bluuebunny/crossref_metadata_2025 huggingface dataset:
TGFβ2 is present in infant formula, resists digestion in vitro and is biologically active
Authors: Lonnerdal Bo , Du Xiaogu , Morris Kristin , Jouni Zeina , Rai Gyan , Waworuntu Rosaline
Published: June 2021

TGFβ2 is present in human milk and believed to be important for IgA production and oral tolerance. Concentrations are lower in bovine milk, but by utilizing milk protein sources high in TGFβ2, formula TGFβ2 can be increased. We investigated whether TGFβ2 in human milk and formula can resist proteolysis under conditions similar to those in the infant gut. Human milk and formulas were exposed to pepsin at pH 5.0, 3.5 or 2.0 (reflecting increasing maturity of the infant gut) at 37C for 30 min, neutralized to pH 7.0 and incubated for 30 min with pancreatic enzymes. TGFβ2 was analyzed by ELISA. TGFβ bioactivity was assessed by a Smad2 redistribution assay using a Cellomics ArrayScan system and by inhibition of IL‐2 stimulated growth of HT‐2 cells. TGFβ2 levels in formula were variable, but in some cases exceeded those of human milk samples. Digestion with pepsin at pH 2.0 or 3.5 substantially increased immunodetectable TGFβ2 in formula, and digestion with pancreatic enzymes increased it further. This suggests that acidification and/or proteolysis play important roles in liberation of immunodetectable TGFβ2. TGFβ2 in digests (pepsin+pancreatin) was highly bioactive by both assays used. In summary, TGFβ2 in infant formula continues to be immunodetectable and retains activity after in vitro digestion, strongly suggesting that TGFβ2 can survive in the infant gut and exert its biological activities. Supported by Mead Johnson.
Changes in liver weight are independent of hepatic lipid content in guinea pigs fed a high‐cholesterol low‐carbohydrate diet
Authors: deOgburn Ryan C , Leite Jose O , Ratliff Joseph C , Volek Jeff S , McGrane Mary M , Fernandez Maria Luz
Published: June 2021

We have used high cholesterol (HC) diets to study mechanisms of atherosclerosis in the guinea pig and shown that low carbohydrate diets (LCD) mitigate aortic cholesterol accumulation and inflammatory expression. We have also shown that HC diets induce fatty liver in guinea pigs. We evaluated whether LCD could improve hepatic steatosis in guinea pigs. Male Hartley guinea pigs were fed a HC diet (0.25g/100g) and randomly assigned (n=10/group) to consume either a LCD or high‐carbohydrate diet (HCD) for 12 wk. Plasma and whole livers were collected for lipid analysis. Plasma LDL cholesterol was lower in animals fed the LCD (P < 0.01) diet while plasma triglycerides (TG) were higher (P < 0.05). There were no differences in plasma free fatty acids or plasma glucose between groups. However, glucagon‐like peptide‐1 (GLP‐1) was lower in the LCD group (P < 0.01). Liver weight was 40% higher in the LCD compared to the HCD group. Interestingly despite the increased liver weight in LCD there were no differences in hepatic TG, cholesterol or non‐esterified fatty acids between groups. Histological analysis performed by hematoxylin and eosin staining confirmed there were no differences in hepatic lipid accumulation or steatosis between groups. Collectively these results suggest that in the presence of a HC‐LCD, the increase in liver mass is unrelated to lipid accumulation. An increased number of hepatocytes through an accelerated regenerative process may be responsible for these observations in the LCD group.
High Protein Diet and DHA Supplementation was Effective to Recover the Impaired Nutritional Status in Irradiated Rats
Authors: Kim Kyoung‐Ok , Jung Ji‐Hye , Lim Sung‐Hee , Park Su‐Jin , Chun Mi‐Son , Kang Seung‐Hee , Kim Hyun‐Sook
Published: June 2021

Radiation has been widely used as therapeutic methods of various malignancies. This treatment induces the nutrition‐related symptoms such as vomiting, weight loss and anorexia‐cachexia. Cancer patients on radiation therapy are supervised with the food intake and the supplementation of specific nutrients, such as amino acids and n‐3 polyunsaturated fatty acids. The major goal of this study was to determine the effects of high protein diet (HP) and DHA supplementation in minimizing the adverse effects of radiation for cancer treatment. Rats were divided into four groups: normal protein diet (NP) and HP groups and NP + DHA and HP + DHA groups. Each diet group was further divided into treatment groups that received radiation (RT group) and one that did not (non‐RT group). The diet administration was supplied from 12th day prior to radiation treatment with irradiation dose of 17.5 Gy. The diets were continued until 10th day after radiation treatment and animals were sacrificed. Radiation treatment caused the significant fall in serum protein, albumin, alkaline phosphatase and HDL‐C, while LDL‐C and TG levels were elevated. The most significant observation of this study was that high protein diet and DHA supplementation was effective to improve the distorted nutritional status by decreasing elevated lipid levels in irradiated rats.
Regulation of vascular calcification by endogenous ATP release coupled to extracellular pyrophosphate accumulation: Modulation by cAMP signaling
Authors: Prosdocimo Domenick Anthony , Dubyak George R.
Published: June 2021

Extracellular pyrophosphate (ePPi) inhibits vascular smooth muscle cell (VSMC) calcification and we have found that extracellular ATP (eATP) constitutively released from VSMC is coupled to ePPi generation via the NPP1/PC‐1 ecto‐ATPase. Recent studies have indicated that cAMP/PKA‐induced calcification modulates ePPi homeostasis. Thus, we tested whether such cAMP signaling pathways also perturb ePPi accumulation via attenuation of eATP release and/or the NPP1‐mediated hydrolysis of eATP. We report that forskolin (Fsk)‐induced activation of cAMP/PKA in primary rat VSMC suppresses eATP release and coupled ePPi accumulation in the absence of decreased NPP1 activity. This Fsk‐induced attenuation of endogenous eATP release and ePPi potentiates phosphate (Pi)‐induced VSMC calcification, an effect rescued by exogenously added ATP. These data provide direct support for a model wherein constitutive ATP release is efficiently coupled to PPi generation (via NPP1) to inhibit aberrant mineralization in VSMC.

(Supported by P01‐HL18708, RO1‐GM36387, and T32‐HL07887).
FROM MONOMER STRUCTURE TO FIBRIL POLYMORPHISM: COMPUTATIONAL AND EXPERIMENTAL STUDIES OF THE ALZHEIMER'S ABETA PEPTIDE
Authors: Head‐Gordon Teresa
Published: June 2021

I will describe the development and application of all‐atom and coarse‐grained models for the investigation of disease protein aggregation for the Alzheimer's Aβ systems, in which we show how the aggregate equilibrium, formation kinetics, and structural ensembles depend on sequences ad structure of the monomeric peptide. We discuss the implications of our results in regards to interpretation of NMR observables and the kinetics of fibril assembly using chromatography and dye‐binding experiments.
Bacterial quorum sensing
Authors: Hughson Frederick
Published: June 2021

Bacteria sense and respond to small molecule signals in a cell‐cell communication process termed quorum sensing. We are investigating recently identified quorum sensing circuits in several different bacterial species. To develop a molecular understanding of how quorum sensing signals (called autoinducers) are detected, and how sensory information is transduced to control behavior on a community‐wide scale, we have adopted an integrated approach combining synthetic organic chemistry, bacterial genetics, biochemistry, and x‐ray crystallography. The results further our mechanistic understanding of transmembrane signal transduction via two‐component sensor kinases, of which these quorum sensing receptors represent particularly tractable examples. Supported by NIH AI054442 and HHMI.
Aged adipose derived stem cells are more proliferative and less responsive to differentiation cues
Authors: Stern Matthew Michael , Fearing Bailey V. , Soker Shay , Kritchevsky Stephen B. , Van Dyke Mark
Published: June 2021

Adipose derived stem cells (ADSCs) are mesenchymal stem cells that reside within the microvasculature of adipose tissue. Two questions regarding ADSCs that have not been adequately addressed are 1) whether the aging process adversely affects the potential of ADSCs for use in therapeutic applications and 2) whether age‐related changes in ADSCs contribute to increased adiposity observed in aging individuals. To address these questions, we obtained ADSCs from young and old rats and compared their ability to proliferate and differentiate into multiple cell lineages. ADSCs obtained from young and old animals exhibited the same phenotype; however, old ADSCs were more proliferative. When induced to undergo adipogenic or osteogenic differentiation, young ADSCs exhibited greater levels of staining for indicators of terminal differentiation and greater changes in the expression of several genes involved in differentiation. Our results suggest that ADSCs are subject to cell‐intrinsic aging mechanisms that render aged cells more proliferative and less responsive to differentiation cues than young cells even when cultured under identical conditions. This raises concern over the potential use of autologous ADSCs in therapeutic applications for elderly individuals and suggests that hyperproliferative ADSCs could play a role in the accumulation of adipose and adipose‐like tissue in aged individuals.
Structures of RB69 DNA polymerase ternary complexes reveal multiple modes of metal ion coordination to correct incoming dNTPs
Authors: Wang Mina , Konigsberg William H. , Steitz Thomas A. , Wang Jimin
Published: June 2021

We have observed three different modes of metal ion coordination to the triphosphate tails of all correct dNTPs in the crystal structures of 14 ternary complexes of the RB69 pol triple mutant L561A/S565G/Y567A. These include the classic tridendate coordination to all three α,β and γ phosphates and two novel modes of bidendate metal ion coordination, to α,β phosphates and β,γ phosphates. The bidendate dNTP complexes have not been previously observed in any of the ternary crystal structures of RB69 pol reported to date. We have identified residues D684, E686, and E716 as being specifically involved in α,β bidendate coordination. We also showed that replacement of E686 and D684 with alanine residues reduced the catalytic efficiency by 70 fold and 20 fold respectively. These results provide the first direct evidence that the α,β bidendate coordination complex is a kinetically important intermediate in the nucleotidyl transfer reaction pathway.
TGF‐β superfamily type‐II receptor regulation of telomerase and telomeres in human breast cancer cells
Authors: Nicholls Craig Daniel , Cassar Lucy , Li He , Liu Jun‐Ping
Published: June 2021

Transforming growth factor‐beta (TGF‐β) plays an important role in regulating telomerase activity in cancer cells. Signalling by TGF‐β superfamily ligands is initiated by ligand binding to one of four transmembrane receptor kinases and subsequent phosphorylation of target proteins, resulting in intracellular signalling cascades. Whereas the downstream target protein Smad3 represses the gene coding for telomerase reverse transcriptase (TERT), the roles of the specific receptors of the TGF‐β family in mediating regulation of telomerase activity remain to be established. Many recent studies indicate a key role for the TGF‐β family receptors in cancer cell proliferation, with truncating mutations detected in many cancers ‐ particularly those of breast, colorectal and prostate origin. Such mutations inactivate the kinase ability of these receptors and abolish downstream signalling. This work focuses on the effect of dominant‐negative (DN) type‐II receptors on telomerase and telomeres in breast cancer cells.

Human breast cancer MCF‐7 cells were transfected with plasmid constructs expressing truncated type‐II receptors fused to GFP. After approximately 2.5 months stably transfected cells were analysed for telomerase activity, telomere length, cell proliferation and senescence markers. Cells expressing DN‐BMPRII exhibited increased telomerase activity coupled with increased cell proliferation relative to control. Conversely, cells expressing DN‐ACVRIIA/B or DN‐TGFβRII all had decreased telomerase activity, reduced telomere length and an increase in cellular senescence. Taken together these results indicate that BMPRII signalling mediates inhibition of telomerase activity and cell proliferation in MCF7 breast cancer cells, whereas TGFβRII and ACTRIIA/B signalling may be required to sustain telomerase activity and cell proliferation.
The structural insight into the biological role of PHD fingers
Authors: Kutateladze Tatiana G , Champagne Karen S , Pena Pedro V , Musselman Catherine A , Hung Tiffany , Saksouk Nehme , Cote Jacques , Gozani Or
Published: June 2021

Covalent modifications of histone tails and protein domains that bind these epigenetic marks are essential components of the chromatin remodeling and gene expression machinery. The PHD (plant homeodomain) finger is found in many transcriptional regulators and chromatin‐associating complexes, however the overall biological role of this module remains unclear. Recently, it was found that a subset of PHD fingers recognizes histone H3 tri‐methylated at lysine 4 (H3K4me3). In this study, we characterize the binding specificity of the PHD finger family using a set of biochemical, crystallographic and spectroscopic approaches. We compare the crystal structures of the H3K4me3‐bound PHD fingers, identify the binding sites for methylated histone H3 tails by resonance perturbation analysis and mutagenesis, and measure the binding affinities by tryptophan fluorescence. Our results provide novel insight into the molecular mechanisms underlying the activity of the histone‐binding PHD fingers, and further our understanding on how the histone code is read.
Analysis of archaeal histone interactions from M. fervidus.
Authors: Stencel Katherine Ann , Tims Hannah S.
Published: June 2021

Archaea are prokaryotes with cellular characteristics of eukaryotes. Homologues of eukaryotic histones are found in some archaea, however, the structural and functional characteristics of archaeal nucleosomes are not well understood. Archaea can have 0‐6 histones, which are able to form hetero/ homodimers. Histones from the archaea, Methanothermus fervidus (HMfA, HMfB) were expressed and purified from E. coli plasmids containing clones of the histones (gift of Kathleen Sandman, The Ohio State University). Cloned archaeal high affinity DNA sequences were purified from E. coli to be used to assemble nucleosomes. The histones were tagged with fluorescent dyes to be used in Fluorescence resonance energy transfer (FRET) experiments to study histone‐histone and histone‐DNA interactions. To allow specific placement of the dyes, the histones were mutated to introduce cystine residues as attachment sites. Kathleen Sandman (OSU) provided mutated HMfB. HMfA will be mutated with a QuickChange mutagenesis kit (Stratagene) for hetero‐histone complex investigation.

This work was supported by a grant to The College of Wooster from the Howard Hughes Medical Institute through the Undergraduate Science Education Program.
Proteome analysis of three mouse models for Alzheimer's disease suggests impairment of adolescent hippocampal plasticity
Authors: Hartl Daniela , Zabel Claus , Rohe Michael , Mao Lei , Klose Joachim
Published: June 2021

We have analyzed three mouse models for Alzheimer's disease (AD) using our high‐resolution 2‐dimensional gel electrophoresis technology in combination with fluorescent dyes and mass spectrometry. We found numerous protein expression changes in brain regions of transgenic mice which largely preceeded the occurrence of amyloid plaques. Interestingly, we also found a considerable peak in the number of hippocampal protein changes of adolescent transgenic mice. Furthermore, the absence of normal developmental proteome alterations as well as a down‐regulation of proteins related to synaptic plasticity were observed in this early phase. Together, our results suggest the disturption of hippocampal plasticity during adolescence in APP transgenic mice. Our findings are in line with the observation that AD is preceded by a clinically silent period lasting several years to decades.
Delineation of VEGF‐regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis
Authors: Boone Thomas Hunter , Howarth Byran James , Mowa Chishimba Nathan
Published: June 2021

VEGF‐regulated genes in the cervices of pregnant and non‐pregnant rodents were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein. Tissues were analyzed by genome‐wide DNA microarray analysis, Real‐time and gel‐based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffe's F‐test for multiple comparisons. Of the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular‐ and non‐vascular‐related processes were found to be regulated by VEGF in the cervix, including immune response, cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes were altered. VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. We show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM. These data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near‐term, including cervical remodeling.
Purification and Reconstitution of Recombinant Myostatin Latent Complex
Authors: Zhang Hong , Trill John , Johnson Kathleen , Scott Gilbert , Kull Frederick , Johanson Kyung
Published: June 2021

Myostatin (GDF‐8) is a member of the TGF b super family. It is expressed in skeletal muscle and acts as a negative regulator of skeletal muscle growth. Myostatin is synthesized as pre‐pro‐myostatin and secreted as an inactive pro‐myostatin, where pro‐domain is processed from mature myostatin by a furin‐like protease. In vivo, myostatin is in circulation as an inactive latent complex (LC) which consists of pro‐domain and mature myostatin. It was shown that active mature myostatin was released from inactive latent form upon cleavage of pro‐domain by BMP‐1. To study the proteolytic processing of LC, full length myostatin was expressed as a fusion protein with an N‐terminal 6His/GB1tag with Tev cleavage site in a CHO secretion system. Myostatin was captured using Ni‐NTA chromatography. However, it was a mixture of various forms, 1) incomplete furin cleavage, 2) intact or cleaved pro‐domain at BMP‐1 cleavage site or C‐terminus truncated pro‐domain, and 3) intact or C‐terminus truncated mature domain. Every component was tightly aggregated together. To obtain an intact recombinant latent complex (rLC), the mixture was treated with furin and Tev, adjusted to 6M Gdn‐HCl to dissociate the aggregation, then passed through Ni‐NTA to both remove the His/GB1 tag and to release the tag‐cleaved pro‐domain. Using C8 RP‐HPLC, mature myostatin was isolated from the mixture. When the pro‐domain and mature myostatin were mixed in 6M Gdn‐HCl, dialyzed, then loaded onto a sizing column in PBS, only the intact pro‐domain was complexed with mature myostatin forming an intact rLC. This rLC was used as a substrate for proteolytic study.
Affordable nutritious foods identified using the Nutrient Rich Food (NRF) Index and CNPP Food Prices Database
Authors: Drewnowski Adam , Monsivais Pablo , Eichelsdoerfer Petra
Published: June 2021

The USDA Center for Nutrition Policy and Promotion's Prices database includes the mean national price per 100g of food ($/100g), edible portion for 6640 foods listed in the Food and Nutrition Database for Dietary Studies 1.0. The Nutrient Rich Food (NRF) Index, a unitary metric reflecting the total nutrient content of a given food, is calculated as the sum of the Daily Values (DVs) of 9 nutrients to encourage (protein, fiber, vitamins A, C, E, and calcium, iron, potassium and magnesium) minus the sum of the DVs of 3 nutrients to limit (saturated fat, added sugars, and sodium), all per 100 kcal. Analyses of the database showed an inverse relation between energy density (MJ/kg) and energy cost ($/MJ) and a positive relation between nutrient density and energy cost. The energy cost of refined grains, added sugars and fats was less than the cost for perishable fresh produce. Analyses of NRF value per unit cost helped to identify affordable nutritious foods both across and within major food groups. Each food group included foods and food categories that provided high nutritional value at an affordable cost. The integration of nutrient profiling with food prices databases offers new insights into the tradeoffs between food quality and food cost and a new approach to price‐ and nutrient‐related research. Supported by the National Dairy Council and NCCAM fellowship 2 T32AT000815
Diet Quality of Low‐income and Higher‐income Americans in 2003‐2004 as Measured by the Healthy Eating Index‐2005
Authors: Guenther Patricia M. , Juan WenYen , Lino Mark , Hiza Hazel A , Fungwe Thomas V , Lucas Richard
Published: June 2021

The Healthy Eating Index‐2005 (HEI‐2005) has 12 components designed to measure diet quality in terms of compliance with the 2005 Dietary Guidelines for Americans. Mean HEI‐2005 scores were estimated using one 24‐hour recall from 8,272 participants in the National Health and Nutrition Examination Survey, 2003‐2004. Long‐term intakes were estimated using the population ratio method. For the population age 2 and older, the total score was 57.5 out of a possible 100. Scores were particularly low for Dark Green and Orange Vegetables and Legumes, Whole Grains, Sodium, and Calories from Solid Fats, Alcoholic beverages, and Added Sugars. Total scores of the low‐income and higher‐income populations were not significantly different; however, compared to higher‐income people, low‐income people (family income less than 130% of poverty) had significantly lower scores for Total Vegetables, Dark Green and Orange Vegetables and Legumes, and Whole Grains; and a higher score for Sodium (indicating a lower intake) (p<.05). There was no significant difference in total HEI‐2005 scores for children ages 2 to 18 by income level. Low‐income children (less than 185% of poverty) had a higher score for Total Vegetables. All Americans, regardless of income level, could benefit from increased intake of fruits, vegetables, whole grains, and fat‐free or low‐fat milk; choosing more nutrient‐dense forms of foods; and lower sodium intake.
Omega‐3 Index as a risk factor of ischemic and haemorrhagic stroke: a pilot case‐control study
Authors: Park Yongsoon , Park Seonhye
Published: June 2021

Previous studies suggested that fish consumption and blood level of omega‐3 fatty acids negatively correlated with ischemic stroke, but the relation to the risk of haemorrhagic stroke remains unclear. This study was investigated the effect of Omega‐3 Index (EPA + DHA in red blood cell, RBC) on hemorrhagic and ischemic stroke.

Hemorrhagic (n=24) and ischemic stroke (n=31) cases, and controls (n=20) were included in the present study. Omega‐3 Index (8.36 ± 0.58 vs. 10.66 ± 0.68 %), total n‐3 fatty acids (15.30 ± 0.57 vs. 12.61 ± 0.78 %), and C22:6n3 (DHA; 8.93 ± 0.55 vs. 7.09 ± 0.38 %) in RBC were significantly higher in control than ischemic and haemorrhagic stroke patients. Omega‐3 Index and total n‐3 fatty acids, and DHA were significantly increased the risk for haemorrhagic stroke, but not for ischemic stroke. However, among the ischemic stroke subgroups, lacunar infarction patients had the significantly lower level of Omega‐3 Index and EPA, suggesting that low level of omga‐3 fatty acids might be a provocating factor on lacunar infarction. There was no significant difference in intake of nutrients and foods between control and patients, except low intake of brown rice, seaweed soup, bean sprouts soup, and bean paste pot stew in stroke patients. In conclusion, n‐3 fatty acids may have beneficial effect on haemorrhagic stroke and lacunar infarction, a subgroup of ischemic stroke.
Effect of Storage Time and Temperature on Indicators of Nutritional Quality or Spoilage in Pumped Human Milk
Authors: Fogleman April D. , Tully Mary Rose , Allen Jonathan C.
Published: June 2021

Objectives were to study the effect of storage for 3 wk at ‐20, 4, and 24°C on protein (TP), free amino ends (FAE), fat (F), free fatty acids (FFA), bacterial growth (B), secretory immunoglobulin A (SIgA) and lysozyme (L) activity in freshly expressed human breast milk (HM) and to examine bacterial growth in pasteurized (P) and unpasteurized (UP) HM in a bottle contaminated by an infant and stored at 24°C for up to 24 hr and at 4°C up to 6 days. HM samples were collected from 12 women and stored up to three weeks. There was a decrease in TP in HM stored at 24°C at 1 wk (p < 0.01), an increase in FAE at 4°C at 1 wk (p < 0.05) and 24°C at 1 wk (p<0.001), an increase in FFA at ‐20°C at 2 wk (p < 0.05), 4°C at 48 hr (p < 0.0001), and 24°C at 48 hr (p < 0.0001), and an increase in B at 4°C at 2 wk (p < 0.05) and 24°C at 48 hr (p < 0.01). HM samples were collected from 5 women whose infants partially consumed bottles of P and UP HM. There was an increase in B in P HM stored at 24°C at 24 hr (p < 0.0001) and in UP HM stored at 24°C at 24 hr (p < 0. 001). Infant suckling contributed less B than pumping to HM. The data indicate that HM may be safely stored longer than current recommendations.
Transforming growth factor beta in human milk and some infant formula are biologically active in HT‐2 cells
Authors: Jouni Zeina E , McMahon Robert J , Morrow Ardythe L , Rosales Francisco J
Published: June 2021

Transforming growth factor beta (TGFβ) of human milk (HM) is thought to play an important role in maintaining appropriate immune function in infants. In the present study we assessed and compared total and TGFβ2 specific activity in HM and infant formulas (IF) in HT‐2 cells. This bioassay is used to study regulation of T‐cell growth by TGFβ inhibiting the S‐phase progression of HT‐2 cells stimulated with IL‐4. Mature HM were sampled from mothers in the US and Mexico, while IF samples (Enfamil® LIPIL®/A+®) were obtained from US, China, Mexico, Thailand, and the Philippines from the manufacturer. HM inhibited cell growth in a dose‐dependent manner. HM at a concentration of TGFβ equivalent to that found of Enfamil (4900 ± 1400 pg/ml) exhibited a 2‐folds higher total bioactivity, which may be explained in part by additional functional factors in HM that also inhibit cell proliferation, differences in physiochemical properties of the milks, or potential matrix effects. TGFβ related activity in HM did not differ between US and Mexican mothers; similarly TGFβ related activity in IF did not vary by the manufacturer location. TGFβ2 specific neutralizing antibody decreased activity in IF and HM by 2‐3 folds and 2‐7 folds respectively, suggesting that some, but not all, of the activity is due to TGFβ2. These results demonstrate that both infant formula and HM exerted similar TGFβ related activities in HT‐2 cells. Supported by MJN.
Effects of dietary calcium and milk on oxidative and inflammatory stress and lifespan in wild‐type and aP2‐agouti transgenic mice
Authors: Bruckbauer Antje , Sobhani Marilyn T , Zemel Michael B
Published: June 2021

Dietary Ca reduces oxidative and inflammatory stress, while milk exerts a greater effect than supplemental Ca. To examine the effects on lifespan and related biomarkers, aP2‐agouti transgenic mice (model of diet‐induced obesity) and wild‐type littermates were fed a low Ca (0.4% Ca), high‐Ca (1.2% Ca from CaCO3), or milk (1.2% Ca from non‐fat dry milk) obesigenic diets until death. Randomized subgroups were sacrificed at 28, 52 and 78 weeks of age for biomarker analysis. The milk diet attenuated weight and adiposity gain and prevented the age‐related muscle loss (p<0.04) and rise in reactive oxygen production (ROS) in adipose tissue (p=0.005). There was a corresponding increase in SOD3 (in wild‐type, p=0.04) and glutathione peroxidase gene expression (p<0.02) in soleus, and in liver SOD activity (p<0.04). The milk diet decreased adipose tissue IL6 (p<0.05) and TNFα (p<0.03) expression in male mice only. The survival analysis showed an overall shorter lifespan in transgenic vs. wild‐type mice. Although no diet effect was found on lifespan, the milk diet resulted in a significant increase in early (75%) survival rate in wild‐type mice (p<0.02). These data demonstrate that milk diet attenuates adiposity, protects against muscle loss and reduces oxidative and inflammatory stress. Although these effects did not influence maximum lifespan, they did suppress early mortality.

Grant Funding Source

The National Dairy Council
Neonatal birth weight according to maternal serum lipid levels and dietary intakes
Authors: Choi Hyung In , Kim Hyesook , Kim Ki Nam , Ha Eun‐Hee , Park Hye‐Sook , Ha Mina , Kim Yangho , Hong Yun‐Chul , Chang Namsoo
Published: June 2021

The relationship of neonatal birth weight with maternal serum triglyceride (TG) level has been reported. This hospital‐based cohort study examined the relationship between maternal dietary intake and serum lipid levels in relation to neonatal births weights in 460 pregnant women and their babies. Maternal serum TG and HDL‐cholesterol (HDL‐C) were correlated with intakes of food and nutrients such as cereals and cereal products, meat and meat products, carbohydrates, and polyunsaturated fatty acid. After controlling for maternal age, babies' sex, income and history of low birth weight, the adjusted odds ratio of low birth weight (<2.5kg) delivery was 6.1 (95% confidence intervals, 1.202‐30.707) in women with below the median serum TG level at parturition. The adjusted odds ratio of macrosomia (=4kg) delivery was 0.4 (95% confidence intervals, 0.133‐0.931) in women with above the median serum HDL‐C level. We suggest that maternal lipid levels at parturition of which dietary intake may have partial contribution throughout the pregnancy is associated with birth weight.

*This work was supported by the Ministry of Environment and the 2nd stage of Brain Korea 21 project in 2008
Dietary Supplementation with Dried Plum Prevents Ovariectomy‐Induced Bone Loss in C57BL/6 Mice and Modulates the Immune Response
Authors: Rendina Elizabeth , Lim Yin F. , Marlow Denver , Wang Yan , Clarke Stephen L. , Kuvibidila Solo , Lucas Edralin A. , Smith Brenda J.
Published: June 2021

Due to recent evidence demonstrating the link between the immune system and skeletal health, this study was designed to determine the osteoprotective effects of dried plum (DP) on bone metabolism and the immune response. Twelve‐week old female mice (n=59) were assigned to 5 treatment groups for a duration of 4 wks: sham‐operated (Sham) control diet (AIN‐96M), ovariectomized (OVX)‐control diet and OVX animals receiving diet supplemented with 5%, 15%, or 25% DP (w/w). Bone mineral density and content of the spine was decreased (8%) by OVX, which was totally prevented by 25% DP. µCT analyses revealed the OVX compromise in spine and proximal tibial bone volume was prevented by higher doses of DP. Similar results were also observed in the trabecular number and thickness of the spine. Simulated biomechanical tests (finite element analyses) showed the improved structural properties observed with DP (15% & 25%) resulted in enhanced bone strength and stiffness. These alterations in bone mass and architecture coincided with increased serum IGF‐I and reduced bone turnover (i.e. serum PINP) in OVX mice consuming DP. Following ex vivo Con A stimulation, splenocyte TNF‐α production was suppressed by DP, but no change in IL‐6 occurred. This study supports DP's positive effects on bone in the OVX mouse model; however the role of the immune system in this response warrants further investigation. (Supported by California Dried Plum Board)

Grant Funding Source

California Dried Plum Board
Effects of conjugated linoleic acid supplementation on inflammatory mediators and immunoglobulins in overweight Korean females
Authors: Kwak Ho‐Kyung , Kim Ok Hyun , Jung Hana , Kim Jung Hee
Published: June 2021

Conjugated linoleic acids (CLAs) are a group of fatty acids shown to have anti‐adipogenic and anti‐inflammatory properties, and beneficial effects on immune function. In a double‐blind placebo‐controlled study, 81 overweight premenopausal Korean females (BMI ≥ 23 kg/m2) aged 20‐50 years were randomized to one of 4 groups. Each group was received placebo (olive oil), a CLA mixture 1.5g/d, 2.25g/d or 3g/d for 12 weeks. The major isomer content of tested CLA mixture (78.4% free fatty acids) was 36.8% cis‐9 trans‐11 CLA and 37.3% trans‐10 cis‐12 CLA. With 12 weeks of supplementation, mean C‐reactive protein was increased by 35% in a group with olive oil, while groups with CLA at 2.25 and 3mg/d showed 1 and 11% elevation, respectively (p>0.05). Plasma interleukin‐10 (IL‐10), an anti‐inflammatory cytokine, was significantly increased with 3g/d CLA supplementation for 12 weeks (P<0.05). Although mean plasma IL‐10 was found to be increased after 12 weeks in all groups, the groups with CLA supplementation showed greater changes (187‐232%) compared with placebo (146%). Elevated plasma immunoglobulin M was observed in 73% of individuals who received 3mg/d CLA for 12 weeks. These results suggest that CLA supplementation might have a potential to modulate markers related with inflammation and immune function. However, the effects observed in the current trial seem to be much more subtle than those found in animals and few other clinical studies, and a clinically useful level of CLA dose should be further elucidated.
Xanthohumol decreases adipocyte differentiation
Authors: Teixeira Diana , Mendes Vanda , Pestana Diogo , Calhau Conceição , Azevedo Isabel , Monteiro Rosário
Published: June 2021

Lately, some phytochemicals have been demonstrated to possess anti‐obesity properties. In this regard we investigated the effect of xanthohumol (XN), a flavonoid from hops (Humulus lupulus L.) in preadipocyte differentiation. For this, we tested the effect of XN on 3T3‐L1 cells in lipid accumulation in several periods of preadipocyte differentiation, PPARγ expression and NFκB activation. Differentiation was measured using oil red O incorporation or PPARγ expression and NFκB activation was assessed by ELISA. XN reduced culture lipid content and this effect was more pronounced when the compound was present before (46% of control) or during differentiation induction (47% of control). Supporting these results, PPARγ transcription was also reduced by treatment with 10 μM XN, corroborating an inhibitory effect of the flavonoid in adipocyte differentiation. NFκB activation was detected in preadipocytes which may account for part of the effects on differentiation, although it was unaltered in adipocytes. These results suggest that XN is able to reduce adipocyte differentiation which may contribute to increase adipocyte size. Since adipocyte hypertrophy may result in obesity‐related inflammatory events, XN may not improve the metabolic profile associated with obesity.

Supported by FCT (POCI, FEDER, Programa Comunitário de Apoio, SFRH/BPD/40110/2007) and Instituto de Bebidas e Saúde (iBeSa).
Measurement of deuterium‐labeled phylloquinone in plasma by LC‐APCI‐MS
Authors: Fu Xueyan , Dolnikowski Gregory , Peterson James , Booth Sarah L
Published: June 2021

Deuterium‐labeled vegetables were fed to humans for the measurement of both unlabeled and deuterium‐labeled phylloquinone in plasma. We developed a technique to determine the quantities of these compounds using liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC‐APCI‐MS). Sample preparation involved liquid‐liquid and solid‐phrase extraction techniques. The internal standard (K1(25)) was used to quantify both unlabeled and labeled phylloquinone. The standard calibration curve had a linear correlation coefficient (R2) of 0.998 and 0.999 for unlabeled and labeled phylloquinone, respectively. The minimal detectable concentration of unlabeled and labeled phylloquinone was 0.05 and 0.08 pmol/injection. For pooled plasma samples spiked with 0.5 to 32 nmol phylloquinone/L, average recoveries were 95 +/‐ 4.0 % for unlabeled phylloquinone and 96 +/‐ 7.9 % for labeled phylloquinone. The phylloquinone concentrations determined by LC‐ fluorescence and LC‐APCI‐MS methods from healthy subjects (n=20) were correlated (R2=0.983, P=0.053). This new method allows us to measure labeled phylloquinone concentrations in healthy subjects (n=3) in response to ingesting deuterium‐labeled food. The LC‐APCI‐MS method is a sensitive and accurate technique for determination of both labeled and unlabeled phylloquinone, and can be applied to bioavailability studies.
β2 INTEGRIN COMPLEMENT RECEPTOR 3 (CR3, CD11b/CD18) REGULATION OF NEUTROPHIL FUNCTION
Authors: Byrd Angel S. , Morin Nicole A. , Lavigne Liz M. , Reichner Jonathan S.
Published: June 2021

Complement receptor 3 (CR3, CD11b/CD18) is a β2 integrin that is unique among integrins in that it contains a polysaccharide binding lectin‐like domain (LLD), in addition to the conventional binding domain (I‐domain). The ligands for the I‐domain are many, such as iC3b, ICAM‐1, fibronectin and fibrinogen. The LLD is a polysaccharide binding site to which a structure known as beta (β) (1‐3) D‐glucan can bind. A number of neutrophil functions have been shown to be altered, including adhesion, diapedesis, chemotaxis and cytotoxicity, depending on single or dual ligation of CR3. Our goals are to analyze downstream signaling pathways once the I‐domain alone is bound; once the LLD alone is bound; once both domains are bound. We hypothesize that single or dual ligation of CR3 will result in stimulation of different intracellular pathways, determining the character of the cell. Experiments will be done using neutrophils isolated from human blood and stimulated with antibodies or ligands directed against the I‐domain and/or LLD. Once the domain specific stimuli have been thoroughly distinguished, mass spectrometric analysis will be done to analyze downstream signaling pathways upon binding of individual or both domains. Ultimately, these studies are expected to render new signaling pathways for CR3. These various pathways will provide fundamental targets for anti‐inflammatory pharmacological therapies.
Opposing effects of carbon monoxide (CO) releasing molecule (CORM3) on activation of polymorphonuclear leukocytes (PMN) and endothelial cells (HUVEC) in sepsis.
Authors: Mizuguchi Shinjiro , Stephen Jancy , Bihari Relka , Markovic Nevena , Katada Kazuhiro , Capretta Fred , Potter Richard F. , Cepinskas Gediminas
Published: June 2021

Recently, it has been shown that CORM‐released CO can modulate inflammatory response. In this study we assessed the effects and potential mechanisms of water‐soluble CO‐releasing molecule, CORM3, in experimental model of sepsis.

Sepsis in mice was induced by cecal ligation/puncture (CLP). CORM3 (3mg/kg; iv) was administered 15 min after CLP induction. PMN accumulation (MPO assay) in the lung was assessed 6 hr later. In in vitro experiments human PMN were primed with LPS (10ng/ml) and stimulated with fMLP (100nM). PMN production of ROS (oxidation of L‐012/DHR123 over 30 min period of time) and release of elastase (AAPV‐pNA; 1hr) were assessed. In parallel, HUVEC were stimulated with LPS (1µg/ml) and assessed for ROS production, E‐selectin/ICAM‐1 expression (RT‐PCR) and 51Cr‐PMN adhesion/migration. Some experiments were performed in the presence of CORM‐3 (1‐100 µM).

The obtained results indicate that CORM3 significantly reduced PMN accumulation in the lung of septic mice. In parallel, LPS‐induced increase in HUVEC ROS production, E‐selectin/ICAM‐1 expression and PMN adhesion/migration were attenuated by CORM‐3. On the contrary, CORM3 markedly increased ROS production and potentiated release of elastase by LPS‐primed PMN.

These data indicate that CORM3 reduces inflammation in the septic lung by differentially modulating PMN and endothelial cell activation (HSFO‐ NA6171 and MOP‐68848)
Cross Reactivity of Human Recombinant thrombin (Recothrombin) to Antibovine Thrombin Antibodies. Clinical Implications
Authors: Zhu He , Hoppensteadt Debra , Adiguzal Cafer , Jeske Walter , Messmore Harry , Fareed Jawed
Published: June 2021

Bovine thrombin preparations are widely used topical hemostatic agents. Whereas older bovine thrombin preparations were reported to produce immunologic responses upon repeated exposure, the newly introduced recombinant human thrombin preparation (Zymogenetics, Seattle, WA, USA) and bovine thrombin preparations purified by membrane filtration are claimed to be less immunogenic. Despite their reduced immunogenic nature, repeated administration of purified bovine thrombin preparations can result in the generation of antibovine thrombin antibodies. In this study, groups of rabbits were challenged repeatedly with bovine thrombin over a 6 months period and purified IgG preparations were obtained using protein G columns. In order to determine their cross reactivity with human recombinant thrombin (Recothrombin), two batches of this agent were evaluated by immunoblotting techniques using the rabbit IgG fractions. Recothrombin preparations cross reacted with the rabbit antibovine thrombin IgG. The cross reactivity of Recothrombin with antibodies was time dependent and a progressive increase in the immunoreactivity was evident. These results demonstrate that human recombinant thrombin can cross react with antibodies to bovine thrombin. Thus, the use of Recothrombin may not be recommended in patients previously treated/exposed to bovine thrombin.
Nitric Oxide from commensal bacteria protects intestinal epithelial cells from apoptosis by protein S‐nitrosylation
Authors: Kwon Young Man , Neish Andrew Scott
Published: June 2021

Commensal bacteria mediate a variety of homeostatic effects on the gut epithelium and may protect mucosal surfaces against pathogens and other potentially injurious stimuli. How commensal bacterial mediate these function are not known. Nitric oxide (NO) is small molecule involved in many signaling pathways that acts by post‐translational modification (S‐nitrosylation) of regulatory proteins and has been shown to have anti‐apoptotic (or immunomodulatory) effects. We demonstrate that most strains of commensal bacteria produce NO, and this NO is efficiently transferred into cultured model epithelia. Transfer of NO is increased when the bacteria and epithelia are cocultured in presence of exogenous nitrate. In model epithelia colonized with commensal bacterial strains, we observed S‐nitrosylation of thioredoxin1, which has redox regulatory and anti‐apoptotic functions, and the key apoptotic signaling intermediates, ASK1 and JNK. As expected, this modification resulted in the functional inactivation of the pro‐apoptotic JNK pathway, and accordingly, commensal treated cells were resistant to exogenous proapoptotic stimuli. These observations suggest that the S‐nitrosylation of these proteins in epithelia by NO produced by commensal bacteria increases survival of intestinal cells, and explain how epithelial mucosa can show tolerance to environmental stresses.
RIP1 and Inflammation Mediated Septic Liver Injury
Authors: McNeal Sam , Chung Chun‐Shiang , Ayala Alfred
Published: June 2021

RIP1 is a death domain containing kinase that interacts with the death domains of Fas (CD95) and TNF. RIP1 is understood to play a role in caspase independent cell death and has been reported to increase concomitantly with increased CD95 expression in response to a variety of insults such as ischemia/reperfusion injury and in experimental stroke. Thus, it was our hypothesis that as signaling through both Fas and TNF play pathologic roles in experimental sepsis (cecal ligation & puncture, CLP), that in some fashion up regulation of RIP1 would be involved down stream signaling contributing to septic liver damage observed in this model. To assess the contribution of RIP1 to this process we attempted block its expression with RIP1 specific siRNA as well as look at concomitant alterations in its expression in the liver as well as the development of liver damage. Surprisingly, no change in septic mortality was observed when RIP1 was silenced using RIP1 siRNA vs. scrambled seq. Furthermore, while we have demonstrated increased CD95 levels on hepatocytes during sepsis, when we attempt to determine the extent of RIP1 expression in CLP (24 h post) mouse hepatocytes, the RIP1 signal (as assessed by western blot) was lost in these same cells. Subsequently, we attempted to antagonize RIP1 activity using the selective inhibitor necrostatin 1 (Nec1). Interestingly, our preliminary data indicate that when Nec1 is injected into mice subjected to CLP we observe a decline in the blood levels of pro‐inflammatory cytokines; however, an increase in Alanine aminotransferase (ALT) levels (an index of liver damage) was also seen. Together these findings suggest that signaling via RIP1 may not be a central pathway involved in mediating pathological signaling for Fas or TNF in the septic liver (supported by NIH GM53209).
TNF‐α induces VCAM‐1 expression via PKCδ/Src/EGFR transactivation linking to AP‐1 and NF‐¿B in human renal mesangial cells
Authors: Chou Chih‐Hsing , Hsieh Hsi‐Lung , Yang Chuen‐Mao
Published: June 2021

In glomerular cells, TNF‐α can induce adhesion molecule expression and then attracts monocytes adhesion. However, the mechanisms underlying TNF‐α ‐induced VCAM‐1 expression in human renal mesangial cells (HRMCs) remain unclear. Western blot, RT‐PCR, and monocytes adhesion analyses showed that in HRMCs, TNF‐α induced VCAM‐1 mRNA and protein expression in a time‐dependent manner, which were attenuated by the inhibitors of PKC (GF109203X and rottlerin), c‐Src (PP1), EGF receptor (AG1478), PI3K (LY294002), p38 MAPK (SB202190) and JNK (SP600125), or transfection with siRNA of c‐Src, Akt, p38 MAPK, and JNK2. These results suggest that EGFR transactivation participates in VCAM‐1 expression induced by TNF‐α. Accordingly, TNF‐α ‐stimulated phosphorylation of p38 MAPK and JNK was inhibited by pretreatment with GF109203X, rottlerin, PP1, AG1478, LY294002, SB202190, or SP600125. TNF‐α induced VCAM‐1 expression was blocked by selective inhibitors of AP‐1 (tanshinone IIA) and NF‐¿B (Bay11‐7082). Moreover, TNF‐α ‐stimulated activation of VCAM‐1 promoter activity was blocked by these selective inhibitors. Taken together, these results suggest that in HRMCs, PKCδ/c‐Src‐dependent EGFR transactivation, PI3K/Akt, p38 MAPK, JNK activation linking to AP‐1 and NF‐¿B is involved in TNF‐α‐induced VCAM‐1 expression and monocytes adhesion.