bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional consequences of ROS‐induced ROS release in vascular endothelium
Authors: Roos Carolyn M. , Zhang Bin , Miller Jordan D.
Published: June 2021

We have previously shown that global reductions in mitochondrial antioxidant capacity and induction of reactive oxygen species (ROS)‐induced ROS‐release (RI‐RR) do not impair endothelial function in young mice due to compensatory increases in an apparent NAD(P)H oxidase‐derived endothelium‐derived hyperpolarizing factor (EDHF). In the present study, we hypothesized that endothelial MnSOD is a major determinant of the functional consequences of RI‐RR, and NAD(P)H oxidase‐derived H2O2 is the major compensatory EDHF released from endothelium when MnSOD is reduced. MnSOD was reduced in endothelium by crossing MnSODFL/FL mice with mice carrying cre‐recombinase driven by a Tie‐2 promoter (Cre+). Aorta from Cre+/MnSOD+/+ and Cre+/MnSODFL/+mice were used for studies of endothelial function. Maximum relaxation to acetylcholine (MR, ACH) was not affected between Cre+/ /MnSOD+/+ and Cre+/MnSODFL/+ mice (61%±1.9, 61%±2.6; respectively). Incubation with apocynin did not change MR, ACH in Cre+ /MnSOD+/+ mice (63%±2.0), but significantly impaired MR, ACH in Cre+/MnSODFL/+ (42%±4.7). Incubation with catalase did not effect MR, ACH in Cre+/MnSOD+/+ mice, but substantially impaired MR, ACH in Cre+/MnSODFL/+ mice (61%±1.2, 48%±1.7). We conclude that inducing RI‐RR in endothelium by reducing MnSOD elicits a compensatory increase in EDHF production, most likely from an increase in NOX2‐derived H2O2.

NHLBI
Pulmonary Endothelial Barrier Disruption via Bicarbonate Stimulation of Endogenous Soluble Adenylyl Cyclase, AC10
Authors: Sayner Sarah , Kunstadt Ryan , Nix Jessica
Published: June 2021

Transmembrane adenylyl cyclase (AC) generates subplasma membrane cAMP that strengthens the pulmonary endothelial barrier. In contrast, P. aeruginosa inserts the toxic soluble AC, ExoY, into pulmonary microvascular endothelial cells (PMVECs) where it generates cytosolic cAMP that phosphorylates the microtubule associating protein, tau, destabilizes microtubules and disrupts the endothelial barrier. A mammalian soluble AC, AC10, has been identified, which also localizes to the cytosol. We tested the hypothesis that bicarbonate stimulation of AC10 increases pulmonary endothelial cell permeability via tau phosphorylation. Western analysis revealed PMVECs have greater AC10 expression than pulmonary artery endothelial cells (PAECs). While adjusting CO2 tension to maintain constant media pH, bicarbonate dose‐dependently increased the phosphodiesterase 4 sensitive cAMP pool in PMVECs, but not in PAECs. RT‐PCR and Western analysis revealed heterogeneous expression of carbonic anhydrase and bicarbonate cotransporters in pulmonary endothelial cells. Further, bicarbonate increased tau serine214 phosphorylation and decreased resistance across the PMVEC monolayer. These data reveal bicarbonate stimulates AC10 activity generating a phosphodiesterase 4 regulated cAMP pool in PMVECs, which increases tau phosphorylation and compromises PMVEC barrier integrity. Funding AHA11GRNT7430039
Effects of acute systemic inhibition of nitric oxide synthase on plasma levels of pro‐and anti‐inflammatory cytokines in anesthetized mice
Authors: Singh Purnima , Castillo Alex , Islam Mohammed T. , Majid Dewan S. A.
Published: June 2021

As demonstrated earlier that nitric oxide synthase (NOS) inhibition induced TNF‐α production both in‐vivo and in‐vitro, we examined further the hypothesis that NOS deficiency is primarily linked with induction of various inflammatory cytokines. Levels of pro‐(IL‐6 & MCP‐1) and anti‐inflammatory (IL‐10) cytokines were measured (using ELISA kits) in plasma collected from anesthetized mice (C57BL6; ~10 weeks age) infused with a NOS inhibitor, nitro‐L‐arginine methyl ester (L‐NAME; 200 μg/min/kg bw iv for 85 min; n=5). Compared to the values in vehicle treated mice (n=5), there were reductions in plasma levels of IL‐10 (2.5±0.7 vs 0.7±0.2 ng/mL), an increase in MCP‐1 (1.6±0.05 vs 3.1±0.4 ng/mL) without altering IL‐6 level (1.2 ± 0.1 vs 1.3±0.1 ng/mL) in L‐NAME treated mice. To assess the role for oxidative stress in these responses, L‐NAME was also infused in mice pre‐treated with a superoxide scavenger, tempol (2 μg/min/kg, iv; n=4). L‐NAME treatment in tempol pre‐treated mice also showed similar plasma IL‐6 (1.1±0.6 ng/mL) and IL‐10 (0.6±0.1 ng/mL) levels but lower MCP‐1 level (0.6±0.3 ng/mL). These data indicate that NOS inhibition induced production of pro‐inflammatory cytokine, MCP‐1 possibly via enhanced oxidative stress, but not affecting IL‐6 generation. These data also indicate that the production of anti‐inflammatory cytokine, IL‐10 is primarily linked to NOS activity. (Funded by NHLBI grant #66432)
Role of NO in the Organization of Ciliated Cells in the Lateral Brain Ventricle
Authors: Fowajuh Ann-Desdemonia Neh , Peunova Natalia , Enikolopov Grigori
Published: June 2021

Cilia beating in brain ventricles, lungs, trachea and oviduct are important for normal physiology of these organs. Ciliopathies are diseases associated with the dysfunction of ciliogenesis and cilia function. Low nasal nitric oxide (NO) levels are characteristic of ciliopathies; however, the link of NO with ciliogenesis is unknown. The rationale behind the project was to find the link between medical observations of low NO levels in ciliopathies and molecular mechanisms of NO action in ciliogenesis. We hypothesize that NO is critical to ciliogenesis and function and low levels of NO results in dysfunction in the mucociliary tissue in the brain ventricles. Using immunochemistry, the integrity of the pattern of ciliated cells and the polarity of the cytoskeleton in neuronal NO synthase knock out (nNOS KO) and wild type (WT) mice were assessed. The role of NO in cilia function was investigated by recording the movement of fluorescent beads; in the brain ventricles of WT and nNOS KO mice using Imaris Software. Pinwheel structures were found at the ventricular surfaces in neurogenic areas of the adult brain in both WT and KO. However, ciliated cells surrounding neuro stem cells had a higher planar polarity in WT versus nNOS KO mice. Results also show that NO controls cilia planar polarity in the lateral ventricle of mice and a deficiency of NO causes a decrease in cilia driven flow
Role of Protein Disulfide Isomerase during vascular repair after injury
Authors: Tanaka Leonardo Yuji , Araújo Haniel Alves , Csordas André Alcantara , Hironaka Gustavo Ken , Takimura Celso Kiyochi , Laurindo Francisco Rafael Martins
Published: June 2021

Endoplasmic reticulum(ER) redox chaperone protein disulfide isomerase(PDI) regulates vascular/phagocytic NADPH oxidase and supports cell migration. We investigated the role of PDI during vascular repair after injury(AI) induced by balloon in rabbit iliac artery. There was marked increase of PDI mRNA and protein(5–10‐fold) at 4, 7 and 14 days AI vs. intact control(CT). PDI immunostaining was greater in intima = neo‐endothelium > media. Increased cell‐surface PDI was also evident. ER stress‐related KDEL chaperones also increased with similar time‐course AI. PDI siRNA(siPDI) transfection in cultured vessel rings collected 14 days AI enhanced KDEL expression vs. scrambled siRNA(siScr) (siScr 2.1±0.9 vs. siPDI 5.0±2.3‐fold vs. CT, p<0.05), apoptosis (siScr 4.6±0.2 vs. siPDI 6.2±0.9 %TUNEL + nuclei, p<0.05) and proliferation marker PCNA (siScr 1.2±0.3 vs. siPDI 4.0 ±0.2 AU, p<0.05), and decreased differentiation marker calponin‐C (siScr 0.52±0.04 vs. siPDI 0.36 ±0.04 AU, p<0.05). siPDI in CT rings did not alter such variables. PCR array analysis showed analogous pattern of mRNA changes. Also, siPDI 14 days AI upregulated Nox1 and downregulated Nox4 NADPH oxidase, while siPDI attenuated oxidant production (in situ hydroethidine) only in CT vessels. Thus, strongly‐overexpressed PDI 14 days AI protects against apoptosis and ER stress and sustains VSMC differentiation. Support: FAPESP, CNPq/INCT Redoxoma
Physical activity blunts oxidative stress response to exercise in sickle cell trait carriers
Authors: Pialoux Vincent , Chirico Erica N , Faes Camille , Aufradet Emeline , Féasson Léonard , Messonnier Laurent , Martin Cyril
Published: June 2021

Sickle cell trait (SCT) is characterized by hemorheological and endothelial abnormalities, possibly related to an increase in oxidative stress. Habitual exercise training has been shown to decrease oxidative stress through an up‐regulation of antioxidant defences. The aim of this study is therefore to test if regular physical activity can affect oxidative stress and nitric oxide (NO) metabolism in SCT carriers compared to sedentary counterparts. Plasma levels of 4 oxidative stress markers, antioxidant enzymes activities (SOD and GPX), NO metabolism (NOx) and adhesion molecules were assessed at baseline and following a maximal incremental exercise test (Tex) in 40 trained (T) or sedentary (S) subjects, carriers (SCT) or not (CON) of the sickle cell trait. At Tex, T‐SCT had a lower level of all the oxidative stress markers compared to the 3 others groups. Regardless the disease status (SCT or CON), trained subject had a higher activity of SOD, GPX and NOx at Tex. Finally, the changes in oxidative stress induced by exercise were correlated positively with changes in adhesion molecules and negatively with changes in NOx. Our findings indicate that regular physical training can decrease oxidative stress and improve nitric oxide metabolism and vascular adhesion in response to exercise in sickle cell trait carriers. This is likely explained by the improvement of antioxidant enzymes activities in trained subjects.
Endurance (ET), Resistance (RT), and Combined (CT) Training Increase Mixed Muscle Protein Synthesis Independently of Age
Authors: Irving Brian A , Lanza Ian R , Henderson Gregory C , Weymiller Audrey , Sun Yiquan , Nair K S
Published: June 2021

The effects of different modes of exercise on mixed muscle protein synthesis (MMPS) in young (Y) and older (O) adults are not fully understood. We determined the effects of ET, RT, and CT on MMPS in 33Y (25±1y) and 31O (70±1y) adults who were randomized to one of three 8wk interventions: (i) Control: no exercise, (ii) ET: cycling 1h @ 65% VO2 peak, 5x/wk, (iii) RT: RT 4x/wk. After completing the control period, the controls completed an 8wk (iv) CT program (ET & RT). Subjects were studied at baseline and 48h following the last training bout. [13C6]Phe was infused for 8h, with serial v. lateralis biopsies. Isotopic enrichments (IE) were measured by LC/MS for free Phe from tissue fluid (TF) and from that incorporated into MMP. MMPS (%/h) = (ΔIEMMP)/(IETF)/t*100. A rank sum test was used to test baseline differences between Y and O. 2‐way ANCOVA was used to test for differences between groups by treatment, with baseline values serving as the covariate. MMPS (%/h) tended to be higher in Y (0.060±0.003) compared to O (0.053±0.003) (P=0.06). ET [57%, P<0.0001], RT [21%, P=0.042] and CT [42%, P<0.0001] increased MMPS independently of age (P>0.4). The ET‐induced increase in MMPS was greater than the RT‐induced increase (P<0.05), although ET unlike RT does not increase muscle mass suggesting that muscle protein turnover was increased. In conclusion, ET, RT and CT were effective at increasing MMPS independently of age. R01AG09531, KL2RR024151
The Effect of Treadmill Exercise on the Regulation of Protein Synthesis during IL‐6 Induced Cancer Cachexia
Authors: Puppa Melissa , White James , Sato Shu , Carson James
Published: June 2021

Cachexia is associated with the loss of skeletal muscle mass and a reduction in myofibrillar protein synthesis related to the suppression of the IGF‐1/mTOR signaling pathway. The purpose of this study was to determine if treadmill exercise training could offset IL‐6 over‐expression induced suppression of IGF‐1/mTOR signaling pathway. ApcMin/+ mice, an IL‐6 dependent model of colon caner cachexia, were trained on a treadmill 6d/wk 1h/d at 18m/min starting at 6wk of age. Between 12–14wks of age mice were electroporated with either an IL‐6 overexpression (IL6+) or control (CON) vector. Muscle IGF‐1 mRNA was reduced 50% by IL6+, while exercise increased muscle IGF‐1 mRNA 1.3 fold regardless of IL‐6 overexpression. Phosphorylation of mTOR(S2448), p70(T389), and 4EBP‐1(S65) were decreased 50% by IL6+. Exercise was able to attenuate the decrease in mTOR, p‐70, and 4EBP‐1 phosphorylation independent of changes in p‐AKT. These data show that treadmill exercise training can prevent IL‐6 induced skeletal muscle loss by attenuating the suppression of IGF‐1/mTOR signaling in skeletal muscle.

Funded by RO1CA121249‐01
Cranberry bioactives augment the activation status of THP‐1 monocytes/macrophages
Authors: Creasy Rebecca A , Khoo Christina , Percival Susan S
Published: June 2021

Cranberries contain proanthocyanidins (PAC), which have been suggested to interact with immune cell pathogen recognition receptors. We hypothesized that recognition of cranberry PAC would alter cellular function toward an activated state. THP‐1 monocytes were incubated with cranberry fractions and analyzed by flow cytometry with FITC‐CD49a. Cranberry fractions activated monocytes in a time and dose dependent manner as shown by increased expression of CD49a on the surface. Cytokines and chemokines secreted in the culture medium were analyzed by multiplex technology. MIP‐1a, MIP‐1β, and IL‐8 were secreted by the cells without marked secretion of pro‐inflammatory cytokines, IL‐1a, IL‐1β, TNF‐a, and IL‐6. Significantly more MIP‐1a mRNA was present. This suggests compounds found in cranberry can activate monocytes to help regulate immune cell trafficking. A second experiment was designed to determine if cranberry primes immune cells. THP‐1cells were differentiated into macrophages and treated with cranberry fractions or DMSO vehicle for 16 hr. Cells were then washed and challenged with LPS for 6 hr. A trend toward increased MIP‐1a protein was found in cranberry‐treated cells compared to vehicle‐treated cells. Taken together, this suggests that cranberry bioactives prime THP‐1 macrophages for a heightened response upon pathogen challenge.

Grant Funding Source : Ocean Spray Cranberries, Inc. and AES, University of Florida
MicroRNA‐143 regulates the expression of pref‐1 in adipocyte
Authors: Kim Sang Hoon , Kim Yoon-Jin
Published: June 2021

Adipose differentiation is the result of transcriptional remodeling that leads to the activation of a large number of adipose‐related genes. MicroRNA plays a essential role in the progression of adipogenesis, but its role in adipocyte is not well defined. Preadipocyte factor‐1 (Pref‐1) is a transmembrane EGF repeat domain‐containing protein encoding 385 amino acids. Rref‐1 activates the MEK/Extracellular signal‐Regulated kinase pathway to inhibit adipocyte differentiation through inhibition of the expression of both PPAR¥ã and C/EBP¥á. In the present study, we found that during adipogenesis the expression of miR‐143 was increased, whereas the expression of pref‐1 was decreased. Both mRNA and protein level of pref‐1 were downregulated in 3T3‐L1 preadipocyte ectopic expressed miR‐143. The activity of luciferase reporter including pref‐1 mRNA 3ˈ UTR was decreased after co‐transfection with miR‐143. Although pref‐1‐mediated adipogenic inhibition is occurred by the activation of erk1/2, overexpression of miR‐143 resulted in repression of phophorylation of Erk1/2. Therefore, these results suggest that microRNA‐143 induces the differentiation of adipocyte by down‐regulating the post‐transcription of pref‐1.
Inactivation of glycogen‐synthase kinase 3 beta in Madin‐Darby Canine Kidney cells increases epithelial resistance
Authors: Jouret Francois , Rajendran Vanathy , Caplan Michael J
Published: June 2021

Glycogen‐synthase kinase 3‐beta (GSK‐3β) is a ubiquitous serine‐threonine kinase involved in numerous biological processes. We have recently demonstrated that pharmacological inhibition of GSK‐3β activity leads to the assembly of tight junction (TJ) components in epithelial cells, even in the absence of extracellular Ca2+ (Ca2+ e). Here, we generate and characterize a Madin‐Darby Canine Kidney cell line stably knocked‐down for the expression of the gene encoding GSK‐3β. At steady‐state, GSK‐3β inactivation is associated with an increased expression of ZO‐1 and occludin, at both the mRNA and protein levels. Transepithelial electrical resistance (TEER) is significantly higher in GSK‐3β KD (763.4 ± 54.5 ohm.cm2) than in control (332.6 ± 38.6 ohm.cm2) cells. Following Ca2+ switch from low (5μM) to normal (1.8mM) Ca2+e, the relocation of ZO‐1 to cell‐cell contacts is significantly faster in GSK‐3β KD versus controls. Conversely, Ca2+e deprivation in control cells for 2 hours induces the internalization of the GSK‐3β substrate, β‐catenin, as well as a loss of measurable TEER. In GSK‐3β KD cells similarly exposed to low‐Ca2+e, β‐catenin stays localized predominantly at the basolateral membrane, and TEER remains measurable (197.1 ± 23.9 ohm.cm2). These results support the conclusion that GSK‐3β activity modulates TJ assembly and stability in epithelial cells.
TRPA1‐activated anion secretion is linked with PGE2 receptor EP4 in human and rat colon
Authors: Kaji Izumi , Karaki Shin-ichiro , Kuwahara Atsukazu
Published: June 2021

The transient receptor potential A1 (TRPA1) channel is considered a chemosensor in several sensory tissues. In the present study, we investigated the secretory effect of potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colons using the Ussing flux chamber. The mucosal application of AITC (10−6–10−3 M) induced Cl− and HCO3 − secretion in a concentration‐dependent manner, while the serosal application induced weaker effect. AITC‐evoked anion secretion was attenuated by the pretreatment with cyclooxygenase inhibitor piroxicam and prostaglandin (PG) E2, but the secretion was not affected by tetrodotoxin, atropine, or extracellular Ca2+ depletion. These results indicated that TRPA1 activation induces anion secretion through PG synthesis independent of neural pathway in the colon. Further analysis also indicated that AITC‐evoked anion secretion is mainly mediated by EP4. In addition, RT‐PCR using isolated colonic crypts, in situ hybridization and immunohistochemistry supported the TRPA1 expression in epithelial cells. Therefore, we conclude that the activation of TRPA1 activity on the colonic epithelial cells is likely involved in host defense mechanism through rapid anion secretion.
Feeding of probiotic formulation protects from obesity and diabetes
Authors: Yadav Hariom , Rane Sushil G
Published: June 2021

The role of gut flora in obesity and diabetes is an area of active debate. We found that feeding of probiotic formulations (i.e. Culturelle, Primal, VSL#3, Pb8 and Ultimate 10 probiotics) protect high fat diet (HFD)‐induced obesity and insulin resistance in mice. VSL#3 exhibited the best anti‐obese/diabetes potential in terms of reducing weight gain, fat mass and dyslipidemia. VSL#3 treated mice exhibit improved glucose homeostasis characterized by enhanced insulin sensitivity and glucose tolerance. Interestingly, VSL#3 treated mice dramatically decreased daily food/energy intake. Moreover, VSL#3 treatment modulated the gut‐brain axis by altering the levels of gut hormones and adipokines that regulate food intake and energy homeostasis. Specifically, VSL#3 treatment decreased ghrelin levels and increased PYY, GLP‐1, leptin and adiponectin levels in HFD‐fed mice. In line with these results, VSL#3 treatment enhanced phosphorylation levels of the leptin signaling mediator, STAT3. Further, we observed changes in expression of hypothalmic genes that regulate food intake. Thus, reduced expression of Npy and Agrp and increased Pomc expression was seen in the hypothalamus of VSL#3‐treated mice. These results provide a proof of concept that feeding of probiotics can be potential therapeutic approach to protect from obesity and diabetes via modulation of gut‐brain axis and adipose biology.
Co‐culture supernatants of toxigenic Clostridium difficile 43285 with specific Lactobacillus and Bifidobacterium species significantly attenuated IL8 and MIP3α induction and restored epithelial viability
Authors: VijayaKumar DeepakKumar , Nanthakumar Nanda
Published: June 2021

Interleukin‐8 (IL8) and macrophage inflammatory protein 3α (MIP3α) are chemokines secreted by the intestinal epithelium to attract innate immune cells during the onset of inflammatory responses. During C. difficile infection in human, these chemokines are significantly induced to attract the neutrophils to the site of infection. In this study, we investigated the impact of culturing specific probiotic (Lactobacillus or Bifidobacterium species) and commensal (B. fragilis) bacterium with virulent C. difficile 43285 and assayed for IL8 and MIP3α responses in CaCo‐2 cells. CaCo‐2 cell is a model for human enterocytes. Only the co‐culture of C. difficile with L. paracasei, L. casei, L. plantarum, L. rhamnosus LGG, L. delbruikii, B. infantis and B. adolescentis significantly attenuated IL8 and MIP3α responses compared to the monoculture supernatant of C. difficile. This suggests specificity of few probiotics and may suggest that coculture with these probiotics may act to neutralize the toxins and other virulence factors of C. difficile or create a metabolic environment that makes it less favorable for C. difficile to become virulent. Cell viability assay showed that CaCo‐2 cells treated with these specific probiotic co‐cultured supernatants showed enhanced viability as compared to the supernatant of C. difficile. This suggests that the culture supernatants of specific probiotic co‐cultures may possess metabolites that are likely to prevent inflammatory response and ultimately cell death in human enterocytes.

Supported by NIH grant RO1HD059126; Contact: Dr. Nanda Nanthakumar; e‐mail: nanthaku@helix.mgh.harvard.edu
A genome‐wide methylation study of vitamin D deficiency in African American adolescents
Authors: Zhu Haidong , Wang Xiaoling , Shi Huidong , Su Shaoyong , Harshfield Gregory A , Gutin Bernard , Snieder Harold , Dong Yanbin
Published: June 2021

Emerging in vitro evidence suggests that histone modification plays a role in vitamin D‐mediated immune‐regulatory and anti‐inflammation effects. We hypothesized that DNA methylation is also involved in vitamin D deficiency mediated immune cell regulation. A genome‐wide methylation scan was performed using the Illumina HumanMethylation 27 BeadChip on leukocytes DNAs of 11 vitamin D deficient cases (serum 25(OH)D ≤25 nmol/L) and 11 age‐matched controls (serum 25(OH)D>75 nmol/L); the subjects were African American normal‐weight males aged 14–19 years. The Limma package was used to analyze each CpG site for differential methylation between cases and controls. To correct for multiple testing, the set of raw p values were converted to false discovery rates (FDR). Seventy nine CpG sites achieved statistical significance (raw p<0.001). The top two CpG sites (cg16317961 and cg04623955) survived multiple testing using a FDR of 10% (FDR=0.078). Moreover, half of the top ten genes have been implicated in immune cell activation, regulation and inflammatory response. In summary, our pilot data suggest that vitamin D deficiency is associated with methylation differences in blood leukocyte DNA in African American adolescents, suggesting that DNA methylation may be involved in vitamin D‐mediated immune regulation. Larger genome‐wide studies are warranted to confirm these findings.
CCN1 induces death of esophageal adenocarcinoma: Trick or TRAIL?
Authors: Chai Jianyuan , Norng Manith , Pham Jennifer , Wu Sing-Yung , Jamal M Mazen
Published: June 2021

Background

Selectively to kill cancer cells has always been a dream of all oncologists. TRAIL has shown promising effect in several types of cancer. A recent study demonstrated a synergistic apoptotic effect of TRAIL and CCN1 in prostate cancer. This study is to investigate the role of CCN1 in esophageal adenocarcinoma (EAC).

Methods

Expression of CCN1 and TRAIL were assessed in EAC cells (OE33) and the tumor tissue by quantitative PCR. Normal esophageal cells (Het‐1A) and tissue were used as control.

Results

(1) CCN1 expression was down by 65% (P < 0.01, N = 15) in OE33 cells compared in Het‐1A cells; and by 95% (P < 0.05, N = 3 normal and 12 EAC) in EAC tumors compared in normal esophageal tissue. (2) OE33 cells expressed TRAIL receptors but not TRAIL ligand, while Het‐1A cells just the opposite. (3) CCN1 up‐regulated pro‐apoptotic genes (e.g. TRADD 181 fold) in OE33 cells, but promoted anti‐apoptotic genes (e.g. BIRC5 137 fold) in Het‐1A cells. (4) TRAIL induced apoptosis in OE33 cells but not in Het‐1A cells. TRAIL and CCN1 combination increased apoptosis by 35% (P < 0.01, N = 3) in OE33 cells. (5) Mixed culture of OE33 and Het‐1A cells caused massive death of OE33 cells.

Conclusions

TRAIL can selectively kill EAC cells without harming normal esophageal cells due to the lack of receptor, and CCN1 can enhance the effect. Expression of TRAIL receptors but not the ligand is a strategy of EAC to escape potential cell death. The study was supported by the VA Merit Review.
Phase II randomized trial of walnut supplementation and subsequent influence on male reproductive health
Authors: Carpenter Catherine L , Henning Susanne M , Seltzer Mollie , Peterson Erica , Xun Lin , Robbins Wendie A
Published: June 2021

Protective walnut benefits have been studied in relationship to heart disease and cancer prevention but little is known about whether walnuts influence male reproductive health. Walnuts contain both α‐linolenic acid (ALA) that could impact sperm membrane integrity, and, folate, selenium, and tocopherols that function in antioxidant and other capacities. We conducted a three‐month phase II randomized controlled trial on 117 young adult males (age 20–35) attending a major university. The intervention group received 75 grams whole‐shelled walnuts per day compared to controls who avoided tree nuts. Both groups were instructed to consume an ad libitum diet. Our objective was to determine if walnut supplementation would influence male fertility and sperm parameters, studied against a background diet consumed by American college students. College‐aged men were selected at their peak of reproductive fitness inferring that walnut effects observed in this group would be applicable to men of all ages. Baseline and follow‐up measures included weight, height, BMI, exercise, semen analysis, serum fatty acids, selenium, and other micronutrients, seminal fluid anti‐oxidant capacity, hormones, completion of a baseline NCI (National Cancer Institute) Diet History Questionnaire, and 3‐day Food Records at baseline and the study end. We conducted 24‐hour recalls every 2 weeks using the ASA24 on‐line assessment provided by NCI to evaluate whether dietary compensation occurred. Compared to controls, men in the walnut intervention group did not gain weight from baseline (p=0.9), and, serum ALA (p=0.001) and sperm motility (p=0.04) increased. Our results suggest that walnuts impart a protective influence on male fertility that may result from constituents found in walnuts or the whole walnut itself. Funding was provided by the California Walnut Commission.
Effects of whole and refined grains on cardiometabolic risk factors in a controlled‐feeding, weight loss study: Preliminary findings
Authors: Harris Kristina Arline , West Shiela , Heuvel Jack Vanden , Jonnalagadda Satya , Kris-Etherton Penny
Published: June 2021

Making “half your grains whole” is recommended to reduce risk of chronic disease; yet, evidence from clinical trials is mixed. Our aim was to study the effects of chronic whole (WG) and refined grain (RG) intake on cardiometabolic risk factors in a controlled‐feeding setting. Overweight or obese (BMI 25–42 kg/m2) men (n=25) and women (n=25) aged 35–55 with increased waist circumference and at least 1 other Metabolic Syndrome criterion were randomized to a healthy diet containing either WG or RG. An isocaloric diet was provided for 6 weeks followed by a hypocaloric diet (~500 kcal/day) for 6 weeks. Both groups had reductions (p for time <0.001) in body weight, waist circumference, abdominal % body fat, C‐reactive protein, insulin, triglycerides, HDL‐C, LDL‐C, and blood pressure, but group differences for these endpoints were not significant. Fasting plasma glucose (FPG) tended to decrease more with WG compared to RG (−1.2±0.9 vs −4.0±0.9 mg/dL, p=0.1) while controlling for baseline FBG and weight loss. In a subgroup analysis, there was a greater decrease in the number of prediabetic participants (FPG ≥100 mg/dL) from baseline to study end in the WG (10 to 1) compared to the RG group (8 to 7). In conclusion, a healthy diet led to significant improvement of cardiometabolic risk factors in an “unhealthy overweight” population while WG provided extra benefits for glycemic control, particularly in prediabetic individuals.

Grant Funding Source : Bell Institute for Health and Nutrition, General Mills, Inc.; NIH Grant M01RR10732; ClinicalTrials.gov NCT00924521
Problem solving strategies and the relationship between visualization ability and spatial anatomy task performance
Authors: Nguyen Ngan , Mulla Ali , Nelson Andrew J , Wilson Timothy D
Published: June 2021

Background

In a previous study, we showed that visualization ability (VZ) is related to performance on a spatial anatomy task (SAT). Low VZ learners demonstrated consistently poorer task performance than high VZ learners. In the current study, we explored the processing commonalities and differences of learners in order to determine which problem solving strategies distinguish those of high VZ from those of low VZ.

Methods

Forty‐two students completed a standardize measure of VZ, the SAT, and a questionnaire involving self‐analysis of the processes and strategies used while performing the SAT.

Results

Consistent with our previous study, high VZ learners performed better on the SAT than low VZ learners. Concerning problem solving strategies, more low VZ learners reported using movements of body parts and/or surrounding objects while performing the SAT. These learners also stated they were more concerned about time, that is finishing all SAT questions, than they were about answering correctly.

Conclusion

The tendency for low VZ learners to offload cognitive work onto external perceptual‐motor processes suggests that they have problems with mental manipulations, which may contribute to their poor SAT performance. Furthermore, low VZ learners are more prone to errors while performing the SAT, as suggested by their tendency to focus on the quantity, rather than the quality, of their answers.

Grant Funding Source: none
Phenolic Acid Concentrations in Plasma and Urine from Men Consuming Green or Black Tea and their Chemopreventive Properties for Colon Cancer
Authors: Henning Susanne M , Wang Piwen , Vicinanza Roberto , Abgaryan Narine , de Oliveira Daniela Moura , Zhang Yanjun , Lee Ru-Po , Carpenter Catherine L , Aronson William , Heber David
Published: June 2021

Green (GT) and black tea (BT) polyphenols are metabolized to phenolic acids (PAs) by colonic microflora and may contribute to teas’ chemopreventive activity. The concentration of PAs was quantified in urine and serum from men drinking 6 cups daily of GT, BT or water by high performance liquid chromatography and electrochemical detection. 3,4‐dihydroxyphenyl acetic acid (3,4‐ DHPAA), 4‐hydroxyphenylacetic acid (4‐HPAA), 3‐ hydroxyphenylacetic acid (3‐HPAA), 3‐methoxy‐4‐ hydrozyphenylacetic acid (3M4‐HPAA) and hippuric acid were increased significantly in urine from men drinking GT. The consumption of BT was associated with a significant increase in urine 4‐HPAA and 3‐HPAA only. No significant changes were observed in serum. Fecal water contains a mixture of tea polyphenols such as epigallocatechin gallate (EGCG) and their PA metabolites. We therefore investigated the antiproliferative effect and intracellular concentration of EGCG, 3,4‐DHPAA and their methylated forms in HCT116 and HT‐29 colon cancer cells. Incubation with both EGCG and 3,4‐DHPAA exhibited an additive antiproliferative effect, while methylation of 3,4‐DHPAA was significantly decreased. In summary the consumption of GT was associated with a higher increase in urinary phenolic acids compared to BT. Combining EGCG with 3,4‐DHPAA enhanced the antiproliferative activity in colon cancer cells. Supported by RO1CA116242.
The role of membrane shaping proteins, MTs and interorganelle contacts in regulating ER structure
Authors: Voeltz Gia , Friedman Jonathan , West Matt , DiBenedetto Jared , Rowland Ashley , English Amber
Published: June 2021

The endoplasmic reticulum (ER) has an elaborate and dynamic structure that is conserved throughout eukaryotes. We are interested in how ER shape is regulated to generate a functional organelle. We have studied three major mechanisms that influence the 3‐D structure of the ER. ER shape is determined by membrane shaping proteins, dynamics on the cytoskeleton, and interactions with other organelles. These forces work together to distribute this organelle throughout the cytoplasm and generate the complexity of ER functional domains. In turn, we have recently demonstrated that ER shape and dynamics also influence the distribution and dynamics of other organelles. I will discuss the dynamic interplay between the various mechanisms that regulate the elaborate structure of the ER and the organelles that it contacts.
Assessment of whole body cholesterol pool size in Smith‐Lemli‐ Opitz syndrome children using liquid chromatography tandem mass spectrometry
Authors: Myrie Semone B , Merkens Louise , Yang Haifeng , Roullet Jean- Baptiste , Steiner Robert D , Jones Peter J
Published: June 2021

Smith‐Lemli‐Opitz syndrome (SLOS) patients typically exhibit abnormally low cholesterol, high 7‐dehyrocholesterol (DHC) and 8‐DHC levels in tissues and plasma. Therapeutic intervention for SLOS is aimed at maximizing whole body cholesterol pool size (WBCP) while down‐regulating biosynthesis to decrease the buildup of potentially toxic precursors. The study aim was to develop a sensitive liquid chromatography‐tandem mass spectrometry (LC/MS/MS) method for detection of changes in blood isotope cholesterol enrichment over time, for assessment of WBCP in SLOS patients receiving supplemental dietary cholesterol. Subjects (n=13; mean age: 7±1 yr) were given iv [18O]cholesterol (1.0–1.4 mg/kg bodyweight) or D7‐cholesterol (0.9–1.4 mg/kg). Blood samples were collected at baseline and over a 10 wk period, cholesterol extracted from RBC, derivatized with piconyl ester and analyzed using LC/MS/MS. The [M+H] +>[M‐picolinic acid] + transition was chosen for monitoring, with 18O‐cholesterol at m/z 494>369 and D7‐cholesterol at 499>376. WBCP was 427.3 ±52.0 mg/kg bodyweight. Plasma cholesterol level was not related to WBCP (p>0.05), but inversely related to 7DHC and 8DHC (p<0.05). In summary, LC/MS/MS is a sensitive method for stable isotopic assessment of WBCP in humans. This study is the first assessment of WBCP in children and in SLOS patients. The data will be a useful reference for a longitudinal SLOS study.

Grant Funding Source : NIH
ABCG5/G8 deficiency results in reduced intestinal cholesterol and fatty acid transport
Authors: Zhang Linda Shuo , Kohan Alison B , Tso Patrick
Published: June 2021

Adenosine triphosphate‐binding cassette (ABC) half transporters G5 and G8 are critical in protecting the body from the accumulation of dietary phytosterols, which is associated with an elevated risk of coronary heart disease. Expressed in the liver and intestines, they are proposed to promote phytosterol excretion and to limit phytosterol absorption. However, the role of G5/G8 in cholesterol and fatty acid (FA) absorption is unclear. The aim of this study was to investigate the effects of G5/G8 deficiency on intestinal lipid transport in chow‐fed mice. We employed the conscious lymph fistula model to examine radiolabeled dietary cholesterol and FA transport into the lymph using wild‐type (WT) and G5/G8 knockout (KO) mice. Since it has been thought that G5/G8 promote sterol excretion, we were surprised to find that G5/G8 KO mice displayed a ~53% reduction in dietary cholesterol transport into the lymph compared to WT controls. In addition, G5/G8 KO mice also displayed a ~45% reduction in FA transport into the lymph. This reduction in cholesterol and FA transport was also accompanied by an accumulation of cholesterol and FAs in the intestinal lumen, suggesting that the loss of G5/G8 inhibit transport across the enterocyte. We conclude that G5/G8 deficiency results in reduced intestinal cholesterol and FA absorption in mice, and therefore may play a previously unknown role in intestinal lipid absorption.

Grant Funding Source: University of Cincinnati
Feeding Blueberry Diets Dose‐Dependently Inhibits Bone Resorption in Young Rats
Authors: Zhang Jian , Lazarenko Oxana P. , Badger Thomas M. , Ronis Martin J. , Chen Jin-Ran
Published: June 2021

Nutritional status is a critical factor that influences bone development. We previously reported that weanling rats fed AIN‐93G semi‐purified diets supplemented with 10% whole blueberry (BB) powder for only two weeks beginning on postnatal day 21 (PND21) significantly promoted bone formation. However, whether there is an effect of BB diet on bone at lower levels of supplementation or if there are additional effects on bone resorption is unknown. The current study examined the effects of three levels of BB diet supplementation (1, 3, and 5%) for 14 days beginning on PND21on osteoclastic bone resorption. Using peripheral quantitative CT scan of tibia, we demonstrated that bone mineral density and content were dose‐dependently increased in BB rats compared to control AIN‐93G fed animals (p < 0.05). Proteins were isolated from long bone of femurs, and Western blot revealed that RANKL (the cytokine receptor activator of nuclear factor‐κB ligand essential for osteoclast formation) protein expression was dose‐dependently decreased (p<0.05). This was accompanied by decreased expression of PPARγ, RANKL and TRAP (Tartrate‐resistant acid phosphatase) mRNA expression (p<0.05). These results indicate significant stimulation of bone growth by dose‐dependent inhibition of bone resorption after BB consumption during early development. Supported in part by ARS CRIS #6251‐51000‐005‐03S.

Grant Funding Source: USDA / ARS CRIS
Pediatric Overweight Prevention through a Parent Training Program for 2–4 year old Latino Children
Authors: Slusser Wendelin , Neumann Charlotte , Cumberland William , Renenger Kristel , Fischer Heidi , Frankel Fred
Published: June 2021

The objective of this study was to measure over a one‐year period, whether a parent training based on social learning theory combined with evidenced based interventions to promote optimal nutrition and physical activity will reduce the upward trend of BMI z‐scores in groups of 2–4 year old Latino children living in low‐income households.

Methods

A randomized controlled pilot study evaluated the effectiveness of the intervention contrasting 61 children whose mothers were randomized to receive Parent Training (PT) with 60 Wait‐list (WL) subjects. 7 weekly classes with 2 booster classes were delivered to mothers. Forty subjects did not attend the one‐year follow up assessment resulting in 81 subjects who have measurements for both baseline, and one‐year follow up assessments. To adjust for differential dropout rates and missing observations, imputation of missing data was done using a carefully constructed model.

Results

There were no significant subject differences between groups at baseline for family characteristics and BMI categories for child and mother. Children in the intervention group decreased their z scores significantly on average by .20 (SE= .08) compared to children in the control group who increased z scores on average by .04 (SE=.09) at one year (P<.05).

Conclusion

PT is effective to reduce the risk of overweight in preschool Latino children living in low‐income households.
Phenolics in mulberry leaves protect Hep G2 cells against hyperglycemia‐induced oxidative damage
Authors: Zou Yu-Xiao , Liao Shen-Tai , Liu Fan , Blumberg Jeffrey , Chen C-Y. Oliver
Published: June 2021

Hyperglycemia is associated with increased risk of type 2 diabetes, partially due to increases in oxidative stress. Phenolics in mulberry leaves (MLP) may protect tissues from hyperglycemia‐induced oxidative damage. We examined the effect of MLP on hyperglycemia‐induced oxidative damage in hepatic HepG2 cells. HepG2 were treated with glucose (5.5 or 50 mM) with or without MLP at 10 or 100 μM GAE. Hyperglycemia‐induced oxidative damage in HepG2 was demonstrated by a 666% increase in O2.− production at 0.5 h, 400% increase in malondialdehyde (MDA) at 24 and 48 h, mitochondrial membrane hyperpolarization (MMHP) at 2 h followed by depolarization, and 474% increase in nucleus NFκB‐P65 at 4 h relative to the control cells (P ≤0.05). Both MLP doses inhibited hyperglycemia‐induced O2.− production by 40%. Hyperglycemia‐induced MDA production at 24 h was diminished by 34 and 54% by MLP at 10 and 100 μM, respectively. MLP treatment protected HepG2 cells from hyperglycemia‐induced MMHP in a dose‐dependent manner with the highest dose maintaining mitochondrial polarization status. MLP at 10 μM inhibited 35% hyperglycemia‐induced NFκB‐P65 translocation and at 100 μM completely prevented the NFκB activation. Our data show that MLP in vitro protects hepatoctyes from hyperglycemia‐induced oxidative damage.
Estrone‐3‐Sulfate is a substrate to verify functionality of uptake transporters in primary hepatocytes
Authors: Keiser Markus , Radebold Jennifer , Ullrich Anett , Damm Georg , Nüssler Andreas , Siegmund Werner , Runge Dieter
Published: June 2021

Background

Primary mammalian hepatocytes are used for several in vitro applications like testing of drug metabolism, toxicity and transporter assays. However, little is known about species specific differences or similarities in the activity of uptake and efflux transporter. Therefore, we started a species specific characterization and comparison of uptake transporters functionalities in relevant species used for drug testing. Here, we report the uptake of estrone‐3‐sulfate (E3S) in primary hepatocyte cultures of human, rat and monkey.

Methods

Hepatocytes were incubated in serum free media. Two ‐ four days after cell isolation a time and concentration dependent uptake of [3H]‐E3S was measured using liquid scintillation counting. Rifampicin was added in various concentrations to measure its effects on E3S uptake.

Results

All hepatocytes showed at 37 °C a time‐dependent and saturable increase in E3S uptake compared to the uptake at 4 °C. E3S revealed a high affinity to human (Km = 12.9 ± 10.1 μmol/l; Vmax = 84.2 ± 30.3 pmol/mg × min) and monkey (Km = 8.2 ± 2.3 μmol/l; Vmax = 31.3 ± 3.6 pmol/mg × min) but not to rat hepatocytes. Rifampicin clearly inhibited the E3S uptake in monkey hepatocytes while rat hepatocytes were not influenced.

Conclusion

Our data suggest that [3H]‐E3S is a suitable substrate to verify the functionality of uptake transporters in primary hepatocytes of humanoids but not of rats.
Down‐regulation of desmoglein‐2 in colonic epithelial cells suppresses proliferation and reduces tumor growth via a desmocollin‐2‐dependent mechanism
Authors: Kolegraff Keli , Kamekura Ryuta , Hilgarth Roland , Nava Porfirio , Parkos Charles , Nusrat Asma
Published: June 2021

Expression of the desmosomal cadherins desmoglein‐2 (Dsg2) and desmocollin‐2 (Dsc2) is altered in epithelial cancers, and we have shown that loss of Dsc2 in colonic epithelial cell lines enhances proliferation and tumor formation, thus providing evidence of a tumor suppressor function for Dsc2. To determine if loss of Dsg2 has similar effects, we used shRNA to down‐regulate Dsg2 expression in colonic epithelial cell lines. Interestingly, loss of Dsg2 decreased cell proliferation and inhibited xenograft tumor formation in mice. Furthermore, down‐regulation of Dsg2 in a Dsc2‐negative epithelial cell line had no effect on proliferation or tumor growth, indicating that Dsc2 may promote growth suppression following loss of Dsg2. To examine whether Dsc2 contributes to growth suppression in Dsg2‐deficient cells, we assessed the effect of Dsg2 loss on Dsc2 expression. Our results show that Dsg2‐deficient cells have increased Dsc2 protein levels compared to controls, suggesting that compensatory Dsc2 up‐regulation may mediate growth suppression. Consistent with this notion, siRNA knockdown of Dsc2 restored proliferation in Dsg2‐deficient colonic epithelial cell lines. Taken together, our data demonstrate that, unlike Dsc2, loss of Dsg2 reduces cell growth and inhibits tumor formation in vivo. Thus, anti‐Dsg2‐targeted therapies may be useful for inhibiting growth of tumors expressing both Dsc2 and Dsg2.

This work was supported by CCFA Fellowship and AGA Research Scholar Awards (PN) and NIH grants DK61379 (CP), DK55679 and DK59888 (AN).
Mucin Protects against Trypsin‐mediated Increases in Intestinal Epithelial Permeability
Authors: Chang Marisol , Alsaigh Tom , Kistler Erik B , Schmid-Schönbein Geert W
Published: June 2021

The intestinal mucosal barrier, composed of epithelium and a mucus layer, normally compartmentalizes pancreatic digestive enzymes in the lumen. However, during early periods of gut ischemia trypsin and other enzymes appear in the wall of the small intestine initiating autodigestion. The role of mucin, the main component of the mucus layer, as a trypsin barrier is unknown. To investigate whether mucin provides a barrier against trypsin transport, IEC‐18 cells (on Transwell plates) were coated with and without a mucin film (400μm, 10%, porcine stomach). FITC‐dextrans (20kDa, 5mg/ml) with and without trypsin (50 μM) was added to the apical side and the rate of FITC accumulation on the basal side over 60 min was measured. To determine if trypsin cleaves the adherent junctions of the epithelium, we measured by Western blot the levels of intra‐ and extracellular domains of E‐cadherin. The results indicate that without a mucin film, E‐cadherin is cleaved and epithelial permeability is increased by 42% after incubation with trypsin. In contrast, when mucin is present cleavage is reduced and permeability is decreased by 30% (p<0.05). These results suggest that in the absence of mucin in the mucus layer such as occurs in ischemia; trypsin perturbs the epithelial layer by cleavage of E‐cadherin and increases its permeability, resulting in transport of enzymes and macromolecules into the intestinal wall. Supported by GM85072
Healthy stores intervention associated with psychosocial improvements in adult American Indian consumers
Authors: Gittelsohn Joel , He Siran , Kim Elizabeth , Delrow Betti , Pardilla Marla
Published: June 2021

Obesity and diet‐related chronic diseases are at epidemic levels in American Indian (AI) populations. We conducted a 14 month intervention (Navajo Healthy Stores (NHS)) to change the food environment in parts of the Navajo Nation (NN), in partnership with the NN Special Diabetes Program. We worked in large and small food stores in 7 randomly selected areas of the NN, matched with 6 comparison areas. NHS involved encouraging stores to stock healthier foods, point of purchase promotions (shelf labels, posters, giveaway items) interactive sessions), and community media (radio and newspaper). A random sample of 146 adult Navajo community members were interviewed pre and post intervention, in both intervention and comparison areas. No intervention impact was seen by treatment group. When we stratified by level of exposure to the intervention, we found that more highly exposed respondents visited intervention stores more frequently, significantly improved their healthy food self‐efficacy and behavioral intentions (p<0.05), and had a positive trend toward improved frequency of purchase of healthy foods. Changing the food environment by working with retail food stores is a potentially effective means for addressing the obesity epidemic in rural AI communities.