bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Probiotic Supplementation on Canine Colon Health
Authors: Hussein Hussein S , Ngwenya Anele , Bollinger Laurie M. , Sakuma Toshie , Yobi Abou , Fernandez George C. J. , Boileau Thomas W.‐M. , Sunvold Gregory D.
Published: June 2021

To determine effects of probiotics on canine colon health, 30 dogs (averaging 29.5 kg in body weight [BW]) were used. The dogs were allotted to three similar groups and were fed a premium diet twice daily to maintain their BW. After 31 d of adjustment to the diet, two groups (probiotic groups) were given a daily oral dose (109 CFU/dog) of Bifidobacterium pseudolongum or Lactobacillus murinus ruminis and the third group (control) was given a placebo during the evening feeding. These treatments continued for 84 d with fecal samples being collected on d 0 (the day before probiotic dosing; covariate), 21, 42, 63, and 84. The experimental design was a completely randomized design with the treatments being arranged as a 3 (treatments) × 4 (sampling times) factorial. Significant (P < 0.05) interactions between the probiotic treatments and sampling times were detected for fecal numbers of Bifidobacterium, Lactobacillus, and Clostridium difficile. The numbers of these bacterial groups or species fluctuated over time without a clear trend. Across sampling times, the numbers of Clostridium perfringens were altered by probiotics. In comparison to the control and Lactobacillus groups, dogs receiving Bifidobacterium had the lowest (P < 0.05) fecal numbers of C. perfringens (8.46, 8.80, and 7.52 CFU log10/g dry matter, respectively). The results suggest supplementation of Bifidobacterium as a strategy to improve the microbial ecology of canine colon by decreasing fecal numbers of the pathogen C. perfringens.
Antioxidant dietary supplements enhance DNA repair capacity in free‐living humans
Authors: Neuhouser Marian L. , Gu J. , Schmezer P. , Ulrich C. , White E.
Published: June 2021

Dietary antioxidants, particularly from dietary supplements, provide protection against free radical‐induced oxidative stress. We used the comet assay (single cell gel electrophoresis) on viable lymphocytes from fasting blood to assess DNA damage and repair capacity in users of antioxidant dietary supplements in Washington State. Participants (n = 121) were aged 50–74 years, 55.4% male, 93% never/former smokers and 64.5% were overweight/obese. Detailed inventories obtained information on supplement use; 85.9% reported using at least one antioxidant‐containing dietary supplement; over half used > one supplement; 8.3% were long‐term high‐dose users of either vitamin C or E (defined as ≥ 1000 mg of vitamin C or 800 IU vitamin E/day for ≥ 5 years). In multivariate‐adjusted regression models, the highest doses of supplemental vitamin C, E or beta‐carotene (vs. no supplements) were associated with enhanced DNA repair capacity (all p< 0.05). Associations were attenuated when antioxidants from food + supplements were considered. Long term use (> 5 years) of vitamins C or E was associated with better DNA repair capacity. Compared to non‐users of dietary supplements, long‐term users of vitamin C or E had a 1.7 fold (p = 0.007) and 1.5 fold (p = 0.01) increases in DNA repair capacity, respectively.

Conclusion: use of DNA antioxidant dietary supplements may enhance DNA repair capacity. Supported by R03CA105336,R01CA74846.
Nutritional Management of Very Low Birthweight Infants: Effects of Different Feeding Regimens on Calcium Absorption
Authors: Hicks Penni Davila , Grifin Ian J , Hawthorne Keli M , Abrams Steven A
Published: June 2021

Background: Adequate nutrition is a key aspect of care for very low birth weight (VLBW) infants. However, it is difficult to provide adequate nutrition to VLBW infants who have health problems which require fluid restriction and increased caloric density feedings. The effects of these changes on growth, calcium (Ca) metabolism and bone mineral status are unknown.

Objective: To compare the effects of different feeding strategies on Ca absorption in VLBW infants.

Methods: Stable isotopes were used to evaluate Ca absorption in preterm infants fed fortified human milk (HM) (n=8) or preterm formula (PF) (n=8). Infants were fed at either 24 or 27 kcal/oz HM or PF. After 2 weeks on the study diet 2 mg of 44Ca was given with a feeding and 20μg of 46Ca was given IV.

Results: There was no difference in gestational age for HM or PF (mean = 27.1 weeks) or birthweight for HM, 1050 ± 179g and PF, 1085 ± 301g (mean ± SD). Mean Ca absorption was 50.5 ± 19.9% for HM and 54.1 ± 17.6% for PF (p = 0.4). Ca intake was 223 ± 14 mg/kg for HM and 221 ± 10 mg/kg for PF (p = 0.6). Total Ca absorption was 113 ± 46 mg/kg for HM and 119 ± 37 mg/kg for PF (p = 0.6).

Discussion: Currently used feeding approaches provide Ca comparable to the in utero accretion rates.
Non‐responsive feeding styles are related to maternal mental health symptomatology and low rates of breastfeeding
Authors: Hurley Kristen Marie , Black Maureen M , Papas Mia A , Caulfield Laura E
Published: June 2021

Objectives: To investigate whether feeding styles are related to maternal mental health and breastfeeding. Method: Analyses are based on 702 mother‐infant pairs from a statewide telephone survey of WIC mothers administered in English and Spanish; 64.1% had initiated breastfeeding. Results: In bivariate analyses, breastfeeding was related to responsive feeding (β =0.13; CI: 0.03, 0.22). In multivariate analyses, adjusting for maternal education, breastfeeding, race/ethnicity, and infant age and gender, maternal stress symptomatology was associated with forceful (β =0.03; CI: 0.02, 0.05) and uninvolved (OR=1.4; CI: 1.1,1.7) feeding styles, maternal depression symptomatology was associated with forceful (β=0.03; CI: 0.004, 0.05), indulgent (β=0.03; CI: 0.004, 0.06), and uninvolved (OR=1.5; CI: 1.001, 2.2) feeding styles, and maternal anxiety symptomatology was related to restrictive (β=0.11; CI: 0.01, 0.21), forceful (β=0.04; CI: 0.02, 0.06), and uninvolved (OR=1.4; CI: 1.01, 1.9) feeding styles. Conclusion: Mothers who report non‐responsive feeding styles report mental health symptomatology and low rates of breastfeeding. Findings provide support for broadening the focus of child nutrition programs to include feeding styles.
Well‐fed and sleeping: motivations driving infant feeding practices in rural Appalachia
Authors: Habibi Mona , Anderson Kirsten , Spence Marsha , Kavanagh‐Prochaska Katherine
Published: June 2021

The objective of this project is to inform the development of educational strategies to modify infant feeding practices that may predispose the child to obesity. Data pertaining to caregiver's infant feeding perceptions and practices were collected through focus groups with participants of the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) in two East Tennessee counties. Participants had infants 0–6 months of age. Focus groups replaced the participants’ required, pre‐scheduled nutrition education class, and so included a representative cross‐section of this population. Thirty‐two focus groups were convened, in which a total of 121 caregivers participated. Out of these 32 transcripts, 8 main subject domains emerged: foods introduced at < 4 mo of age, reasons for introducing new foods, advice given to caretakers, ability to perceive hunger cues, knowing when to stop a feed, crying tolerance, sleep patterns, and perceptions of overall infant health. Using cluster analysis, subtopics have been established and patterns of behavior have surfaced within each domain. In conclusion, preliminary data suggests patterns linking early introduction of cereal to reflux, longer periods of sleep, and attempts to extend infant satiation. Source of support: The University of Tennessee, Knoxville, Professional Development Award.
Overweight and Obesity in elderly people adults in Mexico
Authors: Nasu Lucia Cuevas , Levy Teresa Shamah , Hernandez Salvador Francisco Villalpando , Aguilar Alejandra Jimenez
Published: June 2021

This study describes the prevalence of overweight (OW) and obesity (O) (BMI 30 kg/m2) and assesses its association with socioeconomic (SES), demographic, and nutritional status indicators. Data from the National Health and Nutrition Survey in Mexico (ENSANUT 2006) on 5480 elderly people (aged 60–99 y) were analyzed. Models of logistic regression were used and they were adjusted by design of the survey. Prevalence of OW on men was 43.9 and women 38.9% (p< 0.05) and O on men was estimated on 20.2 and women 35.5% (p<0.05); high waist circumference: (men 69.5 and women 73.7%). The risk of OW+O was positively associated with urban residence (OR=1.4, p<0.05); level of education (OR=1.4, p<0.05), waist circumference (OR=16.9, p<0.001) and previous diagnostic of obesity (OR=4.2, p<0.001). Smoking (OR=0.7, p<0.05), being beneficiary of food assistance programs (OR=0.9, p>0.05), and age (OR=0.9, p<0.001) were not associated with the risk for OW+O. Results demonstrate a high prevalence of overweight and obesity in elderly adults in Mexico. It is a priority to enact strategies and programs to prevent and control it. Although, smoke diminishes the possibility of suffering overweight and obesity, we are far from endorsing it.

Supported by Ministery of Health, Mexico.
Bone mass is associated with vitamin D status in lactating adolescent mothers
Authors: Bezerra Flávia F. , Mendonça Laura M.C. , Donangelo Carmen M.
Published: June 2021

The aim of the present study was to investigate the association between serum concentrations of vitamin D [25(OH)D] and bone mass in 28 lactating Brazilian adolescents (15–18y). Total body mineral content (TBMC), total body (TBMD) and lumbar spine (L2–L4, LSBMD) bone mineral density were assessed by DXA. Serum 25(OH)D and iPTH were assessed by radioimmunoassay and chemiluminescent assay, respectively. 25(OH)D varied in a range between 49–75.5 nmol/L and correlated negatively with iPTH (r= −0.56, P<0.01). Positive correlations were observed between 25(OH)D and the following bone measurements: TBMC (r= 0.48, P<0.01), TBMD (r= 0.40, P<0.05), TBMD z score (r= 0.53, P<0.01) and LSBMD z score (r= 0.40, P<0.05). In a stepwise linear regression, 25(OH)D appeared as a modest, but significant factor influencing TBMD and TBMD z score (Table).

Our results indicate that the lactating adolescent mothers studied had serum 25(OH)D concentrations below the proposed cutoff (≤ 80nmol/L) to define deficiency. Moreover, these results suggest that bone mass may benefit from improving vitamin D status during lactation in adolescent mothers.

Financial support: CNPq and FAPERJ (Brazil).
Increasing entrée portion size does not always increase children's energy intake at a meal.
Authors: Johnson Susan L , Ross Erin Sundseth , Grunwald Gary , Burger Kyle , Fisher Jennifer O
Published: June 2021

Children's energy intake has been reported to increase as a function of the portion size of foods offered to them. However, these studies have routinely used one “amorphous” entrée, macaroni and cheese, to test these effects. We investigated the effects of increasing the portion size of two entrees (one unit and one amorphous food; hotdogs and macaroni and cheese, respectively) on children's (3–6 y) energy intake at lunch. Entrées were provided in small or large portions along with fixed amounts of other foods. We measured entrée and total food intake (in g and kcal) across 5 presentations of each portion size of each entrée. A linear mixed model with repeated measures was used to test main effects for portion size and presentation and a portion*presentation interaction. Overall, the portion size of the entrée marginally influenced the amount of entrée consumed (kcal, p<.12; g, p<.01) however it did not impact total energy consumed at the meal (p<.96). When examined by type of entrée, children ate significantly more (in kcal and g) of the large size of the amorphous entrée (p<.02) but not of the unit entrée (p<.15). Increases in portion size resulted in a large increase in entrée intake (25–34%) but only a 4% increase in total energy intake at the meal. Our findings suggest that children do not always respond to increases in portion size by increasing total energy intake.

(Research Support: The National Cattlemen's Beef Association)
Potential effects of 5‐caffeoylquinic acid and caffeine found in coffee on suppressing P‐selectin expression.
Authors: Park Jae B
Published: June 2021

Platelet activation is an essential process in hemostasis. However, platelet activation and its related processes are often involved in pathophysiological development of several inflammatory and cardiovascular diseases such as atherosclerosis, angina, acute myocardial infarction, and ischemic cerebral stroke. Platelet activation can be greatly attenuated by compounds inhibiting COX enzymes and/or modulating cAMP production. Chlorogenic acids and caffeine found in coffee (Coffea species) are capable of inhibiting COX enzymes and modulating cAMP production, respectively. In this study, a chlorogenic acid (5‐caffeoylquinic acid) found in coffee was investigated to determine its effects on P‐selectin expression on platelets in mice. First, the effect of 5‐caffeoylquinic acid on COX I and II enzymes was investigated, because COX enzymes are deeply involved in regulating P‐selectin expression on platelets. At the concentration of 0.05 μM, 5‐caffeoylquinic acid was able to inhibit COX‐I and COX‐II enzymes by 63% (P < 0.013) and 59% (P < 0.015), respectively. At the same concentration, 5‐caffeoylquinic acid was able to inhibit P‐selectin expression on platelets by 33% (P < 0.011). The inhibition by 5‐caffeoylquinic acid was also modulated depending on the concentration of caffeine. This study points to the possible potent effects of chlorogenic acids and caffeine found in coffee on inflammatory and cardiovascular diseases. This study was funded by ARS/USDA.
New hypothesis concerning pathogenesis of systemic sclerosis using a tissue engineering method
Authors: Corriveau Marie‐Pier , Boufaied Ines , Lessard Julie , Chabaud Stéphane , Grodzicky Tamara , Senécal Jean‐Luc , Moulin Véronique
Published: June 2021

Scleroderma is a connective tissue disease characterized by skin and internal organs fibrosis, vasculopathy of small arteries and activation of the immune system. The objective of this project is to study the capacity of scleroderma fibroblasts to induce fibrosis using a model of tissue engineered dermis. Fibroblasts were isolated from skin biopsies of patient with scleroderma for less than one year (early stage) or for more than ten years (late stage). For each patient, two biopsies were taken, one from affected area and the other one from a non‐affected area. Control fibroblasts were obtained from healthy donors. Only fibroblasts isolated from the skin biopsies from affected area of patient at late stage of scleroderma were able to reconstruct dermis thicker than that obtained with control fibroblasts. All of the other fibroblasts tested formed dermis thinner than the one obtained with control fibroblasts. These observations are not the consequence of a change in the amount of fibroblast present, but due to a different turnover of extracellular matrix (matrix metalloproteinase‐1/collagen I) according to the stage and the area. These results suggest the necessary presence of exogenous factor(s) to induce fibrosis in the early stage of the disease, while in late stage these factor(s) would not be essential. This study was supported by CIHR, by the “Fondation de l’Hôpital St‐Sacrement” and a scholarship from FRSQ (VM).
Desmocollin 2 regulates proliferation in the intestinal epithelial cell line SK‐CO15 through activation of the small GTPase Rap1
Authors: Kolegraff Keli , Nava Porfirio , Severson Eric , Koch Stefan , Laukoetter Mike , Parkos Charles , Nusrat Asma
Published: June 2021

Desmosomes (DMs) are essential for maintaining tissue integrity. In simple epithelial cells, two DM transmembrane cadherins referred to as desmoglein‐2 (Dsg2) and desmocollin‐2 (Dsc2) associate with underlying cytokeratin filaments via cytoplasmic plaque proteins. While DM regulation of epithelial mechanical properties is well established, their role in regulation of cell growth and morphogenesis is incompletely understood. Our studies were focused on defining the function of Dsc2 in simple intestinal epithelial cells (IEC). By knocking down Dsc2 with shRNA in a model IEC cell line SK‐CO15, we observed that loss of Dsc2 expression induced IEC proliferation as determined by BrdU incorporation and 3D cyst formation assays. Studies to determine the mechanism by which Dsc2 knockdown induced proliferation revealed increased activation of a small GTPase, Rap1, and down‐regulation of cell‐cell and cell‐matrix adhesion proteins JAM‐A, E‐cadherin, and β1‐integrin. Furthermore, over‐expression of Dsc2 decreased Rap1 activity. These findings support a role for Dsc2 in controlling intestinal epithelial cell adhesion and proliferation through regulation of Rap1.

Supported by NIH DK‐59888 (A.N.) and CCFA fellowship award (P.N.)
PI3kinase mediates HGF and EGF induced hepatocyte to biliary epithelial cell transdifferentiation in organoid cultures
Authors: Limaye Pallavi B. , Bowen William C. , Luo Jianhua , Tseng George C. , Michalopoulos George K.
Published: June 2021

We have previously demonstrated that HGF and EGF can stimulate hepatocyte transdifferentiation into biliary epithelium (BE) in hepatocyte organoid cultures. To elucidate the mechanisms underlying this process rat hepatocyte organoid cultures were exposed to various growth factors (GF) including VEGF, PDGF, stem cell factor (SCF), macrophage stimulating protein (MSP), FGF‐a, FGF‐b, and FGF‐8b. None of the growth factors tested other than HGF and EGF were able to induce hepatocyte transdifferentiation despite the presence and activation of their cognate receptors. These findings indicate that HGF and EGF regulate a distinct set of genes required for transdifferentiation, which other GFs fail to activate. Microarray analysis of the organoid cultures identified upregulation of key HGF and EGF target genes such as osteopontin, CD44, CXC chemokine LIX, cyclin D1, and β‐catenin, which was confirmed by Western blot. Analysis of the signaling kinases downstream to HGF and EGF indicated increased activation of AKT. However, addition of AKT inhibitor to organoid cultures failed to prevent hepatocyte transdifferentiation. Interestingly inhibition of PI3kinase by LY294002 completely prevented development of biliary epithelium. These data indicate that the mechanism of hepatocyte to biliary transdifferentiation in hepatic organoid cultures induced by HGF and EGF is AKT‐independent PI3kinase activation.
Regulatory Mechanism of Soluble Epoxide Hydrolase by Post‐Translational Modifications
Authors: Luria Ayala R , Tsai Hsing‐Ju , Lu Anthony , Morisseau Christophe , Hammock Bruce D
Published: June 2021

Sumoylation is an important post‐translational modification that provides a rapid and reversible means for controlling the activity, subcellular localization, and stability of target proteins. We have examined the covalent attachment of the small ubiqutin‐like modifier (SUMO) proteins to a consensus sequence ([psi]KXE) on the soluble epoxide hydrolase (EPHX2; sEH) enzyme. This enzyme has epoxide hydrolase activity on the C‐terminal domain and a phosphatase activity on the N‐terminus. Polymorphism in EPHX2 have been recently discovered (K55R) that is associated with risk of incident of coronary heart disease events. Interestingly, features of the sEH N‐terminal catalytic site showing a consensus site for SUMOylation (VKEP) with lysine residue at position 160. This and another human‐specific site of SUMOylation (MKGE with lysine residue at position 55), suggest a post‐translational modification and regulatory mechanism of sEH by SUMOylation. Our primary aim of this study was to determine whether sEH can be in vitro SUMOylated and defines its regulatory role of SUMOylation in its both C and N‐terminus activity. Indeed, SUMOylated process catalyzes the formation of polymeric chains of SUMO‐2 and SUMO‐3 on sEH in vitro, and was detected in vivo. We also identified the SUMOylated site on the sEH sequence and revealed its regulatory mechanism. Supported by NIEHS Grant R37 ES02710, NIEHS SBRP Grant P42 ES04699.
Polyisoprenyl Diphosphate Phosphatase 1 Remodels Cellular Presqualene Diphosphate
Authors: Carlo Troy , Fukunaga Kioichi , Levy Bruce D.
Published: June 2021

Previous work established that presqualene diphosphate (PSDP), a polyisoprenyl diphosphate (PIPP) that blocks pivotal intracellular signals in PMN, is rapidly converted to its monophosphate form (PSMP) upon cell activation. PIPP phosphatase 1 (PDP1) is the first PSDP phosphatase. To investigate if cellular PDP1 activation remodels endogenous PSDP and subsequent signal transduction, we addressed PDP1's contribution to total cellular PSDP phosphatase activity. In response to the protein kinase C (PKC) stimulating agonist PMA, HEK293 cells showed concentration dependent increase in PSDP phosphatase activity. Western and biochemical analysis showed that PDP1 is a substrate for phosphorylation by PKC in an isoform specific manner. HEK293‐PDP1‐siRNA cells with 60% reduction in PDP1 expression displayed 56% inhibition of PSDP phosphatase activity and RS‐[2‐14C]‐mevalonolactone incorporation into these cells showed a dramatic decrease in conversion of cellular [14C]‐PSDP to [14C]‐PSMP. In conclusion, our data are consistent with PDP1 having a critical role in cellular PSDP remodeling and suggest that PDP1 could serve as a regulatory checkpoint for cell responses.
Neutrophil‐selective transgenic RNA interference in vivo
Authors: Cullere Xavier , Lauterbach Michael , Tsuboi Naotake , Mayadas Tanya
Published: June 2021

Tissue specific silencing of genes has applications for genetic engineering. Current strategies rely on Cre/Lox technology in mice requiring the generation of multiple transgenic lines and breeding strategies. Here we pursued a method to silence genes in neutrophils using an miRNA strategy. As proof of principle, we chose CD18 as the target gene since it is an abundantly expressed surface protein. CD18 specific miRNA hairpin driven by the myeloid specific MRP8 promoter resulted in a >90% reduction in CD18 protein levels in neutrophils and monocytes with no observable decrease in T cells and only a partial diminution in macrophages. Neutrophil CD18 silencing resulted in mild neutrophilia, splenomegaly and significant defects in neutrophil trafficking in irritant dermatitis. Our data demonstrate the utility of using miRNA approaches to silence genes in neutrophils which are terminally differentiated cells with a short half life that largely precludes their genetic manipulation in vitro. Furthermore, the mouse model provides a valuable tool to examine the contribution of CD18 on neutrophils to Leukocyte Adhesion Deficiency I (LADI), a complex inherited disorder in which reduced or absent CD18 expression in multiple leukocyte subsets leads to impaired innate and adaptive immune responses.

Note: XC, ML and NT contributed equally. Supported by: NIH KO1AR054984 (XC), HL065095 and AR050800 (T.N.M), and a Arthritis Foundation fellowship (NT).
Pyruvate‐enhanced cardioplegia preserves myocardial Hsp 70 and endothelial NOS in a porcine model of cardiopulmonary bypass
Authors: Flaherty Devin C , Ryou Myoung Gwi , Hoxha Besim , Sun Jie , Ferlitch Heather R , Olivencia‐Yurvati Albert H , Mallet Robert T
Published: June 2021

Background: The use of pyruvate‐fortified cardioplegia to arrest the heart during cardiopulmonary bypass (CPB) enhances post‐surgical recovery of cardiac function, compared with conventional cardioplegia solutions. Pyruvate's energy‐yielding and antioxidant effects wane as pyruvate is cleared from the circulation, yet its functional benefits persist, suggesting a protein‐mediated cardioprotective mechanism.

Hypothesis: Pyruvate cardioplegia during CPB prevents subsequent depletion of the cytoprotective proteins eNOS and Hsp‐70. Methods: After sternotomy, swine were subjected to 60 min cardioplegic arrest and 30 min myocardial reperfusion on CPB, then were weaned from bypass. The crystalloid portion of the 4:1 blood:crystalloid cardioplegia contained 24 mM lactate or pyruvate. Left ventricular myocardium was sampled at 4 h post‐CPB for extraction and immunoblot detection of Hsp70 and eNOS; actin served as loading control. Sham pigs underwent the same surgical and analytical procedures, but without CPB.

Results: Actin‐normalized myocardial eNOS and Hsp‐70 contents fell by 32 and 68%, respectively, and NOS activity fell from 132 ± 10 to 91 ± 6 nmol/min/g protein in the lactate cardioplegia group, vs. respective sham values (P < 0.05). Pyruvate cardioplegia prevented loss of eNOS and Hsp70, and maintained NOS activity at 134 ± 8 nmol/min/g protein (P < 0.05 vs. lactate group).

Conclusions: Cardioplegic arrest on CPB can deplete myocardium of the cytoprotective proteins eNOS and Hsp70. Protection of these proteins by pyruvate‐enriched cardioplegia may support improved post‐CPB cardiac performance.

Support: Osteopathic Heritage Foundation 02‐18‐522
VEGF‐A Induces a CD36low Endothelial Cell Phenotype by Activating a MAPK‐Dependent Pathway: Novel Mechanism for Sustaining Proliferation in the Presence of TSP‐1
Authors: Anderson Christopher Richard , Hastings Nikki , Blackman Brett R. , Price Richard J.
Published: June 2021

We have shown that, in a rat model of inflammation‐induced angiogenesis, capillary sprout endothelial cells (EC) markedly downregulate CD36, a receptor for the anti‐angiogenic molecule thrombospondin‐1 (TSP‐1). The expression of VEGF‐A, a known regulator of angiogenic phenotype, increases in this model (2.5‐fold by ELISA), so we tested the hypothesis that VEGF‐A directly regulates the CD36‐phenotype. Human umbilical vein endothelial cells (HUVECS) exposed to VEGF‐A (50 ng/ml) exhibited a significant 60% decrease in CD36 mRNA and protein expression after 24 h. This effect was dependent on VEGFR‐2 and MAPK, as pharmacological inhibitors of these signaling molecules (SU5416, UO126, PD98058) restored CD36 expression in VEGF‐A treated cells. Importantly, HUVECS that had been pre‐conditioned to a CD36low phenotype became insensitive to inhibitory TSP‐1 signaling due to the loss of CD36 expression. These results identify the downregulation of CD36 via the activation of a MAPK‐dependent signaling pathway as a novel mechanism through which VEGF‐A can sustain EC proliferation in the presence of TSP‐1. Supported by NIH HL74082 and NIH HL65958.
Cdc42/alpha2beta1 integrin/membrane type‐1 metalloproteinase complexes regulate endothelial lumen formation in 3D collagen matrices
Authors: Koh Wonshill , Sacharidou Anastasia , Fisher Kevin E. , Davis George E.
Published: June 2021

Endothelial cell (EC) lumen formation is a critical step during new blood vessel development either by vasculogenesis or angiogenesis. Our laboratory has previously shown that EC tube morphogenesis in 3D collagen matrices is regulated by the development and coalescence of intracellular vacuoles and lumens, and that integrins, Rho GTPases, and matrix metalloproteinases (MMPs) play a key role in this process. Here, we show that EC lumen formation in 3D collagen matrices requires a coordinated interaction by integrin alpha2beta1, the Rho GTPase Cdc42, and membrane type‐1 MMP (MT1‐MMP). Treatment of ECs with siRNAs targeting alpha2 integrin subunit, Cdc42, or MT1‐MMP blocks EC vacuole and lumen formation. We further demonstrate that these three molecules exist as a complex to regulate EC lumen formation and that a disruption of this complex results in blockade of EC lumen formation. Inhibition of protein kinase C or Src family kinases, which have been shown to regulate EC lumen formation, leads to disassembly of this complex thereby inhibiting EC lumen formation in 3D collagen matrices. Our findings reveal that tight coordination of Cdc42, integrin alpha2beta1, and MT1‐MMP controls signaling pathways necessary for EC lumen and tube formation in 3D matrices. A detailed examination is underway to identify and examine additional signaling molecules involved in this morphogenic response.
Natural and small molecule regulators of cardiogenesis
Authors: Mercola Mark
Published: June 2021

I will present our recent advances in understanding mediators of the Dkk1, Nodal and Notch pathways that control differentiation and cell cycle of cardiomyocytes as well as our high throughput screening approaches to identify small molecules that control cardiomyocyte differentiation and physiology. New structure/function studies of the Dkk family of secreted proteins will be presented demonstrating a novel signal that stimulates heart induction and tissue morphogenesis. Dkk proteins have been known solely as antagonists of Wnt activity because of their ability to disrupt the Wnt signaling complex; however, this new activity is independent of Wnt signaling. Second, it is possible to stimulate cell cycle entry of committed precursors through the actions of the Notch pathway, which functions in this context by both an RBP/CSL‐dependent transcriptional action and also by a novel activity that is indpendent of downstream transcription. Third, our high content screening (HCS) approach for stem cell differentiation has yielded several classes of novel synthetic compounds that promote early steps of ESC cardiogenesis will be presented. HCS strategies and phenotypic screening constitute a feasible chemical biological approach for the identification of cellular targets for discovery of drugs to enhance cardiomyocyte regeneration.
Mopping up after messy polymerases and more…
Authors: Hingorani Manju M
Published: June 2021

The DNA mismatch repair system (MMR) helps maintain the integrity of the genome by locating and correcting base pair mismatches and other replication errors in DNA, and suppressing recombination between non‐identical DNA sequences. Given its critical role in controlling mutagenesis, it is not surprising that defects in MMR protein function underlie an estimated 15 – 25% of a broad spectrum of sporadic cancers as well as a high fraction of the hereditary non‐polyposis colon cancer syndrome. MMR has another important protective function, and that is to mediate cytotoxic effects of DNA damage, e.g., from alkylating agents found in tobacco smoke or chemotherapy. We are interested in understanding the workings of protein components of the MMR system, and to this end we are currently studying MutS protein (from S. cerevisiae and T. aquaticus), which recognizes defects in DNA and utilizes ATP to signal repair or apoptosis. By measuring kinetic and equilibrium constants that define individual steps in the MutS‐catalyzed reaction, we aim to develop a comprehensive model describing the mechanism of action of this protein, and thus establish a basis for understanding how it detects and mounts selective responses to irregularities such as mismatches or lesions in DNA.
The reconstruction of the genome‐scale transcriptional regulatory network in e. coli
Authors: Palsson Bernhard O.
Published: June 2021

Regulation of gene expression is widely considered to be a core issue in biology. Normally such studies have focused on a single gene or a group of genes. Advances in chip‐chip technology now enable us to study this issue on a genome scale. We have obtained over 40 chip‐chip data sets for e. coli that include; rnap, 4 sigma factors, most of the broad acting transcription factors, and the majority of the dna binding protein. This data enables us to reconstruct the transcriptional regulatory network in e. coli at a genome‐scale. This first draft reconstruction can be represented mathematically and its systems properties studied.
ABCA1, ABCG1 and the Athero‐protective Effects of HDL
Authors: Tall Alan R.
Published: June 2021

Cholesterol efflux from macrophage foam cells to HDL or its apolipoproteins is thought to have an important anti‐atherogenic role. Two major ABC transporters are involved in this process: ABCA1 which promotes cholesterol efflux to lipid‐poor apolipoproteins, and ABCG1 which promotes cholesterol efflux to HDL particles. While bone marrow transplantation studies have indicated an anti‐atherogenic role of macrophage ABCA1, results with ABCG1 deficient bone marrow have been mixed. We reported a decrease in atherosclerosis in LDLR−/− mice transplanted with ABCG1−/− bone marrow. This unexpected result appeared related to compensatory up‐regulation of ABCA1 and apoE secretion in ABCG1−/− macrophages. To assess this hypothesis we recently transplanted double KO ABCA1−/−ABCG1−/− bone marrow into LDLr deficient mice, resulting in a dramatic increase in atherosclerosis compared to WT or single KO BM recipients. This indicates overlapping roles and mutual compensation of ABCA1 and ABCG1 in macrophage cholesterol efflux and athero‐protection. However, ABCG1 appears to have a specific role in protecting macrophages from apoptosis induced by oxidized LDL, related to the ability of ABCG1 (but not ABCA1) to promote efflux of specific oxysterols such as 7‐ketocholesterol from macrophages to HDL. Large HDL that accumulates in CETP deficiency is enriched in apoE and LCAT and is highly efficient at promoting macrophage sterol efflux via the ABCG1 pathway. It is interesting to speculate that HDL/ABCG1 could reverse oxysterol‐mediated macrophage apoptosis and inflammatory gene expression in atherosclerotic lesions. In addition, ABCG1 is high expressed in endothelial cells suggesing a possible role of HDL/ABCG1 in maintenance of endothelial function.
Human Amniotic Fluid‐Derived Stem Cells: A Novel Source of Dopaminergic Neurons?
Authors: Santos Cesar C. , Klorig David , Chun So Young , Atala Anthony , Soker Shay
Published: June 2021

The aim of our study is to investigate whether human amniotic fluid derived stem cells (hAFSC) can be used for regenerative applications in neurodegenerative diseases like Parkinson's disease (PD).

To yield dopaminergic cells, hAFSC were cultured in a 2 stage culture media systems that include growth factors to mimic the normal midbrain dopaminergic development. At different time points during differentiation differentiated cells were analyzed using RT‐PCR and immunostaining. At the end of differentiation the amount of cellular dopamine quantified and a whole‐cell voltage clamp recording was performed.

The hAFSC at end of the expansion phase expressed nestin and Nurr1 and their expression was enhanced by exposure to hypoxic culture condition. At the end of terminal differentiation, cells look bipolar and pyramidal and showed expression of Girk2. After recording the inward currents, barium was added to the bath to block the Girk‐specific inward current. A reduction in the amplitude of the currents evoked by the same hyperpolarizing voltage step protocol in the presence of barium indicated the presence of Girk‐mediated potassium channel. Terminally differentiated cells secreted dopamine in a dose response manner, yielding approximately 2nM/100,000 cells.

In this study we showed that hAFSC can be successfully differentiated into dopamine secreting neuron‐like cells that express neuronal genes.
Peripheral blood‐derived multipotent adult progenitor cells differentiate to neural phenotypes in vivo and in vitro
Authors: Spitzer Nadja , Butts Heather M. , Samuel Sarah A. , Price Elmer M.
Published: June 2021

Cell therapy offers a promising approach in treating neurodegenerative disorders such as Parkinson's Disease (PD), and therapies utilizing adult‐derived stem cells are highly desirable. We have derived novel multipotent progenitor cell lines (PBD‐MAPCs) from the peripheral blood of adult green fluorescent protein (GFP) transgenic swine. Undifferentiated PBD‐MAPCs proliferate as suspended spheroids through >100 doublings. To demonstrate the neuronal potential of these cells, we injected them into the brains of neonatal (P0) rats. After 8 weeks, GFP+ cells with neuronal‐like morphology were localized primarily to the hippocampus and expressed β‐tubulin III, NeuN and other neuron‐specific proteins. Therefore, given appropriate differentiation signals provided by the environment in the neonatal brain, PBD‐MAPCs can develop neuronal phenotypes. In an effort to predifferentiate PBD‐MAPCs to a neural lineage in preparation for transplantation into animal models of PD, we cultured cells in media with defined factors. High proportions of these cells develop a neuronal morphology and express neuronal markers. Finally, PBD‐MAPC cells injected into the striatum of 6‐hydroxydopamine‐lesioned rats reduce apomorphine‐induced rotations; the injected cells therefore act to rectify PD‐like deficiencies. Supported by NSF EPSCoR EPS‐07‐02#2#EPS0554328.
Progenitor‐Derived Endothelial Cell Therapy Restores Erectile Function
Authors: Rodríguez Sergio , Chen Kuo‐Liang , Atala Anthony , Yoo James
Published: June 2021

Introduction

We examined whether endothelial cell therapy could restore normal erectile function in a diabetic rat model. We investigated the feasibility of using endothelial progenitor cells (EPC), obtained from peripheral blood, as a source of endothelial cells for therapy.

Materials and Methods

EPC, isolated from peripheral blood of donor rats, were induced into an endothelial cell lineage. The culture expanded endothelial cells were labeled with red fluorescent dye and injected directly into the dysfunctional corpora of diabetic rats, and followed for up to 12 weeks. Erectile function was assessed by intracavernous pressure and the corporal tissues were retrieved for analyses.

Results and Discussion

The progenitor and differentiated endothelial cell phenotypes were confirmed with cell specific antibodies. The animals injected with cells showed a substantial improvement in intracavernous pressure and their erectile function was restored to normal levels. Histologically, the implanted cells that were labeled with the fluorescent dye tracer PKH26 survived and integrated into the corporal tissue within the injected region.

Conclusions

This study demonstrates that the EPC‐derived endothelial cells are able to restore normal erectile function in a diabetic rat model. This cell‐based technology may be a viable treatment modality for diabetic patients with erectile dysfunction.
Erythropoietin Producing Cells for the Treatment of Renal Failure Induced Anemia
Authors: Egydio Fernanda , Aboushwareb Tamer , Stern Kathryn , Gyabaah Kenneth , Moore Philip , Zhang Yuanyuan , Atala Anthony , Yoo James
Published: June 2021

Introduction

Anemia is an outcome of chronic renal failure due to the kidney's decreased ability to produce erythropoietin (EPO). We investigated whether supplementation of erythropoietin producing cells would be a possible treatment option for renal failure induced anemia.

Materials and Methods

Mice renal cells were culture expanded and characterized for EPO expression. The levels of EPO production was measured in the cells grown under normoxic and hypoxic conditions. Renal cells mixed in collagen gel were implanted subcutaneously in athymic mice followed by retrieval at 14 and 28 days after implantation for analyses.

Results and Discussion

The cultured renal cells expressed EPO at each subculture stage. Western Blots detected EPO protein in the kidney cells of all passages tested. ELISA Assay showed that the renal cells grown under hypoxic condition produced EPO. Histologically, the retrieved implants showed cell survival and tissue formation in vivo. Presence of EPO producing cells were confirmed using EPO specific antibodies.

Conclusions

These results demonstrate that EPO producing renal cells can be grown, stably express EPO in culture, and are able to form tissue in vivo. This study shows that EPO producing cells may be used as a potential treatment option for anemia caused by chronic renal failure.
Muscle Progenitor Cells for the Restoration of Irreversibly Damaged Sphincter Function: A Pre‐Clinical Study
Authors: Eberli Daniel , Yoo James , Soker Shay , Atala Anthony
Published: June 2021

Background:

We explored the possibility of achieving functional recovery of the urinary sphincter muscle using cell‐based therapy. We investigated whether irreversibly damaged urinary sphincter function could be fully restored using autologous muscle progenitor cells (MPC).

Methods:

A model of urinary sphincter insufficiency was created by excising approximately 80% of the sphincter muscle in 26 dogs. Sphincter function was assessed by urodynamic studies on normal and damaged sphincters. Autologous MPC were injected into the damaged sphincter muscle and the animals were followed for up to 6 months after injection.

Results:

Animals with MPC injection were able to recover the sphincter pressure to approximately 80% of normal, while the pressures in the control animals dropped and remained at 20%. Histologically, the implanted cells that were labeled with the fluorescent dye tracer survived and formed new innervated muscle fibers.

Conclusion:

Autologous muscle progenitor cells are able to restore otherwise irreversibly damaged sphincter function in dogs. The injected cells are able to survive and form mature tissue within the damaged sphincter. This study demonstrates the feasibility of using autologous muscle precursor cells for the functional restoration of urinary sphincter muscle in patients with sphincter insufficiency.
Notch pathway activation in lentiviral encephalitis
Authors: Kofler Julia Konstanze , Wang Guoji , Bonneh‐Barkay Dafna , Murphey‐Corb Michael , Wiley Clayton Alexander
Published: June 2021

HIV encephalitis is characterized by infiltration of the brain by infected macrophages, formation of microglial nodules, multinucleated giant cells, and subsequent synaptic and neuronal loss. The Notch signaling pathway plays an important role in cell fate decision during development, but more recently has also been shown to be activated under various pathologic conditions of the adult brain. We performed immunohistochemical studies to investigate activation of the Notch pathway in lentiviral encephalitis and found strong upregulation of Notch‐4 in HIVE and SIVE brains, but not in infected but non‐encephalitic cases. Notch‐4 reactivity was restricted to perivascular macrophages and microglial nodules. Variable Notch 4 expression was also seen in vessels. No upregulation of Notch‐4 in microglia/macrophages was observed in non‐infected control brains from patients with Alzheimer‘s disease, subacute stroke, and HSV encephalitis. Our results suggest that Notch‐4 activation in microglia/macrophages may be a process specific to lentiviral infection, and further studies are warranted to evaluate the contribution of Notch activation to neuronal degeneration in HIVE.

NIH funding: MH01717, MH64921 and MH071151
Novel Genotypes Relevant to Enhanced Resistance to γ‐irradiation in Escherichia coli
Authors: Popelars Michael C. , Hao Li , Hamilton Lindsay L. , Klugman Sarit A. , Paider Jessica J. , Wood Elizabeth A. , Battista John R. , Cox Michael M.
Published: June 2021

To better understand the extraordinary resistance to ionizing radiation displayed by bacterial species such as Deinococcus radiodurans, we have generated a series of E. coli populations that also have a greatly elevated capacity to survive exposure to ionizing radiation. One single colony isolate from each population was obtained and given a designation: CB1000, CB2000, and CB3000, taken from the first, second, and third evolved population, respectively. These isolates were independently subjected to comparative genome resequencing, revealing in each case the presence of 60–70 mutations1. To determine which of the CB1000 mutations was relevant to the radiation resistance phenotype, we subjected the population from which CB1000 was derived to ensemble sequencing. DNA from that population was PCR amplified at each region where a mutation was identified in CB1000. Those mutations that were common in the population could be identified in the resulting ensemble sequences. Those mutations incidental to the phenotype were generally not detectable in the ensemble sequences. Less than 5 mutations were found via the ensemble approach. These mutations and their effects on otherwise wild type E. coli strains will be reported on in this poster.
study on the regulation of intestinal lactase gene during postnatal development in rats
Authors: kaur jaspreet , Mahmood Safrun , Mahmood Akhtar
Published: June 2021

Maturation decline in lactase activity is complex phenomenon which is either due to shift in lactase synthesis and processing to the inactive 100Kda and Increased degradation of the lactase activity of 130kda subunit, or decline in the enterocyte life span.lactase phlorizin hydrlase shifts from a high sialylation status during weaning to high fucosylation level in adult rats. The activities of brush border lactase were assayed. sialic acid and fucose were estimated. Membrane protein were resolved by SDS‐PAGE on to nitocellulose paper/PVD membrane and immunostained with polyclonal antibodies against lactase. Total RNA was isolated and transfered RNA to nylon membrane.Determination of fucose to sialic acid ratio revealed close parllelism between fucosylation and sialylation of microvillus membranes with decline in intestinal lactase activity with development age in rat. Upstream region of prolactase gene, suggesting the absence of functional role in mature lactase mRNA and also mRNA is poorly translated to active lactase in the adult intestine which may be due to its interaction with 100kda protein product of prolactase hydrolysis. Western blot analysis revealed the presence of 100kda protein in rat intestine during postnatal development.
Regulation of hnRNP A1 Expression by the Schizophrenia Candidate Gene Quaking
Authors: Ryder Sean P. , Zearfoss N. Ruth
Published: June 2021

Schizophrenia (SCZ) is a debilitating psychiatric disorder with no known cure. Two lines of evidence suggest that splicing defects contribute to the disease. First, numerous aberrantly spliced mRNAs are found in SCZ brains. Second, SCZ is associated with reduced expression of Quaking (QKI), a sequence‐specific RNA binding protein that regulates alternative splicing of myelin mRNAs by an unknown mechanism. Here, we demonstrate that QKI regulates splicing indirectly by modulating expression of the splicing factor hnRNP A1. We show that QKI associates with hnRNP A1 transcripts and have localized the binding site to a conserved element in the 3′UTR. A single U‐to‐G mutation in this element completely abrogates binding. Using a dual‐color fluorescence reporter assay in live oligodendrocyte precursor cells, we demonstrate that QKI regulates hnRNP A1 through two distinct mechanisms: (1) it increases hnRNP A1 mRNA levels, and (2) it decreases the translational efficiency of hnRNP A1 transcripts. The U‐to‐G binding site mutation eliminates QKI‐dependent regulation, as does reduction of QKI by RNAi. Finally, over‐expression of QKI in Cos‐7 cells dramatically reduces endogenous hnRNP A1 protein without decreasing mRNA levels. Together, our results demonstrate that hnRNP A1 is regulated by QKI and suggest that hnRNP A1 deregulation underlies the splicing defects observed in qkI mutants and in SCZ.