bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of NOX‐1 on Real‐Time Blood Nitric Oxide and Hydrogen Peroxide in Acute Hyperglycemia
Authors: Mawhinney Ashley Elizabeth , Bertolet Michael , Minni Michael , Barsotti Robert , Young Lindon , Chen Qian
Published: June 2021

Hyperglycemia has been associated with vascular endothelial dysfunction in part by a reduction in nitric oxide (NO) production and increased oxidative stress (e.g., increased superoxide (SO) and hydrogen peroxide (H2O2). Endothelial‐derived NO can be significantly reduced by increased SO/H2O2 in part by the activation of NADPH oxidase during hyperglycemia. Of the 7 NADPH oxidase isoforms, NOX1 is mainly expressed in the vasculature and may play a major role in hyperglycemia induced oxidative stress and vascular endothelial dysfunction. To test this hypothesis, we measured blood NO and H2O2 levels in real time via NO and H2O2 microsensors inserted into femoral veins of rats. Hyperglycemia (e.g., 200 mg/dl) was maintained by infusion i.v. of 30% glucose solution for 3 hours with or without a selective NOX1 inhibitor, ML171. We found that hyperglycemia for 3 hours significantly increased blood H2O2 levels by 1.88±0.4 μM (n=6) compared to the saline infused control (P<0.05, n=2). By contrast, ML171 (1 and 5 μM) reduced hyperglycemia‐induced H2O2 levels by 1.36±0.61 μM (n=8) and 4.35±1.02 μM (n=5), respectively, at the end of experiment. Meanwhile, hyperglycemia significantly reduced blood NO levels by 82.48±38.12 nM (n=3) compared to the saline control (P<0.05, n=2). By contrast, ML171 (1 μM) attenuated the hyperglycemia induced decrease in blood NO levels and increased blood NO levels by 71.15±24.00 nM (n=5) at the end of experiment. Our preliminary results indicate that NOX1 activation may contribute to hyperglycemia‐induced oxidative stress and NO reduction. Furthermore, inhibition of NOX1 may mitigate the deleterious effects of hyperglycemia.

Support or Funding Information

This study was supported by Division of Research, the Center for Chronic Disorders of Aging, and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.
A Sex‐Specific Role for Egr1 in Mediating Norepinephrine‐Induced Contraction in Mesenteric Arteries
Authors: Thakali Keshari M , Wahl Elizabeth C , Kang Ping , Shankar Kartik
Published: June 2021

The immediate early transcription factor early growth response 1 (Egr1) is upregulated in smooth muscle cells of atherosclerotic plaques and in adipose tissue of obese animals and humans. In a heterologous endothelial cell expression system, Egr1 overexpression upregulated the α2δ accessory subunit of the L‐type voltage gated Ca2+ channel (Fu et al, Gene; 2003). Moreover, Ca2+ influx via voltage‐gated Ca2+ channels increased Egr1 expression, suggesting that Egr1 regulates ion channel flux and vascular tone. In light of these findings, we tested the hypothesis that mesenteric arteries (MA; 2nd order) from Egr1 knockout (KO) mice would exhibit reduced contractility compared to MA from their wild‐type (WT) litter mates. Isolated vessel contractility was assessed using wire myography (DMT 620M). A small rightward shift in cumulative concentration response curves to norepinephrine (NE, 10−9 – 10−5M) in MA from male KO mice compared to WT littermates (−log EC50 = 5.96±0.11 vs −5.68±0.03 M NE, respectively; p=0.08) was observed, while there was no difference in NE‐induced contraction in female KO and WT mice. Neither maximal NE‐induced constriction nor maximal acetylcholine‐induced relaxation in NE‐preconstricted MA was different between male KO and WT mice or female KO and WT mice. Current studies are leveraging RNAseq to assess global transcriptome profiles of aorta from male and female WT and KO mice, to determine which pathways are impacted by Egr1. Our preliminary arterial contractility data suggest that smooth muscle Egr1 plays a role in modulating vascular tone.

Support or Funding Information

This work was funded by USDA‐ARS Project 6026‐51000‐010‐05S.
Benefits of being small? The scaling of flight performance in stingless bees and size‐dependent scaling of insect flight metabolism
Authors: Duell Meghan , Harrison Jon F.
Published: June 2021

Intra‐specific evolutionary studies usually report major advantages to large body size, yet most species in all animal clades are small. Smaller animals have some fundamental disadvantages such as reduced force production and movement speed that inhibit their abilities to effectively compete with larger species. In addition, smaller animals generally have higher mass specific metabolic rates at rest, leading to higher costs of living and per gram food requirements. What are the evolutionary advantages of being small? Across insects, birds and mammals, the cost of flight has been reported to scale hypometrically, with larger species having lower mass‐specific energy costs during hovering flight. We studied the flight physiology of stingless bee species ranging from 1–115 mg in mass using flow‐through respirometry to assess metabolic rate, high speed video and sound analysis to measure wingbeat frequency, and “grad and stab” thermocouple measurements of body temperature. We also measured the masses of head, thorax, and abdomen, wing size, and voluntary load‐lifting abilities. Thorax masses scaled isometrically, and all bees lifted similar fractions of their body mass. Bees less than 7 mg had body segment temperatures 1–3°C above air temperature, independent of mass, while larger bees had substantially elevated body temperatures. Metabolic rate during hovering scaled hypermetrically (scaling slope = 1.26) and hypermetry could not be explained by thermal effects. Wingbeat frequency was constant across size, but larger bees had relatively smaller wings. Combining our data with prior literature, we show that the scaling of flight metabolic rate changes from hypermetric to hypometric at approximately 40 mg body mass in insects. Thus a reduced absolute and per‐gram cost of flight likely provides selective advantages for the evolution of small body size in insects. This research was partially supported by NSF IOS 1122157 and a short‐term fellowship from the Smithsonian Tropical Research Institute.
Base Excision Repair of Bulky DNA Adducts Generated by the Antitumor Drug Yatakemycin
Authors: Mullins Elwood A. , Shi Rongxin , Igarashi Yasuhiro , Eichman Brandt F.
Published: June 2021

Threats to genomic integrity are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. We recently described the first DNA glycosylase mechanism that does not require base flipping for either binding or catalysis1. The DNA glycosylase AlkD recognizes aberrant base pairs through contacts with the phosphoribose backbone, while the damaged nucleobase remains stacked in the DNA duplex, and and uses catalytic CH–π and charge–dipole interactions to preferentially stabilize the transition state. We now show through a combination of crystallographic, biochemical, biophysical, and cellular techniques how this unique mechanism enables AlkD to repair large adducts formed by yatakemycin (Fig. 1), a member of the duocarmycin and CC‐1065 family of antimicrobial and antitumor natural products. Bulky adducts of this, or any type, are not excised by DNA glycosylases that use a traditional base‐flipping mechanism. Hence, these findings represent a new paradigm for DNA repair and provide insights into damage recognition and base excision.

Support or Funding Information

This work was funded by the National Science Foundation (MCB‐1122098 and MCB‐1517695) and the National Institutes of Health (R01ES019625).
Transcriptional and Post‐transcriptional Regulatory Mechanisms Lead to Metabolic Adjustment in Response to Glucose Availability
Authors: Munshi Azad Hossain , Claggett Julia M , Johnson Tracy L.
Published: June 2021

The transcription factor Gcr1 controls expression of over 75% of the genes in actively growing yeast. Yet, despite its widespread effects, regulation of Gcr1 itself remains poorly understood. Here we show that posttranscriptional Gcr1 regulation is nutrient‐dependent. Moreover, GCR1 RNA contains a highly conserved intron, which allows the cell to generate multiple RNA and protein isoforms whose levels change upon glucose depletion. Intriguingly, an isoform generated by intron retention is exported from the nucleus, and its translation is initiated from a conserved, intronic translation start site. Expression of gene products from both the spliced and unspliced RNAs is essential, as cells expressing only one isoform cannot adjust their metabolic program in response to environmental changes. Finally, we show that the Gcr1 proteins form dimers, providing an elegant mechanism by which this one gene, through its regulation, can perform the repertoire of transcriptional activities necessary for fine‐tuned environmental response. So, this study provides a striking example of how alternative splicing in the form of intron‐retention can present the cell with novel mechanisms for regulating gene expression.

Support or Funding Information

This work was supported by the National Science Foundation (MCB‐1051921 to T.L.J.) and the National Institute of General Medical Sciences ( GM‐085474 to T.L.J.)
Vacuolar Turnover of rRNA Mediated by An Autophagy‐Dependent Mechanism Is Necessary To Maintain Cellular Homeostasis
Authors: MacIntosh Gustavo C , Floyd Brice , Morriss Stephanie , Bassham Diane
Published: June 2021

Ribosomes are major components of all cells. Much is understood about their biosynthesis and several pathways have been characterized for the elimination of misassembled ribosomal subunits. However, little is known about the decay of stable ribosomes and recycling of rRNA. RNase T2 ribonucleases represent one of the most widely distributed RNase families, and they are conserved in all eukaryotes, many prokaryotes, and some viruses. Our work showed that RNS2, a class II RNase T2 in Arabidopsis thaliana, is essential for normal ribosomal RNA decay. Null rns2‐2 plants show constitutive autophagy, RNA accumulation in the vacuole, and longer rRNA half‐life. Autophagosomes and autophagic bodies in rns2‐2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA levels increase in the rns2‐2, in the autophagy mutants atg9‐4 and atg5‐1 which are deficient in core autophagy machinery, and in rns2‐2atg9‐4 and rns2‐2atg5‐1 double mutants. Interestingly, rns2‐2atg5‐1 had significantly higher total RNA than the single mutants and the rns2‐2atg9‐4 double mutant. These results suggest that, together with RNS2, autophagy has a role in RNA turnover. In addition, rRNA accumulates in the vacuole in rns2‐2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2‐2atg5‐1 double mutant but not by an rns2‐2atg9‐4 double mutant. Furthermore, atg5‐1 vacuoles had significantly lower amounts of rRNA than WT or atg9‐4. Thus, it is clear that ATG5 and ATG9 have different roles in the turnover of rRNA. Our results suggest that an ATG5‐dependet selective autophagic process transport rRNA to the vacuole for degradation by RNS2. Disruption of this process in the rns2‐2 mutant triggers general macroautophagy which depends on ATG9 and ATG5, as a compensatory mechanism to continue the regular turnover of ribosomes and to re‐establish cellular homeostasis.

Support or Funding Information

This work was supported by grant no. MCB‐1051818 from the United States National Science Foundation to G.C.M. and D.C.B.
Structure‐Function Studies of the ADPGlucose Pyrophosphorylase from Agrobacterium tumefaciens: Defining the Roles of the K310 and P288 Residues in Allosteric Regulation
Authors: Duong Vanessa , Butz John , Nguyen Tuong , Ochoa Jackie , Meyer Christopher R
Published: June 2021

ADP‐glucose pyrophosphorylase (ADPG PPase, EC 2.7.7.27) is the enzyme that catalyzes the rate limiting step in the glucan biosynthesis pathway. By identifying important residues of the protein a more active enzyme may be engineered thus yielding more renewable carbon. Agrobacterium tumefaciens (Ag. t) ADPG PPase is activated by fructose‐6‐phosphate (F6P) and pyruvate and is inhibited by inorganic phosphate and sulfate. It is believed these effectors bind at a common allosteric site. Based on previous experiments it is believed the P288 residue, which is part of the loop region connecting the N‐terminus domain to the C‐terminus domain, is involved in allosteric regulation. The P288D mutant displayed higher activity than the wild‐type (WT) in the absence of activators and it displayed desensitization to effectors. Molecular modeling suggests reveals the K310 residue could potentially be forming a salt bridge with the aspartate residue at the substituted P288 site. It had been previously shown that the K310A enzyme displayed very low activity that was similar to WT in its response to activators; the K301A enzyme activity was increased by the presence of the P288D mutation which also desensitized the enzyme to effectors. To further probe the roles of these two residues with respect to charge and size, the K310Q, K310R, P288D/K310Q and P288D/K310R were successfully generated. The altered proteins were generated by site‐directed mutagenesis (QuikChange Kit, Stratagene) and expressed in E.coli glgC− cells. The K310Q and K310R mutants were successfully generated and purified using Phenyl Sepharose, Diethylaminoethanol, and Cibracon Blue chromatography. Substitution of the P288 residue resulted in changes in allosteric behavior with the largest effects being desensitization to activators. Preliminary studies on the crude extract of the K310Q and K310R mutants show a 2.85 and 17.46 fold increase activity compared to the WT, respectively, indicating the importance of shape and charge for optimal activity. For the each double mutation the activity was diminished, for P288D/K310Q and P288D/K310R, a 6.5 and 1.6 fold decrease in activity compared to WT, respectively. Complete kinetic and structural characterization of the single and double mutant enzymes will allow for a comprehensive comparison to the WT enzyme and a more detailed understanding of structure‐function relationships.

Support or Funding Information

Supported in part by NSF and NSF BIO MCB grant #0448676.
Endothelial function assessment in nutritional research studies: a comparison of two arterial occlusion sites for assessing reactive hyperemia using a peripheral arterial tonometry device
Authors: Woodhouse Leslie R , Adkins Yuriko , Bonnel Ellen , Peerson Janet M
Published: June 2021

Endothelial function assessment can be an important tool in evaluating artery health in humans, and can be an important phenotyping tool in nutritional research studies as well as an early indicator of cardiovascular disease. We conducted a study of endothelial function in healthy adults using reactive hyperemia peripheral arterial tonometry (RH‐PAT) by the EndoPAT™ device (Itamar Medical Ltd., Caesarea, Israel) in order to compare two arterial occlusion sites. Brachial artery (upper arm) occlusion is the most common occlusion site, yet a forearm occlusion site may be a potentially valid site for endothelial function testing. RH‐PAT testing is an operator independent, non‐invasive technique that measures the arterial pulse wave amplitude using pneumatic fingertip probes during reactive hyperemia allowing a RH‐PAT index (RHI) to be calculated. Reactive hyperemia was achieved by occluding either the upper arm (brachial artery) or the forearm (ulnar and radial arteries) in the individual using a blood pressure cuff inflated to ~200 mmHg for 5 minutes. Twenty healthy adults (14 female, 6 male), age 38 ± 12 years (mean ± SD) and BMI 24 ± 3 kg/m2 participated in the study. RH‐PAT measurements were determined in the morning after a 12 hour fast. Participants were tested approximately 3 days apart using a crossover design with randomization of upper arm and forearm as occlusion sites. The participant's non‐dominant arm was always the occluded arm. Participants were tested in a quiet, temperature‐controlled (21–24°C) room in a supine position for at least 15 minutes prior to the 15 minute test: 5 minute baseline, 5 minute occlusion, and 5 minute post‐occlusion. A self‐reported numeric pain intensity scale (0–10) was used to evaluate pain during the occlusion and post‐occlusion (hyperemic period) portion of the measurement. The RHI is the post‐to‐pre occlusion PAT signal ratio in the occluded arm, relative to the same ratio in the control arm, corrected for baseline vascular tone of the occluded arm. The mean RHI for the two occlusion sites: 2.15 ± 0.78 (upper arm) and 2.36 ± 0.75 (forearm) was not different (p=0.18, paired t‐test). A Pearson correlation of the RHI from both sites (r=0.602, p=0.005) was positive and significant. The mean reported pain intensity for the upper arm was 3.7 ± 1.3 compared to 2.7 ± 1.8 for the forearm; these indices were significantly different (p=0.002, signed‐rank test). In summary, occlusion of the forearm appears to be a potentially valid alternate occlusion site for RH‐PAT measurement and is less painful than using the upper arm as an occlusion site. In addition, the occlusion site should be standardized in the study protocol. Human study approved by the UC Davis Institutional Review Board.

Support or Funding Information

USDA, ARS Projects: 2032‐51000‐022; 203253000‐001
Long‐term Intermittent Leucine Pulses during Continuous Feeding Impact the Plasma Metabolome of Neonatal Pigs
Authors: Suryawan Agus , Boutry Claire , Manjarín Rodrigo , El‐Kadi Samer W. , Steinhoff‐Wagner Julia , Nguyen Hanh , Fiorotto Marta L. , Davis Teresa A.
Published: June 2021

Infants who are unable to maintain oral feeding can be nourished by continuous administration of feed via orogastric tube. We have shown in a neonatal piglet model that intermittent pulses of leucine administered during continuous feeding enhanceslean tissue growth compared to continuous feeding alone. This response was consistent with the attainment of higher rates of muscle protein synthesis in the leucine supplemented piglets. To determine the long‐term effects of intermittent leucine pulses on metabolomic markers in the plasma of neonates, piglets (n = 23, 7‐d‐old) were fed continuously a milk replacement formula via orogastric tube for 21 d with an additional parenteral infusion of either a leucine (800 μmol·kg−1·h−1, CON+LEU)or alanine (CON+ALA) supplement for 1 h every 4 h; blood samples were collected at d 21. Plasma samples were analyzed by Metabolon (Durham, NC, USA) using GC‐MS and LC‐MS. There were 483 compounds identified in the analysis matching known chemical structures. Following log transformation and imputation of missing values, Welch's two‐sample t‐test was used to identify metabolites that differed significantly between experimental groups. The results showed that in the CON+LEU compared to the CON+ALA group, the BCAA‐related changes in metabolites was consistent with increased leucine metabolism. Concentrations of leucine‐containing dipeptides, an indicator of protein synthesis, were elevated in the CON+LEU group compared to the CON+ALA group (P < 0.05). Concentrations of dimethyl arginine, a marker of proteolysis, were reduced in CON+LEU compared to CON+ALA group (P <0.05). These results are consistent with the observed differences in the growth responses to the two treatments. Altogether, these data support the notion that, in addition to increasing protein synthesis, leucine supplementation ameliorates muscle protein degradation, and therefore may be useful as atherapeutic agent to promote growth in neonates. Thus, plasma metabolomics profiling may be a useful tool to identify biomarkers of protein synthesis and degradation pathways that are modulated by leucine supplementation in neonates.

Support or Funding Information

NIH AR444474, NIH HD072891, USDA NIFA 2013‐67015‐20438, and USDA/ARS 6250‐51000‐055
Native African Plant Materials Modify in vitro Bioaccessibility of Provitamin A Carotenoids from Blended Cereal Products
Authors: Debelo Hawi , Ndiaye Cheikh , Ferruzzi Mario , Hamaker Bruce
Published: June 2021

Native plant materials can play an important role in addressing deficiency of key shortfall micronutrients including vitamin A (VA), iron (Fe) and zinc (Zn) in Sub‐Saharan Africa. However, information on the micronutrient composition of these plants and strategies for their incorporation into viable consumer products is scarce. In particular, limited information is available on the interaction of materials on the bioavailability of key nutrients such as proVA carotenoids. In the current study, 28 distinct plants and traditional cereal grains (millet and sorghum) from Senegal and Niger were characterized for their carotenoid and mineral content. Among the plant materials analyzed, Adansonia digitata (Baobab), Moringa oleifera (Moringa), and Hibiscus sabdariffa (Hibiscus) were identified as potential sources of key shortfall nutrients, Fe (54.5–237.2 μg/g) and Zn (4.2 – 72.38 μg/g). Dried carrot and mango materials common to West Africa were identified as provitamin A sources. Following initial screening, plant materials were formulated in model instant cereal based porridges designed to mimic thin porridge products common to West Africa. Proportions of instant cereal and plant materials were dry blended to deliver ~25% of the RDA for proVA, Fe and Zn as follows: extruded cereal based flour (45–65%), dried proVA rich plants (30% as carrot and mango) and dried mineral rich plants (0–25% as Hibiscus, Moringa or Baobab). Porridges were reconstituted with hot water (100°C), 5% oil was then added and the final product was introduced to an in vitro digestion model to assess the impact of mineral rich plant materials and grain background on the bioaccessibility of proVA carotenoids. Baobab inclusion was found to significantly increase (p<0.05) bioaccessibility of total proVA (TPVA) carotenoids from 18.26±1.8% to 31.68±4.1% (mean ± SEM) in dose‐dependent manner from 5% to 25% baobab. Conversely, carotenoid bioaccessibility was reduced with increased amount of Moringa and Hibiscus. Interestingly, Baobab at 5% had the lowest (p<0.05) digestive recovery of TPVA carotenoids (24.86 ± 5.96%) whereas Hibiscus at 5% had the highest recovery (55.35 ± 2%) suggesting that interactions between plant materials may impact both digestive stability and micellarization of proVA carotenoids. Based on bioaccessible proVA content of digested porridges, 200g of porridge with baobab formulated at 25% of dry mix) would provide 30% of the vitamin A EAR for children 1–3 years old. These results suggest that native plant materials selected to enhance mineral content may also promote interactions that can impact availability of proVA carotenoids in finished products.

Support or Funding Information

This research was supported by USAID Food Processing Innovation Lab (AID‐0AA‐L‐14‐00003).
Activation of ubiquitin‐proteasome system components in heat‐stressed pig skeletal muscle
Authors: Zhao Lidan D , Zhang Zhenhe , Xie Guohao , Selsby Joshua T , Baumgard Lance H , Rhoads Robert P
Published: June 2021

Hyperthermia causes metabolic changes and leads to lower lean tissue accretion and increased adipose tissue deposition in animals. The direct effect of heat stress on muscle breakdown has been reported in poultry, rabbits and lactating cows. Muscle breakdown occurs by activating the ubiquitin‐proteasome system and the autophagy‐lysosome pathways. To study the mechanism by which heat stress induces muscle breakdown, the ubiquitin‐protease system in the pig skeletal muscle was examined. Pigs (35 ± 0.8 kg) were assigned to one of three treatments: thermal neutral fed ad libitum (TN, 21°C, n=8), heat stress fed ad libitum (HS, 35°C, n=8), or thermal neutral, pair‐fed to HS intake (PF, n=8) for 7 days. Longissimus dorsi muscle was biopsied on day 3 and 7 relative to start of treatments for real‐time PCR and immunoblotting assays. Heat stress increased activating transcription factor‐4 (ATF4), a stress‐response transcription factor, mRNA abundance by more than 1.5 fold regardless of days (P < 0.05). Heat stress increased growth arrest and DNA damage‐inducible 45a (Gadd45a) mRNA abundance by about 1.5 fold compared with PF muscle (P < 0.05). Similarly, the protein abundance of skeletal muscle Gadd45a was elevated in HS pigs by 50% as well as in PF pigs compared with TN pigs (P < 0.05). The phosphorylation of GSK3 β was similar between groups on day 3 and not different in HS versus PF and TN muscle. However, PF pigs had significantly higher phosphorylated GSK3β compared with TN pigs (P < 0.05). HS pigs exhibited intermediate atrogin‐1 mRNA abundance, which was not different from either TN or PF pigs. However, HS pigs had a significantly higher muscle ring finger‐1 (MuRF1) mRNA abundance (approximately 2 fold) compared with PF pigs (P < 0.05). In conclusion, heat stress activated components of muscle breakdown by inducing AFT4, which increased Gadd45a expression, and subsequent apparent activation of the ubiquitin‐protease system.

Support or Funding Information

Results described herein were supported by Agriculture and Food Research Initiative Competitive Grant nos. 2011‐6700330007 and 2014‐67015‐21627 from the USDA National Institute of Food and Agriculture.
Bioaccessibility of Vitamin D from Bread Fortified with UV‐Treated Yeast is Lower than Bread Fortified with Crystalline Vitamin D2 and Bovine Milk
Authors: Lipkie Tristan E , Ferruzzi Mario , Weaver Connie M
Published: June 2021

UV‐treated yeast may be used as a vitamin D fortificant in bakery products, yet the vitamin D status of rats fed bread fortified with vitamin D2 enhanced yeast was lower than those fed a diet with crystalline vitamin D3. The purpose of this study was to determine whether the digestive release and micellarization (bioaccessibility) of vitamin D from yeast might be a limiting factor. White wheat and whole wheat bread fortified with vitamin D2‐enhanced yeast or crystalline vitamin D2 and vitamin D3 fortified bovine milks (skim, 2%, and whole) were subject to in vitro digestion, and analyzed by HPLC‐MS. Bioaccessibility was greater (p<0.05) from bovine milks (70–85%) than from breads fortified with vitamin D2 enhanced yeast (6.0–7.5%). Bioaccessibility was about 4× greater from breads fortified with crystalline vitamin D2 than those with vitamin D2 enriched yeast. Presence of intact yeast cells after simulated gastric and upper intestinal digestion suggests that vitamin D may be entrapped within yeast cell membranes. These data suggest that low digestive release of vitamin D from yeast may partly explain the low apparent bioavailability as compared to crystalline vitamin D2 and D3 and additional processing may be required to enhance the digestive release of vitamin D from yeast.

Support or Funding Information

Support provided by Purdue University and the USDA National Needs Fellowship Program.
Plasma Biomarkers Of Glycocalyx Shedding After Hemorrhage And Resuscitation: Correlation With Microvascular Events
Authors: Filho Ivo Torres , Torres Luciana N , Salgado Christi , Valdez Celina , Dubick Michael A
Published: June 2021

The microvascular endothelial glycocalyx plays an essential role in several physiological functions but can be damaged after hemorrhage and fluid resuscitation. Many studies report plasma levels of syndecan‐1 and heparansulfate as indirect markers of glycocalyx degradation. However, it is unknown how much these measures are representative of the changes occurring at microvascular level. To fill this void, we studied quantitatively the relationship between plasma biomarkers and changes in various microvascular parameters, including glycocalyx thickness, using hemorrhage and a wide range of resuscitation fluids. Rats were bled 40% of estimated total blood volume and resuscitated with different fluids(fresh whole blood, packed red blood cells:lactated Ringer's, fresh frozen plasma, 5% albumin, lactated Ringer's, 3% hypertonic sodium chloride solution, and normal saline). Control animal groups were either not hemorrhaged or not resuscitated. Intravital fluorescence video microscopy was used to estimate glycocalyx thickness and permeability in 270 post‐capillary venules from 58 cremaster preparations in 9 animal groups while other microvascular parameters were measured using non‐invasive techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. In hemorrhaged rats, mean arterial pressure decreased from 100 ± 2 mmHg to 50 ± 3 mmHg after bleeding, and remained at intensities significantly lower than baseline after resuscitation. Exposure of animals to hemorrhage and different resuscitation fluids resulted in various glycocalyx degradation levels as expressed by different levels of glycocalyx thickness and plasma concentrations of syndecan‐1 and heparan sulfate. Glycocalyx thickness changes were correlated with plasma level changes of syndecan‐1 (r = −0.9, p<0.01) and heparansulfate (r = −0.9, p<0.01). Microvascular permeability changes were correlated with changes in both plasma biomarkers (r = 0.7, p<0.05). Syndecan‐1 and heparan sulfate were also correlated (r = 0.7, p<0.05). Except for diameter and permeability, changes in microvascular parameters (red blood cell velocity, blood flow, wall shear rate) did not correlate with plasma biomarkers or glycocalyx thickness changes. Concurrent measurements of microvascular parameters, glycocalyx thickness and levels of plasma biomarkers, coupled with evaluation of systemic parameters, allowed a comprehensive evaluation of glycocalyx degradation after hemorrhage and resuscitation. The quantitative framework provided here suggests that monitoring plasma biomarkers of glycocalyx shedding may be useful in guiding resuscitation strategies following hemorrhage.

Support or Funding Information

Supported by US Army Medical Research & Materiel Command.
Stoichiometry of Novel Protein Modifications in Livers of Warm‐adapted Threespine Sticklebacks from Baja California (Mexico)
Authors: Levitan Bryn Bo , Gómez‐Jiménez Silvia , Li Johnathon , Kültz Dietmar
Published: June 2021

The purpose of this study was to investigate molecular phenotypes that have enabled the survival of a species above its typical lethal temperature limit. A unique population of threespine sticklebacks (Gasterosteus aculeatus) from Baja California, Mexico are surviving and able to function normally at temperatures close to 30 degrees Celsius, a temperature that is higher than normal for this species. Understanding how this warm‐adapted population is able to function at high temperatures at the molecular level may give insight into the mechanisms that have allowed for their survival, and the potential for other populations to adapt to increasing temperatures due to global climate change. Livers from sticklebacks of this unique warm‐adapted population (Baja California, Mexico) were compared to those of a typical cold‐adapted population (Anchorage, Alaska) using gel‐free quantitative proteomics by liquid chromatography‐mass spectrometry (LC‐MS/MS) in order to identify post‐translational modifications (PTMs) in proteins involved in redox and detoxifying biochemical pathways. The stoichiometry of these proteins were then compared between the two populations. Nineteen novel PTMs were identified in 11 different redox proteins, with major differences in stoichiometry detected between the two populations for catalase, alcohol dehydrogenase 1, three glutathione‐s‐transferase isozymes, and carbonyl reductase [NADPH] 1. Further investigation into the functional significance of these PTMs has the potential to reveal novel mechanisms by which adaptation to high temperatures affects redox regulation and detoxification mechanisms. It is well known that adaptation to high temperature affects gene and protein sequences at evolutionary time‐scales and the expression of abundances of particular mRNAs and proteins. Our study provides evidence that specific covalent protein modifications also represent important targets of evolutionary selection during high temperature adaptation of fish. Such modifications result in modulation of protein structure and function leading to concomitant changes in molecular and higher‐order phenotypes that provide an apparent adaptive advantage at elevated habitat temperature.

Support or Funding Information

This work was supported by the National Science Foundation, IOB 1355098.
Obesity Prevention Intervention Recruitment and Implementation by Home Visitation Staff (HVS): Characteristics Associated with Success and Implications for Research Staff Training
Authors: Eck Kaitlyn , Byrd‐Bredbenner Carol
Published: June 2021

HomeStyles is a childhood obesity prevention program disseminated to families with young children via home visitation staff (HVS) who are non‐nutrition professionals (NNPs), but received program‐related training. Little is known about HVS’ abilities to recruit and implement obesity prevention intervention research with high‐risk families. Thus, the purpose of this study was describe the demographic characteristics, attitudes towards weight‐related parenting practices, personality traits, and lifestyle characteristics of home visitation staff (HVS) who are non‐nutrition professionals (NNP) and compare those who successfully recruited families to participate in an obesity prevention intervention to those who did not with the goal of better targeting and tailoring NNP HVS childhood obesity training. HVS were surveyed to describe their characteristics, attitudes towards weight‐related parenting practices, and personality traits. Unpaired t‐tests indicated that HVS who recruited families had significantly (p<.05) more education (93% vs. 49% had baccalaureate degrees) and were younger (age=31.00±8.26SD vs 39.90±8.23SD) than HVS not recruiting families. Successful recruiters were significantly more culturally aware (4.31±0.45SD vs 3.97±0.55SD, 5‐point scale) and had greater needs for cognition (i.e., propensity for enjoying problem solving, (4.31±0.61SD vs. 3.56±0.78SD, 5‐point scale). Successful recruiters also had significantly higher disinhibited (2.22±0.59SD vs 1.92±0.61SD, 4‐point scale) and restraint (2.68±0.65SD vs 2.35±0.59SD, 4‐point scale) eating scores, indicating they were less able to self‐regulate intake and restricted intake to control weight. Attitudes toward weight‐related parenting practices also differed significantly; successful recruiters felt significantly more strongly that parents should not use food rewards (4.40±0.64SD vs 3.92±0.73SD, 5‐point scale), but supported non‐food rewards to get children to eat healthy foods (4.07±0.82SD vs 3.21±1.13SD, 5‐point scale). Findings suggest that to increase HVS recruiting success for childhood obesity prevention programs, staff training should build cultural awareness, align personal eating behaviors with obesity prevention best practices, and improve attitudes toward recommended child feeding practices.

Support or Funding Information

USDA NIFA #2011‐68001‐30170
Kaposi's sarcoma‐associated herpesvirus reduces cellular myeloid differentiation primary response gene‐88 (MyD88) expression via modulation of its RNA
Authors: Zhang Luwen
Published: June 2021

Kaposi's sarcoma (KS)‐associated herpesvirus (KSHV) is a human gamma herpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Interleukin 1(IL1) is a major mediator for inflammation and plays an important role in both innate and adaptive immunity. Myeloid differentiation primary response gene 88 (MyD88) is an essential adaptor molecule for IL1 as well as most Toll‐like receptors signaling. In this report, we identified a novel mechanism by which KSHV interferes with host inflammation and immunity. KSHV RTA specifically reduces the steady state protein levels of MyD88, and physiological levels of MyD88 are down regulated during KSHV lytic replication when RTA is expressed. The N‐terminal region of RTA is required for the reduction of MyD88. Additional studies demonstrated that RTA targets MyD88 expression at the RNA level, inhibits RNA synthesis of MyD88, and may bind MyD88 RNA. Finally, RTA inhibits interleukin 1(IL1)‐mediated activation of NF‐kappaB (NF‐kB). Because IL1 is abundant in KS microenvironment and inhibits KSHV replication, this work may expand our understanding on how KSHV evades host inflammation and immunity for its survival in vivo.

Support or Funding Information

This work was supported in part by grants from the National Institute of Health (CA138213, RR15635), Department of Defense (W81XWH‐12‐1‐0225), and National Multiple Sclerosis Foundation (PP3446)(LZ).
Safety of combined bambuterol and theophylline as a potential treatment of high altitude‐induced fatigue in humans
Authors: Schroeder Thies , Khiabani Hasse Z , Noveck Robert J , Bell Christopher , Hamilton Karyn , Irwin David C , Strand Trond‐Eirik
Published: June 2021

Theophylline is moderately effective against high altitude‐induced malaise, but may be more valuable if combined with other compounds. We hypothesized that theophylline can be combined with a β2 adrenergic receptor (β2AR) agonist in humans to treat high altitude‐induced fatigue. We demonstrate that the β2AR agonist bambuterol alone (high dose = 0.05 mg/kg i.p.) significantly improves exercise performance in rats at simulated high altitude. Combined with theophylline (15 mg/kg), low‐dose bambuterol (0.01 mg/kg) was ergogenic in the same model, whereas theophylline or low‐dose bambuterol alone was not. In a phase‐1 human safety trial, neither single‐dose oral theophylline and bambuterol (Theo‐Dur 400 mg and Bambec 20 mg) nor their combination produced any SAEs. Bambuterol alone decreased plasma potassium (from 3.78 mM to a base average of 3.37 mM), which was not aggravated further by the addition of theophylline (3.83 mM to 3.33 mM). Most frequent AEs were K+<3.5mM and tremor. There was a small influence of each drug on the metabolism of the co‐administrate, decreasing Tmax of the bambuterol metabolite terbutaline and delaying t1/2 of theophylline. We conclude that the combination of theophylline and bambuterol is promising and feasible in humans and should undergo testing for safety and efficacy at high altitude.

Support or Funding Information

This work was supported by grants from the Norwegian Armed Forces Medical Service, Institute of Aviation Medicine, the US Defense Advanced Research Projects Agency (DARPA), prime Award Number N66001‐10‐C‐2134, and by the US Office of Naval Research, prime award number N0014‐14‐0699
The Fibronectin III‐1 Domain Activates a PI3‐Kinase/Akt Signaling Pathway Leading to αvβ5 Integrin Activation and TRAIL Resistance in Human Lung Cancer Cells
Authors: Cho Christina , McKeown‐Longo Paula
Published: June 2021

Fibronectin is a mechanically sensitive protein which is organized in the extracellular matrix as a network of interacting fibrils. The lung tumor stroma is enriched for fibronectin which is thought to contribute to metastasis and drug resistance. Fibronectin is an elastic, multi‐modular protein made up of individually folded domains, some of which can stretch in response to increased mechanical tension. Very little is known about the relationship of fibronectin's unfolded domains to lung cancer resistance to chemotherapy. In the present study, we evaluated the impact of unfolding the first Type III domain of fibronectin (FnIII‐1c) on TRAIL resistance. FnIII‐1c inhibited TRAIL‐induced activation of caspase 8 and subsequent apoptosis in H460 lung cancer cells. FnIII‐1c treatment was associated with the activation of the PI3K/Akt pathway and the αvβ5 integrin receptor for vitronectin, both of which were required for TRAIL resistance. Immunohistochemical staining of sections from non‐small cell lung cancers showed that vitronectin was localized around blood vessels and in the tumor‐stroma interface. These findings suggest that unfolding of Type III domains within the fibronectin matrix may promote TRAIL resistance through the activation of a PI3K/Akt/αvβ5 signaling axis and point to a novel mechanism by which changes in secondary structure of fibronectin contribute to cancer cell resistance to apoptosis.

Support or Funding Information

This study was supported by NIH CA058626 and by DoD LC090169 to PM‐L
Chronic Hypoxia uncouples Ca2+ and eNOS in bradykinin‐induced relaxation of Ovine pulmonary arteries
Authors: Blum‐Johnston Carla M , Romero Monica M , Wee Chelsea , Blood Quintin , Wilson Rachael H , Blood Arlin B. , Francis Michael , Taylor Mark S , Longo Lawrence D , Wilson Sean M
Published: June 2021

Bradykinin‐induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+, which activates a nitric oxide (NO)‐dependent signaling pathway. This pathway leads to vasodilation, thereby regulating pulmonary blood flow and 02 uptake. This vasodilation process includes stimulation of endothelial nitric oxide synthase (eNOS) and downstream activation of large‐conductance K+ (Maxi K+) channels. Chronic hypoxia (CH) is known to increase pulmonary pressures and restrict arterial relaxation, and can contribute to the development of pulmonary hypertension. We thus examined the effects of CH on bradykinin‐induced Ca2+ signals, bradykinin‐induced vasorelaxation, and the roles of eNOS and Maxi K+ channels in this relaxation. Wire‐myography and confocal microscopy studies were performed on pulmonary arteries (PA) from nonpregnant ewes that lived in a normoxic state at low altitude or a hypoxic state at high altitude (3,801 m) for >100 days. eNOS was inhibited with 100 μM NG‐nitro‐L‐arginine methyl ester (LNAME), and Maxi K+ channels were blocked with 1mM tetraethylammonium (TEA). The data show CH augmented endothelial Ca2+ signals, but restricted bradykinin relaxation. Further, CH caused bradykinin‐induced contraction. LNAME sensitivity was restricted, which suggests eNOS dysfunction is central to the uncoupling of Ca2+ signals and bradykinin relaxation. CH also abolished TEA‐sensitivity in bradykinin relaxation, suggesting there is loss of Maxi K+ function following CH. Overall, these results suggest that CH causes uncoupling of endothelial Ca2+ signaling and eNOS function and mediates major changes in the mechanisms of membrane hyperpolarization. It follows that CH‐induced Ca2+‐eNOS uncoupling and Maxi K+ channel dysfunction are important mechanisms to examine as future therapeutic avenues for pulmonary hypertension.

Support or Funding Information

Support from NIH, NSF and Loma Linda University School of Medicine
The Effect of Aging and Rapid Saline Infusion on Compensatory Reserve during Whole‐Body Passive Heat Stress
Authors: Gagnon Daniel , Romero Steven A , Ngo Hai , Poh Paula YS , Convertino Victor A , Crandall Craig G
Published: June 2021

The compensatory reserve is defined as the physiological capacity to compensate for reductions in central blood volume. It is measured by a machine learning algorithm, called CRI, which analyzes continuous changes in the features of a photoplethysmographic signal obtained by finger pulse oximetry. The purpose of this study was 2‐fold. First, we examined the effect of healthy aging on compensatory reserve during normothermic and heat stress conditions. Second, we examined the effect of rapid saline infusion during heat stress on compensatory reserve. Eleven young (6 males/5 females; mean ± SD: age, 25 ± 4 years; height, 165 ± 6 cm; weight, 63 ± 9 kg) and ten aged (4 males/6 females; age, 69 ± 4 years; height, 165 ± 9 cm; weight, 68 ± 9 kg) adults were exposed to central hypovolemia via 15 and 30 mmHg of lower‐body negative pressure (LBNP) under normothermic and heat stress (increase in core temperature of 1.5°C) conditions. Approximately 10 min after the heat stress LBNP challenge, warm saline was rapidly infused (15 ml/kg in ~6 min) while subjects remained hyperthermic. Absolute CRI values (Table) range between 0 and 1, where 0 represents little to no reserve for cardiovascular compensation and 1 represents maximal physiological reserve to compensate for central hypovolemia. Under normothermic conditions, an age × LBNP level interaction was observed (P<0.01). Relative to baseline, CRI was reduced during 15 mmHg LBNP in young (P<0.01) but not aged (P=0.21) adults. During 30 mmHg LBNP, CRI was reduced in both groups (P≤0.01). During whole‐body passive heat stress, CRI was lower relative to normothermic baseline in both groups (P<0.01). During subsequent LBNP, CRI was further reduced in young adults during 15 and 30 mmHg (P<0.01). In aged adults, CRI was further reduced at 30 mmHg during heating (P=0.06), but not at 15mmHg (P=0.27). Relative to heat stress, rapid saline infusion did not affect CRI in young (P=0.47) or aged (P=0.99) adults. These results demonstrate that CRI responses at low levels of central hypovolemia (i.e. 15 mmHg ≈ 250 ml) differ between young and aged adults under normothermic and heat stress conditions. Furthermore, CRI is unaffected by rapid saline infusion performed under heat stress conditions in both young and aged adults.

Support or Funding Information

Funded by the Department of Defense (W81XWH‐12‐1‐0152).

CRI responses in young and aged adults.

Normothermic baseline
LBNP15
LBNP30
Heat stress
LBNP15
LBNP30
Infusion

Young
0.88±0.06
0.77±0.11*
0.53±0.17*
0.43±0.27*
0.31±0.20†
0.18±0.13†
0.40±0.18

Aged
0.84±0.20
0.79±0.21
0.67±0.24*
0.45±0.20*
0.40±0.20
0.35±0.23†
0.41±0.15

Values are mean ± SD.

different from baseline at P≤0.05;

different from heat stress at P≤0.06.