bluuebunny/crossref_metadata_2025 huggingface dataset:
Nogo‐B limits intima‐media thickening during mouse vein graft adaptation
Authors: Muto Akihito , Yi Tai , Fitzgerald Tamara N , Fancher Tiffany , Magri Dania , Yu Jun , Sessa William C , Dardik Alan
Published: June 2021

Successful vein graft adaptation to the arterial environment is critical to maintain long‐term graft patency. However, the mechanism of vein graft adaptation remains unclear. Since Nogo‐B protects against vascular injury, we determined whether Nogo‐B plays a mechanistic role, limiting intima‐media thickening, during vein graft adaptation.

Intrathoracic inferior vena cava was harvested from wild‐type (WT) or Nogo‐B knockout (Nogo‐KO) mice and placed as an interposition vein graft into the infrarenal aorta of WT mice. Vein grafts were harvested and analyzed after 2 or 3 weeks.

In WT mouse vein grafts Eph‐B4 immunoreactivity decreased without increased Ephrin‐B2 immunoreactivity (n=4).

Nogo‐KO vein grafts had increased intima‐media area compared to WT vein grafts (.25 mm2 vs .16 mm2, p<.05). However, Nogo‐KO vein grafts also had diminished Eph‐B4 immunoreactivity, similar to the decrease in WT vein grafts.

Mouse vein grafts exhibit decreased markers of venous identity, recapitulating changes in humans and rats. Nogo‐KO vein grafts have loss of venous identity, similar to WT vein grafts, but increased intima‐media thickening. This data suggests that Nogo‐B plays a critical role during venous adaptation to an arterial environment independently of mechanisms that determine venous identity, and is also consistent with a general protective role of Nogo‐B in response to injury of the vasculature.
How to attach the limb to the body axis
Authors: Streicher Johannes , Pomikal Christine
Published: June 2021

While the question “How to make a limb” is extensively addressed in terms of pattern formation, morphogenesis, and genetic background, the ontophyletic scenario of attaching the limb to the axial skeleton via girdles has been paid much less attention. In a comparative study we have investigated pelvis morphogenesis and axio‐appendicular linkage in species from different vertebrate classes. Series of developmental stages from frog tadpoles1, chick embryos and mouse embryos2 were serially sectioned, and 3D‐reconstructed3. The 3D‐models were aligned to 4D‐visualizations of developmental dynamics and further analyzed.

Our data reveal, that in all three species ‐ at the morphological level ‐ the pelvic element originates from a single anlage in close relation to the femur. Shape and orientation, however, differ already at initial stages and are followed by divergent subsequent morphogenesis. The timing and patterns of ossification are significantly different. In frog tadpoles a connective tissue strand serves a guiding function for attachment to the sacral vertebra1.

These results demonstrate substantial differences in the establishment of the axio‐appendicular linkage among vertebrates and prompt for clarification of their genetic and epigenetic background.
Mechanisms of Estrogen Receptor Neuroprotection in Dopamine Neurons
Authors: Roberts James
Published: June 2021

Estrogen involvement in neuroprotection is now a widely accepted research area, although the mechanisms of estrogen action in neuroprotection remain unclear. To investigate the neuroprotective effects of 17‐β estradiol on dopaminergic neurons we utilized primary mesencephalic neurons cultured from embryos of time‐mated C57 Bl/6 mice. The primary mesencephalic cultures allow for analysis of both direct estrogen‐mediated neuorprotection on the dopamine neuron and/or indirect actions through surrounding glial cells. We demonstrate the existence of both ERα and ERβ with a predominance of ERα on both dopamine neurons and astrocytes. In primary cultures of mouse mesencephalic neurons estrogen protects dopamine neurons from injury induced by 1‐methyl‐4‐phenyl pyridium (MPP+). ERα mediated the protection afforded by estrogen since only the ERα agonist HPTE protected against dopamine cell loss. The ERβ agonist DPN, alone did not protect against MPP+ induced neuronal death. Since glial cells were shown to express ERα we investigated an indirect mechanism of estrogen action on dopamine neuron survival. Treating the cultures with the mitotic inhibitor, 5‐Fluoro‐2’‐deoxyuridine (dFUR) abolished glial cells and significantly reduced the amount of neuroprotection seen with estrogen. These data indicate that neuroprotection provided by estrogen is mediated by ERα and involves a interplay among several cell types.
Diffraction Enhanced Computed Tomographic Imaging
Authors: Muehleman Carol , Li Jun , Zhong Zhong
Published: June 2021

Articular cartilage and its defects, characteristic of osteoarthritis (OA), are not visible through conventional radiography. DEI is a novel x‐ray technology in which the x‐ray beam transmitted through the specimen is imaged after diffraction in the vertical plane by means of a silicon crystal analyzer placed between the subject and the detector. This analyzer crystal detects the object's x‐ray scattering and refraction at the microradian level, a sensitivity beyond that of conventional radiography. At the midpoint of the x‐ray intensity curve subtle angle variations are converted into intensity variations which make refraction effects visible in DEI images. We have previously shown the efficacy of planar DEI for simultaneous bone and soft tissue imaging. Here we have tested DEI in the computed tomographic mode (DEI‐CT) for imaging intact human cadaveric knee joints at the X15 beamline of Brookhaven National Laboratory at 51 keV. Images were acquired using a 30 micron resolution detector and processed with IDL software. Examination of 30 μm refraction images demonstrate unsurpassed soft tissue and bone contrast, simultaneously, and without contrast agents. Articular cartilage along with its degenerative defects were readily visible as were the cruciate and other synovial joint ligaments. Importantly, the radiation dosage to the specimen is lower than for conventional radiography. Of note is that DEI does not innately require the use of a synchrotron – a portable DEI setup using a conventional x‐ray tube has recently been developed and is being tested.
Hox genes and the control of limb bud outgrowth
Authors: kmita marie , grégoire damien , scotti martina
Published: June 2021

Gain‐ and loss‐of‐function experiments have demonstrated that the establishment of the limb architecture relies upon the function of HoxA and HoxD genes. Moreover, we have shown that the complete inactivation of these two gene clusters results in severe limb truncation. During limb development, there are two phases of Hox expression: At early limb bud stages, Hox genes are sequentially activated in time, with expression of ‘late’ genes (from group 10 to 13) being progressively restricted to the posterior mesenchyme. At later developmental stages, transcription is maintained for only a subset of the HoxA and HoxD genes (group 9 to 13), with a collinear nested pattern along the proximal‐distal axis. While phenotypes associated to individual gene loss‐of‐function correlates with the second wave of transcriptional activation, we found that the function of Hox genes is necessary from early limb bud stages onwards. For instance, by removing both HoxA and HoxD gene clusters, we have uncovered a requirement for these transcription factors in Shh activation and maintenance. We have further analyzed HoxA/HoxD conditional mutants and found that decreasing the Hox dosage within developing limb buds impairs cell survival. Our results suggest that, in addition to patterning functions, Hox genes are needed for the proper outgrowth of developing limbs.
Role for the Werner syndrome protein (WRN) in replication fork regression
Authors: Orren David K , Machwe Amrita
Published: June 2021

Loss of function of human RecQ family member WRN causes the premature aging disease Werner syndrome. WRN has weak helicase and 3′ to 5′ exonuclease activities that preferentially act on complex DNA structures. Like many RecQ helicases, WRN also helps anneal complementary DNA strands. Combined unwinding and annealing likely promotes branch migration and strand exchange by WRN. In general, RecQ members act to suppress illegitimate recombination events. Specifically, WRN‐deficient cells show chromosomal instability, hypersensitivity to hydroxyurea and certain genotoxins, and replication abnormalities including asymmetric fork progression, suggesting a role for WRN in overcoming replication blocks. Resolution of such blockage may be initiated by fork regression–i.e., nascent daughter strand pairing and parental strand re‐annealing to form Holliday junctions. Since regression involves coordinated unwinding and annealing, we examined WRN's action on several model replication forks. We find that WRN readily regresses replication forks to form Holliday junctions. Further, WRN's exonuclease activity enhances regression of some fork substrates by digesting the leading daughter strand. Thus, the multiple enzymatic activities of WRN cooperate to catalyze efficient fork regression, theoretically as part of a pathway that accurately overcomes replication blocks. Supported by NCI grant R01‐CA113371.
Fluorescent biosensors for cell pathways
Authors: Waggoner Alan
Published: June 2021

We are developing a toolkit of fluorescent probes for quantifying the protein regulation pathways of living cells at high resolution. This biosensor development program blends genetics, protein structure, nucleic acid structure and fluorescent dye chemistry. Our initial biosensor platform consists of two well characterized single chain antibody classes that bind fluorogen dyes with high affinity and large fluorescence increases. One antibody binds the green‐fluorescing TO1 fluorogen and the other binds Malachite Green to give red fluorescence. We are using this platform to create sensors for protein‐protein interactions, protease activity and membrane surface labeling. Imaging and informatics technologies are integrated with the new fluorescent probe technologies so that these combined tools to obtain spatial and temporal information about pathways in living cells.
The effects of high‐fat feeding on the hepatic transcriptome of human apoE2 transgenic mice
Authors: Jun Hee‐jin , Dawson Kevin , Rodriguez Raymond L. , Lee Sung‐joon
Published: June 2021

Human apolipoprotein E2(ε 2/ε 2) (apoE2) leads type III hyperlipidemia (HLP) by an impaired clearance of cholesterol‐enriched remnant lipoproteins. The HLP is significantly influenced by diet, and thus high‐fat diet exacerbates it. However, the consequences of high‐fat feeding on the gene expression profile is likely to be complex, and therefore we performed genomic analysis of the response of apoE2 transgenic mouse to high‐fat feeding using two‐color microarray. The mRNA from normal and high‐fat diet‐fed apoE2 mice livers was labeled with Cy3 and Cy5, and then hybridized onto 40K‐oligonucleotied arrays. The 5,062 and 1,434 genes were significantly up‐ and down‐regulated by high‐fat feeding (Significant Microarray Analysis, false discovery rate=0%). We found that high‐fat feeding suppressed regulation of transcription activity and induced chromatin remodeling by histone modification and histone deacetylase superfamily. The expression of cytochrome P450 (CYP) enzymes and glutathione metabolism were suppressed by high‐fat feeding, which induces inhibition of detoxification of chemicals present in food and the environment. Especially, expression of CYP3A which plays an important role in drug metabolism was significantly down‐regulated. In addition, interestingly, transcriptional regulation of telomerase RNA component gene was induced by high‐fat feeding, which may induce aging in HLP state.
Folate status response to chronic folic acid doses in large‐scale population‐based randomized intervention trial in young Chinese women
Authors: Hao Ling , Yang Quan‐He , Li Zhu , Bailey Lynn B , Zhu Jiang‐Hui , Hu Dale J , Zhang Bao‐Lan , Erickson J D , Zhang Le , Gindler J , Li Song , Berry R J
Published: June 2021

The study objective was to evaluate changes in folate and Hcy in response to different daily or weekly dosages of folic acid. Women (N=1108) were randomly assigned to 1 of 6 folic acid intervention groups for 6 mo (100 μg once daily, 25 μg 4 X daily, 400 μg once daily, 100 μg 4 X daily, 4,000 μg once daily, or 4,000 μg once weekly. Plasma folate, Hcy, and RBC folate were measured at 0, 1, 3, and 6 mo, and 3 mo post‐supplementation. Plasma folate plateaued at 3 mo with 108% (95% CI 97.7–120), 259% (95% CI 240–279), 460% (95% CI 417–503), and 142% (95% CI 123–162) observed increases for 100 μg/d, 400 μg/d, 4,000 μg/d and 4,000 μg/wk folic acid groups, respectively. Hcy increased (p<0.001) at 1 mo in response to the lowest folic acid doses (100 μg/d and 4000 μg/wk). The rate of reduction in blood folate during the 3 mo post‐supplementation acid was dose‐dependent, with a higher folic acid intake associated with a faster reduction. Changes in folate and homocysteine were unaffected by different dosing schedules of the same amount of daily folic acid. After cessation of folic acid, the decrease in blood folate was rapid and dose dependent. These findings are important to evaluate optimal folic acid intervention strategies in northern China. This study was supported by a cooperative agreement from CDC.
Retroconversion of docosahexaenoic acid (22:6) to eicosapentaenoic acid (20:5) is undetectable in serum of young women
Authors: Brenna J. Thomas , Zhang Ying , Vandal Milène , Cunnane Stephen C.
Published: June 2021

Background

. The polyunsaturated fatty acid (PUFA) 22:6 is widely accepted to be synthesized from 20:5 via these steps: 20:5 → 22:5 → 24:5 → 24:6 → 22:6. Many papers report apparent retroconversion of 22:6 to 20:5, presumed to be by reverse of this pathway, but this hypothesis has not been directly tested in human adults.

Methods

. Four young adult women were given a single oral dose of [U‐13C]‐22:6, and serum was collected several times on day 1 and weekly until 28 days post‐dose. FAME were prepared and analyzed by high precision GC‐combustion isotope ratio mass spectrometry, and data are expressed as nmol 13C‐FA per ml serum.

Results

. On average, 22:6* peaked at 24 h and dropped smoothly until day 28, when it remained well above baseline. In contrast, no other n‐3 PUFA showed an increase in labeling of more than a few percent above baseline, and there were no trends attributable to retroconversion. 16:0* peaked at 11% of the 22:6* peak on day 1, dropped to baseline, then gradually increased to a plateau at 4 days, while 18:0* peaked on day 2 at about 7% of the 22:6* peak. These changes were driven primarily by one subject.

Conclusions

. Increases in serum 20:5 often ascribed to increasing dietary 22:6 may not be due to retroconversion but to other mechanisms such as sparing of 20:5 oxidation. Alternatively, retroconversion may be driven by much higher levels of dietary 22:6. Funding: Canadian Institutes of Health Research.
Obesity‐associated mouse adipose stem cell secretion of monocyte chemotactic protein‐1 (MCP‐1)
Authors: Claycombe Kate J , Zhou Hui Ren , Kim Eun‐Kyoung
Published: June 2021

Studies showed that MCP‐1 concentrations are increased in obesity. In our current study, we demonstrate that plasma MCP‐1 level in leptin deficient ob/ob mice is significantly higher than in lean mice. Furthermore, we determined that basal adipose tissue MCP‐1 mRNA levels are significantly higher in the ob/ob mice compared to lean mice. In order to determine the mechanisms underlying obesity‐associated increases in plasma and adipose tissue MCP‐1 levels, we determined adipose tissue cell type sources of MCP‐1 production. Our data show that adipose tissue stem cells (CD34+), macrophages (F4/80+) and stromal vascular fraction (SVF) cells express significantly higher levels of MCP‐1 compared to adipocytes under both basal and lipopolysaccharide (LPS)‐stimulated conditions. Furthermore, basal and LPS‐induced MCP‐1 secretion levels were the same for both adipose F4/80+ and CD34+ cells while adipose CD34+ cells have two‐fold higher cell numbers (30% of total SVF cells) compared to F4/80+ macrophages (15%). Our data also show that CD34+ cells from visceral adipose tissue depots secrete significantly higher levels of MCP‐1 ex vivo when compared to CD34+ cells from subcutaneous adipose tissue depots. Taken together, our data suggest that adipose CD34+ stem cell may play an important role in obesity‐associated increases in plasma MCP‐1 levels.
“Causal” effects of dieting on obesity: what is the impact of adjustment for time dependent confounding by prior weight?
Authors: Boone Janne E , Gordon‐Larsen Penny , Field Alison E , Adair Linda S
Published: June 2021

Prior analyses show associations between dieting behaviors and weight gain, but confounding by prior body weight is possible. In longitudinal analysis, body weight may be a time dependent confounder: a confounder (associated with current dieting and obesity) and a mediator (in the causal pathway between prior and current dieting). Unlike traditional multivariate adjustment (MVA), marginal structural models (MSM) can control for time dependent confounding. To illustrate, we compared longitudinal effects of self‐reported dieting on incident obesity (BMI=30 kg/m2 or BMI=95th percentile CDC/NCHS; self‐reported height and weight) estimated by MSM versus MVA using National Longitudinal Study of Adolescent Health data (Wave I: 1995, ages 11–21; II: 1996; III: 2001; total N=7407), adjusting for sociodemographics and physical activities and excluding underweight and obese adolescents. MVA and MSM estimates of dieting at Waves I and II on incident Wave III obesity were positive, with stronger associations estimated by MSM [OR (95% CI): Wave I: 3.54 (2.07, 6.07); Wave II: 1.90 (1.19, 3.03)] than MVA [OR (95% CI): Wave I: 1.49 (1.10, 2.04); Wave II: 1.05 (0.84, 1.32)]. These results show that the effect of dieting on obesity is sensitive to the method used to adjust for prior body weight, and suggests that weight may be a time dependent confounder that requires appropriate analytical methods. Funding: R01‐041375
Unexplained decline in the prevalence of anemia among US children and women between 1988–1994 and 1999–2002
Authors: Cusick Sarah E , Mei Zuguo , Freedman David S , Looker Anne C , Ogden Cynthia L , Gunter Elaine , Cogswell Mary E
Published: June 2021

We used data from the 1988–1994 and 1999–2002 National Health and Nutrition Examination Surveys to examine recent anemia changes in US children and women. We calculated the prevalence of anemia [hemoglobin <11.0 g/dL (<24 m), <11.1 g/dL (24–59 m), <12.0 g/dL (women)], iron deficiency anemia (anemia + abnormal value ≥2: serum ferritin, transferrin saturation, erythrocyte protoporphyrin) and high blood lead (≥10 μg/dL) and anemia in children 12–59 m and women 20–49 y. In women we also calculated the prevalence of folate deficiency (erythrocyte folate <317.2 nmol/L) and anemia and high C‐reactive protein (>10 mg/L) and anemia. Multiple logistic regression was used to compare total and type‐specific anemia between surveys, controlling for race, age and sex. Anemia declined significantly in children (from 8.0% to 3.6%, OR=0.4; 95% CI: 0.3, 0.7) and women (10.8% to 6.9%, 0.6; 0.4, 0.7). The prevalence of iron deficiency anemia did not change significantly in children (1.5% vs. 1.2%, 0.7; 0.4, 1.5) or women (4.9% vs. 4.1%, 0.8; 0.6, 1.1). Folate deficiency and anemia declined significantly in women (4.1% to 0.5%, 0.1; 0.1, 0.2), but none of the types of anemia explained the decline in total anemia when included in multivariate models. Anemia declined significantly in US women and children between 1988–1994 and 1999–2002, but the decline was not associated with changes in iron or folate deficiency, inflammation or blood lead.
Biotransformation of soy isoflavones and enhanced cholesterol lowering effect with an oligofructose‐enriched inulin in equol producers
Authors: Wong Julia MW , Kendall Cyril WC , de Souza Russell , Liu Zhen , Emam Azadeh , Jenkins David JA
Published: June 2021

Objective: To assess whether the hypocholesterolemic effect of soy can be enhanced with an oligofructose‐enriched inulin (OF‐IN) by promoting the biotransformation of colonic soy isoflavones (i.e. equol production). Method: 23 hyperlipidemic adults (11M, 12F; 58±7y; LDL‐C, 4. 1±0.7mmol/L) completed three 4‐week phases: OF‐IN (10 g/d) and low‐fat dairy (control), and soy foods (isoflavones, 61 mg/d) with or without OF‐IN (10 g/d). Fasting blood lipids and anthropometrics were measured biweekly. Urine (24h) was collected at baseline and at the end of each phase. Results: A stepwise reduction in TChol:HDL‐C was observed for OF‐IN (0.25±0.13, P=0.08), soy (–0.07±0.08, P=0.41) and soy plus OF‐IN (–0.36±0.14, P=0.02), compared to baseline. Preliminary analysis suggested that this effect was seen most clearly in equol producers (n=7). TChol:HDL‐C for equol producers was 0.09±0.16 on OF‐IN, –0.23±0.14 on soy, and –0.72±0.42 on soy plus OF‐IN, compared to baseline. For non‐equol producers, TChol:HDL‐C was 0.41±0.17, 0.04±0.09, and –0.17±0.08, respectively. Conclusion: Intake of soy in conjunction with OF‐IN resulted in greater improvements in the lipid profile than their intake alone, suggesting a synergistic effect. This association was more prominent in equol producers possibly related to increased biotransformation of soy isoflavones.

Funding: Heart and Stroke Foundation of Ontario, ORAFTI Group
Leucine and Calcitriol Modulation of Mitochondrial Biogenesis in Muscle Cells and Adipocytes
Authors: Zemel Michael B. , Sun Xiaocun
Published: June 2021

The anti‐obesity effect of dairy is mediated, in part, by leucine which exerts significant effects on both adipocyte and skeletal muscle energy metabolism, and by dietary calcium suppression of calcitriol. We recently demonstrated that leucine and calcitriol participate in the regulation of fatty acid oxidation (FO) in skeletal muscle cells in vitro, as leucine promotes FO while calcitriol exhibits the opposite effect. Moreover, leucine coordinately regulated adipocyte lipid metabolism to promote flux of lipid to skeletal muscle and regulate metabolic flexibility. We have now investigated the role of mitochondrial biogenesis in mediating these effects. Leucine (0.5 mM) increased mitochondrial number by 30% and 53% in C2C12 myocytes and 3T3‐L1 adipocytes, respectively, while calcitriol (10 nM) decreased mitochondrial number by 37% and 27% (p<0.02). Leucine stimulated PGC‐1α, NRF‐1 and UCP3 expression by 3–5 fold in C2C12 myocytes (p<0.003) while calcitriol reduced their expression by 11%, 22% and 14% respectively. Adipocyte‐conditioned medium reduced mitochondrial number by 81% in C2C12 myocytes (p<0.001) and decreased UCP3 expression by ~29% but increased PGC‐α1 by 50%, suggesting feedback stimulation of mitochondrial biogenesis. These data suggest that leucine and calcitriol may regulate muscle and adipocyte energy metabolism, in part, by modulating mitochondrial biogenesis.
Cytochrome P450s CYP26A1, CYP26B1 and CYP2c22/70, LRAT and RARß gene expression are regulated nutritionally by vitamin A and induced concomitantly by retinoic acid (RA) during first pass metabolism in rat liver
Authors: Ross A. Catharine , Cifelli Christopher J. , Li Nan‐qian , Zolfaghari Reza , Qian Linxi
Published: June 2021

All‐trans‐RA, a principal metabolite of VA, is a ligand for RAR nuclear receptors. Several genes involved in retinoid homeostasis are also regulated by RA, and this autoregulation is thought to function as a sensing mechanism to control the formation and/or catabolism of RA itself, as well as downstream RA‐regulated genes involved in cell differentiation, development, and metabolism. The objective was to evaluate, over a wide range of VA status, the basal expression levels of several genes that are potentially involved in the control of RA metabolism: CYPs26A1, 26B1, 2C22/70, lecithin:retinol acyltransferase (LRAT), which esterifies retinol and has been shown to be physiologically regulated by RA in liver, and RARß, a gene known to be autoregulated directly by RA. RBP4 and GAPDH were measured as controls. In dietary studies, CYP26A1 exhibited the most graded expression and widest dynamic range, up to >20 times higher in VA‐supplemented vs. adequate rats. On each diet, CYP2c22/70 mRNA greatly exceeded either CYP26A1 or CYP26B1. In response to RA treatment under first pass conditions (20 μg, i.v.), mRNAs for CYP26A1, CYP26B1, and CYP2c22/70, whose activities may be redundant, as well as LRAT and RARß, all significantly increased very rapidly (generally by 30 min), suggesting that physiologically they are all early response genes, even though they may be regulated by different molecular mechanisms. (NIH CA‐90214)
Signaling Downstream of Mammalian Target of Rapamycin Complex 1 (mTORC1) is Derepressed Following Treatment with L‐Asparaginase in the Liver of GCN2 Knockout Mice
Authors: Bunpo Piyawan , Dudley Allison , Cundiff Judy K , Anthony Tracy G
Published: June 2021

Asparaginase reduces liver protein synthesis by increasing the phosphorylation of eukaryotic initiation factor 2 (p‐eIF2) and inhibiting signaling downstream of mTORC1. The objective of this study was to assess the relationship between the eIF2 kinase, GCN2, and mTOR in the hepatic response to the anti‐leukemic drug, asparaginase. GCN2+/+ and GCN2−/− mice (n=6 per group) were administered PBS or asparaginase (3 IU g/kg Elspar) 30 min after injection with either rapamycin (2 mg/kg) or PBS. Mice were killed and livers collected 8h later. Asparaginase, alone or in combination with rapamycin, significantly increased p‐eIF2 in the liver of GCN2+/+ but not GCN2−/− mice, whereas rapamycin alone reduced p‐eIF2 in GCN2+/+ mice only. In the livers of GCN2+/+ mice, the phosphorylation states of two markers of mTORC1 activity, namely p‐4E‐BP1 and p‐S6K1, were slightly reduced by asparaginase, moderately reduced by rapamycin, and maximally inhibited by the drug combination. In the livers of GCN2−/− mice, both p‐S6K1 and p‐4E‐BP1 were significantly reduced by rapamycin. On the other hand, p‐S6K1 and p‐4E‐BP1 were derepressed following asparaginase treatment, with values similar to PBS‐injected controls. These results demonstrate that asparaginase inhibits mTORC1 signaling in liver by a GCN2‐dependent and rapamycin‐independent pathway. Funded by AICR and IUSM
Program/Abstract # 307.1
Authors: Kung'u Jacqueline K , Juma Hamad J , Goodman David , Wright Victoria , Tielsch James M , Bickle Quentin D , Stoltzfus Rebecca J
Published: June 2021

Helminths aggravate anemia and malnutrition among school children, but their relation to malnutrition in children under 2 years has not been studied. We investigated whether early helminth infections were associated with anemia and malnutrition in a cross‐sectional study of 6‐23 mo old Zanzibari children (n=2124). Ascaris, Hookworms, and Trichuris infections were diagnosed by examination of 3 stool samples using kato‐katz and sedimentation methods to maximize detection of light infections. Recent fever, malaria, mid upper arm circumference (MUAC) and hemoglobin concentration (Hb) were also assessed. The prevalence of infection was 9.8% Ascaris, 3.7% Hookworms, 16.8% Trichuris and 23.6% any helminth. Contrary to expectation, infected children had higher Hb (Î² =4.7g/L, p<0.001) and MUAC age Z score (Î² =0.22 Z score, p<0.001) after adjusting for sex, age, malaria and fever. Helminth infected children were also less likely to be infected with malaria [(adjusted odds ratios 0.64(95%CI: 0.50, 0.82)] but this association did not explain the Hb or MUAC differences. In conclusion, we put forward two hypotheses. Firstly, anemic, malnourished or malaria‐infected children may be less motorically advanced or exploratory of their environments and therefore limit their exposure to soil‐transmitted helminths; or secondly, that helminths may find it more favorable to establish infection in a better‐nourished child.
Synergistic Combinations of Active Dietary Components for the Prevention of Pancreatic Cancer
Authors: Zhou Jin‐Rong , Li Linglin , Blackburn George
Published: June 2021

Pancreatic cancer (PANC) is the 4th leading cause of cancer death among men and women in the United States. PANC is often dubbed the silent killer because it seldom causes symptoms until advanced. As a result, the morbidity rate equals the mortality. It is now clear that the effective control of this deadly disease is by prevention. The role of diet and nutrition in the prevention of PANC has not been adequately studied. Diets contain a diverse array of active components that have variety types of biological activities, and the potent cancer prevention activity may be due to the interaction between active components in a synergistic or an additive manner. The objective of this research was to identify the synergistic combination regimen of dietary active components for PANC prevention. We applied cell‐based bioassays to compare the relative potencies of a panel of dietary components that are commonly present in fruits and vegetables to identify the potent components for growth inhibition of PANC cell lines in vitro. We further determined the combined effects of candidate components to identify potential synergistic combinations. Our studies showed that the combination of epigallocatechin gallate and myricetin had a synergistic effect on PANC cell growth via combined modulation of cell cycle progression and apoptosis. The identified combination regimen will be further verified in clinically relevant animal models.
Regulation of Estrogen Receptor Alpha Expression in Breast Cancer Cells by Sulforaphane
Authors: Ramirez Marcela Cortes , Singletary Keith W
Published: June 2021

Sulforaphane (SUL, 1‐isothiocyanato‐4‐(methylsulfinyl) butane) is an isothiocyanate derived from glucoraphanin that is abundant in broccoli and broccoli sprouts and which has a variety of potential chemopreventive actions. SUL is capable of inhibiting cancer at both early (initiation) and late (promotion) stages of experimental tumorigenesis. In this study we analyzed the effects of SUL on proliferation of several human breast cancer cell lines and evaluated the effect of SUL on estrogen receptor alpha (ERα) protein and mRNA expression in MCF‐7 cells. SUL at doses between 10 and 30 μM inhibited proliferation of neoplastic breast cell lines to a greater extent than non‐neoplastic MCF‐10F breast cells. SUL at doses between 2.5 and 30 μM decreased ERα protein expression in MCF‐7 cells. This effect of SUL on ERα expression was apparent under complete and estrogen‐depleted growth conditions and was accompanied by a decrease in progesterone receptor protein expression. At SUL doses of 30μM and greater, SUL treatment was associated with suppression of ERα mRNA transcription. In contrast, at doses of SUL less than 30μM, there was no effect of SUL on ERα mRNA expression. However, the proteasome inhibitor MG132 reversed the suppression of ERα protein levels in MCF‐7 cells treated with SUL at the lower SUL doses. Thus, the downregulation of ERα protein expression appears to be due to multiple dose‐dependent mechanisms involving changes in mRNA expression and proteasome activity. The effect of SUL on hormone‐responsiveness of human breast cancer cells warrants further study.
Dietary protein intake is an independent and dose‐dependent regulator of hepatic anabolism in rats fed high protein diets
Authors: Chevalier Laure , Bos Cécile , Azzout‐Marniche Dalila , Fromentin Gilles , Tomé Daniel , Gaudichon Claire
Published: June 2021

We previously observed that drastic increases in protein consumption greatly modify hepatic protein anabolism in rats, yet the confounding effects of the other macronutrient changes and the ability of a moderate protein increase to produce the same effects are not established. This study examined the metabolic and hormonal responses of rats submitted to 14‐d isoenergetic diets with normal, intermediary or high protein levels (NP:14% of energy, IP: 33%, HP: 50%) and different carbohydrate (CHO) to fat ratios within each protein level. Fasted or fed rats (n=96) were killed after injection of a flooding dose of 13C‐valine. Hepatic protein content increased with the dietary protein level (P<0.05). The hepatic fractional synthesis rates (FSR) of protein were significantly influenced by both the protein level and the nutritional state (fasted vs. fed) (P<0.0001) but not by the CHO/fat ratio and reached on average 110%/d, 92%/d and 83%/d in rats fed the NP, IP and HP diets, respectively. Muscle FSR did not differ among diets. Insulin levels significantly decreased with both increasing protein level and decreasing CHO/fat ratio. Our results reveal that excess dietary protein inhibits hepatic protein synthesis rates independently of the other macronutrients and the related changes in insulin levels. This response is observed at moderate levels of protein intake (33%) that are plausible in human consumption.
Impacts of dietary supplemental phytase and inulin on bone mechanical and chemical properties of young pigs
Authors: Faber Catherine Ann , Roneker Karl R. , Lei Xin Gen
Published: June 2021

Enhancing peak bone mass and integrity early in life may reduce or delay osteoporosis. We explored benefits of supplementing phytase and inulin in a P‐adequate, corn‐soybean meal basal diet (BD) on bone development in young pigs. The present study was conducted with 35 weanling pigs (8.5 ± 1.6 kg) to compare the effects of supplementing a bacterial phytase at 3,500 units/kg (OptiPhos, JBS United, Sheridan, IN) and inulin at 4% (Synergy‐1, Orafti, Tienen, Belgium) on bone mechanical and chemical properties. Pigs were fed BD, BD + phytase, BD + inulin, or BD + phytase + inulin for 5 wk. Five pigs were euthanized from each diet group to collect the femur. Supplemental inulin exerted marginal (P = 0.06 to 0.08) effects on femur height, modulus elasticity, and cancellous bone ash concentration. Cortical bone concentrations (ash basis) of Mg, Na, Sr, and Zn were altered (P < 0.05) by both supplements, whereas the concentration of S was affected only by phytase and those of Ca and P were affected only by inulin. In cancellous bone, phytase enhanced (P < 0.05) concentrations of Sr and Zn while inulin enhanced (P < 0.05) concentrations of Ca and Mg. The two supplements affected (P < 0.05) S concentration and displayed an interaction (P < 0.05) on Mg and P concentrations in cancellous bone. In conclusion, a short‐term supplementation of phytase and inulin to the plant‐based diet was able to alter the mineral proof of femur bone ash in young pigs.
Whole mushrooms inhibit in vitro monocyte binding to human aortic endothelial cells
Authors: Martin Keith R
Published: June 2021

Cardiovascular disease (CVD) is a leading cause of mortality in the US. Studies show that fruit and vegetable consumption reduces CVD risk, in part, due to antioxidant activity and immunomodulation since oxidative stress and inflammation are features of atherogenesis. We focused here on commonly available dietary mushrooms since ~59% of the annual 5 million metric tons of cultivated edible mushrooms are estimated to contain functional neutraceutical or medicinal properties. In this study, human aortic endothelial cells (HAEC) were incubated overnight (20 h) with control media alone or with whole dehydrated mushrooms resuspended in DMSO as a vehicle (0.1 mg/mL). Test mushrooms included Agaricus bisporus (white button and crimini), Lentinula edodes (shiitake), Pleurotus osteratus (Oyster), and Grifola frondosa (maitake). After 20 h, monolayers were washed and incubated with medium alone or with the pro‐inflammatory cytokine IL‐1 beta (5 ng/mL) for 6 h. Monolayers were assayed for adhesion molecule expression and binding of pre‐labeled human monocytes. White button mushrooms consistently reduced (p < 0.05) VCAM‐1, ICAM‐1, and E‐selectin expression; other mushrooms also reduced expression, but sporadically. All mushrooms significantly reduced monocyte binding to both quiescent and cytokine‐stimulated monolayers providing evidence that dietary mushrooms can protect against cardiovascular disease.
Role of macrophage scavenger receptor MARCO in lung innate defenses against influenza
Authors: Ghosh Sanjukta , Kobzik Lester
Published: June 2021

The class A scavenger receptor MARCO is expressed by lung macrophages and mediates clearance of inhaled bacteria and particulate matter. We sought to investigate whether MARCO contributes to early innate defenses against another type of inhaled pathogen, the influenza virus. We compared the inflammatory response of normal or MARCO‐deficient mice following intranasal infection with H1N1 strain of influenza A virus A/PR8/34 (200pfu). At 24 and 48 hours after infection MARCO−/− mice showed 5–8 fold more neutrophils in the bronchoalveolar fluid (BALF) compared to WT controls. Concomitant with the increase in neutrophils, there was a >4‐fold increase in mRNA expression of the potent neutrophil chemoattractant molecule, CXCL1, in the MARCO−/− BALF cells. Analysis of gene expression in BALF cells 48 hours after infection revealed greater expression in MARCO −/− mice of several chemokines and cytokines including interferon‐gamma. Infected MARCO−/− mice also exhibited greater loss of body weight indicative of greater morbidity. These findings suggest that lung macrophages contribute to clearance of virus in the early phase of infection, and that the scavenger receptor MARCO mediates uptake of virus by alveolar macrophages, thereby reducing early injury and acute inflammation. NIEHS 11008, 00002.
Modulation of host apoptotic signaling by the Salmonella effector protein AvrA
Authors: Jones Rheinallt , Wu Huixia , Luo Liping , Neish Andrew
Published: June 2021

Certain enteric pathogens stably establish themselves within host cells because they have evolved mechanisms to evade elimination. For example, the intracellular pathogen Salmonella secretes the AvrA protein which has potent suppressive effects on innate immune and apoptotic pathways. To determine the mechanism by which AvrA mediates these effects, we created transgenic Drosophila harboring avrA, allowing directed expression of AvrA in Drosophila tissues. We show that AvrA has potent and specific inhibitory activity against the JNK pathway in Drosophila and in cultured mammalian cells. Intriguingly, we also found that AvrA expression in a background of constitutive JNK pathway activation resulted in increased proliferation of Drosophila tissue whereas AvrA expression in a wild type background did not. Furthermore, reporter gene assays in cultured cells showed AvrA mediated inhibition of constitutive JNK pathway activation resulted in increased ERK signaling. We also show that the mechanism by which AvrA mediates JNK pathway inhibition is via the acetylation of the activation loop threonine of MKK4 ‐ a residue necessarily phosphorylated during activation of this enzyme. Together, these studies show Salmonella has evolved a protein that inhibits the pro‐apoptotic JNK pathway, with the secondary effect of dysregulating normal tissue growth. Research funding provided by Burroughs Welcome Fund.
Platelet microparticles enhance Plasmodium falciparum‐parasitised red blood cell cytoadherence and activate human brain endothelial cells
Authors: Combes Valery , Faille Dorothee , Mitchell Andrew , Juhan‐Vague Irene , Chimini Giovanna , Fusai Thierry , Grau Georges Emile
Published: June 2021

One of the mechanisms of cerebral malaria (CM) is the sequestration, within brain microvessels, of P. falciparum‐parasitised red blood cells (RBC), leucocytes and platelets. Besides, CM is characterised by an overproduction of pro‐inflammatory cytokines and endothelial activation with upregulation of adhesion molecules and microparticle (MP) release from vascular cells. MP are submicron membrane elements carrying on their surface proteins from their cell of origin. Platelets have been shown to modulate PRBC cytoadherence, thus we analysed the role of platelet MP in this binding. Confocal microscopy showed that MP adhered to and penetrated in human brain microvascular endothelial cells (HBEC). MP upregulated ICAM‐1 and VCAM‐1 expression on HBEC. Furthermore, MP adhered to parasitised, but also to normal RBC, as revealed by detection of CD41 on RBC. MP binding to RBC was not affected by temperature or phosphatidylserine blockade by annexin V, but was significantly reduced when PRBC were incubated with MP in the presence of blocking antibodies against platelet‐specific antigens. Lastly, MP, while interacting with the two other cell types, dramatically increased the binding of parasitised but also of normal RBC to HBEC and altered endothelial functions. MP thus appear to be an important element in RBC sequestration and in endothelial pathology, suggesting a novel mechanism in CM pathogenesis.
Acculturation level and dietary intakes among Hispanic women in the EFNEP program
Authors: Winham Donna M. , Florian Traci Armstrong
Published: June 2021

A convenience sample of 168 female Hispanics in the Extension Food and Nutrition Education Program (EFNEP) completed the standard EFNEP demographic and food behavior forms, an acculturation scale, and food frequency questionnaire (FFQ) for fruit/vegetable and fat intake validated for use in Hispanics (Wakimoto et al., 2006). We hypothesized that nutrition quality would decline with acculturation. Mean age in years, body mass index, fruit and vegetable and fat FFQ scores were not significantly different by acculturation category (Spanish‐dominant, Bicultural, English‐dominant). Examination of the individual food items within the FFQ showed significantly higher consumption frequencies by the less‐acculturated women of fruit juices, vegetable soups, green salads, green peppers, plantains, tomatoes, and corn tortillas (p= 0.00). More‐acculturated women ate potatoes, hamburger, French fries, and used fat or oils more often than less‐acculturated women. Despite differences on individual food items, only 28% of the less‐acculturated and 23% of the more‐acculturated women consumed 5 or more servings of fruits and vegetables per day, and 55% in all acculturation categories exceeded the FFQ dietary fat recommendation categories. Our findings suggest that Hispanic EFNEP participants are consuming excessive amounts of dietary fats and insufficient fruits and vegetables regardless of acculturation level.
Cell behavior in three dimensions
Authors: Schwarzbauer Jean E. , Engler Adam , Hunt Geoffrey
Published: June 2021

The architecture of the extracellular matrix (ECM) dictates cell morphology, proliferation and differentiation in many tissues. However, its role in controlling stem cell behaviors remains poorly characterized. We are using natural three‐dimensional (3D) matrices to determine the regulatory effects of ECM architecture in ES cell self‐renewal and differentiation decisions. The switch from self‐renewal to differentiation is monitored with ES cells expressing GFP driven by the promoter for Nanog, a self‐renewal gene. Self‐renewing ES cells express integrin receptors and assemble a matrix rich in fibronectin, a major fibrillar ECM protein. When ES cells are cultured in suspension, they increase fibronectin production as they form embryoid bodies and reduce Nanog expression. Plating of embryoid bodies on a 3D network of fibronectin matrix fibrils induces dissociation of GFP‐negative cells from the cell aggregates. Cells migrate into the matrix and up‐regulate expression of differentiation markers. Thus, there is a correlation between increased ES cell‐ECM interactions, loss of cell‐cell contacts, and cell differentiation. These results suggest that the balance between cell‐ECM and cell‐cell interactions is a critical control point for the self‐renewal to differentiation decision and that modulation of the ECM environment can be used to shift ES cells into a differentiation mode.
Creation of long‐lasting blood vessels in vivo
Authors: Fukumura Dai , Au Patrick , Tam Joshua , Koike Naoto , Jain. Edwin L Rakesh K.
Published: June 2021

A major challenge in tissue engineering and regenerative medicine is the creation of stable vasculature. One approach in vascularizing an engineered tissue is to add the cellular components of blood vessels (endothelial and perivascular cells) directly in the tissue‐engineered construct. We have shown the formation of long‐lasting vascular networks in vivo by co‐implanting human umbilical cord vein endothelial cells (HUVECs) and 10T1/2 mesenchymal precursor cells in a three‐dimensional fibronectin‐type I collagen scaffold. Because mesenchymal precursor cells differentiate into perivascular cells and fortify blood vessels in vivo, the resulting vessels are stable and functional for one year. In contrast, the HUVEC‐alone constructs showed minimal perfusion and disappeared by two months. These findings indicate that fostering endothelial‐perivascular cell interactions in vivo offers a promising approach for engineering blood vessels. Furthermore, the tissue‐engineered blood vessel model provides an ideal platform to study cellular and molecular mechanisms of vessel formation and maturation. The potential of different sources of vascular cells in the creation of engineered vessels in vivo can also be tested with this model.

Supported in part by NIH grants (P01CA80124, R01CA96915).
Transcriptional pathways in heart development
Authors: Black Brian L , Kang Jione , Harris Ian S , Schachterle William
Published: June 2021

The second heart field (SHF) is a cardiac progenitor population that resides in the splanchnic and pharyngeal mesoderm and is progressively added to the linear heart tube at the time of cardiac looping to gives rise to the outflow tract (OFT), right ventricle (RV), ventricular septum, and portions of the left ventricle. Although much has been learned regarding the contributions of the SHF to the developing heart, several crucial questions remain regarding the molecular pathways that control SHF development. The LIM‐homeodomain transcription factor ISL1 is required for SHF development and sits at or near the top of a transcriptional cascade for the development of this progenitor population. Likewise, the MADS domain transcription factor MEF2C is required for RV and OFT development. We identified Mef2c as a direct transcriptional target of ISL1 via a novel SHF‐specific enhancer, establishing a direct link between these two essential factors.

We have recently identified a novel mesoderm‐specific enhancer from the Isl1 gene, which we are analyzing to define early steps in SHF specification. This enhancer directs expression to the SHF from early cardiac crescent stage through pharyngeal mesoderm development. We are defining the pathways upstream of this novel Isl1 enhancer, and we are continuing our analyses of the Mef2c SHF enhancer with the goal of defining the mechanism through which ISL1 results in sustained activation in the RV and OFT after Isl1 expression itself is extinguished. These data and a SHF transcriptional network involving ISL1, GATA4, Nkx2‐5, MEF2C and other factors will be presented.