bluuebunny/crossref_metadata_2025 huggingface dataset:
CTLA4 Polymorphisms are associated with Severe Pulmonary Tuberculosis in a Chinese Han population
Authors: Wang Chong , Jiang Tingting , Wei Liliang , Zhong Jianping , Liu Jiyan , Zhang Xing , Xu Dandan , Chen Zhongliang , Li Zhongjie , Li Ji‐Cheng
Published: June 2021

The cytotoxic T lymphocyte antigen‐4 (CTLA4) is a key negative regulator in T lymphocyte activation. Polymorphisms on CTLA4 are associated with susceptibility to many autoimmune diseases, resulting in down‐regulation of the inhibition of cellular immune response. The present study aimed to investigate potential associations of CTLA4 +49A>;G (rs231775), +6230G>;A (rs3087243), and 11430G>;A (rs11571319) polymorphisms with pulmonary tuberculosis (PTB) in a Chinese Han population. A case‐control study was performed, and the three SNPs were genotyped by PCR and DNA sequencing methods. We found the frequency of CTLA4 +49AG genotype in 274 PTB cases was significantly lower than that of the 265 controls (Pcor=0.025, 0R 0.613, 95% CI 0.401–0.937). However, no associations were found between the other 2 SNPs (P>;0.05). After divided PTB cases into two groups: with or without cavities, we found a novel relation of +6230AG genotype to cavities (Pcor=0.046, 0R 2.677, 95% CI 1.029–6.965). Lately haplotype analysis showed that haplotype AGG was significantly associated with healthy controls (P=0.002, OR 0.286, 95% CI 0.124–0.657), while haplotype AAG was related to PTB with cavities (P=0.047, OR 2.053, 95% CI 1.000–4.215). Our results suggest the CTLA4 polymorphisms can be associated with PTB in a Chinese Han population, and +49AG may reduce the risk of being PTB, while +6230AG may contribute to cavities formation.

This work was supported by grants from National Natural Science Foundation of China (No.81072724, No.81273882), National Special Sci‐Tech Projects (No.2012ZX10005001–006, No.2008ZX10005–010), and Zhejiang Province Special Sci‐Tech Projects (No.2009C03011–3).
Purification and Analysis of Rho‐1D4‐tagged Peripheral Cannabinoid Receptor CB2
Authors: Locatelli‐Hoops Silvia Cristina , Gorshkova Inna , Gawrisch Klaus , Yeliseev Alexei A
Published: June 2021

Human peripheral cannabinoid receptor CB2 is involved in regulation of the immune response. For studies requiring surface immobilization, pure and functional receptor with a reliable tag is required. The goal of this project is to test the usefulness of the C‐terminal epitope of rhodopsin (rho‐1D4‐tag) for purification and surface immobilization of recombinant CB2 using the monoclonal antibody 1D4.

Expression of rho‐1D4‐tagged CB2 as a fusion with maltose binding protein (MBP) in E coli was evaluated in terms of protein levels, accessibility of the rho‐1D4 tag and activity. Highly pure tagged CB2 was obtained by rho‐1D4 and Ni‐NTA affinity chromatography. The reconstituted protein was functionally active by G protein activation assay. The interaction of rho‐1D4‐tagged CB2 with 1D4 antibody was characterized by surface plasmon resonance (SPR). Either purified or fusion CB2 from crude extracts was captured on the 1D4 antibody‐coated surface in a specific and quantitative fashion, and its presence confirmed by biding of an anti‐CB2 monoclonal antibody. Ligand binding to purified receptor in detergents was studied upon immobilization of fusion CB2 on 1D4 antibody resin. This methodology was also used to study thermal stability of the receptor in micelles.

In conclusion, the rho‐1D4 tag was successfully utilized for purification, immobilization, biochemical and biophysical studies of functional CB2.
Obesity: Exploring a link between BMI and Leptin
Authors: Kennedy Shane Patrick , Parmar Nitika
Published: June 2021

The project will seek to determine if there is a correlation between mutations in a protein called leptin and Body‐Mass Index (BMI) of people coming from different racial backgrounds. The study will focus on examining community members for their physical characteristics such as body weight and height and comparing BMI values to implicated biological protein aberrations. This activity will be useful in establishing a relationship between BMI and selfe‐steem of community members based on their ethnic backgrounds. The “community” element is critical here as cultural background invariably impacts how people view obesity and how it can affect their perceptions of themselves in terms of health. People from Caucasian, African‐American, Asian and Hispanic lineages will be targeted and their BMI indices will be investigated. Because obesity is linked to high BMI values and a genetic component has also been implicated behind it, it is necessary to study a relationship between this implicated protein, leptin and BMI. Support: This work will commence in January, 2013 and will be supported by the Hank Lacayo Institute Fellowship Award to Shane Kennedy. The project is expected to be completed by end of March, 2013.
Influence of Dietary Intake on Plasma Metabolites and Lipids in C57BL/6J Mice
Authors: Meidenbauer Joshua , Ta Nathan , Seyfried Thomas
Published: June 2021

Dietary therapy, which includes calorie restriction, ketogenic diets, and diets high in omega‐3 fatty acids, has been used to improve health and to treat a variety of neurological and non‐neurological diseases, including cancer. It is not clear if the therapeutic effects of these diets arise from their composition or from the amount of calories consumed. We therefore investigated the effects of dietary intake on circulating glucose and beta‐hydroxybutyrate (metabolites relevant to dietary efficacy for treating epilepsy and cancer), along with circulating lipids in C57BL/6J mice over a period of 30 days. We evaluated a standard mouse chow diet (SD), a lard‐based ketogenic diet (KD), and a standard mouse chow diet supplemented with fish oil (FO), each administered either in unrestricted amounts (UR) or restricted (R) to reduce bodyweight by 20% (n = 4 mice/group). The KD‐UR increased glucose, beta‐hydroxybutyrate, triglycerides, and cholesterol compared to SD‐UR. The FO‐UR diet, however, increased beta‐hydroxybutyrate while decreasing glucose and triglyceride levels. When given in restricted amounts, all three diets yielded a similar metabolite profile, which included decreased circulating glucose and triglycerides along with increased beta‐hydroxybutyrate compared to SD‐UR. We also performed linear regression analysis to analyze the important predictors of bodyweight, glucose, beta‐hydroxybutyrate, triglyceride, and cholesterol levels. We found that circulating glucose most strongly predicts bodyweight (R2 = 0.850) and triglyceride levels (R2 = 0.883). Calorie intake moderately predicts glucose levels (R2 = 0.500) and strongly predicts beta‐hydroxybutyrate levels (R2 = 0.771). Dietary fat intake strongly predicts cholesterol levels (R2 = 0.701).
Quantitative proteomics reveals regulation of KPNA2 and its potential novel cargo proteins in non‐small cell lung cancer
Authors: YU CHIA‐JUNG , Wang Chun‐I , Chien Kun‐Yi , Wang Chih‐Liang , Liu Hao‐Ping , Cheng Chia‐Chen , Chang Yu‐Sun , Yu Jau‐Song
Published: June 2021

The process of nucleocytoplasmic shuttling is mediated by karyopherins. Dysregulated expression of karyopherins may trigger oncogenesis through aberrant distribution of cargo proteins. Previously, we identified karyopherin subunit alpha‐2 (KPNA2) as a potential biomarker for non‐small cell lung cancer (NSCLC). Herein, we applied gene knockdown, subcellular fractionation and SILAC‐based quantitative proteomic strategies to systematically analyze the KPNA2‐regulating protein profiles in NSCLC cell line. Interaction network analysis revealed that KPNA2 involved in signaling for cell cycle, cellular component movements and cell migration. Importantly, E2F1 was identified as a potential novel cargo of KPNA2 in the nuclear proteome. The mRNA levels of potential effectors of E2F1 measured using quantitative PCR indicated that E2F1 is one of the “master molecule” responses to KPNA2 knockdown. Immunofluorescence staining and immunoprecipitation assays disclosed co‐localization and association between E2F1 and KPNA2. An in vitro protein binding assay further demonstrated that E2F1 interacts directly with KPNA2. Moreover, knockdown of KPNA2 led to subcellular redistribution of E2F1 in lung cancer cells. Our results collectively demonstrate the utility of quantitative proteomic approaches and provide a fundamental platform to further explore the biological roles of KPNA2 in NSCLC.
Comparison of Free Radical Scavenging Abilities of Various Chalcone Antioxidants
Authors: Lee Sharon , Rossi Miriam , Caruso Francesco
Published: June 2021

Free radicals are highly reactive chemical species found in cells; various reactive oxygen species (ROS) are produced as a result of normal cell metabolism, air pollution, smoking, UV radiation, and inflammation. Oxidative stress may be the cause of many neurodegenerative and cardiovascular diseases, and continued oxidative stress in cells leads to loss of cell membrane integrity, DNA damage, and apoptosis. Chalcone antioxidants, found in plants and foods, stabilize ROS. Chalcones have antifungal, anti‐inflammatory, antimalarial, antitumor, and antiviral activities. The present study concerns relative efficacies of various hydroxylated chalcones in comparison with one another and with synthetic antioxidants. Free radical scavenging abilities of seven hydroxylated chalcones were analyzed using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assays. Triplicate results confirmed previous results by our group that chalcones with 5′‐hydroxyl groups performed significantly better. Synthetic chalcones performed with less efficacy than natural chalcones. Butylated hydroxytoluene (BHT), a synthetic antioxidant found in processed foods, was used as a standard and had the least efficacy. In addition, the analysis of single crystal X‐ray studies on these chalcones solidifies the role of intermolecular interactions of hydroxyl groups in antioxidant activity. Supported by HHMI 52006322 and VC URSI 2012.
TGF‐β regulated miRNAs: therapeutic targets for cardiac hypertrophy
Authors: Yang Xiao , Wang Jian
Published: June 2021

Heart failure (HF) is one of the most frequent causes of death worldwide. The underlying causes of HF are diverse but often relate to cardiac hypertrophy. The role of TGF‐β signaling in cardiac hypertrophy has been extremely contradictory. We have previously showed that targeted deletion of Smad4 unexpectedly leads to cardiac hypertrophy, demonstrating that the endogenous cardiomyocyte Smad4‐dependent TGF‐β pathway protects heart from cardiac hypertrophy and fibrosis.

Recently, we have revealed that the function of endogenous TGF‐β/Smad signaling in maintaining cardiac homeostasis involves the downregulation of miRNAs inducing cardiac hypertrophy. We show that TGF‐β1 inhibits the expression of miR‐23a/miR‐27a/miR‐24–2 and miR‐23b/miR‐27b/miR‐24–1 clusters at the transcriptional level. Transgenic mice with cardiomyocyte‐specific overexpression of miR‐27b exhibit cardiac hypertrophy and dysfunction by directly targeting PPAR‐¦Ã. Most importantly, in vivo silencing of miR‐27b in a pressure‐overload‐induced mouse model of HF attenuates cardiac hypertrophy and dysfunction. The function and mechanisms of other miRNAs regulated by TGF‐β/Smad signaling in cardiac hypertrophy have also been investigated. All these results provide critical genetic evidence showing that TGF‐β‐regulated miRNAs might serve as efficient therapeutic targets for cardiac diseases.
Adaptation of a compact rapid vitamin A assay device (iCHECK™) to simulated field conditions and relevant substrates in Guatemala
Authors: Zambo Zsofia Magdolna , Sanchez‐Mena Jose David , Orozco Monica N , Solomons Noel W
Published: June 2021

Background

Guatemala has mandated fortifying granulated table sugar with vitamin A at 10–20 ppm. Children in the interior commonly continue partial breast‐feeding through the first 2 y of life.

Objective

To adapt the preparation of samples of sugar and bovine milk for a rapid assessment of vitamin A using nonlaboratory items for mixing and measuring.

Methods

Sugar samples were mixed for homogenization in plastic bags, with 4 g delivered into a 20 mL syringe to prepare a 20 w/v% solution. Heavy cream was diluted in plastic bags. Samples were injected into iEX™ extraction vials and measured by fluorometry in an iCHECK™(BioAnalyt, Telbow, Germany) rapid assay unit, providing digital read‐outs in μ/L.

Results

Intra‐sample CVs ranged from 0.3–7.8% and intraobserver CVs ranged from 2.1– 15.0% for dairy. The r value for the inter‐observer assay association for 12 sugar specimens was 0.86 (p<0.001). Inter‐observer accord on median vitamin A values was excellent for both substrates. Plausible values for sugar vitamin A, with a median of 11 ppm, were found for 58 samples of sugar gathered from across 4 regions.

Conclusion

The iCHECK™ system provides plausible values for vitamin A content of fortified sugar and bovine milk and cream. It offers important promise for a rapid, on‐site, field‐level evaluation of materials for timely public health responses. Supported by a donation from Sight & Life
Main food sources of sugar in Brazil: the National Dietary Survey, 2008–2009
Authors: de Moura Souza Amanda , Bezerra Ilana Nogueira , Peterson Karen E. , Sichieri Rosely
Published: June 2021

In order to evaluate major food sources of added, free and total sugars in Brazil according to sex, age and income, day‐1 food records from 34,003 participants (≥10yrs) in the 2008–2009 Brazilian National Dietary Survey were analyzed. Foods were classified into 40 groups according to nutrient profile. The mean percentage of energy intake from total, added and free sugar intake was 19.6%, 13.5% and 18.5%, respectively. Women had higher intake of sugar than men; sugar consumption decreased with age and increased with income quartile. Juices, coffee and soft drinks were the major sources of total, added and free sugar in the Brazilian diet. These three food groups together contributed to 58%, 65% and 73 % of the percent of calories from total, added and free sugar, respectively. For all age‐sex groups those reporting consumption of soft drinks and cookies had higher mean energy intake, indicating these food groups as markers of excessive intake of sugar and energy in the Brazilian population. Our results emphasize the importance of public health efforts to limit sugar sweetened beverage and cookie consumption, particularly among adolescents, as an effort to curb obesity.
Plain water and total moisture intakes and subsequent risk of all‐cause mortality in a national cohort
Authors: Kant Ashima , Graubard Barry I.
Published: June 2021

Few studies have examined the association of water intake with health. We examined the association of plain water and total moisture reported in the NHANES 1988–94 and the continuous NHANES 1999–2004 with all‐cause mortality in respondents aged >;25 years at the time of the initial survey (12015 men and 12635 women). The plain tap or bottled water intake was estimated from a question asked after the 24‐h recall and total moisture was the sum of plain water, beverage and food moisture estimated from a 24‐hour recall. The baseline plain water and 24‐hour total moisture intakes were 946+0.62 g and 3030+20 g, respectively. Mortality status from the date of examination in each NHANES survey until Dec. 31, 2006 was determined by the NCHS from probabilistic matching with the National Death Index. Over median 6.7 years of followup, 2332 men and 1935 women in the analytic cohort died from a known cause. In Cox proportional hazards regression analyses, with age the underlying time metric, plain water and total moisture intakes were not associated with all‐cause mortality in either sex (p>;0.05). These analyses accounted for survey design, were weighted, and were adjusted for race/ethnicity, education, body mass index, smoking status, alcohol use, history of chronic diseases, and recreational physical activity. The results were unchanged when plain water and total moisture were examined as grams/1000 kcals or as grams/kg of body weight.
The mammalian O‐mannosylation pathway: protein substrates, enzymes, and structures
Authors: Wells Lance , Stalnaker Stephanie , Dobson Crissy , Stuart Ryan , Praissman Jeremy
Published: June 2021

The O‐linked mannose modification imparts essential functions to targeted glycoproteins. The protein substrates, enzymes, and glycan products of the O‐mannosylation pathway underlie a variety of cellular processes and their perturbation contribute to diverse pathophysiological conditions including congenital muscular dystrophy, viral‐host interactions, and tumor metastasis. We are currently identifying the protein substrates and sites of modification for O‐mannosylation in mammalian brain and muscle using chemical biology approaches coupled to novel tandem mass spectrometry approaches. We are also developing genotype/phenotype correlations for POMGnT1 that when mutated can generate phenotypes ranging from Muscle‐Eye‐Brain disease to the much less severe limb‐girdle muscular dystrophy. Also, we have recently established the presence of O‐mannose structures on leukocytes and have demonstrated that we can use a mass spectrometry‐based blood test to differentiate between controls and patients with Walker‐Warburg syndrome, the most severe of the congenital muscular dystrophies. Finally, using analytical biochemical methods, we are attempting to determine the complete O‐mannose‐initiated structure(s) and the enzymes responsible for synthesis that are used for binding components of the extracellular matrix.
Impact of a Combination of Medium‐Chain Triglycerides and Dietary Fiber in a Form of Meal Replacement Shake on Postprandial Energy Expenditure
Authors: Amagase Harunobu , Handel Richard , Nance Dwight
Published: June 2021

To investigate the interaction of medium‐chain triglycerides (MCT) with other nutritional ingredients on postprandial energy expenditure (PPEE), 4 different commercially available meal replacement shakes were compared in a randomized, double‐blind clinical trial. Major differences from 1 serving of Product A, B, C and D were: MCT content, 5.5, 2.2, 0.4 and 0.2%; dietary fiber content, 5, 5, 3 and 0 g; and energy content, 158, 90, 90 and 200 kcal, respectively. These were individually mixed with water and taken orally following an overnight fast. Samples were randomly tested multiple times in the morning at weekly intervals. PPEE was measured by breath oxygen volume immediately before (baseline), 1, 2 and 4 h after sample intake. There was a significant rise in PPEE over baseline following consumption of product A (6.2±0.6 SEM %) during the 4 h testing period (P<0.05). This rise in PPEE was greater than that observed after consumption of products B, C, and D, which were 1.7±0.4, 1.6±0.4 and 3.7±0.5%, respectively (P<0.05). PPEE after intake of Products B, C and D had returned to the baseline level by the 4‐h measurement point. Although PPEE is usually dependent upon the caloric load consumed, PPEE per energy content in Product A was 0.59±0.06, which was about twice that of the other test diets. These results suggest that a combination of nutrients, including MCT and dietary fiber, may enhance meal‐induced energy expenditure.
Fructose reduction improves CVD risk in adolescents with NAFLD
Authors: Vos Miriam , Jin Ran , Welsh Jean , Le Ngoc‐Anh
Published: June 2021

Objective

Nonalcoholic fatty liver disease is associated with increased CVD risk. We have previously reported abnormal post‐prandial TG response after fructose‐enriched meals in adolescents with NAFLD suggesting that fructose may contribute to the increased CVD risk. We hypothesized that fructose reduction would improve CVD risk factors in adolescents with NAFLD.

Methods

Overweight, Hispanic adolescents who self‐reported high consumption of sugar beverages (>;24oz/day) were recruited for hepatic fat screening by MRS. 24 adolescents had high hepatic fat (HHF, ≥5%) and were randomized to either 4 weeks of fructose reduction (home beverages replaced with 3 glucose study provided beverages per day) or 4 weeks of continued high fructose (home beverages replaced with 3 fructose study provided beverages per day). MRS, fasting labs, and anthropometrics were repeated at baseline, 2 and 4 weeks.

Results

After 4‐weeks of glucose beverages (fructose reduction), subjects had improved adipose IR (p=0.034), hs‐c reactive protien (p=0.026), circulating oxidized LDL (p=0.056), and LDL oxidation susceptibility (p=0.009); which were not observed in fructose beverage group. No improvement was seen in BW, hepatic fat, and fasting TG.

Conclusion

Reduction of fructose containing beverages for 4 weeks improved CVD risk profile in adolescents with HHF despite a lack of change in hepatic fat.
Corn Peptide Ingestion with Exercise Attenuate Body Fat Mass and Enhance Lean Body Mass in obese rats
Authors: Lou Shujie , Shi Rengfei , Wang Ru , Wang Xiaohui , Xu Leiting
Published: June 2021

Obesity is a serious health problem. Moreover, Loss of lean mass also is a problem caused by negative energy balance. Thus, the treatment of obesity requires both the reduction of fat and maintenance of lean mass. Corn peptides (CP) were shown to decrease body fat and increase lean mass in animal studies. DIO rats were randomly divided into five groups, DIO, DIO+Casein, DIO+CP, DIO+exercise(Ex) and DIO+CP+Ex group(n=8/group). Supplement of CP and Casein(200g/kg diet) and Ex (Treadmill running in 15 m/min for 60 min/d, 5d/wk) protocol executed for 4 wk. Perirenal fat and soleus were harvested and western blotting was used for phosphorylations of p70S6k and rpS6. Compared with DIO Control, CP or Casein dietary has no significantly effect on body weight(BW) (p>;0.05), BW was attenuated by CP+Ex in the beginning of the 2th wk(p<0.05). BW was significantly reduced by Ex just in the 4th wk (p<0.05). Compared with DIO Control, perirenal fat mass index in DIO+CP+Ex group has a significant decrease (P<0.05) and soleus mass index has a significant increase(P<0.05). Phospho‐p70S6k (Thr389) and phospho‐rpS6 (Ser235/Ser236) in soleus were significantly higher in DIO+CP+Ex, but not in CP or Casein supplementation. CP+Ex had a BW reduction effect by lowering adipose tissue and improving lean body mass in DIO rats. These novel findings indicate that a CP‐enriched diet may potentially enhance growth of muscle.
Effects of topically applied Platycarya strobilacea S. et Z. extract on hair growth in C57BL/6 mice
Authors: Lee Bog‐Hieu , Kim Eun Jin , Choi Joo‐Yeon , Yu Miri
Published: June 2021

The study was conducted to evaluate the effect of Platycarya strobilacea S. et Z. extract on hair growth. Four‐week‐old male C57BL/6 mice were assigned to three experimental groups (n=5/group); the dimethyl sulfoxide (DMSO) group (NC), the minoxidil group (PC), and the Platycarya strobilacea S. et Z. extract group (PSE). Hairs of animals were shaved on the dorsal skin before the study and 100 μℓ of test samples were topically applied for 3 weeks everyday, while the dorsal skin was taken pictures on the 0th, 7th, 14th, 21st day. We observed morphology, length of hair follicle, and no. of mast cells and stem cell factors (SCF) on skin tissues. PSE group markedly promoted the hair growth than did NC and PC groups. Depth of hair follicle in PSE group(843.33±84.33 μm) was the deepest the deepest in PSE group, while those of NC or PC group were 758.33±150.05 μm and 655.00±145.45 μm, respectively. In case of number of mast cells, PSE group have shown the lowest in number(1.50±0.29) in comparison to NC(6.33±0.88) and PC(7.00±0.71)(p<0.001). Also, SCF staining in follicles, mast cells, skin layer indicated that PSE‐treated group have shown the strongest immune response than did the NC and PC groups. From the findings, topical application of PSE had a positive effect on hair growth and improved hair follicle length, mast cell, and immune response. Further research should be conducted to clarify the action mechanism related to Platycarya strobilacea S. et Z. extract.

Grant Funding Source: This research was supported by Basic Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology(2011–0013949)
Antioxidant activities of the EtOAc fraction and active compounds from Taraxacum coreanum under cellular system and in vivo
Authors: Cho Eun Ju , Lee Ah Young , Lee Sanghyun , Kim Hyun Young , Kang Ki Sung , Yamabe Noriko
Published: June 2021

We evaluated protective activity of the EtOAc fraction and active compounds, luteolin (LT) and luteoloside (LS), from Taraxacum coreanum NAKAI, one of the native plant in Korea, against nitric oxide (NO) and Aβ25–35. The LLC‐PK1 renal epithelial cells showed declines in cell viability and increase in NO formation through oxidative stress induced by sodium nitroprusside (SNP). However, the treatment of the EtOAc fraction, LT and LS significantly increased cell viability and decreased NO formation. Furthermore, NO formation and lipid peroxidation were significantly elevated in the mouse injected with Aβ25–35, whereas the groups administered the EtOAc fraction of T. coreanum, inhibited dose‐dependently lipid peroxidation and NO production in the brain, liver and kidney. These results indicated that the EtOAc fraction contains the bioactive compounds with protective effect against NO and Aβ25–35. In addition, LT and LS from T. coreanum are beneficial in preventing oxidative damage from free radical.
The effects of Actigen™ and dietary threonine on nursery pig gut morphology
Authors: Good Lindsay , Samuel Ryan S , Graugnard Daniel E , Pescatore Anthony J , Jolliff James S , Mahan Donald C , Brennan Kristen M
Published: June 2021

Adequate dietary threonine (Thr) is necessary for proper gut health in nursery pigs. Supplementing with a mannan oligosaccharide product, such as Actigen™ (ACT, Alltech Inc.), can aid in preventing bacterial colonization in the gut. This study was designed to determine whether supplementation with Thr and ACT had beneficial effects on intestinal morphology. Weaned pigs (17 d old) were randomly assigned to one of six factorial treatments: three ratios of true ileal digestible threonine:lysine (Thr:Lys TID): 57% (low), 67% (requirement), or 77% (high) and with or without 400g/T ACT. On d 42 of treatment, pigs (n=6) were euthanized and jejunal sections were fixed and embedded in paraffin wax for histological analysis. Sections (5 μm) were stained with Alcian blue‐periodic acid Schiff stain. Image analysis revealed that piglets fed 77% Thr:Lys with or without ACT supplementation had increased villi height (P<0.01), villus height‐to‐crypt depth ratio (P<0.05), and goblet cell density (cells/μm2; P<0.04) compared to 57% or 67% Thr:Lys. Total goblet cell area (μm2) and goblet cell density were increased (P<0.05) in treatments containing ACT. There were no significant interactions, indicating that adequate Thr levels and ACT supplementation positively influenced gut morphology characteristics independently to increase absorptive capacity and efficiency. The study was funded by Alltech Inc. (Nicholasville, KY).
Ceramide mediates smoking‐induced insulin resistance
Authors: Thatcher Mikayla O , Brassfield Eric S , Smith Melissa E , Nelson Michael B , Prince John T , Reynolds Paul R , Bikman Benjamin T
Published: June 2021

Environmental toxins are increasingly implicated in adverse health outcomes. Considering the lung's apposition with the environment, we sought to test the hypothesis that ceramide functions as an intermediate between smoking and systemic insulin resistance. To determine the effect of cigarette smoke on lung ceramide metabolism, common lung cell lines including A‐549 and Beas2B were exposed to cigarette smoke extract (CSE). CSE treatment elicited a robust increase in ceramide and elevated expression of several enzymes involved with de novo ceramide synthesis. Moreover, myoblasts incubated with conditioned medium from CSE‐treated lung cells became completely insensitive to insulin stimulation, suggesting the presence of an insulin antagonist in the medium of CSE‐treated lung cells. Lastly, we measured systemic lipids in mice following nasal instillation of CSE and found an increase in muscle and hepatic ceramides; however, such findings were not observed in lung‐RAGE KO mice. This work identifies ceramide as a possible intermediate between the lung and peripheral tissues. Future work will determine the role of lung‐secreted ceramides on systemic effects in vivo, such as cardiovascular function and insulin signaling. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI; PRR) and a BYU Mentoring Environment Grant (BTB).
Proliferation and Differentiation Potential of Adipose‐Derived Stem Cells Isolated from Dystrophin and Utrophin Double Knockout mice
Authors: Sohn Jihee , Oyster Nick , Tang Ying , Lu Aiping , Wang Bing , Huard Johnny
Published: June 2021

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive weakening of the skeletal, cardiac, and diaphragmatic muscles. Recent evidence has emerged implicating adult stem cell dysfunction in DMD histophathogenesis. Using utrophin/dystrophin double knockout (dys−/−utro−/−, dKO) mice, a DMD model that better emulates the human phenotype of the disease in mice, we determine if adipose derived stem cells (ADSCs) from dKO mice are affected in their proliferation and differentiation capacities compared to C57BL (wt) ADSCs. ADSCs were isolated from subcutaneous fat tissue of 6 week old dKO and wt mice and flow analysis confirmed positive expressions of Sca‐1, CD34, CD44 and CD29. Cell proliferation assay using a Live Cell Imaging system showed that dKO‐ADSCs were more active in proliferation than wt‐ADSCs. dKO‐ADSCs showed increased adipogenic, osteogenic, and chondrogenic potentials compared to wt‐ADSCs in vitro. Additional experiments revealed that transplanted dKO‐ADSCs displayed increased proliferation and survival and formed/accumulated more adipocytes in dKO gastrocnemius muscle (GM) compared to that in wt GM. These results suggest that dystrophic muscle milieu affects the stem cell niche, impacting stem cell function to be more active in proliferation and adipogenesis and this may partially explain ectopic fat deposition in skeletal muscle seen in advanced cases of DMD.
A Synopsis of Chromosomal Microarray Cases at Dartmouth‐Hitchcock Medical Center
Authors: Tayoun Ahmad N Abou , Tsongalis Gregory J. , Lefferts Joel A.
Published: June 2021

Background

Chromosomal microarrays (CMA) used to assess DNA copy number are currently considered first‐tier clinical diagnostic tests for postnatal evaluation of individuals with intellectual disability (ID), developmental delay (DD), autism spectrum disorders (ASD), and/or multiple congenital anomalies (MCA). Here, we provide a summary of the CMA cases performed at Dartmouth‐Hitchcock Medical Center during the first month that clinical CMA testing was offered.

Methods

We used the CytoScan®HD microarray containing 750,000 SNP probes and 1.9 million non‐polymorphic probes with an average intragenic spacing of 880 base pairs and 384 base pairs for 340 genes recommended by the International Standards for Cytogenomic Array (ISCA) consortium. Several databases of copy number variants were used during interpretation including ISCA, OMIM, DECIPHER and the Database of Genomic Variants (DGV).

Results

16 cases (9 females and 5 males) with an average age of ~7yrs were analyzed and reported. The clinical indication for testing in 14/16 cases (87.5%) was ID, DD or ASD. Clinically significant copy number variants were detected and reported in 3/16 cases, a diagnostic yield of 18.75%. Additionally, two variants of uncertain significance (VOUS) were also reported in two cases with unexplained developmental delay.

Conclusions

Despite the small number of cases, our diagnostic yield matches the well‐established CMA yield (15–20%) for genetic testing of individuals with ID, DD, ASD or MCA. This further emphasizes the fact that CMA provides higher sensitivity compared to G‐banded karyotyping albeit with the slight disadvantage of increased VOUS detection.
Disruption of TRPV4 Ca2+ signaling at myoendothelial projections (MEPs) contributes to endothelial dysfunction in Ang II hypertension
Authors: Sonkusare Swapnil K , Bonev Adrian D , Dalsgaard Thomas , Santana Luis F , Kotlikoff Michael I , Nelson Mark T
Published: June 2021

Endothelial cell (EC) dysfunction is a hallmark of hypertension, but the molecular mechanisms underlying this dysfunction remain unclear. We recently discovered elementary Ca2+ signals (“sparklets”) through single TRPV4 channels at MEPs (Sonkusare et. al., Science, 2012). Activation of TRPV4 sparklets at MEPs by muscarinic agonists is responsible for 70% of EDH (endothelium derived hyperpolarization)‐dilation in 3rd order mouse mesenteric arteries (MAs). Three weeks of angiotensin II (Ang II) infusion (1.4 mg/kg/day) leads to hypertension (HT) (systolic blood pressure, 178 mm Hg) and to a complete loss of the EDH‐mediated dilation without an effect on the NOS‐dependent component (20%). The maximal current density (i.e. channel number) of the ion channels (TRPV4, IK, SK, Kir) involved in the EDH dilation were unaffected in HT. Muscarinic receptor activation with carbachol increased TRPV4 sparklets at MEPs by 5‐fold in MAs from normotensive (NT) mice, but had no effect on TRPV4 sparklets from HT mice. IP3‐mediated increases in global Ca2+ were unaffected. TRPV4‐activation of IK and SK currents in freshly isolated ECs was reduced by 50% in HT mice. Our results support the concept that endothelial dysfunction in this form of hypertension does not result from a loss of the signaling elements, but it reflects crippled local communication.
Adiponectin vasodilates small arteries by increasing BK activity
Authors: Greenstein Adam S , Bonev Adrian , Sonkusare Swapnil , Heppner tom , Nelson Mark
Published: June 2021

We have previously shown that human perivascular adipose tissue (PVAT) vasodilates adjacent arteries, in part by release of adiponectin. This study was designed to further elucidate this adipose‐coupling at a cellular level. Adiponectin caused a small vasodilation of 3rd order mesenteric arteries constricted with pressure‐induced myogenic tone. The vasodilation was substantially reduced by pre‐incubation of arteries with ryanodine (Apn: 5.5%±0.7 vs Apn&Ryr: 2.2%±1, p=0.03). Confocal microscopy of Fluo‐4 loaded mesenteric arteries showed that the presence of intact PVAT increased vascular smooth muscle cell (VSMC) calcium (Ca) spark frequency in mesenteric arteries. However, there was no effect on VSMC Ca spark frequency following incubation with adiponectin. Using patch clamp of mesenteric VSMC, we found that adiponectin significantly increased the BK current when cells were incubated with ryanodine (20μM). Consistent with this, adiponectin increased amplitude but not frequency of spontaneous transient outward currents in untreated cells. Adiponectin had no effect on the Kv component of the whole cell current. It is concluded that adiponectin vasodilates small arteries predominantly by modulating VSMC BK activity, independently of Ca sparks. Intact PVAT appears to have an additive effect by increasing Ca spark frequency, suggesting the contribution of alternative adipose derived vasodilators.
Taxol alters gene expression of drug metabolizing enzymes by TLR4 dependent mechanism in vitro
Authors: Mallick Pankajini , Shah Pranav , Ghose Romi
Published: June 2021

Toll‐like receptors (TLRs) are pattern recognition receptors that are key components of the innate immune system. We have shown activation of TLR2 and TLR4 leads to down‐regulation of drug metabolizing enzymes (DMEs). Recently, TLR agonists are increasingly used in poly‐pharmacy settings. These medications can alter DME expression and cause drug‐drug interactions with co‐administered drugs. Taxol is a ligand for TLR4 and pregnane X receptor (PXR). Therefore, taxol may (a) increase DME expression by activating PXR and/or (b) reduce DME expression by activating TLR4. We hypothesize that the effect of taxol on DMEs will be higher in the absence of TLR4. Primary hepatocytes isolated from adult male C57BL/6 mice were treated with taxol (1–20μM). RNA was analyzed by real‐time PCR. To determine the role of TLR4, the cells were incubated with a TLR4 inhibitor, TAK‐242 (1μM) for 1h, followed by treatment with taxol for 24h. Taxol induces Cyp3a11 (~9–19 fold), Cyp2b10 (~7–8 fold) and Ugt1a1 (~3–4 fold). Taxol significantly induced cytokines interleukin (IL)1‐β, IL‐6 and tumor necrosis factor (TNF)α. Ugt1a1 induction by taxol significantly increased (~2.5 fold) upon treatment with TAK‐242 but it did not affect the induction of Cyp3a11 and Cyp2b10 by taxol. The results indicate that TLR4 has a role in regulating expression of Ugt1a1 by taxol in vitro. Thus TLR4 may mediate drug interactions between taxol and Ugt1a1 substrates
Do pericytes represent the cellular switch between kidney health and disease?
Authors: Peppiatt‐Wildman Claire M
Published: June 2021

Pericytes are pluripotent cells with numerous functional roles. We focus on the role of pericytes in kidney (patho)physiology and describe data identifying pericytes as being pivotal in both settings. Using the live kidney slice model [1], we have demonstrated i) that pericytes respond to various endogenous vasoactive mediators to regulate vasa recta diameter and thus medullary blood flow (MBF) [1], ii) neuro‐vascular coupling between pericytes and sympathetic nerves and iii) hypotonicity‐induced regulation of vessel diameter by pericytes, which is indicative of pericyte‐mediated tubulovascular crosstalk [1]. Collectively these represent novel mechanisms by which pericytes match MBF with the high metabolic demand of the tissue.

Additionally, we describe a critical role for pericytes in drug‐induced nephrotoxicity that is established for various commonly prescribed drugs. We demonstrate pericyte‐mediated vasoconstriction in response to immunosuppressant antibiotics and non‐steroidal anti‐inflammatory drugs, which may cause exacerbated local ischemia responsible for the acute kidney injury observed clinically. Our data, in combination with studies highlighting a key role for pericytes in renal diseases, suggests pericytes are the cellular switch underpinning the onset/progression of renal disease.
Does a compensatory formation of nitric oxide during inhibition of prostanoid synthesis in skeletal muscle explain the redundancy between these vasoactive systems?
Authors: Nyberg Michael , Jensen Lasse Gliemann , Hellsten Ylva
Published: June 2021

In skeletal muscle blood flow regulation a redundancy among vasodilators appear to exist, where a compensatory formation of one vasoactive compound may preserve adequate blood flow when the formation of another is compromised. To examine whether inhibition of prostanoid synthesis leads to a greater NO formation, skeletal muscle cells and skeletal muscle specific microvascular endothelial cells were treated with adenosine without and with inhibition of the enzyme cyclooxygenase to prevent prostanoid formation. The results showed that the adenosine‐induced formation of NO (as indicated by DAF‐FM fluorescence) was augmented (P<0.001) in microvascular endothelial cells (n=10) when the formation of prostanoids was inhibited, whereas no difference was detected in skeletal muscle cells (n=10). Samples from the interstitial fluid of human skeletal muscle obtained during exercise with the microdialysis technique will reveal to what extent inhibition of prostanoid synthesis leads to a compensatory formation of NO in vivo. These results indicate that an increase in NO formation in microvascular endothelial cells could compensate for a compromised prostanoid concentration in skeletal muscle.
TRPC channels are involved in calcium‐sensing receptor mediated inhibition of renin release from Juxtaglomerular cells
Authors: Ortiz‐Capisano M. Cecilia , Harding Pamela , Beierwaltes William H
Published: June 2021

Renin containing juxtaglomerular (JG) cells express the calcium‐sensing receptor (CaSR), whose activation increases intracellular calcium (Ca). Increases in intracellular Ca decrease renin release. Canonical transient receptor potential channels (TRPC), a seven‐member subfamily of the transient receptor potential (TRP) family of ion channels, can function either as receptor‐operated or as store‐operated Ca channels, whose activation increases intracellular Ca. However, it is not known if these channels are expressed in JG cells and are involved in CaSR‐mediated regulation of renin release. We hypothesized that TRPC channels are expressed in JG cells and participate in the Ca‐mediated inhibition of renin release in response to activation of the CaSR. RT‐ PCR revealed mRNA expression of TRPC isoforms ‐1, ‐2, ‐3, ‐4, ‐5 and ‐6 in freshly isolated mouse JG cells. Incubation of primary cultures of JG cells with the TRPC blocker (SKF 96365, 50μM) did not change control values (0.46±0.09 vs. 0.46±0.08 μg ANGI/ml/hr/mg prot.) CaSR agonist cinacalcet decreased renin release by 46% to 0.25±0.05 μg ANGI/ml/hr/mg prot.(p<0.04). Addition of SKF 96365 completely reversed the CaSR‐mediated inhibition by cinacalcet. Our data suggests that JG cells contain multiple isoforms of TRPC channels, and at least one of these is linked to down‐stream signaling of CaSR‐mediated renin inhibition. (NIH 5PO1HL090550–04)
The Role of ENaC in Hypernatremia
Authors: Mironova Elena , Bugay Vladislav , Stockand James
Published: June 2021

ENaC in renal physiology controls sodium excretion to achieve blood pressure regulation through the renin‐angiotensin‐aldosterone system. Our recent findings suggest an additional aspect to this channel's regulation of sodium and water homeostasis. AVP stimulates ENaC in low aldosterone states, indicating that sodium reabsorbed through this channel may contribute to the hypertonic medulla drawing free water out of the distal nephron to concentrate urine. To test whether the consequences of activating ENaC depends on simultaneous AQP2 activation, we forced mice to hypernatremia by providing them only with 3% NaCl drinking solution. The activity of ENaC was robust in these animals. This surprising result was dependent on AVP, which also was elevated due to an increase in serum tonicity. Benzamil compromised the ability to concentrate urine of both control mice and mice maintained with 3% saline, appearing as a benzamil‐dependent increase in urine output and a decrease in UOsm. Thus, elevated AVP counters hypernatremia by stimulating ENaC activity to facilitate free water reabsorption through AQP2. In this context, activation of ENaC in the distal nephron is protecting POsm by promoting anti‐aquaresis rather than influencing serum [Na+] via anti‐natriuretic effects.
A Model of Calcium Transport along the Rat Nephron
Authors: Edwards Aurelie , Thomas S. Randall , Perthame Benoit , Seguin Nicolas , Tournus Magali
Published: June 2021

We developed a mathematical model of calcium (Ca2+) transport along the rat nephron to investigate the factors that promote hypercalciuria. The model is an extension of the flat medullary model of Hervy and Thomas (Am J Physiol Renal Physiol 284: 65, 2003). It explicitly represents all the segments beyond the proximal tubule, and distinguishes between short‐ and long‐looped nephrons. It solves dynamic conservation equations to determine NaCl, urea, and Ca2+ concentration profiles in tubules, vasa recta, and interstitium. Calcium is known to be reabsorbed passively in the thick ascending limb, and actively in the distal convoluted (DCT) and connecting (CNT) tubules. Our simulations suggest that urinary Ca2+ excretion is strongly modulated by water and NaCl reabsorption along the nephron. Our model also predicts that the passive diffusion of Ca2+ from the loop of Henle generates a significant cortico‐medullary interstitial Ca2+ concentration gradient. Finally, our results suggest that the DCT and CNT can adapt to upstream variations in Ca2+ transport, but not always sufficiently to prevent hypercalciuria. This work was supported by the program EMERGENCE (EME 0918) of Université Paris 6.
Gating modulation of the sodium‐activated potassium channel KCa4.1 (Slack) by bithionol and niclosamide
Authors: Fleming Matthew R. , Singh Vikrant , Jenkins David Paul , Wulff Heike , Kaczmarek Leonard K.
Published: June 2021

The Na+‐activated K+ channel Slack (also known as KCNT1, Slo2.2, or KCa4.1) is widely expressed throughout the central nervous system, where it contributes to the slow afterhyperpolarization and is involved in controlling bursting behavior and spike frequency adaptation. Gain‐of‐function mutations in Slack have recently been shown to cause malignant migrating partial seizures of infancy. KNa channels further have long been known to protect cells from hypoxic injury. Slack activators therefore should be therapeutically useful for reducing neuronal excitability in epilepsy and ataxia as well as for the prevention of hypoxia associated neuronal damage in stroke and cerebral palsy. Slack channel activity is increased by the antiparasitic compounds bithionol and niclosamide. However, while bithionol is a positive gating‐modulator that requires intracellular sodium to be elevated to exert an effect, niclosamide is a true opener that activates Slack currents in whole‐cell patch‐clamp experiments even with a KF containing intracellular solution in the nominal absence of both Na+ and Ca2+. Both types of Slack channel activators can be detected by resonance wavelength grating optical biosensors (the SRU Biosystems BIND), a high‐throughput assay measuring changes in mass at the plasma membrane. We are currently using this assay to screen for both Slack channel activators and inhibitors.

Supported by R21 NS073943
Ammonium ion trapping in mouse distal colon: Role of NHE3
Authors: Engevik Melinda A. , Worrell Roger T.
Published: June 2021

Despite its critical role in the development of hepatic encephalopathy, ammonium transport in the gastrointestinal tract is poorly understood. It is well known that overall in the gut net ammonium absorption occurs. However, we have shown that net secretion occurs in the distal colon of mice and thus limits overall net absorption. Ammonium secretion occurs in colonic epithelial cells via basolateral NH4+ uptake mediated by NKCC1 and Rhesus glycoprotein B (RhBG). Our data suggests apical ammonia exit occurs via Rhesus glycoprotein C (RhCG) combined with ion trapping via H+ transport on cH/K‐ATPase and/or NHE2/3. In the human colonic cell line T84, inhibition of NHE2/3 by amiloride produced a 50% decrease in secretory NH4+ flux, implicating NHE2/3 in ion trapping of ammonia. In NHE3−/− mice portal vein ammonium levels were 1.9±0.4 fold higher than in WT, while no significant change was observed in NHE2−/− or cH/K‐ATPase−/− mice. qRT‐PCR, Western blot and immunohistochemistry was used to define distal colon transporter expression in various transporter null mice to establish co‐operative function in ammonium secretion. In NHE2−/− mice NHE3−/− was up‐regulated, in cH/K‐ATPase−/− mice both RhBG and NKCC1 were upregulated but not NHE3. In the NHE3−/− mice, cH/K‐ATPase was up‐regulated. Taken together these data suggest that NHE3 plays a principle role in ammonia ion trapping. Supported by NIH DK079979.