirect calorimetry studies were conducted after acute (A; d 0), semi‐chronic (SC; d 4) and chronic (C; d 13) dietary exposure. Data were analyzed by repeated measures ANOVA and Proc Mixed.

When cats consumed the HC and LC diet, Ox of the restricted nutrient exceeded intake while Ox of the nutrient in excess matched intake. Mean max energy expenditure (EE) of cats consuming the LC and HC diet were 107 and 102 kcal/kg0.67/d and occurred at a mean of 245 and 712 min post feeding, respectfully. Mean max fat (0.90 g/h) and CHO (1.42 g/h) Ox were attained at 26 and 626 min post feeding, respectfully. The changes in Ox and EE suggest that cats adapt to changes in dietary nutrient content, but may require longer than 14 d to adapt to restricted nutrient intake. Supported by: The Procter and Gamble Co., Pet Care, Ohio, USA
Additive Inhibitory Effects of Tart Cherry Anthocyanins and Atorvastatin on Interleukin‐6 Production in Adipose Stem Cells
Authors: Claycombe Kate J , Zhou Zhou , Hsueh Hui Wen , Schroeder Nicholas , Nair Muraleedharan G. , Moustaid‐Moussa Naima
Published: June 2021

Studies have shown positive correlations between inflammatory cytokines interleukin‐6 (IL‐6) and the development of chronic diseases including cardiovascular disease. The objective of our study was to investigate effects of tart cherry extract, a mixture of tart cherry anthocyanins, and an isolated tart cherry anthocyanin in reducing IL‐6. Since we have previously shown that adipose tissue stem cells secrete higher levels of IL‐6 when compared to adipocytes and macrophages, we investigated the effect of tart cherry extract in primary adipose stem cells isolated from obese, leptin deficient ob/ob mice. Since atorvastatin calcium (lipitor) has been independently shown to reduce inflammatory responses, using in vitro stem cell cultures, we also tested the additive effects of tart cherry and lipitor on IL‐6 secretion. Results showed that LPS‐induced adipose stem cell secretion of IL‐6 is reduced with the tart cherry extract, a mixture of tart cherry anthocyanins, and tart cherry cyanidin‐3‐O‐glucoside (C3G) in a dose‐dependent manner. In addition, lipitor added together with tart cherry extract and tart cherry anthocyanins mixture exhibited additive inhibitory effects. These results support potential benefits of using dietary phytochemicals in conjunction with pharmacological therapies to decrease adipose inflammation, drug doses, and ultimately, drug‐induced adverse effects.
Positive association of total protein intake and bone mineral density (BMD) in women from the Framingham Offspring Study
Authors: Sahni Shivani , Broe Kerry E , Tucker Katherine L , McLean Robert R , Kiel Douglas P , Cupples L Adrienne , Hannan Marian T
Published: June 2021

Objective

The effect of protein on bone is controversial; further calcium intake (CA) may modify protein's effect. We evaluated the cross‐sectional association of % total energy from protein intake (TP%) with bone mineral density (BMD) at femoral neck (FN), trochanter (TR) and L2‐L4 spine (LS) in the Framingham Offspring Study, also whether CA modified any association.

Methods

1639 women and 1280 men completed a food frequency questionnaire and had BMD measured in 1991–95 or 1995–98. We used multivariable linear regression to examine the association of TP% with each BMD, adjusting for covariates.

Results

Mean age was 60 years (SD:9.3) in women and 61 years (SD:9.4) in men. In women, positive associations were seen between TP% and each BMD [FN: β=0.0012(SE=0.0009), P=0.04, TR: β=0.0002(SE=0.0008), P=0.01, LS: β=0.002(SE=0.001), P=0.04]. No associations were seen in men (P range: 0.28–0.72). We stratified (a priori) by CA: women with <800mg/d CA showed strong associations [FN: β=0.002(SE=0.001), P=0.07, TR: β =0.002(SE=0.0009), P=0.04; LS: β=0.003(SE=0.001), P=0.07]. In women with ≥ 800mg/d CA the positive associations were not significant (P range: 0.27–0.42). No associations were seen in men (low CA group: P range= 0.15–0.50; high CA group: P range= 0.43–0.91).

Conclusion

In this study higher protein intake was positively associated with BMD at multiple sites in middle aged women but not men. Longitudinal studies are needed especially to examine the role of CA in these associations.

Grant Funding Source: NIH R01 AR053205, NIH R01 AR/AG 41398; and N01‐HC‐25195
Lactose intolerance in adult celiac patients from north India.
Authors: Rana S.V. , Sharma S. , Ola R.P. , Kaur J , Sinha S.K. , Singh K.
Published: June 2021

Celiac disease is common in North India. Diarrhea and flatulence are common symptoms in these patients. Lactose intolerance may be one of the reasons of diarrhea & flatulence in celiac patients. This study was planned to find out the incidence of lactose intolerance in celiac disease patients and apparently healthy controls. 42 adult confirmed celiac patients were enrolled for this study. Celiac disease was confirmed by anti‐tissue transglutaminase (ttgA) measured by ELISA kit. 50 age sex matched apparently healthy controls were also enrolled. Non‐invasive lactose hydrogen breath test was done in all subjects after over night fast. Hydrogen concentration was measured by using SC Microlyzer from Quintron, USA. Out of 42 adult celiac patients, 20 were females and 22 were males while out of 50 healthy controls, 23 were females and 27 males. 28 out of 42 (66.6%) celiac patients were lactose intolerant, while in controls 21out of 50 (42.4 %) were lactose intolerant. This study suggests that lactose intolerance was significantly higher in adult celiac patients as compared to healthy controls. This work has been financially supported by the Department of Gastroenterology, PGIMER, Chandigarh, India,
Decursin, decursinol angelate decursinol inhibit pro‐inflammatory cytokine expression on β‐amyloid peptide‐induced THP‐1 cells
Authors: Li Wei , Li Li , Park Sung‐ki , Jung Sang‐won , Kim Yong‐ho
Published: June 2021

One of the characteristic pathological features of Alzheimer disease(AD) is the inflammatory response associated with extracellular deposition of β‐Amyloid Peptide(Aβ). The most important landmark of inflammation in AD is the pro‐inflammatory cytokines, especially IL‐1β. Decursin, decursinol angelate and decursinol are the three main coumarin components purified from Angelica gigas Nakai. The monocytic THP‐1 cells and the THP‐1 derived macrophage cells were pre‐treated with 20μM, 40μM and 60μM of the three kinds of components respectively for 3 hrs, and then treated with 125nM Aβ for 12 hrs. Without cytotoxicity, pre‐incubation with decursin, decursinol angelate or decursinol has elevated the cell viability compared with the Aβ group in lactate dehydrogenase(LDH) cell viability assay. Also the three components have blocked the pro‐inflammatory cytokine IL‐1β production in western blot with a time‐dependent and dose‐dependent manner in both monocytes and macrophages, respectively. The results of these studies suggested that the three coumarin compounds isolated from Angelica gigas Nakai have great anti‐inflammatory potential, and they could be considered as therapeutic reagents in the Alzheimer disease therapy.

This research is supported by Biohealth Products Research Center(BPRC) of Inje University.
SOY PEPTIDES ACTIVATE LDL‐R: IN VITRO EXPERIMENTS AND DOCKING DATA
Authors: Lovati Maria Rosa , Manzoni Cristina , Parolari Anna , Daniels Jessica , Hecht David , Buehler Lukas , Bakhiet Raga
Published: June 2021

The alpha prime region of the soybean 7S globulin subunit has been associated with a decrease in cholesterol both in in vitro and in vivo studies. One possible mechanism of action could be the binding of the protein or its digested fragments either to the LDL receptor itself or to proteins associated with regulation of LDL receptor sequestering and recycling, resulting in enhanced fusion of the LDL receptor storage vesicles with the cell surface. These regulatory proteins include proprotein convertase subtilisin‐like/kexin type 9 (PCSK9). We found that synthetic peptides from 7S soy globulin such as HKQEKHQGKE, NPDNDE and ENLIKSQSES exerted a statistically significant up‐regulation (+ 43%, +54% and +48% at concentration of 10−5M, respectively: P<0.05) of LDL receptors in Hep G2 cells vs controls. In order to explore the hypothesis that the LDL‐R regulation is due to an interaction of these peptides with the regulatory proteins, as above mentioned, molecular modeling experiments (e.g., protein‐ligand docking) were performed using publicly available x‐ray crystal structures of the LDL receptor, PCSK9. Analysis of potential protein ligand interaction generated from these docking studies identified NPDNDE as potentially having a higher binding affinity to PCSK9 than the other peptides. This is consistent with experimentally generated activity data.
Infant growth chart information influences parental feeding practices among the WIC population.
Authors: Mathai Rose Ann , Shim Jae Eun , Kim Juhee
Published: June 2021

The objective of this research is to determine whether infant's growth chart information affects parental feeding practices. Parent‐infant dyads were recruited at the WIC office in Champaign, IL with the infant ages of 2–5 months (n=26). Parent perception of their child's growth was determined by a survey question “How much does the growth chart information influence your feeding practice?” Parent feeding practices were assessed by a 3 day food record. Approximately, 19% infants were exclusive breastfed, 61% formula fed, 7.7% fed water, 7% juice, and 42% were already introduced to solids contrary to the current infant feeding recommendations. About 42% of parents reported that child's growth chart information influenced their feeding practices. In the influenced group, daily total and complementary feeding frequencies were 5.33 ± 1.68 and 0.17 ±0.53 times/day, respectively. Infant food consumption pattern of this group was composed of 40% breastmilk, 80% formula, 10% water, 0% juice, and 10% solids. In the non‐influenced group, daily total and complementary feeding frequencies were 6.25 ± 1.22 and 1.0 ± 1.97 times/day, respectively. Infant food consumption of the latter group was composed of 14% breastmilk, 86% formula, 7% water, 7% juice, and 36% solids. Thus, growth chart information could potentially affect infant feeding practices and in consequence the growth of children. Funded by CFAR & UIUC Research Board
Nine‐day weight loss program with high protein shakes supplemented with herbal beverage in dry mix powder or liquid form on fasting days leads to healthy weight loss
Authors: Balliett Mary , Burke Jeanmarie R , Rasmussen Oscar , Rockway Susie
Published: June 2021

Aim

To evaluate a nine‐day weight‐loss program consisting of fasting days supplemented with a novel herbal beverage in dry powder mix or ready‐to‐drink liquid form on body composition and blood parameters in healthy adults.

Design

The study randomized 42 healthy adults to one of two groups. Group 1 received herbal beverage from powder mix while Group 2 received ready‐to‐drink liquid. Both groups fasted with 64 oz of water and 16 oz of herbal beverage in two‐day intervals separated by five non‐fasted days in which subjects ate one meal and replaced two others with a meal‐replacement high‐protein shake. Measurements and blood were taken days 0 and 10.

Statistics

ANOVA for repeated measures was performed initially; however, no differences between groups were detected for any measure, thus, data for both groups were pooled.

Results

The average weight loss of subjects was 6.6 lbs (3.8% of initial body weight) and was statistically significant (p<0.05) from baseline weight (174 ± 42). Waist and hip circumference decreased significantly by 0.6 and 0.7 inches, respectively, from baseline. Both lean and fat body mass decreased by 3.2 and 2.9 lbs, respectively. Total‐, LDL‐ cholesterol and TG decreased significantly by 17.8, 8.3 mg/dl and 25.6 mg/dL, respectively. Subjects lost weight and felt better about their body weight and energy level.

Products and funding were provided by Isagenix International, LLC.
Impact of 2‐week Oral Consumption of Gynostemma pentaphyllum (Jiaogulan)‐containing Liquid Dietary Supplement, JULE of the Orient™ on Various Subjective Symptoms, Blood Circulation, Blood Pressure and Other Anthropometric Parameters: A Randomized, Placebo‐controlled, Double‐blind Human Clinical Study
Authors: Amagase Harunobu , Nance Dwight
Published: June 2021

A randomized, double‐blind, placebo‐controlled human clinical trial has examined the general effects of orally consumed saponin‐standardized Gynostemma pentaphyllum (jiaogulan)‐containing herbal tonic named JULE of the Orient™ (JULE). JULE or a placebo was provided as a beverage to healthy adults for 14 days (120 ml/day). Subjective questionnaire ratings (0–5) were used to measure general feelings of well‐being, neurological/psychological traits, gastrointestinal, musculoskeletal and cardiovascular complaints and any adverse effects. Blood circulation in hands was measured using laser Doppler. Questionnaire data were analyzed between day 1 and day 15 with the nonparametric Wilcoxon matched pairs tests. Significant differences were found in the JULE group (N=12) and consisted of decreased ratings for tiredness, procrastination, stiff shoulder, heartburn, physical discomfort, vision issues, stress, memory loss, daydream and increased stamina. Blood circulation in JULE group tended to be increased by about 30% compared to the pre‐intervention. In contrast, the placebo group (N=12) showed no significant changes. All other parametric data, such as body weight, BMI, blood pressure were not significantly different. These results suggest that JULE intake may increase subjective feelings of general well‐being and neurological/psychological traits by enhancing blood circulation.
Intestinal Expression of Osteopontin and Genes in the Wnt and Bone Morphogenic Pathways Differ between Sow‐Reared and Formula‐fed Piglets with no Effect of Prebiotic Supplementation
Authors: Bares Allyson Leigh , Radlowski Emily C. , Monaco Marcia H. , Wang Mei , Comstock Sarah S. , Donovan Sharon M.
Published: June 2021

Intestinal stem cells are regulated through the Wnt/β‐catenin/Bone Morphogenic Pathway (BMP) signaling web. It is speculated that prebiotics replicate the activity of breastmilk oligosaccharides. We hypothesized that supplementation of prebiotics would align mRNA expression of genes in the Wnt and BMP pathways in piglet intestine with that seen in sow‐reared piglets. Piglets (n=5/group) were sow‐reared (SR) or fed formula without (F) or with (F+P) fructooligosaccharides (2 g/L) and polydextrose (2 g/L). Ileum and colon were collected at 7 and 14 days of age, and β‐catenin and frizzled 6 (Wnt pathway), gremlin (BMP pathway), TNF‐α and osteopontin (OPN) mRNA expression were measured by RT‐qPCR. In the d7 ileum, no differences were observed for any gene. On d14, OPN was higher (p<0.05) and gremlin was lower in SR piglets than in F or F+P. In the d7 colon, OPN expression was lower (p<0.05) and frizzled‐6 expression was higher (p<0.05) in the SR piglets than F or F+P. On d14, no differences were observed for any gene in the colon; however, OPN tended (p=0.08) to be greater in SR piglets. In summary, differences in the expression of frizzled‐6, gremlin and OPN, a regulator of cell proliferation and immune function, were observed between SR and formula‐fed piglets with no effect of supplemental 4 g/L scFOS and PDX. (BMS Freedom to Discover Award)
Combination effect of triol‐ and diol‐enriched fraction of ginseng extract on nitric oxide production in vitro
Authors: Hong Soyoung , Kim Ji Yeon , Ok Elly , Kim Mi Kyung , Kwon Oran
Published: June 2021

This study was designed to evaluate the effect of triol‐ and diol‐enriched fraction of ginseng extract on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVEC). We investigated the effect of these fractions on NO production at different concentrations (triol‐enriched fraction 240 μg/ml with diol‐enriched fraction 0, 30, 60, 90, 120, or 480 μg/ml). The triol‐enriched fraction significantly increased NO production in HUVEC compared with control. On the other hands, diol‐enriched fraction did not affect NO production. We determined NO production at different time points (5, 10, 30, 60, 120, 240, 360min and 24hrs) with triol‐enriched fraction. The ordinate is the ratio of treated to non‐treatment control cell at each time point. NO production at 30 min provided the highest production rate. We examined whether the triol‐enriched fraction regulates the phosphorylation of Akt as determined by Western blot analysis. Treatment of HUVEC with triol‐enriched fraction increased Akt phosphorylation in dose‐dependent manners. Although these findings suggest that triol‐enriched fraction is likely to have beneficial effects on increasing NO production, further study will be required to understand the molecular and biochemical mechanisms for NO signaling cascade.*This work was supported by the second stage of Brain Korea 21 project and grants from CJ Co., Ltd.
trans‐10,cis‐12 CLA suppresses osteosarcoma cells via phosphoinositide 3‐kinase pathway
Authors: Wong Janice Y , Park Yooheon , Lee Seong‐Ho , Baek Seung J , Park Yeonhwa
Published: June 2021

Cancer is one of the leading causes of death in the United States, second only to the heart disease. Osteosarcoma is one of the most common and aggressive types of cancer in adolescents and children. Conjugated linoleic acid (CLA), cis‐9,trans‐11 and trans‐10,cis‐12, have shown isomer dependent anticancer properties in in vitro and in vivo studies. Previously, CLA, particularly the trans‐10,cis‐12 isomer, was shown to reduce tumor development and apoptosis in a canine osteosarcoma model, CCL‐183, via non‐steroidal anti‐inflammatory drug‐activated gene 1 (NAG‐1). Thus, the purpose of the current investigation was to determine the specific apoptotic pathway induced by NAG‐1 mediated effects of the trans‐10, cis‐12 CLA isomer. Major cell proliferation protein enzymes, phosphoinositide 3‐kinases (PI3K), c‐Jun N‐terminal kinases (JNKs), Protein kinase C (PKC), and extracellular signal‐regulated kinases/MAP kinase (ERK/MAPs), all of which act as control point(s) of extrinsic apoptosis pathway, were tested. The trans‐10,cis‐12 CLA isomer effectively suppressed the tumor cell growth after 72 hour treatment via phosphoinositide 3‐kinase (PI3K), which was associated with the expression of NAG‐1. Neither apoptotic effect nor NAG‐1 expression were found in the other three kinases or other fatty acids tested. These results confirm that anticancer effects of the trans‐10,cis‐12 CLA isomer on osteosarcomas are via a PI‐3 kinase pathway.
Insights into DNA damage signaling from the structure of an Mre11:Nbs1 complex
Authors: Hopfner Karl‐Peter
Published: June 2021

DNA double‐strand breaks (DSBs) are highly cytotoxic, can lead to cancerogenic chromosome aberrations and induce a complex cellular response including DSB signaling, DSB repair or apoptosis. The multifunctional Mre11‐Rad50‐Nbs1 (MRN) complex senses and helps to repair DSBs, but also initiates DSB signaling by recruiting and activating the kinase ataxia‐telangiectasia mutated (ATM). To reveal how MRN initiates DSB signaling, we determined crystal structures of the Schizosaccharomyces pombe Mre11 nuclease dimer and its complex with the Mre11 interacting region of Nbs1. Two Nbs1 polypeptides bind to one Mre11 dimer by stretching around the outsides of its nuclease domains. Remarkably, one of the two Nbs1 additionally binds asymmetrically across the Mre11 dimer interface, opposite the DNA binding groove. This interaction is mediated by highly conserved motifs on both Mre11 and Nbs1 and includes insertion of an Nbs1 phenylalanine between asparagines from each Mre11 protomer. These prominent asparagines are mutated in A‐T like disease suggesting that Nbs1 dependent Mre11 dimer bridging is critical for DSB signaling. Comparison of the Mre11‐Nbs1 complex with our structure of S. pombe Mre11 alone and with previously reported DNA bound archaeal Mre11 suggests a plausible mechanism for DSB signaling.
Modulation of Cardiac Metabolism by Beta‐Blockers During Diabetes: A Role in Apoptosis Signaling
Authors: Saran Varun V V , Sharma Vijay , Yuen Violet , Allard Michael F , McNeill John H
Published: June 2021

Diabetic Cardiomyopathy is characterized by problems during diastole, this is due to loss of contractile tissue after apoptosis. Apoptosis may be caused by increases in oxidative stress associated with metabolic modifications.

Beta‐adrenergic receptor antagonists, (β‐blockers) improve heart function. Metoprolol (met) and carvedilol (car) are clinically important β‐blockers that modulate metabolism and reduce apoptosis, car, also has antioxidant properties. We tested whether β‐blockers will reduce apoptosis and improve heart function via oxidative stress dependant and or independent pathways.

We employed a Streptozotocin (STZ) induced rat model of type 1 diabetes. This model has been shown to develop diastolic dysfunction. STZ was delivered at 60mg/kg body weight. Met and car were delivered at a rate of 15 and 10mg/kg/day respectively. In order to assess the significance of car's antioxidant abilities, we included groups where met treatment was supplemented with vitamin C at 1000mg/kg/day.

Analysis indicates induction of diabetes in STZ animals. β‐blocker treatment caused a significant reduction in heart rates. Remaining analysis includes assessment of in vivo and ex vivo heart function, metabolic flux, apoptosis, expression analysis of effecter proteins involved in apoptosis, and measurement of oxidative stress.

Funding for this research was provided by the Canadian Institutes of Health Research
Synchrotron radiation can induce apoptosis in the hippocampus of mouse brains
Authors: Liu Na , He Xin , Wu Zhouyi , Han Jin , Shao Jiaxiang , Shi Shengtao , Chen Chen , Chen Heyu , Xia Weiliang , Ying Weihai
Published: June 2021

Synchrotron radiation(SR)‐based medical imaging has multiple unique merits compared with existing medical imaging technologies. It is of critical importance to establish the safety standards for clinical applications of SR‐based medical imaging, regarding which there has been only very limited information. In this study we assessed the effects of SR on mouse brains under the same experimental conditions as that used for SR‐based brain imaging, the energy of which is approximately 32 keV. We found that SR on the brain over 5 min can induce significant abnormalities of the behaviors of the mice. Three days after the radiation 80% of the mice exposed to 15‐min SR were died, while all of the mice exposed to 10‐min SR survived. We also applied TUNEL assay to show that SR can induce apoptosis in mouse brains, which increases with the increase in SR exposure time. Notably, in the hippocampus there were particularly significant increases in cell apoptosis. Our study has also suggested increased inflammatory reactions in the SR‐exposed brains. Collectively, our study provides first evidence suggesting that SR may induce apoptosis and inflammation in the brain, and hippocampus is particularly susceptible to synchrotron radiation. (Supported by a National Key Basic Research 973 Grant #2010CB834306 (to WY and WX) and a Pujiang Scholar Program Award (to WY)).
Bacterial lipoproteins can disseminate from the periphery to inflame the brain
Authors: Londono Diana , Cadavid Diego
Published: June 2021

The current view is that bacteria need to enter the brain to cause inflammation. However, in mice infected with Borrelia turicatae we observed widespread cerebral inflammation despite a paucity of spirochetes in the brain parenchyma at times of high bacteremia. Here we studied the possibility that bacterial lipoproteins can disseminate from the periphery across the blood‐brain barrier to inflame the brain. For this we injected normal and infected mice intraperitoneally with lanthanide‐labeled variable outer membrane lipoproteins of B. turicatae and measured their localization in blood, various peripheral organs, and whole and capillary‐depleted brain at various times. Lanthanide‐labeled non‐lipidated lipoproteins and albumin were used as controls. Brain inflammation was measured by TaqMan of genes known to be upregulated in response to borrelial infection. The results showed that the two lipoproteins we studied, LVsp1 and LVsp2, were capable of inflaming the brain after i.p injection to different degree. LVsp1 was better than LVsp2 and Bt1 spirochetes at moving from blood to brain. The dissemination of LVsp1 from the periphery to the brain occurred under normal conditions and significantly increased with infection. In contrast, LVsp2 disseminated better to peripheral organs. We conclude that some bacterial lipoproteins can disseminate from the periphery to inflame the brain.
Regulatory role of ATF3 on NO production in LPS/IFN‐γ‐treated macrophages
Authors: Jung Da Hye , Rhee Dong‐Kwon , Pyo Suhkneung
Published: June 2021

Activating transcription factor 3(ATF3), known as a stress inducible gene, is a member of the CREB/ATF family of transcription factors which is expressed only under conditions such as inflammation, cell injury or oxidative stress. LPS‐induced ATF3 has been known to suppress productions of Nitric oxide (NO) and cytokines such as IL‐6, and IL‐12 in macrophages. However, the mechanism of action of ATF3 on the macrophage function has not been characterized in detail. In the present study, we examined the regulatory role of ATF3 on inducible nitric oxide synthase (iNOS) expression in LPS/IFN‐γ‐treated macrophages. ATF3 knockdown enhanced NO in macrophages. Western blot and RT‐PCR analysis revealed that protein and mRNA levels of iNOS were also attenuated in ATF3‐knockdown cells. In addition, activities of NF‐κB and AP‐1 were increased by ATF3‐knockdown. These data indicate that ATF3 acts as s negative regulator on iNOS expression via suppression of NF‐κB and AP‐1 activities. Treatment with casein kinase ¥±(CK¥±) inhibitor decreased iNOS expression. LPS/IFN‐γ induced‐phosphorylation and nuclear translocation of ATF3 were also attenuated by CK¥± inhibitor. Overall, these results suggest that ATF3 regulates NO production through modulation of CK¥±, NF‐κB and AP‐1 signaling pathway.
FAK and endothelial cell isometric tension
Authors: Miede Kimberly , Goeckeler Zoe , Wysolmerski Robert
Published: June 2021

The vascular system is lined by a continuous sheet of endothelial cells (EC) that form a semi‐permeable barrier. Inflammatory stimuli trigger EC contraction and intracellular gap formation, resulting in an increase in vascular permeability. Activation of EC contractile activity and/or alterations in cell–matrix adhesion most likely cause inter‐endothelial cell gap formation. Cell‐matrix adhesion occurs through multi‐protein complexes called focal adhesions (FA). FA tether the cells contractile machinery (actin/myosin) to extracellular matrix proteins forming the structural link necessary for isometric tension generation. To investigate the role of FA in EC contraction, focal adhesion kinase (FAK) was ablated. We propose FAK is essential for isometric tension generation and is necessary for assembly and organization of the EC contractile machinery. Infection of EC with a FAK‐shRNA caused a 90% reduction in endogenous levels of FAK protein as well as a 50% reduction in EC FA compared to vector controls. Up‐regulation of Pyk2 was not detected. FAK knockdown reduces basal EC tension and inhibits LPA induced isometric tension generation. Loss of FAK alters nonmuscle myosin II distribution suggesting a role for FAK in myosin II regulation. These data implicate FAK in the regulation of EC isometric tension. Supported by NIH grants T32‐HL090610, HL‐45788, RR‐16440.
P120‐catenin Suppresses the Inflammatory Response in Endothelial Cells
Authors: O'Donnell James John , Cheng Feng , Zhang Jihang , Zhuge Yan , Holian Oksana , Lum Hazel
Published: June 2021

P120‐catenin (p120ctn) promotes cell‐cell adhesion at the adherens junction of vascular endothelium. Emerging evidence from fields in cancer and development suggests that p120ctn can additionally repress transcription. We therefore investigated whether p120ctn of human endothelial cells has transcriptional function and to explore potential gene targets regulated. Results indicated that siRNA‐mediated depletion of p120ctn doubled promoter reporter luciferase activities of transcription factors NFκB, AP‐1, and Kaiso, which were associated with increased phosphorylation of ERK1/2. Immunofluorescence showed that loss of p120ctn increased NFκB nuclear localization, further confirming transcriptional activation. Treatment of endothelial cells with PD98059, an ERK1/2 inhibitor, abrogated the NFκB translocation, suggesting ERK1/2‐dependence. A known gene target of NFκB and AP‐1 is ICAM‐1, and we found that p120ctn loss doubled ICAM‐1 mRNA and tripled ICAM‐1 protein expression. Our findings provide strong evidence that p120ctn suppresses transcriptional regulation in endothelial cells and may be critical for suppressing pro‐inflammatory activity. The study is supported by NIH HL093715.

we investigated whether this upregulation is through pro‐inflammatory signaling molecules Extracellular Signal‐regulated kinases 1 and 2 (ERK1/2).
Chemically Modified Primers for Improved Multiplexed PCR
Authors: Shore Sabrina , Ashrafi Elena Hidalgo , Lebedev Alexandre , Hogrefe Richard , Le Tony , Paul Natasha
Published: June 2021

Multiplex PCR is an advantageous technique used in PCR applications to amplify multiple targets in a single reaction. As useful as it is, this technique presents a new set of challenges that further complicates PCR setup. For example, reactions are more prone to off‐target amplifications such as mis‐priming and primer dimer due to the increased number of primer pairs. Furthermore, preferential amplification of certain targets leads to an unequal distribution of amplicon products, making quantification and detection of problematic targets extremely difficult. To improve upon the problems specific to multiplex PCR, we evaluated Hot Start modified primers which contain either one or two thermolabile 4‐oxo‐tetradecyl (OXT) modifications to prevent DNA polymerase extension at low‐stringent temperatures, and that are released after a Hot Start activation step. Herein, we find that the singly‐modified primers provide greater amplification efficiency, specificity, and yield in the multiplex amplification of DNA targets. In reverse transcriptase PCR (RT‐PCR), the doubly‐modified primers have been proven to be the optimal choice. The presence of two thermolabile protecting groups allows for an efficient one‐step RT‐PCR reaction that provides high specificity for multiple targets. TriLink's innovative technology represents a convenient tool for multiplex PCR amplification of DNA and RNA samples.
Some biochemical parameters in hemolymph of Garden snail (Helix pomatia L.) in the region of power plants of Kosovo
Authors: Halili Fetah , Bislimi Kemajl , Gashi Agim M , Bajraktari Ismet D , Mazreku Ilir , Aliu Naser
Published: June 2021

In current work the concentration of glucose, total proteins, cholesterol and the activity of the alkaline phosphatase in hemolymph of local population of Garden snail Helix pomatia L.(N=20), in the region contaminated by the power plants of Kosovo in Obiliq, was analyzed. The snails of control group were taken from the region of Gjilan far away from polluted area. The preliminary investigation results have shown that the concentration of the cholesterol in the hemolyph of snails from the polluted region was significantly higher whereas the activity of the enzyme alkaline phosphatase significantly lower in comparison with the snails of the control group. There was no indication of difference regarding the glucose and the total protein concentration between the analyzed snail groups.

The investigation shows that the Garden snail can serve as bioindicator, whereas the changing of the hemolymph chemistry as marker for the biochemical indication of the environmental pollution effects.
A critical role of AMPK, and Akt, mTOR and Wnt survival signals for the growth control of colon and liver cancer cells with selenium
Authors: Yun‐Kyoung Lee , Park Song Yi , Lee Won Sup , Surh Young‐Joon , Kim Young‐Min , Park Ock Jin
Published: June 2021

Cancer cells generally exhibit the increased ability to survive, while the susceptibility to undergo apoptosis is strikingly depressed. Survival signals such as Akt, mTOR and Wnt/GSK3β are known to play an important role of cell's sensitivity to death. Recent studies suggest that AMPK acts as a major controller of cancer cell proliferation integrating growth factor signaling with cellular metabolism. The important ability of AMPK to inhibit mTOR or Akt suggests that there exists an intricate network involving AMPK, Akt and mTOR to control cancer growth. We used selenium to activate AMPK to determine whether AMPK activation can inhibit cancer cell growth through suppressing Akt, mTOR or Wnt/β‐catenin. We have found that selenium lost its ability of inhibiting mTOR of HT‐29 cells and Wnt components of Hep3B cells in the absence of AMPK. However, selenium could inhibit Akt regardless of AMPK. The activation of AMPK and deactivation of mTOR or Akt were observed in selenium‐treated xenograft models. These results suggest that there exists a complex interaction between AMPK and mTOR or Akt, Wnt components and the thorough mechanistic understanding of AMPK and survival signals can point an effective strategy for cancer prevention with dietary origin components. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (NO. R01‐2008‐000‐20131‐0)]
Mechanism of protective function of taurine in neurons and PC 12 cells
Authors: WU JANG‐YEN , Pan Chunliu , Prentice Howard
Published: June 2021

Recently, we have shown that taurine could protect cultured neurons from glutamate‐induced neuronal injury by several related mechanisms including inhibiting calcium channels, inhibiting calpain, shifting the ratio of Bcl‐2 and Bax in favor of anti‐apoptosis (J. Neurosci. Res. 87: 1185‐1194, 2009). Furthermore, taurine also exerts protective function against oxidative stress in PC 12 cells. It was found that at 500uM H2O2, the cell viability of PC 12 cells was restored from 20% to more than 80% by taurine at 25 mM. Treatment with taurine restored the H2O2‐induced reduction of expression of Bcl‐2 close to the control level without any significant effect on Bax. In addition, taurine was also found to restore the change of H2O2‐induced alteration of GRP78 and GADD153/CHOP, two marker proteins of ER stress, to the control level suggesting that taurine may also exert protective function against oxidative stress by reducing the ER stress. In summary, taurine appears to exert its protective function against oxidative stress by shifting the ratio of Bcl 2 and Bax in favor of anti‐apoptosis and also protect the integrity of the ER system. In conclusion, taurine exerts its protective function through at least: 1. Lowing the intracellular free calcium level; 2. Inhibiting calpain activity and hence increase the ratio of Bcl‐2/Bax in favor of anti‐apoptosis; 3. Protecting the integrity of ER and mitochondria.
The use of an engineered thymidylate synthase gene for gene therapy applications
Authors: Morrey Erin L , Spencer H. Trent
Published: June 2021

The effectiveness of many gene therapy applications is limited by the efficiency of genetic modification of target cells. For example, clinical trials have shown that the transduction efficiency of hematopoietic stem cells using recombinant retroviruses is extremely low compared to the efficiency of transducing hematopoietic progenitor cells. One method for increasing the percentage of genetically‐modified cells is to selectively expand the population after transduction. We examined the potential for using an engineered thymidylate synthase construct, designed to be optimally expressed in mammalian cells (OPTecTS), as a drug‐resistance marker to preferentially select genetically‐modified cells. An MSCV‐OPTecTS construct was developed and shown to confer extremely high‐level antifolate resistance to transduced cells using several in vitro assays. OPTecTS transduced cells were enriched to a high percentage of the total cell population after only five days in culture. Also, co‐transfection of OPTecTS with GFP resulted in the selection of GFP+ cells after selection, and PCR confirmed the presence of the OPTecTS DNA in all cases, indicating that the OPTecTS construct was directly protecting the GFP+ cells. These results indicate that OPTecTS can be used for the in vitro selection of modified cells, not only as a selectable marker for gene therapy but also for molecular biology applications.
Tocotrienol and Growth Factors In Acute Gasrtic Mucosal Injury
Authors: Ismail Nafeeza Mohd , Saad Qodriyah M , Yusoff Yasmin A
Published: June 2021

Gastric mucosal damage caused by nonsteroidal anti‐inflammatory drugs is recognized as the most serious adverse reaction for this drug class and has been the stimulus for research aimed at developing more effective gastroprotective drugs. Transforming growth factor alpha (TGFa) and epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) are crucial in the regulation for reconstruction of damaged mucosal structures. We have previously shown that tocotrienol (T3) improved NSAID‐induced AGML. The present study aims to assess the effect of pretreatment of rats, exposed to a single dose of NSAID on the expression of growth factors, with T3. After 8 weeks, they were administered with a single dose of indomethacin. Six hours post insult, measurements of TGF‐α, bFGF, EGF‐R and ERK‐1 were done. There was a significant increase in the TGF‐α, bFGF, EGF‐R and ERK‐1 content the gastric mucosa of rats treated with T3 but not in rats without T3. This findings demonstrated that T3 rapidly stimulated the growth factors involved in cell proliferation and migration through involvement of EGF‐R‐ MAP and Erk1 kinase. We conclude T3 may potentially accelerate healing of AGML through higher expression of growth factors, related receptor and kinase involved in gastric mucosal repair. Source of research fund: Ministry of Science, Technology and Innovation, Malaysia IRPA 06‐02‐02‐0052‐ EA246.
Crosslinking Analysis of the M3 Muscarinic Acetylcholine Receptor/G alpha q Interface in Both the Inactive and Active States
Authors: Hu Jianxin , Wang Yan , Zhang Xiaohong , Lloyd John R. , Li Jianhua , Karpiak Joel , Costanzi Stefano , Wess Jurgen
Published: June 2021

The interaction of G protein‐coupled receptors (GPCRs) with heterotrimeric G proteins represents one of the most fundamental biological processes. Although detailed structural information is now available for several GPCRs and heterotrimeric G proteins, the molecular architecture of the GPCR/G protein complex remains poorly defined, primarily due to the lack of a high‐resolution X‐ray structure. In the present study, we applied a comprehensive GPCR/Gα chemical cross‐linking strategy to map a receptor/Gα interface, both prior to and after agonist‐induced receptor activation. By employing the M3 muscarinic acetylcholine receptor (M3R)/Gαq system as a model system, we examined the ability of ~250 combinations of Cys‐substituted M3R and Gαq proteins to undergo cross‐link formation. We identified many specific M3R/Gαq–contact sites, both in the inactive and the active receptor conformation. Co‐immunoprecipitation and mass spectrometry experiments confirmed the molecular identity of the M3R/Gαq complexes. The observed M3R/Gαq cross‐linking patterns yielded a sufficient number of structural constraints allowing us to generate the first evidenced‐based model of a GPCR/Gα complex. Since heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of great general relevance.
Amiodarone and N‐ethyl‐amiodarone exert opposing allosteric effects via a novel site on muscarinic receptors
Authors: Stahl Edward , Ellis John
Published: June 2021

Allosteric modulation provides several advantages over the classical (orthosteric) approach to receptor activation; further, the characterization of new allosteric sites provides additional opportunities for the development of subtype‐selective and functionally selective compounds. We have been investigating the allosteric actions of amiodarone and N‐ethyl‐amiodarone (NEA) at muscarinic receptors. Previous studies have demonstrated that amiodarone interacts with a site that is distinct from the one that has been defined by gallamine.

We have shown that amiodarone enhances stimulation of [3H]arachidonic acid release induced by agonists acting at Gq‐coupled muscarinic receptors. Evaluation of this action in light of Hall's allosteric two‐state model indicates that amiodarone's enhancement of agonist efficacy is tempered by a concomitant reduction in agonist binding affinity. The enhancement of efficacy is attenuated by NEA but not by the orthosteric antagonist N‐methylscopolamine; further evaluation of this finding using an elaboration of Hall's model suggests that amiodarone and NEA exert their functional effects through a common allosteric site.

In conclusion, we have demonstrated that amiodarone and NEA interact allosterically with a novel, extracellular, site on muscarinic receptors, through which they modulate agonist potency and efficacy in a complex manner.