bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS‐induced post‐translational modifications of hsp90 in pulmonary endothelial cells
Authors: Handa Vaishali , Snead Connie , Black Stephen M , Catravas John D
Published: June 2021

Inflammation plays an important role in the pathogenesis of pulmonary vascular diseases. Heat shock protein 90 (hsp90), which accounts for approximately 1 to 2% of total cellular proteins, exists in multi‐protein complexes with various co‐chaperones and client proteins, many of which are crucial to inflammation. Whereas post‐translational modifications affect hsp90 function, there is no information on how inflammation may bring about such modifications. In bovine pulmonary arterial endothelial cells (BPAE), LPS (1000 EU/ml) produced a significant, time‐dependent increase in tyrosine phosphorylation of hsp90, which was prevented by pre‐treatment with the pp60src inhibitor, PP2 (10μM). Infection of BPAE with adenoviral constructs of constitutively active pp60src, but not of wild type or dominant negative pp60src, caused an increase in hsp90 tyrosine phosphorylation of hsp90 which was further augmented by exposure to LPS. Pretreatment (2hr) with the hsp90 inhibitor 17‐(Allylamino)‐17‐demethoxygeldanamycin (17‐AAG; 1μg/ml) significantly suppressed LPS‐mediated hsp90 tyrosine phosphorylation. Furthermore, pp60src activation (Y418 phosphorylation) in BPAEs was also PP2‐ and 17‐AAG‐ sensitive. We conclude that LPS activates hsp90 via pp60src‐ and 17‐AAG‐ sensitive tyrosine phosphorylation.
Elevated levels of von Hippel‐Lindau protein in human and mouse fibrotic lungs
Authors: Zhou Guofei , Pardo Annie , Koenigshoff Melanie , Dada Laura A , Eickelberg Oliver , Varga John , Selman Moises , Sznajder Jacob I
Published: June 2021

Objective

Fibronectin (FN) deposition and fibroproliferation contribute to pulmonary fibrosis. von Hippel‐Lindau protein (pVHL) regulates assembly of fibronectin matrix. We sought to determine whether pVHL participates in pulmonary fibrosis.

Methods and Results

We found that lungs from idiopathic pulmonary fibrosis (IPF) patients expressed higher levels of pVHL mRNA and protein as measured by quantitative real time RT‐PCR (qRT‐PCR) and Western blot analysis. We also discovered that lungs from IPF patients displayed strong staining of pVHL in fibrotic foci upon immunohistochemistry analysis. In the bleomycin‐induced mouse model, fibrotic lungs exhibited increased levels of pVHL mRNA and protein as determined by qRT‐PCR and immunofluorescence staining. Overexpression of exogenous pVHL stimulated proliferation of primary rat and human lung fibroblasts and increased FN expression, assembly and phosphorylation of focal adhesion kinase (FAK).

Conclusions

Our results suggest that fibrotic lungs have increased pVHL expression and overexpression of pVHL increases FN/integrin/FAK signaling, resulting in fibroproliferation and fibrosis.This study is supported by NIH HL‐071643, AR‐042309 and Parker B. Francis foundation.
Calcium Handling in Postganglionic Sympathetic Neurons is enhanced in Prehypertensive Spontaneously Hypertensive rat
Authors: LI DAN , LEE CHEE‐WAN , BUCKLER KEITH , PATERSON DAVID J
Published: June 2021

Noradrenergic hyperactivity is associated with hypertension that may precede the developing of hypertension itself. We hypothesized that Ca2+ signalling in sympathetic neurons is enhanced in young pre‐hypertensive spontaneously hypertensive rats (SHR). We measured depolarisation (high [K+]0) evoked Ca2+ transient ([Ca2+]i) with fluorescence imaging using fura‐2 in superior cervical ganglia from age and gender matched hypertensive and normotensive Wistar‐Kyoto (WKY) rats. In 4‐7 day old rats, no difference was observed in [Ca2+]i between the SHR and WKY. In contrast, among young pre‐hypertensive (4‐5 weeks old) rats, [Ca2+]i were higher in the SHR (male: 1.39±0.11 μM; female: 1.21±0.05 μM) compared with the gender matched WKY (male: 1.08±0.13 μM, p<0.05; female: 1.07±0.05 μM, p<0.05). In adult (16‐18 weeks old) rats the [Ca2+]i were also increased in the SHR (male: 1.82±0.06 μM; female: 1.58±0.07 μM) compared with the gender matched WKY (male: 1.59±0.09 μM, p<0.01; female: 1.12±0.06 µM, p<0.001). Interestingly, in the adult SHR and WKY, [Ca2+]i was significantly higher in male. The overall responses are consistent with the hypothesis that enhanced Ca2+ signalling may contribute to the noradrenergic hyperactivity that is seen before the onset of hypertension itself.

This work was supported by British Heart Foundation and Welcome Trust.
Gamma‐tocopherol and gamma‐tocotrienol are primarily metabolized to conjugated CEHC and sulfated long‐chain carboxychromanols in rats
Authors: Jiang Qing , Freiser Helene
Published: June 2021

Vitamin E is known to be metabolized to the terminal urinary‐excreted metabolite, 2‐(â ‐carboxyethyl)‐6‐hydroxychroman (CEHC). We recently showed that sulfated long‐chain carboxychromanols are generated from the metabolism of tocopherols. Here we identified similar sulfated long‐chain carboxychromanols by electrospray mass spectrometry from human A549 cells incubated with gamma‐tocotrienol (g‐TE). Sulfation was confirmed by sulfatase digestion which converted sulfated carboxychromanols to carboxychromanols. In the culture media, more than 90% long‐chain carboxychromanols from g‐TE, but less than 45% from tocopherols were sulfated. In rats, six hours after administration of g‐TE or g‐tocopherol (g‐T), the plasma had increased levels of sulfated 9′‐, 11′‐, 13′‐carboxychromanol and 13′‐carboxychromanol. Sulfatase digestion revealed that conjugated CEHC is by far the most abundant metabolite, accounting for >75% of total metabolites, whereas free CEHC is a minor metabolite. Total metabolites from g‐TE were significantly (P<0.05) higher than those from g‐T, while g‐TE was significantly (P<0.05) lower than g‐T in the plasma. These results demonstrate that in rats, sulfation is quantitatively important to vitamin E metabolism, g‐TE is more extensively metabolized than g‐T, and sulfation likely occurs parallel to â‐oxidation. (Supported by NIH R01AT001821)
Understanding the role of A2B adenosine receptor using knockout in the regulation of coronary flow
Authors: Sharifi Sanjani Maryam , Tilley Steve , Teng Bunyen , Mustafa S. Jamal
Published: June 2021

Adenosine, released due to increased oxygen demand of the heart, exerts its effects through 4 subtypes of receptors (AR) namely, A1, A2A, A2B and A3. The contribution of ARs in the regulation of coronary flow (CF) needs to be further investigated in order to better understand the heterogeneity of CF regulation. We performed mouse Langendorff experiments using two different approaches: 1) use of A2B selective and non‐selective agonists and 2) use of A2A and A2B knockout (KO) mice. BAY 606583 (selective A2B AR agonist) shows no effect on CF in A2B KO mice, however, it increases the CF significantly (p< 0.05) in A2A KO and WT mice (211.0 ± 34.5 vs. 227.0 ± 79.9, respectively, n=4). Furthermore, administration of 5′‐N‐ethylcarboxamido adenosine (NECA, non‐selective agonist) increased the CF in A2A KO mice by 122.0±19.72% from the baseline. However, NECA‐induced effect on CF in WT and A2B KO is not significantly different. We propose this effect to be due to increased expression of A2A AR as a compensatory mechanism since there is no significant difference between NECA and CGS 21680 (A2A AR selective agonist). In addition, bradykinin‐(endothelium‐dependent)‐induced effect on CF between WT and A2B KO mice is not significant. Our data strongly supports a role for A2B AR in the regulation of CF. Further investigations are needed to define signaling pathways for this AR. Supported by HL027339, HL094447, HL071802, and T‐32 HL090610.
Contrasting influence of two paradigms of hypoxia on catecholamine secretion from neonatal chromaffin cells
Authors: Souvannakitti Dangjai , Kumar Ganesh , Fox Aaron , Prabhakar Nanduri R.
Published: June 2021

Effects of intermittent (IH) and sustained hypoxia (SH) on low PO2‐evoked catecholamine (CA) secretion from neonatal rat chromaffin cells were examined. CA secretion was monitored by amperometry from chromaffin cells isolated from rat pups exposed to either IH (P0‐P5; 15s hypoxia‐5min normoxia;8h/day) or SH (hypobaric hypoxia; 0.4ATM) or normoxia (Controls). IH facilitated whereas SH attenuated hypoxia‐evoked CA secretion. IH increased the epinephrine and norepinephrine content of the adrenal medulla; whereas SH had no effect. Reactive oxygen species (ROS) levels were increased by IH but not SH. Systemic administration of MnTMPyP, a membrane permeable anti‐oxidant, prevented IH‐evoked a) facilitation of hypoxia‐induced CA secretion and b) increases in ROS as well as CA content. Elevated ROS levels and CA content persisted in P35 rats which were exposed to neonatal IH. These observations suggest that: a) neonatal IH and SH evoke diametrically opposed effects on hypoxia‐evoked CA secretion from neonatal chromaffin cells, b) ROS signaling contributes to IH but not SH evoked changes in CA secretion and c) the effects of neonatal IH on chromaffin cells persist into adult life. Supported by NIH‐HL‐76537, HL‐90554, HL‐86493 (NRP); Philip Morris USA Inc. and Philip Morris International grant (AF). D.S. is supported by fellowship from Royal Thai Government.
Hypoxia and hyperoxia both increase superoxide production in nucleus tractus solitarius (NTS) neurons in rat brain tissue slices
Authors: Dean Jay B. , Landon Carol S. , D'Agostino Dominic P. , Putnam Robert W.
Published: June 2021

Maintaining brain slices in 95%O2 produces hyperoxia, oxidative stress and increased cell death, whereas decreasing control O2 to 20‐40% significantly decreases both oxidative stress and cell death (J. Neurophysiol. 98:1030‐41, 2007) and maintains neuronal excitability (Matott et al., this meeting). In this study, beginning at 20‐40%O2, we tested the hypothesis that acute hyperoxia and hypoxia both increase the rate of superoxide (·O2−) production in NTS neurons. Medullary slices (400μm, weaned rats, 36‐37oC) were maintained using 2‐sided superfusion. Intracellular ·O2− production was measured using dihydroethidium (DHE, 2.5μM) added to the superfusate. The rate of ·O2− production (δfluorescence intensity units/min, δFIU/min) increased during hypoxia (40/20%(0%O2) and was blocked by myxothiazol (10μM; inhibits ·O2− produced by mitochondrial Complex III). ·O2− production also increased during hyperoxia (20(95%O2), but to a lesser extent than during hypoxia, and was not blocked by myxothiazol. We propose that NTS neurons undergo redox stimulation and/or stress via increased ·O2− production during both hypoxia (Complex III) and hyperoxia (Complex I ?). We also hypothesize that the smaller increase in δFIUDHE/min during hyperoxia reflects increased production of ·NO during hyperoxia, which reacts with ·O2− (and thus consumes ·O2−) to yield peroxynitrite. ONR N000140710890, NIH R01 HL 56683‐10
Differential intracellular localization of Sry proteins expressed in Rattus norvegicus
Authors: Underwood Adam C , Turner Monte E. , Ely Daniel L. , Milsted Amy
Published: June 2021

Sry is a transcription factor with a conserved HMG domain that facilitates DNA interactions and nuclear localization, through amino (n) and carboxy (c) terminal nuclear localization sequences (NLS). There are seven Sry loci on the SHR/Akr Y chromosome. Amino acid (aa) differences among Sry proteins are in the nNLS at aa 21 (His vs. Arg), cNLS at aa 76 (Pro vs. Thr), and in the presence/absence of 13 aa in a c‐ terminal activation motif. The objective of this study was to determine if these aa differences between proteins alter nuclear localization. Native, chimeric, mutated and truncated Sry sequences were transfected into CHO cells followed by immunocytochemistry and Western blot analysis of nuclear and cytoplasmic CHO cell fractions. Results showed that Sry2 exhibits both cytoplasmic and nuclear accumulation, while Sry1 and 3 localize only to the nucleus. Sry1 and 3 sequences containing the truncated or deleted activation region exhibit no reduction in nuclear localization, while mutations to the NLS result in cytoplasmic accumulation. Changing His to Arg at aa 21 of Sry1 or Sry3 results in nuclear localization patterns equivalent to native Sry2. Results demonstrate that aa variability in Sry proteins leads to different intracellular localization in vivo and may account for the different phenotypes observed in rats after delivery of exogenous Sry1, Sry2 or Sry3.

Support from NIH HL71579 and the University of Akron.
Characterization of postnatal expression of rat MFG‐E8 gene in intestines, liver and lungs
Authors: Wang Xiao , Bu Heng‐Fu , De Plaen Isabelle G. , Tan Xiao‐Di
Published: June 2021

MFG‐E8 (milk fat globule‐EGF factor 8) plays an important role in tissue remodeling, clearance of apoptotic cells, and intestinal epithelial wound healing. Here, we examined the expression of MFG‐E8 mRNA in the small intestine, liver and lungs during postnatal development. Briefly, Sprague‐Dawley rats were sacrificed at different ages (E20, P0, P5, P10 and P21). Total RNA was extracted from various tissues including the small intestine, liver and lungs. The MFG‐E8 gene expression was quantified by real‐time PCR. We found that the MFG‐E8 gene was expressed at a low level on E20 in the liver. Within 24 h after birth, its expression was increased 2.57 fold (P<0.01). It was then stably expressed at high level during the postnatal period. In contrast, in the small intestine, MFG‐E8 gene significantly decreased 2.88 fold (P<0.01) within 24 h after birth compared to E20, then it gradually regained E20 values on P10. The gene was persistently expressed until P21. In the lungs, MFG‐E8 is constitutively expressed prenatally at E20. Its expression did not change during the postnatal period. In conclusions, our study indicated that MFG‐E8 is extensively expressed in the small intestine, liver, and lungs. As opposed to other organs, the expression of intestinal MFG‐E8 is decreased during the first week of life. However, the physiological meaning of MFG‐E8 gene expression pattern in postnatal stage remains to be determined.
An intact SREBP1 pathway is essential for the t‐10, c‐12 CLA‐induced inhibition of de novo fatty acid synthesis in the murine lactating mammary gland
Authors: Foote Monica R. , Monks Jenifer , Harvatine Kevin J. , Neville Margaret C. , Boisclair Yves R. , Bauman Dale E.
Published: June 2021

Trans‐10, cis‐12 conjugated linoleic acid (CLA) is a potent inhibitor of de novo fatty acid (FA) synthesis in the lactating mammary gland. Inhibition of FA synthesis is accompanied by a coordinated decrease in the expression of key mammary lipogenic genes, including sterol response element binding protein 1 (SREBP1). The functional role of the SREBP1 pathway in CLA‐induced inhibition of milk fat synthesis is unknown. Mice expressing a floxed version of the escort protein SCAP, necessary to activate SREBP pathways, were crossed with mice overexpressing Cre under the mammary specific β‐lactoglobulin promoter. Lactating Cre‐ SCAPfl/fl mice and Cre+ SCAPfl/fl mice were orally dosed with water (n = 8) or with 20 mg of CLA (n = 8) for four days. Litters from Cre+ dams showed decreased growth relative to Cre‐ dams. Analysis of milk composition showed that de novo FA synthesis (C<16:0) was decreased in Cre+ mice. CLA decreased growth of litters from Cre‐ mice but had no effect in Cre+ mice. CLA decreased the proportion of C<16:0 in milk of Cre‐ dams but not in Cre+ dams. In addition, CLA decreased the mammary expression of key lipogenic genes (e.g., SCD1, SREBP1c) in Cre‐ dams but not in Cre+ dams. These results demonstrate that an intact SREBP1 pathway is essential for the t‐10, c‐12 CLA‐induced inhibition of de novo FA synthesis in the mammary gland. Research supported by NRI, USDA grant #2006‐35206‐16643, and by NIH grant #PO1‐HD38129
Diet‐induced obesity impairs the T cell memory response to influenza virus infection
Authors: Karlsson Erik A , Beck Melinda A
Published: June 2021

The global obesity epidemic combined with yearly influenza outbreaks and potential for a pandemic makes it crucial to determine how protection from influenza differs in an obese host. In contrast to a vaccine antibody response which recognizes surface proteins, memory T cells generated during a primary infection target internal viral proteins and are protective against heterologous strains. As our prior work demonstrated that obese mice have an impaired primary response to influenza virus infection, we hypothesized that obese mice would also have an impaired memory response to a secondary influenza infection. To test this hypothesis, obese and lean mice were infected with influenza X‐31. At 31 days post infection, mice were challenged with a lethal dose of a different strain, influenza A/PR/8. While lean mice were protected from lethal infection, obese mice had 25% mortality, increased lung pathology and virus titers, reduced antiviral cytokine expression, and decreased and delayed adaptive immune cytokine expression following secondary challenge. These results indicate that memory response to influenza in obese mice is significantly altered and ineffective and obesity may hinder the generation or function of these protective cells. These results suggest obese populations are at risk for impaired immune memory response, which has significant public health implications for current vaccination protocols.

Grant Funding Source

DK56350
The metabolites of Korean traditional fermented foods and their healthy functions
Authors: Kwon Dae Young , Kim Hyunjin , Kim Min Jung , Yang Hye Jeong
Published: June 2021

The metabolomics approaches for Korean traditional fermented foods are very attracted because they give us two important informatics : microbial metabolomics during fermentation and physiological metabolomics during metabolic digestion. In Korea, recently metabolomics study was studied on the microorganism for Korea traditional fermented foods such as kimchi, sool, chungkukjang, meju/doenjang and kochujang and also we started metabolomics study of Korea traditional fermented foods on animals and humans. Another important metabolomic/metagenomic area in foods is metabolomic/genomic investigation of intestinal microbial flora in human bodies, but this area is not investigated in Korea although many scientists are interested. Here, we are introduced primary microbial metabolites identified from the Korea traditional fermented foods (chungkukjang, meju and kochujang). Kimchi is very most important items for metabolomic study, however, we did not studied due to some restrictions. Also we reported the health effect of Korean fermented soy bean products on anti‐obesity, anti‐inflammation, anti‐diabetes and other healthy effect. In few years, we can discussed physiological metabolites/metabolomes during digestion of Korea fermented foods and nutraceuticals.
Wilms Tumor 1 (WT1) and Foxc2 are required for podocyte development in Xenopus
Authors: White Jeffrey Thomas , Wessely Oliver
Published: June 2021

Single‐gene knockout studies have shown that Foxc2, Lmx1b, MafB, Tcf21 and WT1 are the necessary transcriptional regulators of podocyte development. It is still unclear, however, whether these five transcription factors (TFs) can explain all aspects of podocyte development. To this end, we have used the Xenopus pronephros as a tool to study podocyte development.

To test the roles of these five TFs in podocyte development, each was individually knocked down with antisense morpholino oligomer technology. In situ hybridization for podocyte TFs (Foxc2, Lmx1b, MafB, Tcf21 and WT1) and markers of terminal differentiation (Nephrin, Neph1, Glepp1 and Podocin) was used to analyze the resultant phenotype. These data were compiled to construct a gene regulatory network for podocyte development (PGRN). The PGRN displayed classical characteristics of a gene regulatory network, including master regulators, multi‐gene loops and a high degree of redundancy. Based on the network, we hypothesized that WT1 and Foxc2 are the master regulators of podocyte development. Simultaneous depletion of WT1 and Foxc2 resulted in loss of expression of downstream podocyte TFs (Lmx1b, MafB and Tcf21) and markers of terminal differentiation (Nephrin, Neph1, Glepp1 and Podocin); moreover, these embryos were characterized by a complete loss of the glomus structure. This work was supported by F30DK082121.

Grant Funding Source

NIDDK
Fgfs and organ regeneration in zebrafish
Authors: Poss Kenneth , Lee Yoonsung , Wills Airon , Yin Viravuth
Published: June 2021

Appendage regeneration in salamanders and fish occurs through formation and maintenance of a mass of progenitor tissue called the blastema. A dedicated epidermis overlays the blastema and organizes its proliferation and patterning events, yet this regulation is poorly understood. Here, we dissected epidermal influences during zebrafish fin regeneration using inducible loss‐of‐function and gain‐of‐function transgenes that manipulate Fgf signaling. Our results identify positive and negative regulation that confines potent morphogenetic factors to specific epidermal territories in regenerating zebrafish fins.
Regulation of satellite cell proliferation and adult skeletal muscle regeneration by the Wnt family of signalling molecules
Authors: Patel Ketan
Published: June 2021

Satellite cells represent the stem cell population of adult skeletal muscle. These relatively rare cells are normally maintained in a quiescent, mitotically inactive state. However, during phases of muscle growth or regeneration, they start to divide not only give rise to myoblasts that eventually form muscle but also to daughter cells that replenish the stem cell pool. We have a poor understanding of how the small myoblast population that develops after satellite cell activation is expanded to generate numbers required to repair injured tissue. In this study we show that members of the Wnt family of secreted proteins have a profound, identity specific, influence on satellite cell proliferation. Wnt1, 3a, or 5a protein cause a dramatic increase in satellite cell proliferation. In contrast, exposure of satellite cells to Wnt4 or 6 diminishes this process. A key mechanism dictating whether a satellite cell undergoes proliferation or not is the intracellular localisation of Act‐β ‐Cat. We show that proliferation enhancing Wnt proteins induce premature proliferation along with Act‐β ‐Cat nuclear translocation. Finally, we provide evidence that the dynamics of cellular Act‐β ‐Cat translocation observed during in vitro culture are found in cases of either acute or chronic skeletal muscle regeneration in rodents and humans. In summary, we propose that Wnt proteins may be key factors acting to regulate the rate of satellite cell proliferation on adult muscle fibres during the wound healing response.
Integration, Canalization and Malformation: A conceptual framework for relating phenotypic variability and dysmorphology
Authors: Hallgrimsson Benedikt
Published: June 2021

The measurement and analysis of phenotypic variation within populations is a central concern within evolutionary biology. In developmental biology, however, variation tends to be a peripheral concern and is often viewed as a nuisance that confounds clear experimental results. Recently, the subject of variation has attracted mainstream attention within developmental biology under the headings of phenotypic heterogeneity, genetic background effects and incomplete penetrance. These concepts relate to an established conceptual framework within evolutionary biology which concerns the analysis of phenotypic variability. The central concepts within this field, developmental or morphological integration, modularity, canalization, and developmental stability, all have direct relevance to understanding dysmorphology in humans and animal models. Phenotypic variability is often dramatically increased in mutants compared to the wildtype and phenotypic heterogeneity is a poorly understood component of many human dysmorphologies. Using examples from our own work on cleft lip, we argue that asking questions focused on developmental determinants of canalization and integration does lead to mechanistic insights that might otherwise be missed. In this case, as so often, novel insights can emerge from combining the theoretical perspectives of different fields.
Genetics and environment interact synergistically to modulate craniofacial disease phenotypes
Authors: Eberhart Johann , Swartz Mary
Published: June 2021

Craniofacial disease can result from genetic or environmental factors and disease phenotypes can vary greatly between individuals with common underlying factors. A cause of this variability is gene‐environment interactions; our objective is to determine the nature of these interactions. We performed an "interaction screen", where we treated zebrafish embryos carrying mutant copies of genes that regulate craniofacial development with ethanol, a teratogen known to cause craniofacial defects. We found ethanol strongly interacts with the pdgfra locus. Under normal conditions, homozygous pdgfra mutants have cleft palate while pdgfra heterozygotes develop normally. Following ethanol treatment, pdgfra homozygous mutants lose the majority of the craniofacial skeleton and pdgfra heterozygotes have palatal defects, displaying latent haploinsufficiency. Importantly, the phenotype of ethanol treated fish does not appear to be due to the same mechanism causing defects in untreated pdgfra mutants. Untreated pdgfra mutants have cleft palate due to the failure of crest cells to appropriately migrate to the oral ectoderm. However, craniofacial defects in ethanol treated pdgfra mutants and heterozygotes appear to be due, at least partly, to an increase in cell death. Our results suggest that gene‐environment interactions can be synergistic, adding complexity to the etiology of craniofacial disease.
Generation of Functional Humanized Liver in Sheep by Bone Marrow Cells
Authors: Zanjani Esmail D , Porada Christopher D , Almeida‐Porada Graca
Published: June 2021

Hepatic potential of human hematopoietic stem cells were assessed in the fetal sheep model. Human hepatocytes (HH) comprised 5‐10% of total hepatic cells and were present in numerous sites (~13 sites/section examined). Within each site, HH represented > 80% of total hepatic cells. These HH retained functional properties of normal hepatocytes, constituted hepatic functional units with the presence of human endothelial and biliary duct cells, and secreted human albumin. A direct correlation was found between HH activity, cell dose administered, source of cells used, and time post‐transplant. Human hepatocytes were isolated using a c‐met antibody. Overall, 8% of the cells in chimeric livers were c‐met+, but only a small fraction were of human origin. Culture of chimeric sheep liver cells resulted in isolation of foci of HH that could be maintained/expanded for up to 3 months. The cells were CK19 positive, were able to produce human albumin in culture but were cytochrome P450 negative. These results suggest that transplantation of human HSC or MSC in the fetal sheep model, results in efficient production of large numbers of human hepatocytes that may be harvested and maintained/expanded in vitro.
Regulation of endocytic vesicle targeting by Rab GTPases and phosphoinositides
Authors: Lambright David
Published: June 2021

Regulation of endosomal trafficking by the monophosphoinositide PI3P and Rab GTPases is mediated by effector proteins, including Rabenosyn‐5 and EEA1. Both proteins contain a FYVE domain, which directs membrane targeting through interaction with PI3P, and a C2H2 zinc finger, which binds selectively to GTP‐bound Rab5. To gain insight into the structural basis for Rab recognition and endosome tethering, we determined the structure of the EEA1 C2H2 zinc finger in complex with GTP‐bound Rab5. Despite a topology identical to the classic DNA‐binding module, the EEA1 C2H2 zinc finger recognizes Rab5 through a binding modality distinct from that observed for DNA recognition. In contrast, the mode of recognition is unexpectedly similar to that of the helical hairpin Rab binding modules in Rabenosyn‐5, even though the C2H2 zinc finger and helical hairpins are structurally unrelated. Mutational analyses further implicate a common set of conserved and variable determinants in Rab5. These observations provide evidence supporting convergent evolution of a common recognition modality for structurally distinct Rab binding modules as a consequence of selective pressure to bind specifically to Rab5.
The inclusion of milk fortified with micronutrients increases the effectiveness of a calorie restricted diet to reduce obesity in Women
Authors: Rosado Jorge Luis , García Olga Patricia , Ronquillo Dolores , Hervert Deisy , Rodríguez Guadalupe , Gutierrez Jessica , Caamaño María del Carmen , García Alina , Garcia Sandra
Published: June 2021

The effect of milk consumption on weight loss, body composition, lipids profile and blood pressure was evaluated in a randomized, double‐blind, controlled longitudinal study. 139 women (34 ± 6 y) were assigned to one of the following groups: a) 250mL of low‐fat milk (LFM) 3‐times/day and a calorie restricted diet (‐500 kcal); b) 250mL of low‐fat milk added with micronutrients (LFM+M) 3‐times/day and a calorie restricted diet (‐500 kcal); c) calorie restricted diet (CRD)(‐500 kcal). All treatments were administered for 16 weeks. Weight, height, hip and waist circumferences were measured at 0,4,8,12 and 16 weeks. Body composition and blood pressure were evaluated at 0 and 16 weeks. A fasting blood sample was taken at 0 and 16 weeks to determine lipids profile. 107 women finished the study. ANOVA adjusted for age, initial value and community was used to evaluate the treatment effect. After 16 weeks, LFM+M had significantly more weight loss (‐5.81±3.24 Kg) compared to LFM (‐4.06±3.27 Kg) and CRD (‐4.06±3.25 Kg) (p<0.05). BMI change in LFM+M (‐2.61±1.40 Kg/m2) was significantly different to LFM (‐1.75±1.40 Kg/m2) and CRD (‐1.67±1.39 Kg/m2)(p<0.05). % body fat changes in LFM+M (‐2.67±1.66%) was significantly different to LFM (‐1.79±1.68%) and CRD (‐1.63±1.66%)(p<0.05). No differences were found between groups in waist circumference, blood pressure, and lipids profile. In conclusion, three daily servings of LFM+M together with a calorie restricted diet could be an effective strategy for the treatment of obesity in adult women.
Milk consumption after resistance exercise increases fat loss and increases muscle mass and strength gains in young women
Authors: Josse Andrea R , Tang Jason E , Wilkinson Sarah B , Tarnopolsky Mark A , Phillips Stuart M
Published: June 2021

Post‐resistance exercise consumption of fat free milk in men promoted greater lean mass accretion and fat mass loss than soy or carbohydrate. It is unknown if similar body compositional changes and strength gains occur in women. Twenty young women were randomized to drink 500ml of fat‐free milk (MILK: n=10; BMI=26.2±2.0kg/m2; mean±SE) or carbohydrate (CON: n=10; BMI=25.6±1kg/m2) on 2 occasions; immediately post and 1h post‐exercise. Subjects underwent resistance training 5d/wk for 12‐wk. Body compositional changes were measured by DXA, and subjects' strength was measured by 1RM pre‐ and post‐training. Baseline body mass did not differ between groups and only CON gained weight after training (CON: 68.3±4.1kg to 69.1±4.0kg, P=0.05; MILK: 72.0±4.1kg to 72.5±3.8kg, P=0.24). Lean mass increased with training in both groups (P< 0.01), with a greater net change in MILK vs CON (1.9±0.2kg vs 1.1±0.2kg, respectively; P<0.01). Fat mass decreased with training in MILK only (‐1.64±0.4kg, P<0.01) with no change in CON (‐0.27±0.3kg, P=0.47). Bench press and tricep push‐down strength increased more in MILK than CON (P<0.05). Thus, post‐exercise consumption of milk vs isoenergetic carbohydrate resulted in greater muscle mass accretion, fat mass loss and strength gains in women after 12‐wk of resistance training. Our results parallel those shown previously in men.

Sponsored by NSERC, CIHR and The Dairy Farmers of Canada

Grant Funding Source

CIHR
Impact of Pistachio Nut Consumption on Triglycerides in Obese Subjects over 12 weeks
Authors: Li Zhaoping , Song Rubens , Nguyen Christine , Zerlin Alona , Naowamondhol Kris , Karp Hannah , Thames Gail , Kotlerman Jenny , Heber David
Published: June 2021

There is a widely held view that nut consumption due to its high fat content leads to weight gain and unhealthy lipid profiles. In order to study the effects of pistachio snack consumption in obese subjects under real world conditions, subjects were randomly assigned to two weight reduction diets with a 500 Cal deficit diet based on RMR using whole foods and including an afternoon snack of 3 ounces (240 Cal) of pistachios (PS, n = 27) or two ounces of pretzels (PR, n=26) (220 Cal) over 12 weeks. Both groups lost weight during the 12 week study (time trend p<0.001) and there were no differences in weight loss between the pretzel and pistachio groups (PS 189.6 ± 5.2 to 181.0 ± 6.2 vs. PR 189.8 ± 4.0 to 182.5 ± 4.4 lb). At 6 weeks, triglycerides were significantly higher in the PR group compared to the PS group (132 ± 18 mg/dl vs. 90 ± 9 mg/dl, p< 0.04) and a time trend difference was noted between the two groups over the 12 weeks (p<0.01). There were no significant differences in body fat, lean body mass, cholesterol, HDL, insulin, glucose or blood pressure between the groups. Therefore, by comparison to refined carbohydrates, pistachios may have beneficial effects on blood lipids. Despite claims that energy‐dense foods promote weight gain, pistachios can be consumed as a snack to enhance diet quality and palatability by individuals following a weight loss diet without fear that the ingestion of 3 ounces of pistachios per day will impair weight loss.
Effects of altered zinc availability on proliferation and oxidative stress in cultured cells
Authors: Sankavaram Kavitha , Grattan Bruce J , Walker Robin , Park Hea Jin , Freake Hedley C
Published: June 2021

Previous studies from our laboratory have demonstrated that primary and transformed cells differ in their homeostatic response to zinc deprivation. Altered zinc availability might also influence cellular proliferation and antioxidant defenses. In the present study, we examined the effects of zinc supplementation (15‐100 µM) and zinc deficiency induced by diethylenetriaminepentaacetic acid (DTPA, 25‐200 µM) on cell proliferation and reactive oxygen species (ROS). DTPA (50 µM) reduced cell proliferation in both MCF‐7 and MDA‐MB‐231 human breast cancer cells, but effects were greater and seen more quickly in MCF7 cells. 50 µM DTPA also reduced proliferation of H4IIE rat hepatoma cells, but did not affect cell number in primary hepatocytes. Zinc treatment (up to 50 µM) enhanced cell proliferation in MCF‐7 but not in MDA‐MB‐231 cells. It also increased proliferation of H4IIE cells, whereas a decline in cell number was found with primary hepatocytes. Cells were also observed for the generation of ROS. 48 hr incubation with DTPA significantly increased ROS in primary hepatocytes and MCF‐7 cells but not in H4IIE or MDA‐MB‐231 cells. Zinc induced ROS in both MCF‐7 and MDA cell lines but not in primary hepatocytes. Overall, while cellular responses differed, reduction of available zinc limited proliferation and addition stimulated growth. In some cells ROS were enhanced under both circumstances.
Effects of zinc supplementation on growth of children under 5 years of age: A meta‐analysis of randomized controlled trials
Authors: Nguyen Phuong , Ramakrishnan Usha , Martorell Reynaldo
Published: June 2021

Background

Zinc interventions have received much attention as a cost‐effective and promising strategy to improve child health, but their roles in preventing growth retardation in early childhood remain unclear.

Objective

Meta‐analyses of randomized controlled trials were conducted to evaluate the effect of zinc interventions on the growth of children < 5 y old.

Design

Eligible studies were identified by PubMed database search and other methods. Weighted mean effect sizes and 95% CI were calculated for changes in height, weight and weight‐for‐ height Z‐scores (WHZ) using random effect models. Tests for homogeneity and stratified analyses by predefined characteristics were conducted using meta‐regression techniques. Tests for publication bias were done using funnel plots.

Results

Forty‐three trials (56 datasets) had relevant information. There was no evidence of publication bias. Zinc interventions had a small positive effect on change in WHZ (effect size: 0.06; 95% CI: 0.006, 0.11) but no effect on height or weight gain (effect size: 0.07; 95% CI: ‐0.03, 0.17 and 0.06; 95% CI: ‐0.10, 0.23, respectively). The results were similar across categories of age, duration of intervention, and baseline anthropometric status.

Conclusions

Contrary to the earlier findings, we failed to detect any benefits of zinc supplementation in improving early childhood growth. Other strategies are needed to prevent stunting.
CD11d expression is dramatically increased in white adipose tissue of obese rodents
Authors: Thomas Anthony , Oort Pieter J. , Newman John W. , Dunn Tamara N. , Knotts Trina A. , Stern Judith S. , Stanhope Kimber L. , Havel Peter J. , Adams Sean H.
Published: June 2021

Macrophages (MΦ) infiltrate white adipose tissue (WAT) with increasing adiposity and are primarily localized to necrotic adipocytes. Mechanisms underlying WAT MΦ recruitment, tissue migration/retention, and function in obese animals is unclear. Integrins are cell adhesion proteins critical for leukocyte extravasation, migration, and phagocytosis. CD11d encodes the α‐subunit of the integrin αDβ2 and is expressed predominantly on monocytes/MΦ; however, its role in systemic and WAT inflammation is poorly understood. We have demonstrated for the first time that CD11d expression in retroperitoneal (RP) WAT is increased dramatically (by >200 fold) in obese female Zucker rats (n=7) compared to lean controls (n=9), further confirmed in a polygenic obese SD male rat model (~70 fold). To assess whether this upregulation in WAT is the result of obesity‐induced inflammation or generalized inflammation outside the context of obesity, we measured CD11d expression in RP WAT of non‐obese mice administered lipopolysaccharide (LPS) at 0.5 mg/kg (5 day; n=5) vs. vehicle‐treated controls. CD11d expression was increased by LPS treatment, but the fold increase (~3.6 fold) was far less than observed in obesity. Our results are consistent with the hypothesis that CD11d plays a critical role in WAT MΦ migration, tissue retention, and or phagocytosis of cellular debris and lipid from necrotic adipocytes in obesity.

Grant Funding Source

Western Human Nutrition Research Center
Web‐based dietary assessment software for 24‐hour recall interview
Authors: Park Eunyoung , Shin Sangah , Hwang Soochan , Lee Kyo‐Joon , Lee Myung‐Joo , Joung Hyojee
Published: June 2021

The purpose of this study was to develop computerized dietary assessment software for 24 hr recall interview and evaluate its applicability. Subject management system, standardized interview protocol, food composition table, and recipe databases were established for this program. The client software, developed using Microsoft's ClickOnce technology, can be distributed over the internet and automatically installed on user's PC. The client software communicates with the database system via http server to insert or retrieve its data. The steps of operation consist of subject and researcher tracking, input, calculating, output, and evaluation system. To evaluate the feasibility, 191 adults aged between 24 and 68 participated 24 hr recall interview with software and regular 24 hr recall interview using a questionnaire. On‐line interview took 9.7±4.5 min for one day recall and regular interview did 7.5±3.2 min. There were no significant differences in preferences, satisfaction, and ease of response between two methods. The results indicate that the web‐based software was well accepted to

the subjects and can reduce time‐consuming and error‐causing coding work significantly. Further validation studies for the program could prove usefulness of the software. (This study was supported by a grant of the Korea Centers for Disease Control and Prevention (2007‐E00034‐00) and by the Brain Korea 21 Project in 2008.)
Adolescent' ability to correctly identify foods up to fourteen hours postprandial when prompted with an image of a meal
Authors: Schap TusaRebecca E. , Six Bethany L. , Kerr Deborah A. , Delp Edward J. , Ebert David S. , Boushey Carol J.
Published: June 2021

Mobile telephones are widely used throughout the world and can provide a unique mechanism for collecting dietary information that reduces burden on record keepers. Our belief is that allowing the user to interact with the mobile phone Food Record (mpFR) after the eating occasion removes one layer of burden while maintaining accuracy for nutrient analysis. The purpose of this study is to investigate adolescents' ability to correctly identify foods up to fourteen hours postprandial when prompted with an image of their meals. Fifteen adolescents (ages 11‐18 y) were provided lunch and dinner meals. The participants were asked to identify each food (fifteen foods total) in the image up to fourteen hours after the respective meal. One hundred percent of participants exactly identified five of the fifteen foods, i.e., milk, spaghetti, peaches, ketchup, and french fries. The other foods were identified within their same respective major food groups. For example, 80% of participants identified lettuce as salad and 33% labeled Coke as soda, pop, or cola. Further probing programmed into the mpFR would lead to an accurate identification of these food items. The results provide evidence that it is feasible to delay user interaction with the mpFR and still obtain accurate food identification.

Grant Funding Source

NIH‐NIDDK & NCI
Regulation of Neuron Death in Neonatal Hypoxia‐Ischemia
Authors: Shacka John J , DeWitte Cary , Roth Kevin A , Ness Jayne M
Published: June 2021

Hypoxia ischemia (HI) is a major cause of neonatal brain injury and often results in long‐term neurological disability. We have shown that the pro‐apoptotic molecules such as Bax critically regulate neuronal injury following HI. Alterations in the autophagy‐lysosome pathway (ALP) are known to regulate neuron death following neonatal HI and our goal is to delineate mechanisms by which ALP regulates neuron death following neonatal HI. Postnatal day 7 B57BL6/J mice underwent unilateral carotid ligation followed by 45 min exposure to 8% oxygen, and were euthanized 3‐48h later. Brain samples were processed either for IHC or western blot analysis. Within 12h after neonatal HI, intense cytosolic CD immunoreactivity was observed in the ipsilateral hippocampus and peaked at 24‐48h, suggesting induction of lysosomal membrane permeabilization. Increased CD levels were confirmed by western blot analysis. Cytosolic CD co‐localized with NeuN and cleaved caspase‐3 but not p53 in the ipsilateral hippocampus. Bax deficiency attenuated cleaved caspase‐3 immunoreactivity but this attenuation was incomplete, suggesting an additional role for the extrinsic apoptotic pathway in neonatal HI. These results suggest the existence of multiple neuron death pathways following neonatal HI and studies are ongoing to determine if these pathways are regulated through alterations in the ALP.
Proteinase‐activated receptor (PAR)‐2 stimulates cyclooxygenase‐2 (COX‐2) expression in Caco‐2 colon cancer cells: role of matrix metalloproteinases (MMPs)
Authors: Hirota Christina L , Wang Hongying , MacNaughton Wallace K
Published: June 2021

Colon cancer is associated with elevated levels of trypsin, an activator of PAR2; PAR2 activation stimulates MMP‐9 release and COX‐2 expression, which are both involved in inflammation and cancer. We hypothesize that MMPs participate in PAR2‐induced COX‐2 expression in Caco‐2 colon cancer cells. Cells were treated for 3hr with PAR2#‐activating peptide (fLI) with or without a pan‐MMP inhibitor. Concentrated, conditioned media from treated cells were used to treat separate cells. Other cells were treated directly with activated MMP‐9 or tissue inhibitor of metalloproteinase (TIMP)‐1, an endogenous MMP inhibitor with greater selectivity for MMP‐9. COX‐2 expression was assessed by western blot. Secreted MMPs were analyzed by zymography. The pan‐MMP inhibitor decreased fLI‐induced COX‐2 expression in Caco‐2 cells. Concentrated, conditioned media from fLI‐treated cells induced COX‐2 expression in separate cells. TIMP‐1 did not suppress PAR2‐induced COX‐2 expression nor did direct treatment with MMP‐9 increase its expression. Caco‐2 cells secrete gelatinases and casein‐degrading MMPs. It is unclear yet whether PAR2 activation alters the secreted MMP profile. In summary, PAR2 activation stimulates COX‐2 expression via a MMP other than MMP‐9 in Caco‐2 cells. Understanding this pathway may help define molecules to target in the progression from inflammation to cancer. Funded by CAG/CIHR/Abbott.
Direct detection of nanoparticles and hMW DNA in whole blood “seamless sample to answer diagnostics”
Authors: Heller Michael
Published: June 2021

In medical research and diagnostics, it is a significant challenge to directly isolate and identify rare cells, biomarkers such as high molecular weight DNA nanoparticulates and immunocomplexes, and drug delivery nanoparticles directly in blood. We have now demonstrated the isolation and detection of nanoparticles and high molecular weight DNA (hmw DNA) from whole blood using a microarray dielectrophoretic (DEP) device. Both fluorescent nanoparticles and fluorescent‐stained hmw DNA in undiluted whole blood samples were separated and held in DEP high field regions and then detected after the blood cells were removed by a fluidic wash. In buffy coat blood, with reduced cell numbers, 40nm nanoparticles concentrated into the DEP high field regions while the blood cells concentrated into the DEP low field regions. Overall, this work sets the stage for a new generation of "seamless" sample to answer diagnostic systems; i.e., a process where a complex sample is run through a device, and the specific analytes are rapidly concentrated onto microscopic locations and subsequently detected. Such devices will allow highly complex clinical and other biological samples such as blood, plasma and serum to be rapidly and directly analyzed for rare cells, DNA biomarkers and drug delivery nanoparticles. Work supported by the NIH‐NCI Center Grant (U54‐CA119335).