bluuebunny/crossref_metadata_2025 huggingface dataset:
Variations in Dietary Copper Alter Physiological and Behavioral Function
Authors: Bousquet‐Moore Danielle , Eipper Betty , Prohaska Joseph , Mains Richard
Published: June 2021

Post‐translational α‐amidation by peptidyglycine α‐amidating monooxygenase (PAM), a Cu‐dependent enzyme, is required for the synthesis of many neuropeptides. Homozygous deletion of PAM is lethal and mice heterozygous for PAM (PAM+/−) exhibit decreased peptide amidation, increased anxiety‐like behavior, decreased cold‐induced vasoconstriction and increased sensitivity to pentylenetetrazole (PTZ)‐induced seizures. Dietary and genetic limitations to Cu availability result in phenotypes similar to those observed in PAM+/− mice. We tested the hypotheses that increased dietary Cu could ameliorate deficits identified in PAM+/− mice and decreased dietary Cu could cause similar deficits in wildtype (WT) mice. PAM+/− and WT mice were supplemented with Cu (14d; 70 ppm Cu in drinking water), fed a Cu deficient diet (9 wk; Harlan Teklad 80388) or controls. As assessed in the elevated zero maze, anxiety‐like behavior was sensitive to Cu supplementation in PAM+/− mice and to Cu depletion in WT mice. Laser Doppler monitoring of tail blood flow in cold‐exposed mice revealed the ability of Cu supplementation to restore vasoconstriction in PAM+/‐ mice and Cu depletion to impair vasoconstriction in WT mice. Cu deficiency increased seizure sensitivity of WT mice (PTZ; 30 mg/kg ip). Serum levels of PAM activity rose in Cu deficiency and fell following Cu supplementation. Cu availability may affect similar parameters in man.

Grant Funding Source

National Research Service Award from the National Institute of Mental Health
CCR5 signaling suppresses inflammation and attenuates adverse remodeling in healing myocardial infarcts.
Authors: Dobaczewski Marcin , Bujak Marcin , Gonzalez‐Quesada Carlos , Zymek Pawel , Frangogiannis Nikolaos
Published: June 2021

Chemokine‐mediated interactions may regulate chemotactic recruitment of leukocyte subsets and modulate cellular responses in healing infarcts. We hypothesized that CCR5, a receptor for the CC chemokines MIP‐1α, MIP‐1β and RANTES may play a crucial role in the post‐infarction inflammatory response. Morphometric, molecular and echocardiographic endpoints were compared in WT and CCR5 ‐/‐ mice undergoing reperfused infarction protocols. MIP‐1α, MIP‐1β and CCR5 were markedly induced in WT infarcts; however, RANTES mRNA was not upregulated. Neutrophil and macrophage density was comparable in CCR5 ‐/‐ and WT infarcts. However, in the absence of CCR5, expression of the pro‐inflammatory cytokines TNF‐α, IL‐1β and IL‐6 and the chemokines MIP‐1α and MIP‐1β was significantly increased after 6‐24h of reperfusion. CCR5 ‐/‐ hearts exhibited enhanced ventricular dilation following infarction associated with increased levels of MMP‐8 and MMP‐9 and reduced TIMP‐1 expression. The surprising accentuation of the inflammatory response in CCR5 ‐/‐ infarcts was associated with reduced foxp3 mRNA levels suggesting impaired recruitment of regulatory T cells (Tregs). The anti‐inflammatory effects of CCR5 in healing infarcts may be mediated through selective recruitment of leukocyte subsets with suppressive properties (R01 HL‐76246 and HL‐85440).
Latent Cytomegalovirus (CMV) Infection Exacerbates Severity of Dextran Sodium Sulfate (DSS) Colitis
Authors: Onyeagocha Chukwuma , Hossain Mohammad S , Roback John , Gewirtz Andrew
Published: June 2021

CMV infection positively correlates with severity of inflammatory bowel disease (IBD). This may result from CMV triggering and/or exacerbating flares of IBD and/or reflect that IBD, or immunosuppressive drugs used in IBD, increases susceptibility to CMV infection. Thus, we sought to 1) Determine whether infection with CMV modulated gut inflammation in colitis‐prone mice and 2) Define if underlying CMV infection affected the colitis triggered by the colitogen DSS. CMV (1 x 105PFU/ml/mouse) was administered i.p. to C57BL/6 mice (WT or IL‐10−/−). Some mice were later (28 d) placed on water containing 2.75% DSS. In accordance with previous studies, by 3 d, CMV administration resulted in an acute infection in which the virus was readily detected in numerous organs including the intestine and then, by 30 d, waned to a "latent" infection (PCR‐detectable in liver and salivary gland). Such CMV infection, in the acute or latent stage, did not induce colitis in WT mice nor alter severity of colitis in IL‐10−/− mice. However, underlying CMV infection, in an acute or latent state, increased intestinal cytokine production (KC and IL‐6) and markedly potentiated the severity of colitis induced by DSS. DSS did not induce CMV "reactivation". Thus, in conclusion, underlying CMV infection, in an active or latent state, potentiates gut innate immunity and, consequently, may increase the tendency of CMV‐infected hosts to develop colitis.
Scarring of dermal wounds in the absence of CXCR3 involves both the dermal and epidermal compartments
Authors: Yates Cecelia Chrsitina , Krisha Priya , Whaley Diana , Bodnar Richard , Turner Timothy , Wells Alan
Published: June 2021

Healing of excisional wounds requires coordinated remodeling of two tissue compartments, the ectodermally‐derived epithelial epidermis and the mesodermally‐derived mesenchymal dermis. The cellular effects and timely expression of CXCR3 and its ligands IP‐9 and IP‐10 highlights this signaling system as key to communication between the compartments to end the remodeling phase as persistence of this phase leads to scar formation. In this study we determined the long‐term consequences of failure to activate this receptor and its ligands. The absence of CXCR3 in mice resulted in a visibly hypertrophic scar that was hyalinized, thickened and multidirectional growth of collagen bundles resembling scars of fibroproliferative disease of the skin in humans. Histological assessments confirmed the abnormal healing patterns and revealed a persistent chronic inflammatory response. The CXCR3‐/‐ mice resulted in 50% increase in the content of collagen, lower tensile/burst strength, and a decrease in collagen alignment. Additionally, significantly higher levels of proliferation and apoptosis were observed. Transwells co‐cultures of keratinocytes and fibroblast revealed abnormalities in epidermal‐dermal crosstalk of CXCR3‐/‐ mice. These vivo studies suggest that CXCR3 signaling system acts in a paracrine fashion to modulate epidermal‐dermal maturation with its absence resulting in hypertrophic scarring.
Fifty years of Protein Structure: From Myoglobin to the Innate Immune System
Authors: Davies David R.
Published: June 2021

Protein Crystallography started in Cambridge at the MRC Lab with Max Perutz in the 1930's. The first success was the 2.0 Åstructure determination of Myoglobin under John Kendrew in 1959. Now there are more than 50,000 protein structures deposited in the PDB and structure has become an integral part of understanding the mechanisms of action of biological systems. I shall describe the work done in my Lab on the structure and function of the Toll‐Like Receptors, an essential component of the Innate Immune System. In particular, I shall describe the structures of mouse and human TLR3 which recognizes double stranded RNA and activates an antiviral defense. Also the structure and mechanism of signaling by TLR3 complexed with double stranded RNA.
Mechanosensitive control of VEGFR2 transcription and angiogenesis through antagonism between GATA2 and TFII‐I
Authors: Mammoto Akiko , Ingber Donald E
Published: June 2021

Angiogenesis can be controlled by mechanical interactions between cells and extracellular matrix (ECM), as well as by the soluble angiogenic factor, vascular endothelial growth factor (VEGF) that stimulates capillary blood vessel development by binding VEGFR2 receptors on the endothelial cell surface. Here we show that the Rho inhibitor, p190RhoGAP, regulates angiogenesis by controlling VEGFR2 expression, and that it produces this effect by modulating cross antagonism between two transcription factors ‐ TFII‐I and GATA2 ‐ that regulate VEGFR2 transcription. p190RhoGAP binds to both TFII‐I and GATA2, and sequesters them in the cytoplasm, thereby preventing VEGFR2 expression. TFII‐I decreases VEGFR2 promoter activity and expression as well as migration and capillary tube formation by human microvascular endothelial cells, whereas GATA2 produces the opposite effects, and antagonizes TFII‐I. Importantly, this novel angiogenesis signaling pathway is sensitive to extracellular matrix elasticity as well as soluble mitogens. Thus, the balance between TFII‐I and GATA2 appears to be critical for regulation of vascular network formation, and it appears to be controlled by both chemical and mechanical cues in the tissue microenvironment.
Hedgehog signaling and KLF4 contribute to maintenance of smooth muscle progenitor cells in the arterial adventitia
Authors: Passman Jenna , Dong Xiu Rong , Hoglund Virginia J. , Maguire Colin T. , Chen Zhongming , Tzima Ellie , Majesky Mark W.
Published: June 2021

The arterial adventitia plays important roles in growth and remodeling of the artery wall. We previously reported that sonic hedgehog (Shh) signaling is restricted to the adventitial layer of large and medium‐sized arteries in the perinatal period. Localized within this adventitial domain we found a resident population of Sca1‐positive cells (AdvSca1). Although AdvSca1 cells do not express smooth muscle cell (SMC) markers in vivo, they do express SRF and myocardin, and differentiate into SMCs when removed from the adventitia and placed in cell culture. AdvSca1 cells also express potent SRF‐dependent corepressors including KLF4 and Msx1, which may play critical roles in maintaining the SMC progenitor phenotype. We have tested this possible role of KLF4 by gain and loss of function approaches in vitro. Knockdown of KLF4 expression by siRNA results in a loss of Sca1 expression, consistent with a loss of self‐renewal potential of AdvSca1 cells. Conversely, exposure of AdvSca1 cells to KLF4 adenovirus inhibited SMC differentiation. By seven days after >80% flow reduction in a mouse carotid model, a substantially thickened neoadventitia formed that contained increased numbers of AdvSca1 cells with no evidence of SMC marker upregulation. These results suggest that resident AdvSca1 cells can proliferate in response to a stimulus for arterial remodeling, and that KLF4 plays important roles in AdvSca1 cell self‐renewal.
Functional Heterogeneity of MSC Populations Provides Clues to Their Broad Therapeutic Efficacy
Authors: Phinney Donald G
Published: June 2021

Mesenchymal stem cells (MSCs) have demonstrated therapeutic efficacy in a variety of disease models and human clinical trials. The widespread benefits of MSCs have recently been attributed to their capacity to secrete soluble factors that promote tissue repair by stimulating angiogenesis and suppressing inflammation, apoptosis, and immune cell function. However, these functional attributes are inconsistent with the designation of MSCs as stem cells, which by definition lie at the apex of a hierarchy of lineage specification and cellular differentiation. Previously, we catalogued the MSC transcriptome via serial analysis of gene expression to gain better insight into their basic biology. This analysis revealed that MSCs express a diverse range of proteins regulating angiogenesis, cell motility and communication, hematopoiesis, neural activities, immunity and defense. Furthermore, we reported that different classes of expressed regulatory proteins are restricted to distinct subpopulations of MSCs. Based on these findings we proposed that the molecular heterogeneity of the MSC transcriptome reflects the functional complexity of marrow stroma as an organ system in vivo and this complexity accounts for the broad therapeutic effects of MSCs. Examples are provided to illustrate how unique traits of MSC subpopulations from marrow may be exploited to develop more potent cellular vectors for therapy.
Combining experiments with agent‐based modeling to study microvascular growth at the multi‐cell level
Authors: Peirce Shayn
Published: June 2021

Understanding where new endothelial cells and perivascular cells come from and what signals mediate their recruitment is central to therapeutically manipulating microvascular growth and remodeling. Evidence suggests that vascular cells arise from bone marrow‐derived progenitor cells, from within the microvasculature, and from the surrounding interstitial tissue, but to what extent does each of these cell sources contribute, and what are their spatial and temporal dynamics? Cell trafficking‐cells moving from one tissue compartment to another via the circulation‐is integral to emerging treatments, such as stem cell‐based therapeutic angiogenesis, yet cell trafficking is poorly understood in the in vivo setting because, quite simply, it is technically difficult to follow multiple individual cells in the body and observe them (and the molecular mediators of their behaviors) as they interact with other cells in space and time. We combine in vivo and in vitro experimental assays, with agent‐based computational modeling, which is capable of integrating, synthesizing, and computing the current knowledge base regarding cell‐cell adhesive interactions, cell‐matrix interactions, fluid shear forces, and local tissue heterogeneities, in order to achieve, in essence, multi‐cell "fate mapping" in the adult organism. NIH‐HL‐091312‐01.
Wnt5a regulates directional cell migration and cell proliferation via Ror2‐mediated noncanonical pathway in mammalian palatogenesis
Authors: He Fenglei , Xiong Wei , Yu Xueyan , Espinoza‐Lewis Ramon , Liu Chao , Gu Shuping , Nishita MIchiru , Minami Yasuhiro , Yamada Gen , Suzuki Kentaro , Chen Yiping
Published: June 2021

Tissue and molecular heterogeneities are present in the developing secondary palate along the anterior‐posterior (A‐P) axis in mice. Here we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the A‐P axis of the palate. Wnt5a‐deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histology, cellular, and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the A‐P axis of the developing palate and an essential role of Wnt5a in the regulation of directional cell migration.
Methods for Teaching and Integrating Oral Histology Into the Curriculum
Authors: Spears Robert
Published: June 2021

In most curricula the teaching of histology has undergone a dramatic shift to using many new technologies and methods to better teach in both the classroom and the laboratory. Virtual microscopy has become the standard for teaching the laboratory portion of the course, and many schools have integrated histology into other courses such that structure and function are taught simultaneously. While these efforts have improved comprehension by the student of general histology, few programs have incorporated these new methodologies into the teaching of oral histology, a topic which only gets minimal effort at most institutions despite the growing evidence of its importance in overall health concerns. This presentation will give examples of how innovative methods and technologies are currently being used in different programs around the country to teach this material and present different ideas and techniques that can be incorporated into both the lecture and classroom settings in a typical general histology course. Finally, examples from institutions that have a separate oral histology course will be presented, interesting methods and technologies presented, and finally discussion of how this course can be integrated into areas of the curriculum such as the teaching of pathology.
Calpain 2 is activated downstream of wall shear stress and sphingosine‐1‐phosphate to induce endothelial cell sprout formation in three dimensional collagen matrices
Authors: Kang Hojin , Kwak Hyeongil , Kaunas Roland , Bayless Kayla
Published: June 2021

New blood vessel growth is essential for vascularization of wounded tissues. Endothelial cells are integral for this process and are stimulated by growth factors and platelet‐derived lipids such as sphingosine‐1‐phosphate (S1P), while also integrating mechanical signals, such as wall shear stress (WSS). However the molecular signals activated downstream of these cues are not completely defined. Previously, we have established a system to demonstrate that S1P and WSS combine to stimulate primary endothelial cell invasion of 3D collagen matrices. S1P and WSS induced Akt phosphorylation and MMP‐2 activation that correlated with invasion responses. Here we determined whether calpain regulates S1P and WSS‐induced invasion into 3D collagen matrices. WSS and S1P combined to enhance calpain activation. Pharmacological inhibitors of calpain 2, but not calpain 1, significantly reduced S1P and WSS‐induced endothelial sprout density and thickness. Also, Akt phosphorylation and MMP‐2 activation were attenuated by calpain inhibition. To confirm a functional role for calpain 2, short hairpin RNA was delivered using recombinant lentiviruses. Silencing of calpain 2, but not calpain 1, blocked WSS/S1P‐induced EC invasion. These data support an integral role for calpain 2 in mediating S1P and WSS‐induced invasion responses in 3D collagen matrices.

Grant Funding Source

American Heart Association
Late Neointimal Hyperplasia in Vein Grafts Expands via TGF‐β/CTGF Mediated Fibrosis
Authors: Jiang Zhihua , Tao Ming , Omalley Kerri A , Wang Danlu , Ozaki C. Keith , Berceli Scott A
Published: June 2021

Introduction

In weeks to months following implantation, neointimal hyperplasia (NIH) in vein grafts (VG) transitions from a cellularized to decellularized phenotype. Inhibition of early cellular proliferation failed to improve long‐term VG patency. We have previously demonstrated that TGF‐β /CTGF pathways mediate a conversion of fibroblasts to myofibroblasts in the early VG (<2wks). We hypothesize that these similar pathways drive fibrosis observed in the late VG lesion.

Methods

Within rabbit VGs, real time RT‐PCR, Western blotting, and immunohistochemistry were used to examine TGF‐β /CTGF pathways in late (1‐6 month) NIH.

Results

All VGs exhibited a steady NIH growth (p=0.006) with significant reduction in cellularity (p=0.01) over time. Substantial TGF‐β profibrotic activities, as evidenced by enhanced TGF‐β1 activation, TGF‐β types I to II receptor ratio, SMAD2/3 phosphorylation, and CTGF production, persisted throughout the observation period. Increased matrix synthesis was accompanied by a temporal reduction of MMP2 (p=0.001) and 9 (p<0.001) activity.

Conclusion

VG NIH is characterized by a conversion from a pro‐proliferative to profibrotic morphology. Enhanced signaling via TGF‐β /CTGF coupled with reduced MMP activities promotes progressive fibrotic NIH expansion. Modulation of late TGF‐β /CTGF signaling may offer a novel therapeutic strategy to improve the long‐term VG durability.
Massively‐Parallel Sequencing Of Nascent RNA Sequencing Reveals Widespread Pausing And Divergent Initiation From Mammalian Promoters
Authors: Lis John T , Core Leighton J , Min Irene , Waterfall Joshua J
Published: June 2021

RNA polymerases are highly‐modulated molecular machines. We have developed a highly‐sensitive method (GRO‐seq) that maps the position, amount, and orientation of transcriptionally‐engaged RNA polymerases across the entire genome, and have applied it to a primary human fibroblast cell line, and to mouse embryonic stem (ES) cells and isogenic embryonic fibroblasts (MEFs). In this method, nuclear run‐on reactions allow RNA polymerase to incorporate BrU affinity‐tags into nascent RNA that is fragmented, purified 10,000 fold, and subjected to large‐scale parallel sequencing. Mapping these sequences to the genome in these different cell types shows that 30% of all genes have promoter‐proximal paused polymerase, that transcription continues kilobases beyond the 3' cleavage for many genes, and that antisense transcription is prevelant. Surprisingly, in addition promoter‐proximal paused polymerase, most promoters also have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes, but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.

This research was supported by NIH.
HIV‐1 nef signature sequences and pulmonary arterial hypertension: structure‐function relationships to pathogenesis
Authors: Flores Sonia Castro , Almodovar Sharilyn
Published: June 2021

Little is known about the pathogenesis of pulmonary arterial hypertension (PAH) or factors that contribute to its higher prevalence in HlV‐infected patients. Chronic exposure to viral products in the lung may contribute to vascular disease. HIV‐1 Nef is an accessory protein expressed early in infection whose functions are mediated by highly conserved structural domains including N‐myristoylation signal, proline‐rich (PxxP) motif and six sequence motifs that interact with the endocytotic machinery. Disruptions in Nef amino acid sequences have profound pathophysiological implications. A mutated form of Nef (5 amino acid changes) was associated with pathogenesis in SHIV‐nef‐infected macaques. We demonstrated a significant association between mutated Nef and presence of complex vascular lesions in monkey lungs and hypothesized that nef allelic variants are associated with PAH in HIV‐infected patients. We characterized nef alleles from a well‐described cohort of patients from France, analyzed the translated protein sequences and found that specific residues are over‐represented in hypertensive compared to normotensive controls. Changes occurred in five known structural domains including the MHC‐1/AP‐1 binding area, CD4 downregulating motif, acidic cluster, PKC binding domain and PxxP. These studies suggest a pathogenetic explanation for the higher prevalence of PAH in HIV‐infected patients.
Omega‐3‐producing fat‐1 transgenic mice display reduced complex I‐associated hydrogen peroxide production and enzyme activity
Authors: Ramsey Jon , Weber Kristina , Hwee Darren , Van Eenennaam Alison , German J. Bruce , McDonald Roger , Hagopian Kevork
Published: June 2021

The fat‐1 gene of C. elegans encodes a desaturase, which can convert n‐6 to n‐3 fatty acids. A transgenic mouse that constitutively expresses this gene was used as a mammalian model of increased tissue n‐3 fatty acids. The aim of the study was to evaluate the influence of changes in membrane fatty acid composition on the lipid‐protein interaction, specifically on the production of H2O2 and electron transport chain enzyme activities. Liver mitochondria were isolated by differential centrifugation and the activities of the electron transport chain enzymes determined spectrophotometrically, while H2O2 production was determined fluorimetrically. Fatty acid analyses of mitochondrial membranes were undertaken on Percoll‐purified mitochondria, and performed by Lipomics Technology, Inc. The results indicate that the transgenic mice show a decreased H2O2 production at complex I, as well as decreased complex I enzyme activity. Total H2O2 production was also decreased when mitochondria were respiring on succinate or succinate/glutamate/malate. Fatty acid analysis showed an increased n‐3/n‐6 ratio in mitochondrial PC, PE and LyPC lipid classes, with arachidonic acid (n‐6) decreased and eicosapentaenoic acid (n‐3) increased in all phospholipid classes. The results indicate that decreased complex I‐linked ROS production may provide some protection against oxidative stress in membranes with high n‐3 levels.
Folate‐independent remethylation of homocysteine is increased by dietary egg white protein
Authors: Schalinske Kevin Lee , Luchtel Rebecca A , Garrow Tim A
Published: June 2021

Proper maintenance and regulation of methyl group and homocysteine (hcy) metabolism is essential for optimization of health. Methyl deficiency and/or elevations in plasma hcy concentrations have been associated with numerous pathologies, such as vascular disease. It is important to identify dietary components that positively impact these metabolic pathways; thus, we examined dietary protein sources with varying sulfur amino acid content. Rats were fed either a casein or egg white protein (20%) diet for 2 wk after which half the rats in each group were fed folate‐deficient diets for 4 wk. Compared to casein, egg white protein resulted in: (1) elevated hepatic betaine‐hcy S‐methyltransferase activity, responsible for folate‐independent hcy remethylation; (2) increased hepatic activity of glycine N‐methyltransferase, a key methyl group regulatory protein; and (3) reduced plasma glutathione concentrations. Moreover, folate deficiency was without affect on plasma hcy levels in the egg white group. In a parallel study, soy protein did not differ from casein with respect to altering methyl group and hcy metabolism. In conclusion, dietary egg white protein appears to promote hcy remethylation at the expense of catabolism via transsulfuration. These findings may be attributed to the greater met‐cys content of egg white protein compared to casein and soy (Support: Amer. Egg Board, Egg Nutr. Center, IA Egg Council).
Serum folic acid concentrations in the U.S. population after the introduction of folic acid fortification
Authors: Pfeiffer Christine M , Fazili Zia , Kennedy‐Stephenson Jocelyn
Published: June 2021

Knowing what the serum folic acid (FA) concentrations in the U.S. population post‐fortification are is of great interest because FA might have a dual modulatory effect in carcinogenesis and circulating levels of FA in serum have been associated with reduced natural killer cell cytotoxicity in postmenopausal women. As part of a method comparison study, we analyzed 232 serum samples from NHANES 1999‐2004 (convenience set) for folate species by LC‐MS/MS. The distribution of FA concentrations was right‐skewed, with ~30% and 5% of the samples having levels >10 and >100 nmol/L, respectively. Median FA concentrations (nmol/L) were dependent on the length of fasting (29.0 at ≤3 h; 1.72 at 4‐6 h; 0.98 at 7‐12 h; 0.83 at >12 h), the use of FA‐containing supplements during the past month (4.90 for users; 0.63 for non‐users), and the use of a dietary supplement since starting the fasting for the blood draw (14.8 for users; 1.0 for non‐users). Fifteen percent of the FA variability was explained by these variables in a regression model. Subjects who fasted >3h and used no supplement during the past month or during fasting, had a baseline FA concentration of 3.11 nmol/L. For =3h fasting, use of a supplement during the past month, or use of a supplement during fasting, 21.5, 16.8, and 16.0 nmol/L FA needs to be added, respectively. To better interpret "free" FA serum levels, it is important to understand its determinants.
Evidence that physiological doses of vitamin B12 are metabolized or degraded in the gastrointestinal tract: implications for vitamin B12 bioavailability and fortification
Authors: Green Ralph , Lee Kyung‐Seon , Sutter Syrukh , Allen Lindsay H. , Buchholz Bruce , Dueker Stephen R. , Miller Joshua W.
Published: June 2021

Much of our understanding of vitamin B12 derives from radiocobalt tracer studies conducted decades ago. With new technologies, we can now study B12 absorption and metabolism with unprecedented resolution using almost ambient levels of 14C biosynthetically incorporated into the B12 molecule (14C‐B12). In on‐going studies, a dose of 14C‐B12 (1.3 µg, 50 nCi) in water is administered orally and blood, urine, and feces analyzed for 14C by accelerator mass spectrometry. In 6 subjects, the plasma response was consistent with the expected behavior of peroral B12: 14C‐B12 first appeared in the plasma 3h post‐dose with a peak level within 6‐8h. Urinary and fecal excretion was maximal in the first 24h. The amount of 14C found in the urine (10‐50% of the dose) was 100‐fold greater than in previous reports (0.1‐0.5%), and fecal excretion was lower than expected (<10% vs 30‐70%). Most of the urinary 14C was not associated with intact B12. These results are consistent with metabolism or degradation of the vitamin in the GI tract prior to absorption. The discrepancy between the present and previous findings may be due to the form of radioactive B12 used: 14C labeling the 5,6‐dimethylbenzimidazole moiety in the present study and radiocobalt labeling the corrin ring in previous studies. These findings provide valuable information on the fate of B12 after oral consumption that will inform considerations to fortify the food supply with B12.
Short and long term impacts of diabetes peer counseling on HbA1c among Latinos: Preliminary results
Authors: Perez‐Escamilla Rafael , Vega‐López Sonia , Damio Grace , Segura‐Pérez Sofia , Luz Fernandez Maria , Calle Mariana C , Samuel Grace K , Chhabra Jyoti , D'Agostino Darrin
Published: June 2021

The ongoing Diabetes among Latinos Best Practices Trial (DIALBEST) has randomly assigned Latinos with type 2 diabetes to the standard of clinical care or the intervention group that receives home visits from peer counselors. Intervention group participants receive one weekly visit during the first month, biweekly during months 2 and 3, and monthly thereafter until month 12.Groups were equivalent at baseline with regards to their socio‐economic, demographic, and biomarker characteristics. The intervention group continues to have a greater improvement in HbA1c at 3 months compared to the control group (‐1.1 ± 1.6 % vs. ‐0.5 ± 1.7 %, n=130, p=0.04). Although not statistically significant, because we still have not reached the desired statistical power, by 6 months the between group differences remain in favor of the intervention group (‐0.8 ± 1.6 % vs. ‐0.4 ± 1.4 %, n=82) with similar trends seen at 12 months (‐0.7 ± 2.2 % vs. ‐0.1 ± 1.7 %, n=48). Thus, the short term DIALBEST benefits on glycemic control are likely to continue in the longer term. Funded by The Connecticut Latino Health Disparities NIH EXPORT Center (NIH‐NCMHD grant # P20MD001765).
Increased sensitivity of thioredoxin reductase 1 deficient breast cancer cells against TNF‐α induced apoptosis
Authors: Yoo Min‐Hyuk , Xu Xue‐Ming , Carlson Bradley A , Gladyshev Vadim N , Hatfield Dolph L
Published: June 2021

Thioredoxin reductase 1 (TR1), a major redox regulator in mammalian cells, was reported by several laboratories to occur in increased levels in various cancers. We found that inhibiting TR1 expression in various malignant cell lines altered cancer phenotypes such as morphology, anchorage independent growth, tumor formation, metastasis and self‐sufficiency in growth signals (Yoo et al. JBC 281:13005, 2006; PLoS One 2(10):e1112, 2007). To investigate the role of TR1 in breast cancer, we established a TR1 deficient breast cancer cell line (EMT6) and found that these cells manifested much greater sensitivity to TNF‐α induced apoptosis than control cells. Under a low concentration of TNF‐α (10 ng/ml), only 25% of TR1 deficient cells survived whereas more than 80% of control cells survived suggesting that this selenoenzyme is also involved with another cancer phenotype, anti‐apoptosis. We further examined possible signaling cascades to elucidate the pathway affected by TR1 deficiency in the resistance of cancer cells against TNF‐α induced apoptosis and found that the MAP kinase, ERK, is responsible for this increased sensitivity of TR1 deficient breast cancer cells induced by low concentrations of TNF‐α. This research was supported by the Intramural Research Program of the NIH, NCI, CCR and by NIH grants to V.N.G.
Tomato Product Adherence Dynamics in African‐American Men at Risk for Prostate Cancer
Authors: Park Eunyoung , Stacewicz‐Sapuntzakis Maria , Sharifi Roohollah , Freeman Vincent , Xie Hui , Tampe Carolyn , Bowen Phyllis E.
Published: June 2021

African‐American (AA) men have the highest incidence of prostate cancer (PC) and tomato product (TP) intake is associated with reduced risk. Can AA men significantly increase self‐selected TP intake through diet counseling alone? AA men with PSA ≥ 2.5 ng/mL were randomized to either increase TP intake (intervention group (IG), N = 22) through weekly telephone calls, or to a control group (CG, N=14) receiving no TP coaching over 3 months. Mean dietary lycopene intake was 24.6 ± 27.1 and 5.8 ± 9.6 mg/d, respectively for IG and CG (p = 0.04). The IG consumed a mean of 7.1 TP servings/wk and CG only 3.5 (p = 0.005). The IG increased plasma lycopene level by 53% and 40%, at 1 and 3 months (p = 0.001, p = 0.003, respectively), while there was no increase in the CG. Subjects with high baseline leukocyte DNA damage had significantly less damage after 3 months (22.8%). Incentives/impediments to TP intake were assessed weekly in the IG. Incentives in order of frequency were (1) in study, (2) likability, (3) mass media, (4) social support. Impediments were (1) forgot, (2) run out, (3) hospitalization/sick, (4) tight budget. AA men at risk for PC were able and willing to increase TP intake sufficiently to raise plasma lycopene levels and decrease DNA damage over 3 months. Self‐selected whole food interventions through diet counseling alone may be a feasible approach for efficacy studies.

Support: Roybal Center Health Promotion, UIC
Objective and self‐reported recall measurements for nutrient intakes from dietary supplements: Results from the SURE study
Authors: Steffen Alana , Wilkens Lynne , Monroe Kristine , Albright Cheryl , Yonemori Kim , Morimoto Yukiko , Murphy Suzanne
Published: June 2021

The Supplement Reporting (SURE) Study measured dietary supplement (DS) use using a novel inventory method consisting of five home visits over the course of a year. For 402 participants, staff counted the pills in all supplement containers in order to compute an estimate of daily nutrient intake from DS both in the short‐term (over 3 months) and across the year‐long period. Nutrient intakes were also calculated from two‐week and one‐month open‐ended recalls of DS use which the participants completed. Compared to intakes from the corresponding quarterly inventory, weighted kappa values ranged from 0.43 (for riboflavin) to 0.78 (for iron) using the two‐week recalls, and from 0.69 (for vitamin A) to 0.83 (for both vitamins C and E) using the one‐month recalls. The average kappa value across 15 nutrients was 0.63 for the two‐week period, and 0.76 for the one‐month period. When compared against the one‐year inventory values, the average kappa across nutrients was similar for the two recall periods: 0.75 for two weeks and 0.73 for one‐month. We conclude that the recalls provide an acceptable alternative to the costly inventory method when ranking intakes from DS, and that a one‐month recall is better for estimating current DS nutrient intake while both recall periods are comparable for past year of DS use. Supported by NCI grant R01 CA106744.
Methodologic differences in dietary supplements data collection and processing between the Third National Health and Nutrition Examination Survey (NHANES III) and the continuous NHANES (1999‐2006)
Authors: Johnson Clifford L , Hughes Jeffrey , Radimer Kathy , Wilger Jaime J , Dwyer Johanna , Picciano Mary Frances , Bailey Regan L , Sempos Christopher
Published: June 2021

Dietary supplement data has been collected for many years in the National Health and Nutrition Examination Survey (NHANES). Over time, the methods used to collect self‐reported information on dietary supplement use have changed within NHANES. For example, different questions have been asked at different time periods. In addition, the methods of attaining and coding ingredient information about the supplement products have also changed. An example is the use of different methods for assigning default strengths to single and multiple nutrient/ingredient products. The overall prevalence of dietary supplement use increased from 41.3% (NHANES III) to 53.4% (NHANES 2003‐2006). The purpose of this study was to determine if it is possible to quantify how much (if any) of this observed increase in dietary supplement use might be due to the changes in collection, processing and coding of the data that occurred between the two survey time periods.
Components of Plasma Selenium in Healthy Americans
Authors: Jackson Matthew I , Johnson Luann K , Hoeg Antonia , Hoefig Carolin , Davis Cindy D , Milner John A , Schomburg Lutz , Combs Gerald F
Published: June 2021

In a cohort of healthy adults (106 M, 155 W) in eastern North Dakota, we assessed the effects of supplementation with L‐selenomethionine (SeMet) (0, 50, 100 or 200 mcg selenium [Se]/d for 1yr) on three plasma/serum biomarkers of Se status, which at baseline were: Se, 141.5±23.7 (SD) ng/ml; selenoprotein P (SePP), 3.55 (CI 2.61, 4.51) mg/l; glutathione peroxidase (GPX3) activity, 3.64±0.54 nmoles NADPH/min/mg. We imputed the portion of plasma Se present in selenocysteine (SeCys) in SePP and GPX3 from their known SeCys contents (5.5 and 4 g‐atoms/mole), the molecular weights reported for each (57 kD and 92kD), and the enzyme number reported for purified GPX3 (2.8 x 104 nmoles NADPH/min/mg). This showed that prior to intervention 39.8±6.0 % of plasma Se occurred as SeCys, with the balance, 60.2±10.3 % comprised of other forms, presumably SeMet incorporated nonspecifically into various proteins. SeMet‐supplementation did not affect the size of the SeCys component, but increased that of the non‐specific component in a dose‐dependent manner: baseline, 87.5±25.8 mcg/la; 12 mos.: 93.5±28.0a, 133.0±37.4b, 157.6±40.9c and 223.4±51d mcg/l for subjects receiving 0, 50, 100 or 200 mcg Se/day, respectively. Because SeMet is a dominant form of Se in foods, these results indicate that consumption of Se‐containing foods by non‐deficient individuals affects primarily the non‐specific components of plasma Se.
Low carbohydrate diets reduce aortic lipid accumulation, oxidized LDL and atherosclerosis in guinea pigs
Authors: Leite Jose O , deOgburn Ryan , Ratliff Joseph , Su Randy , Volek Jeff S , McGrane Mary M , Dardik Alan , Fernandez Maria Luz
Published: June 2021

While low carbohydrate (LC) diets are effective in inducing weight loss and favorably affecting plasma lipids, little is known about the effects of LC on atherosclerosis. Twenty male guinea pigs were fed either a LC diet or a low fat (LF) diet in combination with high cholesterol (0.25g/100g) for 12 wk to evaluate the effects of changes in macronutrient composition on lipid accumulation and oxidized LDL in a model of atherosclerosis. The energy distribution of the diets was 10:65:25 for carbohydrate:fat:protein for the LC and 55:20:25 for the LF diet. LDL cholesterol (LDL‐C) was lower in the LC compared to the LF group (71.9 ± 11.0 vs 81.7 ± 8.5 mg/dL, p < 0.05). Cholesterol concentration in aortas was also lower in the LC (4.98 ± 1.31 mg/g) compared to the LF group (6.68 ± 2.05 mg/g), p< 0.05). Histological analyses using the Schultz method confirmed cholesterol accumulation. Plasma oxidized LDL (oxLDL) did not differ between groups, however, aorta oxLDL was 77% lower in the LC (1.56 ± 0.56 ug/g) compared to the LF (4.63 ± 3.26 ug/g) group (P < 0.01). LDL‐C was correlated with cholesterol in aorta in both groups (LC, r= 0.657, p= < 0.05 and LF, r=0.66, p< 0.05) suggesting a major role of this lipoprotein in aortic lipid accumulation. In addition, there was no correlation between oxLDL and cholesterol concentration in the aorta. These results suggest that LC not only decreases lipid accumulation but also prevents the oxidation of LDL in the aorta independent of cholesterol concentration.
Effect of body fat distribution on the physiological response to a dietary fat intervention
Authors: de Graaf Albert Arjaan , Pasman Wilrike , van Schalkwijk Daan , Freidig Andreas , Hendriks Henk , Vaes Wouter , Verheij Elwin , Bobeldijk Ivana
Published: June 2021

We conducted a study to examine the effect of body fat distribution on the physiological response to a dietary fat intervention with medium chain fatty acids (MCFA) vs long‐chain fatty acids (PUFA) in humans. The physiological response was evaluated as fatty acid kinetics (plasma and subcutaneous fat appearance), plasma lipoprotein profiles and clinical chemistry parameters. In a cross‐over trial, 12 male subjects (age 51±7 y; BMI 28.5±0.8 kg.m‐2), divided in 2 groups according to waist‐hip ratio, were supplemented 60 grams/day MCFA (C8:0, C12:0) or PUFA (C18:2n‐6) for three weeks, with a wash‐out period between supplementation periods of six weeks. Fatty acid metabolism kinetics were studied by supplying [1,2,3,4‐13C4]C8:0 and [U‐13C18]C18:2n‐6 tracers together with a meal on the test day at the end of each intervention period. Samples taken at different time points after tracer administration were analyzed by GC‐C‐IRMS for concentration‐ and isotopic analysis. A clear treatment effect on fat metabolism was concluded from lipoprotein profiles as well as subcutaneous fat composition data from adipose tissue biopts. From the stable isotope data we conclude the presence of differences in acute fatty acid uptake influenced by treatment and fat disposition, that are moreover different in upper body obese vs lower body obese.
CRP Impairs Endothelial Glycocalyx: Role in Promoting Endothelial Dysfunction
Authors: Devaraj Sridevi , Duncan‐Staley Catherine , Yun JungMi , Adamson Grete , Jialal Ishwarlal
Published: June 2021

Inflammation and endothelial dysfunction are pronounced in atherosclerosis. Increased C‐reactive protein (CRP) are associated with endothelial dysfunction. The glycocalyx, a layer of proteoglycans covering the endothelium, protects the vessel wall. We show that CRP promotes endothelial dysfunction via decreased eNOS; this is mediated by promoting oxidative stress and uncoupling. Thus, the aim of the study was to examine the effect of CRP on the glycocalyx. Human aortic endothelial cells (HAEC) were incubated with CRP at different concentrations (0,12.5,25,50 ug/mL). Endothelial glycocalyx thickness was examined by transmission electron microscopy. CRP dose‐dependently decreased glycocalyx thickness, C: 0.87 ± 0.33 um, CRP 25: 0.51 ± 0.12 *um and CRP 50: 0.22 ± 0.07 um, Boiled CRP: 0.79 ± 0.21 um, p<0.05)and increased hyaluronan release (C: 72 ± 33 ng/mg protein; CRP 12.5: 87± 21 ng/mg protein; CRP 25: 188 ± 66 *ng/mg protein and CRP 50: 222 ±87 *ng/mg protein, B‐CRP: 65 ± 29 ng/mg protein, p<0.01 compared to C). Also, western blotting for heparan sulfate showed significant reduction in expression of heparan sulfate, one of the main glycosaminoglycans in the glycocalyx, with CRP treatment. These correlated to increased PAI‐1 and monocyte‐endothelial adhesion. These data indicate a potential role for CRP in accentuating glycocalyx degradation and promoting endothelial dysfunction
Differential Cell Binding Of Intact (HK) And Cleaved (HKa) High Molecular Weight Kininogen Is Mediated By The Presence Of Urokinase Receptor, uPAR (CD87)
Authors: Pixley Robin A. , Liu Yuchuan , Espinola Ricardo G , Colman Robert W
Published: June 2021

The patho‐physiologic activation of the plasma kallikrein‐kinin system requires the proteolysis and assembly of receptors on the cell membrane. HK binds to endothelial cells through an interaction with a multi‐protein receptor complex that consists of: uPAR, globular C1q receptor (gC1qR, p33) and cytokeratin 1 (CK1). In a preliminary report (Blood 106, 748a, 2005, Abst 2666), we measured the direct binding of HK & HKa to suPAR (soluble uPAR), gC1qR and CK1 using surface plasmon resonance. The binding of HK and HKa to all of receptors was zinc dependent. The affinity for HK & HKa was gC1qR>CK1>suPAR. The high affinity for gC1qR and CK1 was similar for HK & HKa. However, binding of HKa compared to HK was 2.5‐fold tighter for CK1, but 50‐fold tighter for suPAR. Although suPAR has the weakest affinity of the three receptors, it distinguishes between HK & HKa. We hypothesized that there would be differential binding of HK and HKa to cells lacking the uPAR receptor. Human Embryonic Kidney 293 cells (HEK) lack the uPAR receptor. We compared the differential binding of HK and HKa to non‐ and uPAR‐ transfected (uHEK) cells. With zinc there was more binding of labeled HKa than HK to both types. At HKa concentrations >100 nM, there was additional HKa binding to uHEK. We attribute this to the low affinity binding sites of uPAR for HKa. These results suggest that HK and HKa may play different roles in cell interactions on membranes. 5R01CA83121.
Regulation of SIRPa binding to CD47: Evidence for a role of Cis‐homodimerization
Authors: Lee Winston Y , Weber Dominique A , Laur Oskar , Parkos Charles A
Published: June 2021

Interaction of SIRPα with its ligand, CD47 regulates leukocyte functions including transmigration, phagocytosis, oxidative burst, and cytokine secretion. However, the molecular details of how this transmembrane glycoprotein interacts with CD47 on the cell surface are incomplete. Here we report that SIRPα forms noncovalently linked cis‐homodimers and present evidence to suggest that homodimerization facilitates binding to CD47. Treatment of SIRPα expressing cells with a membrane impermeable crosslinker allowed detection of SIRPα dimers and oligomers on western blots. Biochemical analyses of soluble, recombinant extracellular regions of SIRPα, including domain‐truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRPα formed dimers in solution. Co‐immunoprecipitation experiments using cells transfected with different affinity‐tagged SIRPα molecules revealed that SIRPα may form cis dimers. Notably, in cells treated with tunicamycin, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer may be bivalent and capable of binding CD47 with increased avidity. Lastly, in adherent neutrophils stimulated with chemoattractant, dimerization of SIRPα was enhanced. Collectively, these data suggest that cis dimerization of SIRPα plays an important role in regulating CD47 binding and function in leukocytes.
CCR5 deletion impairs the post‐myocardial infarction inflammatory response
Authors: Zamilpa Rogelio , Cigarroa Joaquin , Dai Qiuxia , Escobar G. Patricia , Jimenez Fabio , Martinez Hernan G. , Ahuja Seema S. , Lindsey Merry L.
Published: June 2021

The inflammatory response following a myocardial infarction (MI) regulates multiple components of the wound healing process. In particular, macrophages regulate wound debridement, scar formation, and neovascularization. The CC Chemokine Receptor 5 (CCR5) is a key chemokine receptor expressed on macrophages, and CCR5 ligands are highly upregulated post‐MI. However, the roles of CCR5 post‐MI have not been investigated. Accordingly, we examined macrophage infiltration and collagen concentration in the left ventricle (LV) and plasma inflammatory markers in wild‐type mice (wt, n=25) and CCR5 null mice (null, n=33). The groups had similar infarct sizes (44±2% in wt and 42±2% in null, p= n.s.). Macrophage infiltration was lower in the null (7±1% of total infarct area compared to 11±1% in wt; p<0.05). The collagen insoluble to soluble ratio was 36.7% higher in the null (p<0.05), suggesting reduced collagen synthesis. In addition, plasma levels of IL‐6, IFN‐ ? and TNFá increased in wt, but not in null mice, post‐MI (p<0.05). We conclude that CCR5 deletion has a net negative effect on remodeling post‐MI, as evidenced by a decreased inflammatory response and collagen synthesis. NIH (R01 HL‐75360) and American Heart Association grant (0855119F) to M.L.L. and T32 (HL07446) to R.Z.