bluuebunny/crossref_metadata_2025 huggingface dataset:
Fis1, Mff, MiD49 and MiD51 facilitate Drp1 recruitment for mitochondrial fission
Authors: Losón Oliver Calvin , Chen Hsiuchen , Chan David C.
Published: June 2021

Several mitochondrial outer membrane proteins—Fis1, Mff, MiD49, and MiD51—have been proposed to promote mitochondrial fission by recruiting the cytosolic GTPase Drp1, but fundamental issues remain concerning their function. A recent study supported such a role for Mff, but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1‐null, Mffnull, and Fis1/Mff‐null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. We employ several independent, quantitative techniques to show that these cell lines have significantly different mitochondrial elongation phenotypes. Using a specialized image analysis technique, we find that Fis1 and Mff are important for regulating the number and size of Drp1 fluorescent puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. We also demonstrate that a significant enhancement of Drp1 phosphorylation at S637 is causal for the elongation phenotype during MiD overexpression. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission, and suggest that these proteins may function in parallel pathways. This work was supported by the National Institutes of Health (GM062967). OCL was supported by a R. L. Kirschstein National Research Service Award (5F31GM089327).
Identification of Lipid Droplet Structure‐like Proteins and Their Function on Lifespan of Caenorhabitis elegans
Authors: Liu Pingsheng , Na Huimin , Zhang Peng
Published: June 2021

Storage of neutral lipids in lipid droplets (LDs) has been linked to many metabolic syndromes in humans, but formation, dynamics, as well as functions of the organelle remain elusive. C. elegans is a useful animal model to study lipid metabolism and storage, especially to conduct genome‐wide screening. But lack of LD marker or/and structure‐like proteins such as mammalian Perilipin family proteins (PLINs) in C. elegans have made the research very difficult. We recently isolated LDs from several strains of C. elegans, carried out comparative LD proteomic studies, and identified 154 LD‐associated proteins including three LD structure‐like proteins. We then determined tissue distributions of these three proteins and found that one was located in intestinal cells and another one was expressed almost all tissues. Interestingly, we identified their LD targeting domains and found that they could also target to LDs of mammalian cells. In addition, deletion or/and reduction of two of them significantly shortened lifespan of C. elegans and their effect on lifespan was not Daf‐16 dependent.
Localization of Opi1pFFAT to lipid droplets during inositol starvation in Saccharomyces cerevisiae
Authors: Chang Yu‐Fang , Gaspar Maria , Henry Susan A.
Published: June 2021

Phosphatidic acid (PA) is a signaling lipid that plays an important role in mediating phospholipid biosynthesis. However, the subcellular distribution of PA remains elusive. In this study, PA levels were manipulated by shifting cells from inositol‐containing medium to inositol‐lacking medium. A mutated form of the Opi1p repressor, Opi1pFFAT, which loses the interaction with the endoplasmic reticulum (ER) protein Scs2p but contains a PA‐binding domain, relocalizes to lipid droplets (LDs) after 2‐h of inositol starvation. Opi1pFFAT localization was also monitored in mutant cells with defects in triacylglycerol (TAG) metabolism, including pah1Δ and lro1Δdga1Δ for TAG biosynthesis, and tgl3Δtgl4Δtgl5Δ for TAG mobilization. Opi1pFFAT localized to LDs in the tgl3Δtgl4Δtgl5Δ mutant, but did not localize to LDs in cells unable to synthesize TAG. Another mutated form of Opi1p, Opi1pFFAT/PAc4m, which carries mutations in both FFAT and PA‐binding domains, was also examined. Opi1pFFAT/PAc4m did not localize to LDs, but remained in the nucleus. These results suggest Opi1pFFAT binds to a specific pool of PA that is actively involved in TAG synthesis. Fld1p, a human seipin homolog in yeast that binds to the junction of LDs and ER, was shown to colocalize with Opi1pFFAT followed by 2‐h inositol starvation, suggesting a pool of PA to the sites of LD biogenesis. Supported by NIH grant GM019629.
The Differentiation of the Plasma Membrane
Authors: Levental Ilya , Levental Kandice , Simons Kai
Published: June 2021

The structure of the plasma membrane is a major determinant of its function, and while recent advances have provided key insights into this relationship, major open questions remain. For example, the diversity of plasma membrane structure and composition in various cell lineages, and the impact of this diversity on cell functions have not been widely explored. Mesenchymal stem cells provide an ideal system to study such changes because they can be directed to undergo differentiation along several lineages (e.g. adipocyte, osteocyte) within a short timeframe (~2 weeks) and with minimal biochemical perturbations. Giant Plasma Membrane Vesicles isolated from the various lineages allow for simultaneous quantification of plasma membrane order, composition, and liquid‐liquid separation into raft and non‐raft domains. We find striking differences in phase separation and overall membrane packing between different cell lineages and correlate them to the differentiation of the plasma membrane lipidomes measured by quantitative, comprehensive shotgun mass spectrometry. In addition to being the first to compare plasma membrane properties and compositions during differentiation, these observations also enable identification of the compositional determinants of the physical properties of biological membranes.
Analysis of the Oligomeric State of the Mating ATPase ConE and its Arginine Finger Mutant
Authors: Morton Georgeanna Marie , Swerdlow Kyle , Berkmen Melanie
Published: June 2021

During conjugation, mating pores allow bacteria to transfer DNA to recipient cells. ConE is one of several proteins that make up the mating pore of Bacillus subtilis. ConE, and its‐characterized homologs VirB4 and TrwK, belongs to the FtsK‐HerA superfamily of ATPases which contain a conserved arginine finger that is a good predictor for ring formation and inter‐protomer interactions. Several superfamily members have been purified and characterized in vitro; they form monomers, dimers, and higher order oligomers in solution and generally form hexamers when crystallized. We tested if ConE oligomerizes and how the mutation of ConE's arginine finger affects its oligomerization. His6‐ConE and mutant His6‐ConE (R726A) were purified via nickel affinity chromatography. Purification was monitored via SDS‐PAGE and multimeric states were determined by BN‐PAGE. We found that both wild type and mutant His6‐ConE form monomers, dimers, and higher oligomers. Addition of ATP, single‐stranded DNA, and double‐stranded DNA did not dramatically alter the oligomeric states.
A defined glycosaminoglycan‐binding surface facilitates endoderm differentiation of human embryonic stem cells
Authors: Hernandez Brandon A , Wrighton Paul J. , Klim Joe R. , Kiessling Laura L.
Published: June 2021

To utilize the full potential of human embryonic stem (hES) cells, defined, reproducible culture and differentiation conditions need to be developed. In previous work, we showed a defined glycosaminoglycan (GAG)‐binding surface can be used for the long‐term culture of hES cells. Here, we show that surfaces presenting the peptide GKKQRFRHRNRKG support efficient differentiation of hES cells to definitive endoderm. The conditions using this synthetic peptide‐modified surface are an improvement over current methods, not only because the surfaces are defined, but also because the efficiency of differentiation is enhanced. Specifically, hES cells cultured on the GAG‐binding surface express significantly more Sox17+ (a transcription factor that serves as a marker of definitive endoderm) than do hES cells differentiated on the undefined surface Matrigel. The superiority of the GAG‐binding surfaces for endoderm differentiation underscores that substrata that engage cell‐surface polysaccharides can be highly effective for hES cell expansion and differentiation. This research was supported by the National Institutes of Health and the University of Wisconsin Hilldale Undergraduate Research Fellowship.
A2b adenosine receptor regulation of adipocyte precursor fate and lineage determination: identification of a novel link to the stem cell factor KLF4
Authors: Eisenstein Anna , Ravid Katya
Published: June 2021

Adenosine signals via the A2b adenosine receptor (A2bAR) to activate adenylyl cyclase. Previously we found that mesenchymal stem cells, precursors to osteoblasts and adipocytes, from A2bAR knockout (KO) mice have reduced capacity for osteoblast differentiation. Thus, we hypothesized that the A2bAR regulates adipogenesis. The stromal‐vascular fraction of mouse fat, which contains adipocyte progenitors, was induced to differentiate into adipocytes. We found that the expression of the A2bAR decreases substantially during adipogenesis. Bay 60–6583, an A2bAR specific agonist, inhibits adipocyte differentiation as shown by reduction in expression of fat differentiation markers and the number of mature adipocytes. In search for mechanisms, a novel link was identified between A2bAR signaling and upregulation of the stem cell factor, KLF4, in association with cellular proliferation. Adipocyte precursor proliferation in A2bAR KO mice differed from wild type mice following high fat diet feeding as visualized by in vivo BrdU labeling. These findings demonstrate a novel function of the A2bAR in adipocyte differentiation and positions this receptor as an important mediator of stem cell fate.
Dynamic regulation of VEGF‐A signaling by co‐receptors and the extracellular matrix
Authors: Teran Madelane , Buczek‐Thomas Jo Ann , Nugent Matthew A
Published: June 2021

Angiogenesis is a highly regulated process orchestrated by the complex vascular endothelial growth factor (VEGF) system, which is comprised of multiple isoforms and cell surface receptors. In addition, heparin/heparan sulfate (HS) proteoglycans and neuropilins (NRP) have been described as co‐receptors. In the present study we have characterized molecular interactions between the two most commonly expressed VEGF isoforms and their receptors and co‐receptors, using surface plasmon resonance (SPR). Our data suggest that heparin/HS plays different roles in the regulation of VEGF‐VEGFR complex formation depending on the receptor type. VEGFR2 is able to interact with VEGF165 bound by heparin, but interacts very weakly with heparin. VEGFR1 and NRP1 on the contrary, bind heparin independently of VEGF165. It appears heparin/HS play selective roles within each complex and could be key players in determining VEGF cellular response. NRP1 on the other hand, can interact directly with VEGFR1 and 2, alone or as a complex with VEGF165. Our results are also correlated to MAPK pathway activation in response to VEGF in endothelial cells. We propose that HS/heparin and NRP1 dictate the specific receptor type activated by VEGF and ultimately determine the biological response of the VEGF system. Supported by HL088572 and AHAF M2012014.
AKT2‐signaling involves transient protein interactions
Authors: Bottermann Katharina , Reinartz Michael , Gödecke Axel
Published: June 2021

AKT is a protein kinase with widespread functions in cell metabolism, cell growth and apoptosis. Despite their high sequence homology of approx. 80 % the three AKT‐isoforms AKT 1, 2 and 3 seem to have distinct and specific functions. To analyze individual functions of the AKT2‐isoform, we examined protein interactions of AKT2 using a combination of tandem affinity purification (TAP) and quantitative mass spectrometry. AKT2 was fused with a 8 kDa TAP‐Tag and stably expressed in HEK 293 cells. Purification with AKT2‐Tag revealed Heat Shock Protein 90 (HSP 90), 78kDa glucose‐regulated protein (GRP 78), Elongation factor 2 (EF2) and Enolase 1 (ENO1) as AKT2‐interacting proteins. Using SILAC we analyzed the complex stability and discovered that AKT2 is stably bound only to the HSP 90/cdc37‐complex. Other proteins like GRP78 or Tubulin showed a low affinity binding to AKT2. In addition we could verify EF2 and ENO1 as new AKT2‐specific protein interaction partners using the proximity ligation assay (PLA) as an independent method. Taken together our data reveal that transient protein interactions play an important role for AKT2‐signaling. The newly discovered AKT2‐binding partners point to new AKT2‐mediated effects in glycolysis (ENO1) and protein synthesis (EF2).
Time‐resolved quantitative analysis of human milk proteome reveals developing milk and mammary‐gland functions during the first year of lactation
Authors: Zhang Qiang , Cundiff Judy K , Maria Sarah D , McMahon Robert J , Woo Jessica G , Davidson Barbara S , Morrow Ardythe L
Published: June 2021

In‐depth understanding of the developing function of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of HM proteome. We analyzed the HM proteome (n =10 women with samples at 1, 4, 12, 24, 36 and 52 weeks) using a quantitative proteomic approach. 1147 proteins were identified with 580 being quantified. Principle component analysis showed that HM proteomes transition throughout the first year of lactation. Abundance changes of IgG, sIgA and IgM display distinct features during the first year. Complement components and other acute‐phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that milk fatty‐acid (FA) metabolism shifts from more direct blood intake to more de novo mammary synthesis, which may alter the lipoprotein profile in lactating women and the composition of milk FAs. Glycoprotein metabolism transitions to catabolism in late lactation, which may increase the presence of free anti‐pathogenic HM oligosaccharides and peptides. Increased metabolic activity in late lactation can lead to a larger amount of deleterious by‐products. Cellular detoxification machinery is also transformed, thereby accommodating increased metabolic activities in late lactation. The multiple functions of the HM proteins become more apparent from this study.
Inflammatory cytokines in human milk are inter‐correlated and may be related to infant growth characteristics and maternal weight status
Authors: Young Bridget E. , Morrow Ardythe L. , Davidson Barbara S. , Geraghty Sheela R. , Krebs Nancy F.
Published: June 2021

This ongoing study aims to longitudinally characterize inflammatory cytokines in human milk (HM) and investigate associations with maternal weight and infant growth characteristics. Morning HM samples from 9 normal weight (NW) and 9 overweight (OW) mothers (BMI range: 21.0 – 30.0 kg/m2) and anthropometry measures of their healthy term infants were obtained at 2‐weeks (T1: 2.1 ± 0.4 wks) and 4‐months (T2: 16.7 ± 1.2 wks) post‐partum. HM % fat was measured by creamatocrit; IL‐6, IL‐8, and TNF‐α concentrations by multiplex. The inflammatory load of HM was calculated as the standardized sum of cytokine concentrations. Infant length for age, weight for age, and weight for length Z‐scores (LAZ, WAZ, WLZ) did not significantly change from T1 to T2. IL‐8, TNF‐α, and inflammatory load at T1 were positively correlated with those at T2 (p < 0.01, R2≥ 0.39). IL‐6 and TNF‐α decreased from T1 to T2 (p < 0.05). IL‐6, TNF‐α, and % fat did not differ between NW and OW mothers. HM IL‐8 and inflammatory load decreased from T1 to T2 in NW (p = 0.03) but tended to increase in OW mothers (p = 0.07). By T2, all 3 cytokines were positively inter‐correlated (p < 0.02, R2≥ 0.33). Infant growth results were inconclusive: HM inflammatory load at T1 inversely correlated with ΔWAZ from T1 to T2 (p = 0.04, R2= 0.26) but not ΔLAZ (p = 0.76) or ΔWLZ (p = 0.05). Maternal BMI status may impact inflammatory cytokines in early HM.

Grant Funding Source: NIH T32DK007658–21, 5K24DK083772‐08, P01HD13021
Effects of fructose overfeeding on intrahepatic lipid accumulation and hepatic insulin sensitivity in healthy humans
Authors: Lecoultre Virgile , Egli Leonie , Boss Andreas , Carrel Guillaume , Theytaz Fanny , Zwygart Karin , Kreis Roland , Schneiter Philippe , Boesch Chris , Tappy Luc
Published: June 2021

Added sugars may contribute to the development of insulin resistance in metabolic diseases. It is further suspected that this is related to fructose‐induced intrahepatic fat deposition. How it relates to daily fructose intake, and what the relative roles of fructose, glucose and excess energy are, remains however controversial. We therefore evaluated the effects of short term overfeeding with various amounts of fructose, glucose, or saturated fat on hepatic insulin sensitivity (HIS) and lipid content (IHCL) in healthy male subjects. Data collected in several studies were pooled. Fasting hepatic glucose production (HGP), HIS and IHCL were measured after both 6–7 days on a weight‐maintenance diet (control, C; n=53) and 6–7 days of overfeeding with 1.5 (F 1.5, n=7), 3 (F3, n=17), or 4g fructose/kg/day (F4, n=10), with 3g glucose/kg/day (G3, n=11), or with 30% excess energy as saturated fat (fat 30%, n=8).

F3, F4, G3 and fat 30% all significantly increased IHCL as compared to C (all p<0.05). F4 and G3 significantly increased HGP, (both p<0.05), and F3 and F4 significantly decreased HIS (both p<0.01). In contrast, there was no significant effect of fat 30% on HGP or HISI.

Overfeeding with large amounts of fructose or glucose impairs fasting hepatic glucose metabolism and increases hepatic fat content within 6–7 days. This indicates short‐term regulation of hepatic glucose metabolism by simple carbohydrates.
A pulse‐based diet intervention in women with polycystic ovarian syndrome (PCOS): Compliance, and effects on body composition, fertility and metabolic measures
Authors: Zello Gordon , Ramachandran Kavitha , Rooke Julianne J , Serrao Shani , Chizen Donna R , Chilibeck Phil D
Published: June 2021

Polycystic ovarian syndrome (PCOS) is an endocrine disorder associated with numerous metabolic complications. We compared body composition (BC) between women with PCOS (n=33) and age‐matched healthy controls (n=16), and also determined the effects of a pulse‐based diet on BC, blood lipids and fertility. Women with PCOS were randomized into groups receiving a pulse‐based diet or the National Cholesterol Education Program therapeutic lifestyle changes (TLC) diet for 16 wk while participating in an aerobic exercise program. Women with PCOS were heavier (+26 kg), had greater fat mass (+23 kg) (both p<0.001), but similar lean tissue mass (LTM) compared to controls. They also had similar bone mineral density, but superior bending strength at the femoral neck (p<0.001) which disappeared after controlling for body mass. After intervention, both diet groups lost a significant amount (p<0.05) of body mass (−4.0 kg), fat mass (−3.1 kg), and trunk fat mass (−1.5 kg) and reduced total cholesterol to HDL ratio (8–10%; p=0.05) with no differences between groups. Both groups maintained LTM mass and bone mineral properties, and number of PC ovaries were reduced. Compliance to meals in the TLC and pulse groups was 79% and 59% respectively. A lower compliance may have been to lack of prior dietary exposure to pulses. A pulse‐based diet is as effective as TLC diet for reducing body fat and improving blood lipids. (Support from Saskatchewan Pulse Growers & Agriculture Agri‐Food Canada)
Impact of extrusion on in vitro digestibility of wheat bran‐ or aleurone‐enriched cereals and on bioactive compounds release
Authors: Garcia‐Campayo Vicenta Manuela , Paeschke Teri , Rieschl Chad , Gaspard Dan , Rasmussen Breann , Aimutis Bill
Published: June 2021

Addition of fiber to foods represents a strategy for increasing nutritional quality and health benefits but food matrix and processing play an important role in the properties of the final product. We investigated the effect of different conditions of extrusion on the digestibility of corn‐based cereal puffs with and without 15% fiber added. Two wheat fractions (bran or aleurone) were used as dietary fiber sources. Our results indicate that in vitro carbohydrate digestion of extruded products was faster and more extended than the non processed mixes, regardless of the formulation (presence or absence of fiber) or the conditions of extrusion. We also evaluated the presence of steryl ferulates (campestanyl ferulate, sitostanyl ferulate, and campesteryl ferulate) after in vitro digestion of the aleurone containing materials and observed increased amounts in solutions from extruded cereals compared to non processed raw mixture; this suggesting that processing by extrusion may increase bioaccessibility of health related compounds present in aleurone. Studies on the kinetics of steryl ferulates release and hydrolysis during in vitro digestion are ongoing. Funded by Cargill Inc
Macrophage apolipoprotein E and proliferation of breast cancer cells MCF‐7: Role of LXR
Authors: Bard Jean‐Marie , El Roz Ali , Valin Sabine , Huvelin Jean‐Michel , Nazih Hassan
Published: June 2021

Objective

Apolipoprotein (apo) E is expressed by macrophages under control of LXR, a transcription factor. LXR agonists may inhibit cancer cell proliferation in vitro. We evaluate the effect of apo E, secreted by macrophages under control of LXR on MCF‐7 cell proliferation, a model of breast cancer cells.

Methods

Macrophages derived from THP‐1 monocytes were incubated with LXR agonists [22(R)‐hydroxycholesterol and TO901317] before (THP‐1 + agonists) and after (THP‐1 + siRNA apo E) deprivation of apo E by siRNA. MCF‐7 cells were incubated with media from each macrophage model, as well as exogenous apo E (20 μg/mL). Cell viability was tested by MTT method and apoptosis gene expression (BAX, Bcl‐2) was analysed by qPCR.

Results

Apo E, as determined by ELISA and qPCR is increased in “THP‐1 + agonsits” medium and decreased in “THP‐1 + siRNA apo E” medium. MCF‐7 cell viability is decreased by 18 % and 40% after 24h and 48h incubation with exogenous apo E. Medium from “THP‐1 + agonists” decreases MCF‐7 cell viability by 30% after 24h incubation, while medium from “THP‐1 + siRNA apo E” has no effect. The pro‐apoptotic gene BAX is overexpressed in MCF‐7 cells treated with medium from “THP‐1 + agonists” and underexpressed when treated with medium from “THP‐1 + siRNA apo E”.

Conclusion

Apo E from macrophages stimulated by LXR agonists may inhibit cell proliferation and induce apoptosis in MCF‐7, a model of breast cancer cells.
Vitamin A‐mediated inhibition of cell cycle progression proteins and cell growth requires protein‐bound retinol in a prostate cancer cell line
Authors: Colley Danielle L , Castonguay Thomas W
Published: June 2021

Stimulated by retinoic acid homolog 6 (STRA6) is a transmembrane receptor specific for retinol‐binding protein 4 (RBP4) that mediates uptake of retinol bound to RBP4 (HoloRBP4). Retinoic acid derivatives of retinol have well‐documented anti‐proliferative actions that are mediated by retinoic acid and retinoid X receptors (RAR and RXR, respectively). Thus, it has long been assumed that the effects of circulating retinol on the regulation of apoptosis are dependent on cellular uptake of retinol and activation to retinoic acid. An additional role of STRA6 has recently been identified in vivo and in cultured hepatocytes and renal proximal tubule cells. Upon binding to holoRBP4, STRA6 has been shown to activate a cascade that led to STAT5‐mediated gene regulation. We have detected and induced STRA6 with all‐trans‐retinoic acid (RA) in PC‐3 prostate cancer cells. In this study, we found that PC‐3 cells were growth inhibited following treatment with holoRBP4 whereas 1 μmol/L RA treatment had no effect. Furthermore, we observed that holoRBP4 treatment reduced mRNA and protein abundance of cell cycle progression proteins (CD1, CDK2, and CDK4), which have been identified as a downstream target of STAT5. Collectively, these results indicate that anti‐proliferative actions of retinol may be produced via a signal transduction mechanism, which appear to occur independently of RA‐mediated gene regulation.

Grant Funding Source: National Institutes of Health
Development of respiratory tract infection could be modified by the interactions between maternal diet during pregnancy and offspring's CD14 (rs#2569190) and VDR (rs#7975232) polymorphisms
Authors: Hong Seo Ah , Kwon Sung Ok , Kim Kyung Won , Shin Youn Ho , Ahn Kang Mo , Yu Jinho , Kwon Ja‐Young , Choi Suk‐Joo , Lee Kyung‐Ju , Won Hye‐Sung , Oh Se‐young , Hong Soo‐Jong
Published: June 2021

Maternal diet during pregnancy can potentially influence disease susceptibility of offspring in addition to maternal atopy and genetic background. The objective was to investigate the association of prenatal antioxidant intakes and the genotype with the risk of respiratory tract infection (RTI) in 12‐month‐old infants. As part of a birth cohort study, the COhort for Childhood Origin of Asthma and Allergic Diseases (COCOA) (n=528), we assessed maternal diet during pregnancy by FFQ and genotyping of 4 genes (CD14, VDR, NRF2 and GSTP1) by the Taqman method. Maternal intakes of vit A and C, Fe, folic acid, and vegetable were negatively associated with the risk of RTI, particularly in the infants with atopic mothers (p<0.05). In combined analyses with genotype, we found significant interactions between the genetic variation in CD14 and vit A and C and fruit and between the genetic variation in VDR and fruit and vegetable regarding RTI (p<0.05). These findings suggest that the role of prenatal intakes of antioxidant nutrients related to lower risk of RTI may be modified by CD14 and VDR polymorphisms in infants.

This study was supported by a fund (2008‐E33030–00, 2009‐ E33033–00, 2011‐E33021–00) from the Research of Korea Centers for Disease Control and Prevention.

Grant Funding Source: Nutrition (ASN)
Changes in Leukocyte Populations Among Children Receiving a Follow‐on Cow's Milk‐Based Formula Containing Beta‐Glucan
Authors: Sarvetnick Nora , Murphy Kevin , Pauley‐Hunter Rosemary , Scalabrin Deolinda
Published: June 2021

Background

β‐Glucan can enhance immune system responses to foreign agents, but mechanisms of action remain unknown.

Objective

We examined changes in immune cell populations and activation markers produced by the addition of β‐Glucan to a children's follow‐on formula.

Methods

This was a double blinded, randomized, controlled, parallel‐designed, prospective study. Data is reported for participants who completed the study. Participants (1–4 years of age) received either a marketed, follow‐on formula (n=8), formula supplemented with 42 mg of β‐Glucan per serving (n=10), or formula with 6 × 109 colony forming units of LGG per serving (n=9), twice a day for a 3 week period. Flow cytometry was used to immunophenotype peripheral blood leukocytes. Thirty serum cytokines were measured via multiplex analysis. Changes from baseline were analyzed by Kruksal‐Wallis. As this was a pilot study, differences with p‐values < 0.10 were considered of interest.

Results

Participants who received β‐Glucan had decreased production of stem cell factor and decreased neutrophil proportions. They also had an increase in the ratio of immature‐to‐mature B cells. Finally, they had reduced CD4 T cell activation and an increase in specific subsets of CD8 T cells.

Conclusion

β‐Glucan promotes a complex pattern of activation and inhibition of distinct T and B cell subsets. To our knowledge, this is the first study to examine the effects of β‐Glucan on immune effects in healthy, young children.
Histone deacetylase (HDAC) inhibition prevents and restores bacterial lipopolysaccharide (LPS)‐induced paracellular hyper‐permeability in human lung microvascular endothelial cells (HLMVEC)
Authors: Joshi Atul D , Thangjam Gagan , Barabutis Nektarios , Shaw Mary , Patel Vijay , Catravas John D
Published: June 2021

Objective

LPS disrupts the barrier function of endothelial cell monolayers and increases their permeability to proteins. We have previously shown that inhibition of hsp90 protects and restores LPS‐mediated hyperpermeability in human lung microvascular endothelial cells (HLMVEC). Inhibition of histone deacetylases (HDAC) hyperacetylates hsp90 and thus suppresses its chaperone function. In this study we investigated the effects of HDAC inhibition on LPS‐mediated endothelial barrier disruption.

Methods

Endothelial permeability was measured by electric cell‐substrate impedance sensing (ECIS), in HLMVEC grown to confluence on gold‐plated arrays.

Results

Pre‐treatment with the HDAC inhibitors (TSA, butyrate or panobinostat) protected HLMVEC monolayers from LPS‐mediated hyperpermeability. Treatment with either TSA (2μM) or panobinostat (1μM) 3–6 hours after LPS exposure, restored endothelial barrier function. In addition, LPS‐mediated phosphorylation of MLC and hsp27 was attenuated by pre‐treatment with TSA or panobinostat, possibly preventing cytoskeleton rearrangement. TSA also disrupted hsp90‐CDC37 interaction, in the presence or absence of LPS, indicating a loss of hsp90 chaperone function.

Conclusion

HDAC inhibition protects and restores LPS‐mediated endothelial barrier function possibly by inhibiting hsp90 chaperone function. (Supported by HL093460 and HL101902)
Giardia duodenalis cysteine cathepsin proteases and their role in intestinal disease
Authors: Bhargava Amol , Cotton James A. , Yates Robin , Buret Andre G.
Published: June 2021

Giardia duodenalis, an enteric protozoan parasite, induces pathophysiological effects shared with chronic gastrointestinal disorders, including barrier function disruption, via unknown mechanisms. Cysteine proteases, also identified in the Giardia genome, are implicated in disease pathophysiology of other protozoan parasites.

Objective

To characterize and determine a role for cysteine cathepsins in the pathogenesis of giardiasis.

Results

In vitro cathepsin B/L activity was detected within Giardia trophozoites and in co‐culture supernatants. Giardia trophozoites increased cathepsin B activity in host enterocytes during co‐cultures; this required trophozoite adhesion to host cells. Pretreating Giardia trophozoites with Ca‐074Me (a cathepsin B specific inhibitor) prevented elevation of cathepsin activity in host cells. Inhibition of Giardia cathepsins with a cysteine protease inhibitor (E64) prevented the alteration of the mucus layer glycoprotein, MUC‐2; however, Ca‐074Me failed to prevent Giardia‐induced ZO‐1 cleavage.

Conclusion

Giardia trophozoites increase cathepsin activity in host cells and express cathepsins that play a role in mucus alteration. Supported by NSERC, AIHS, CCFC.
Disparate features of allergic airway disease between young and old mice
Authors: Brandenberger Christina , Jackson‐Humbles Daven N , Allen Katryn M , Wagner James G , Li Ning , Harkema Jack R
Published: June 2021

The prevalence of asthma in the elderly (>;65) is increasing and associated with higher mortality than in children or young adults. Despite this increase, the effects of age on the development, character and severity of allergic asthma have been understudied. To elucidate the influence of age on allergic airway disease, we compared pulmonary responses of young (10‐week) and old (14‐month) BALB/c mice that were intranasally sensitized and challenged with house dust mite extract (HDM). After challenge, bronchoalveolar lavage fluid (BALF) was collected for cell and cytokine analysis and lung tissue was processed for gene expression analysis and histopathology. Both young and old mice developed HDM‐induced allergic airway disease. This was manifested by the presence of BALF eosinophlis and Th2 cytokines (IL5, IL13), as well as elevated gene expression of eotaxin, chitinase 3‐like 4 and airway mucus regulating genes Gob5 and Muc5AC. Compared to young mice, old mice had a significantly greater pulmonary allergic response (e.g., 10 × more eosinophils and IL13). In addition, only old mice showed a Th17 immune response to HDM characterized by increased BALF neutrophils and IL17, IL6 and KC cytokines. These results suggest that the severity and character of allergic airway disease is age dependent and the elderly may be prone to develop more severe allergic asthma with a mixed Th2/Th17 immune response. USEPA RD83479701
An investigation of morphine‐to‐codeine metabolic ratios in postmortem blood, drug interactions, and cytochrome P450 2D6 (CYP2D6) genotype
Authors: Lam Jessica , Woodall Karen , Solbeck Patricia , Ross Colin JD , Carleton Bruce , Hayden Michael R , Koren Gideon , Madadi Parvaz
Published: June 2021

The objective of this study was to investigate the correlation between CYP2D6 genotype, drug interactions, and morphine‐to‐codeine metabolic ratio (MR) in codeine‐related deaths (CRD). The records of the Office of the Chief Coroner of Ontario were examined to identify all CRD from 2006–2008. Deaths in which codeine and its metabolite, morphine, were quantified on the toxicological screen were included. From these, cases in which the manner of death was undetermined, heroin use was suspected, and/or morphine use was suspected were excluded. A total of 59 CRD were included. Postmortem blood samples were analyzed for 17 polymorphisms in CYP2D6 as well as gene duplication. The frequencies of CYP2D6 ultrarapid, extensive, intermediate, and poor metabolizer (PM) was not different in CRDs and a previously published healthy cohort taking opioids. Alcohol use prior to death was significantly associated with CRD that were deemed to be accidental by the coroner (p=0.05). The presence of a selective‐serotonin reuptake inhibitor was significantly associated with suicides (p=0.014). PM was associated with low MR. These findings suggest that CYP2D6 genotype and drug interactions should considered as part of the postmortem toxicological interpretation for CRD, especially in cases with low or high MR.
Determining the mechanism of inhibition of mitochondrial Ca2+ overload by the cocoa flavanol (‐)‐epicatechin as a means of conferring cardioprotection during ischemia‐reperfusion
Authors: Herrera Andrea , Echeverri Jonathan , Khan Priya , Yamazaki Katrina
Published: June 2021

A diet rich in flavonoids has been shown to have a negative correlation with the incidence of cardiovascular disease. Epicatechin (EPI), a cocoa flavonoid, reduced infarct size and improved long‐term left ventricular structure and function post ischemia reperfusion (IR) by significantly inhibiting mitochondrial calcium (Ca2+) overload, a process that induces apoptosis. However, the mechanism is not known. Our objective is to determine the mechanisms by which EPI inhibits Ca2+overload using an in vitro model of simulated IR. We hypothesize that activation of the nitric oxide‐soluble guanylyl cyclase signaling (NO‐sGC) pathway is involved in EPI's protection. Our results demonstrate that EPI significantly decreased Ca2+overload by 2.5‐fold and preserved mitochondrial structure by 4‐fold post IR in HL‐1 cardiomyocytes compared to controls. This correlated with a 20% decrease in apoptosis as determined by cell counts. Studies are being conducted to assess mitochondrial respiration and expression of key proteins involved in the NO‐sGC pathway and nitric oxide production. We anticipate that an increase in mitochondrial respiration and nitric oxide production will be observed in EPI treated cells. Taken together, these results will contribute to improving human health by identifying a mechanism of action of an inexpensive and safe dietary supplement that will lead to a simple and economic treatment for CVD's.
Helium ion microscopy of the rodent kidney and male reproductive tract
Authors: Paunescu Teodor G. , Huynh Chuong , Lechner Lorenz , Goetze Bernhard , Stern Lewis , Breton Sylvie , Brown Dennis
Published: June 2021

We investigated the epithelia of the rat and mouse kidney and epididymis by helium ion microscopy (HIM), a novel technology that uses a scanning beam of He+ ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning, and dehydration methods were evaluated for their ability to preserve tissue architecture. Samples were subjected to critical point drying and HIM, allowing the visualization of fine cellular and plasma membrane details, including depressions suggesting endo‐ or exocytotic events, cilia, and microvesicles. In the kidney, membranous nanoprojections on podocytes and pores within the filtration slit diaphragm were seen in the glomerulus, and extensive apical microplicae of the intercalated cells in the collecting duct. In the epididymis, stereocilia in principal cells and microplicae in clear cells are clearly distinguishable, and high resolution imaging of sperm reveals their close interaction with the epithelium. Colloidal gold probes were used in conjunction with HIM, allowing us to localize membrane proteins. In conclusion, this technology represents a major breakthrough in imaging of the topographical ultrastructure of biological samples and should provide significant advances in our understanding of cell surface structures and membrane organization. Funding sources: NIH DK42956, DK97124, and HD40793; Carl Zeiss Microscopy, LLC.
Role of FoxO within the microvasculature of the skeletal muscle in diet‐induced obesity
Authors: Roudier Emilie Annie , Alim Karim , Vu Vivian , Liu Ying , Birot Olivier , Sweeney Gary
Published: June 2021

Obesity has emerged as a growing worldwide epidemic. Excessive accumulation of fat alters the adipose tissue as well as the peripheral tissues. In particular obesity induces capillary rarefaction in the skeletal muscle. We recently found that Forkhead box O1 (FoxO1) is an angiostatic factor in the endothelium of the skeletal muscle. Here we aim to study the role of endothelial FoxO1 during diet‐induced obesity (DIO). A significant reduction in capillary to fibre ratio and an increased expression of FoxO1 were observed in the skeletal muscle of high fat‐fed C57Bl6 mice (58% vs. 6% kCal fat for 14 weeks). By knocking‐down FoxO, we identified thrombospondin1, BMP and activin membrane‐bound inhibitor and soluble frizzled‐related protein 1 as FoxO targets in murine microvascular endothelial cells (EC). The expression of these genes also was altered in the muscles of the DIO mice, consistent with the increase in FoxO1 protein. To further study the role of FoxOs, DIO was induced in mice with EC‐directed deletion of FoxO1,3,4 proteins (ΔFoxO; MxCre+:FoxO1,3,4L/L). After 8 weeks, DIO induced a significant increase in weight gain in both wild‐type and ΔFoxO mice. After 14 weeks, we will characterize the metabolic alterations and the changes in the skeletal muscle microenvironment. Our work will determine the role that endothelial FoxO plays in promoting obesity‐induced capillary rarefaction.
Differential role of Complement 5a Receptor in microvascular thrombosis in two models of experimental sepsis.
Authors: Stark Ryan , Rumbaut Rolando
Published: June 2021

Complement 5a plays a key role in the response to bacterial infections and has been implicated in inflammatory and thrombotic responses in sepsis. We reported previously that microvascular thrombosis is enhanced by two models of experimental sepsis: polymicrobial sepsis induced by cecal ligation and perforation (CLP) and endotoxemia, induced by lipopolysaccharide (LPS). Thus, we hypothesized that the receptor for C5a (C5aR) mediates enhanced thrombosis in experimental sepsis. We challenged C5aR‐deficient mice with LPS (or saline control) or CLP (or sham surgery control) and measured the kinetics of photochemical injury‐induced thrombosis and plasma levels of pro‐inflammatory cytokines IL‐1β, TNF‐α and IL‐6. While LPS had no effect on kinetics of thrombosis in C5aR‐deficient mice, CLP promoted microvascular thrombosis in these mice. Despite the lack of prothrombotic response to LPS, C5aR‐deficient mice had a pronounced systemic pro‐inflammatory cytokine response to LPS. This study provides evidence of a differential role of C5aR on microvascular thrombosis and systemic inflammation in experimental sepsis. Support: T32 HL007939–07, and a Merit Review Grant from The Department of Veterans Affairs.
The effect of advanced glycation end products on endothelial cell and platelet activation and their interactions
Authors: Maria Zahra , Yin Wei , Rubenstein David Alan
Published: June 2021

Our goal was to elucidate the effects of advanced glycation end products (AGEs) on endothelial cell (EC) and platelet (Pt) activation as a function of time. ECs, incubated for 5 days with reversibly and irreversibly glycated AGEs, were exposed to Pts for up to 4 hours. Pts were pre‐exposed to the same AGEs for 0.5 hour. EC metabolic activity was measured with a MTT assay. A time course for the EC surface expression of connexin‐43/caveolin‐1, ICAM‐1, tissue factor and thrombomodulin was obtained to quantify network formation and inflammatory and thrombotic potential. Expression of Pt P‐selectin, GPIIb/IIIa and PECAM‐1 was quantified with flow cytometry for 4 hours after EC incubation. We hypothesized that exposure to irreversibly glycated AGEs would increase Pt activation and EC inflammatory and thrombogenic potential and that it would be more susceptible with time. Results indicate an increased metabolic activity, inflammatory/thrombogenic potential and Pt activation after 1 hour of incubation, but the enhanced activity reduced after 4 hours. Thus, the results suggest that irreversibly glycated AGEs can promote EC and Pt activation that may induce cardiovascular disease responses. Thanks to OCAST.
Mitochondrial reactive oxygen species contribute to impaired flow‐induced dilation in visceral but not subcutaneous adipose tissue resistance arteries in human obesity
Authors: Robinson Austin T , Szczurek Mary , Bian Jing‐Tan , Cavka Ana , Grizelj Ivana , Phillips Shane
Published: June 2021

Microvascular dysfunction contributes to cardiovascular disease risk. Previous studies indicate that flow‐induced dilation (FID) is impaired in resistance arteries from visceral adipose tissue (VAT) compared to subcutaneous adipose tissue (SAT), in obese subjects. We hypothesized that mitochondrial reactive oxygen species contribute to altered mechanisms of FID in VAT. Resistance arteries (RAs) were dissected from VAT and SAT biopsies obtained from obese subjects during bariatric surgery (n=17 BMI: 48±2). Intraluminal diameters were measured via videomicroscopy before and during flow (pressure gradient 10 to 100 cmH2O) in the presence and absence of Tempol (100μM), PEG‐catalase (PEG‐CAT; 500 U/ml), L‐NAME 100 μM), and Rotenone (50 μM). Fluorescent quantification of flow‐induced H2O2 and nitric oxide (NO) production was also determined. Rotenone improved FID (77±8% vs. 39±4% P<.001) and increased generation of H2O2 and NO in RAs of VAT. There was no effect of Rotenone on FID or H2O2 production in SAT. The addition of PEG‐CAT or L‐NAME to rotenone eliminated the improved FID in VAT. Tempol improved FID in RAs of both VAT and SAT, but was abolished by PEG‐CAT. Taken together these data suggest that 1) mitochondrial ROS contribute to impaired FID in VAT and 2) H2O2 and NO contribute to the restored dilation induced by rotenone and Tempol in visceral adipose during obesity.
Recovery of synaptic transmission following oxygen glucose deprivation (OGD) in the Syrian hamster hippocampus is enhanced at lower temperatures
Authors: Mikhailova Alexandra , Hamilton Jock , Horowitz John , Horwitz Barbara
Published: June 2021

We previously found neural networks in Syrian hamsters, a hibernating species, to be more tolerant to oxygen deprivation (OD) than rats. Here we describe effects of OGD on hamster neural activity under conditions more closely approximating stroke. We hypothesized that exposure of hamster hippocampal slices to OGD impairs neural activity (slows recovery) more than does OD, and that decreased temperature speeds recovery. Evoked response amplitudes (ERAs) of CA1 neurons to afferent stimulation were measured each minute of a 15 min control period [slices perfused with oxygenated artificial cerebrospinal fluid (O2 aCSF, 10 mM glucose)]; during treatment (perfusate switched to aCSF gassed with nitrogen and no glucose) for either 10 or 15 min; and during recovery (slices returned to O2 aCSF, 10 mM glucose for 30 min). At 25°C, 5 min after 15 min of OD, ERAs recovered to 84.4 ± 9.2% of control while OGD‐treated slices recovered to 61.0 ± 14.0% (P<0.05). Thus, at 25°C, recovery from OGD was more impaired than that from OD. Lower slice temperatures significantly shortened recovery time (P<0.05), such that at 5 min recovery after a 10 min ODG exposure, ERAs returned to 36% of control at 35°C, 77% of control at 30°C, and 96% of control at 25°C. In summary, these data support our hypothesis that OGD exposure is more damaging than OD, and that lowering temperature enhances recovery. (Supported by a President's Undergraduate Fellowship to AM)
Effect of renal denervation in aged spontaneously hypertensive rats
Authors: Phelps Jeremiah , Fink Gregory
Published: June 2021

Catheter‐based renal nerve ablation (CBRNA) in resistant hypertension (HTN) is associated with long‐term blood pressure (BP) reduction as well as improvement of glucose intolerance, left ventricular hypertrophy, and atrial fibrillation. Evaluation of mechanisms underlying these improvements has been limited by lack of an appropriate animal model. Preliminary work in our lab identified the spontaneously hypertensive rat (SHR) as a potential model for such studies. To validate this model, we recorded ambulatory BP in male SHR (13wk) undergoing renal denervation (RDX: n=8) or sham(n=7) surgery. Within 24hrs, ΔMAP was greater in RDX compared to sham(−11.5±4.3 vs. 1.1±0.9mmHg), but was not sustained at 1 week. Sham animals aged to 21wks were subdivided into RDX (n=4) or sham (n=3) groups and the intervention repeated. ΔMAP was greater in RDX compared to sham within the first day (−28.4±2.3 vs −0.8±1.6mmHg). In contrast to young SHR, ΔMAP persisted after 1 week in older RDX animals vs older shams(−10.5±2.5 vs. −2.2±1.8mmHg). ΔMAP in these animals persisted for the duration of the study. These data demonstrate that older SHR may better model the effects of CBRNA, and may support the hypothesis that interruption of renal afferent nerves is critical in chronically lowering BP. Research Support from AHA 12PRE8190002
Celiac Ganglionectomy Attenuates Central Sodium‐Induced Hypertension
Authors: Frielle Jennifer , Stocker Sean D
Published: June 2021

In salt‐sensitive hypertension, increases in cerebrospinal fluid (CSF) sodium activate the sympathetic nervous system (SNS) to raise arterial blood pressure (ABP). However, the sympathetic bed that mediates this hypertensive effect of CSF sodium is not known. The present study tested the contribution of the splanchnic sympathetic bed to central sodium‐induced hypertension. To confirm the participation of the SNS, male Sprague‐Dawley rats (280–350g) received intravenous (IV) infusion of terazosin (3 mg/day) plus atenolol (24 mg/day) or saline (6mL/day) followed by a 7‐day intracerebroventricular (ICV) infusion of 0.8M NaCl (0.5 ul/hr) via osmotic minipump. ICV infusion of NaCl in control rats significantly raised ABP from 98.5±2.0 mmHg to 107.0±1.8 mmHg at day 4 (Δ: 8.6±2.8 mmHg, P<0.05). In contrast, ICV NaCl infusion in SNS‐blocked rats failed to increase ABP from 87.4±2.9 mmHg vs 84.3±3.0 mmHg at day 4 (Δ: −2.6±1.2 mmHg). To determine the role of the splanchnic bed, celiac ganglionectomy was performed at least 1 week before ICV infusion of NaCl. ICV NaCl raised ABP from 98.5±2.0 mmHg to 107.0±1.8 mmHg (Δ: 8.6± 2.8mmHg). However, ICV infusion in rats receiving celiac ganglionectomy did not increase ABP from 86.3±3.6 mmHg to 85.8±36 (Δ: −0.1±0.7 mmHg). These results indicate that SNA, specifically splanchnic SNA, mediates central sodium‐induced hypertension.