bluuebunny/crossref_metadata_2025 huggingface dataset:
Singlet Oxygen Production in the Caudodorsal Medulla of Rat Brain Slices Increases During Hyperoxia at Normobaric Pressure and Following Decompression from Iso‐oxic Hyperbaric Nitrogen
Authors: Roberts Teryn R. , Ciarlone Geoffrey E. , Dean Jay B.
Published: June 2021

Previous studies in brain slices harvested from the medulla oblongata of Sprague‐Dawley (SD) rats indicate that neurons in the caudal solitary complex (cSC) are stimulated by normobaric hyperoxia (NBO2) and hyperbaric O2 (HBO2). We've also reported that during NBO2, cSC cells produce a variety of reactive O2 and N2 species, including superoxide, nitric oxide, peroxynitrite, hydrogen peroxide, hydroxyl radicals, carbonate radicals, & nitrogen dioxide. These findings support the premise that cSC neurons employ redox signaling mechanisms that are activated by increasing pO2. Presumably, this explains, in part, the paradoxical stimulation of breathing by prolonged exposures to NBO2 and HBO2. We have tested the hypothesis that singlet oxygen (1O2) production increases in cSC cells exposed to a) NBO2 in the presence of nominal N2 and b) iso‐oxic hyperbaric nitrogen (HBN2). In the case of HBN2, we wanted to determine if inert gas molecules react with O2 molecules at pressure to form collision complexes that yield 1O2. The fluorogenic dye, Singlet Oxygen Sensor Green (SOSG, 2.5μM), was used to measure SOSG intensity of cSC neurons (P16–40 M/F SD rats, 400μm). Fluorescent images were captured at 1–2.5 min intervals and correlated with 1O2 production using one of two Nikon TE2000 inverted microscopes; one microscope was automated for use inside a hyperbaric chamber. The HCO3−‐buffered nutrient medium was equilibrated at room pressure with either (in atmospheres absolute, ATA) 0.4 O2 + 0.05 CO2 + 0.55 N2 (control) or 0.95 O2 + 0.05 CO2 + “0.0” N2 (NBO2).* For iso‐oxic HBN2, the air‐filled hyperbaric chamber (0.8 ATA N2) was compressed (0.25 ATA/min) with 100% N2 to 80–85 psig (5.4–5.8 ATA N2), thereby increasing atmospheric pN2 to 6.2–6.6 ATA (0.95 O2 + CO2 in the nutrient medium; pN2 increases in the medium and slice from HBN2 diffusing in from the overlying atmosphere). Nutrient media were delivered to the brain slice at 1.5 ml/min using an HPLC pump (30–31°C). Results show that increasing pO2 from 0.4 to 0.95 ATA at room pressure increased the rate of 1O2 fluorescence. 1–4 mM ascorbate, but not <1mM, decreased the rate of 1O2 fluorescence in 0.4 ATA O2 and NBO2 in a dose‐dependent manner. Exposure to iso‐oxic (0.95 ATA O2) HBN2 (10–30 min) did not increase 1O2; however, decompression (0.5ATA/min) back to sea level induced a post‐dive increase in 1O2 fluorescence that persisted for 10–30 min. The post‐dive increase in 1O2 fluorescence was inhibited by 4mM ascorbate. These findings suggest that 1O2 production is activated in cSC cells by hyperoxia, compression to at least 6.2 ATA N2, decompression, or the combination of compression/decompression in hyperoxic HBN2. We postulate that 1O2 generated in cSC neurons may contribute, in part, to CNS O2 toxicity, inert gas narcosis, and decompression sickness.

Support or Funding Information

ONR N000141512572 & N000141612537
The Growth and Differentiation Capacity of Porcine Renal Papilla Progenitor Cells Decline with Passaging
Authors: Burmeister David M. , McIntyre Matthew K. , Gomez Belinda I. , Mongomery Robbie , Dubick Michael A.
Published: June 2021

When challenged by an ischemic insult, such as that accompanying traumatic hemorrhage, the kidney undergoes functional recovery via replacement of cells. Different theories about the cellular mechanisms behind this endogenous regeneration exist, however recent studies have identified a population of renal stem/progenitor cells from the renal papillae. With the increased use of swine in, for example, hemorrhagic shock models, we aimed to isolate and characterize progenitor cells from the renal papillae from pig kidneys. After excision and collagenase digestion of renal papillae, non‐adherent cells were removed after 2 days, and the ensuing renal progenitor cell (RPC) population was expanded under normal culture conditions. At passages 2 and 6, RPCs were examined for their: growth kinetics; expression of markers CD24, CD133, CD90, and NFATC1 via flow cytometry; and potential to differentiate into osteoblasts and adipocytes. Additionally, passage 2 RPCs were used in a mixed lymphocyte reaction to determine immunomodulatory activity. At passage 2, RPCs displayed a characteristic spindle‐shaped morphology with substantial proliferative capacity. By passage 6, accelerated vacuolization and minimal growth was seen in RPCs. Flow cytometry revealed that 96.3% of RPCs at passage 2 expressed CD24, which was dramatically reduced to 23.9% at passage 6. Expression of CD90 at the two passages was similar (44.4% and 47.2% at passage 2 and 6, respectively), while there was low expression (~10%) of CD133 or NFATC1 at both passages. Differentiation studies revealed that RPCs were able to differentiate into both adipocytes and osteoblasts at passage 2, but not at passage 6. Moreover, RPCs from passage 2 increased the amounts of IL‐6 and IL‐8 (p<0.05) released in the mixed lymphocyte reaction, but tended to reduce the amount of Interferon‐γ released. Taken together, these data further indicate that porcine RPCs are heterogeneous and lose their “stemness” properties with serial passaging. However, their immunomodulatory capabilities lend them the potential to be leveraged as therapies or therapeutic targets. In instances of ischemic insult, augmentation of RPCs may potentiate the recovery and regenerative properties of the kidney. Further work exploring the characteristics of renal stem cell populations, their interactions with resuscitation fluids, and their efficacy in pre‐clinical animal models is warranted.

Support or Funding Information

US Army Medical Research and Materiel Command (MRMC) provided funding for this project.
Balance of Conformational States Affect the Intrinsic Hydrolysis of NRas When Compared to Other Ras Isoforms
Authors: Reid Derion F , Mattos Carla
Published: June 2021

Ras GTPases are small proteins that are involved in various signal transduction pathways that regulate cellular proliferation, survival, migration, and apoptosis. Although the first crystal structure of Ras was determined more than two decades ago, biochemical and structural differences in the G‐domain of the three isoforms, H‐, K‐ and N‐Ras is only now beginning to emerge. Each isoform functions as a molecular switch where guanine nucleotide exchange factors (GEFs) facilitate the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) turning its signal on. Ras signaling is turned off via intrinsic hydrolysis or through the aid of GTPase activating proteins (GAP). Ras mutants are involved in 20% of human cancers, yet there are no inhibitors that target these proteins effectively. Most of the effort was originally focused on HRas as the classical representative of all three isoforms and more recently on KRas, the most frequently mutated in cancers. Thus, the structural biology and biochemistry of NRas have been largely overlooked. We have determined hydrolysis rate constants for NRas and determined the crystal structure of wild‐type NRas bound to GppNHp (GNP), a non‐hydrolyzable analogue of GTP, for comparison with H‐Ras and K‐Ras. Although each of the three isoforms follow a one‐step mechanism of hydrolysis, K‐ and NRas have lower rates than HRas, with structural features that promote disorder in switch II near the active site. N‐Ras mutants at G12 and Q61 are commonly found in melanoma tumors. These conserved residues are found in the phosphate‐binding loop (G12) and switch II (Q61) that help make up the active site of NRas. The structures of NRas mutants reveal distinct mechanisms through which they affect catalysis, consistent with lower hydrolysis rate constants found for G12D, G12S, Q61H, and Q61K compared to wild‐type NRas. Biochemical and structural understanding of NRas mutants will help further influence the development of novel therapeutic targets of NRas mutant melanoma.

Support or Funding Information

This research was funded by NSF Grant Number MCB‐1517295.
A High Serum Vanin‐1 Phenotype is Not Unique to Diving Marine Mammals
Authors: Boxall Baylye K. , Prager Katherine , Neely Benjamin A. , Lloyd‐Smith James O. , Gulland Frances , Janech Michael G.
Published: June 2021

Diving marine mammals have elevated levels of antioxidant proteins in their tissues to counter oxidative stress in part due to diving. A proteomic study of bottlenose dolphin serum identified and measured the highest concentration of pantetheinase (Vanin‐1) for any mammal. Vanin‐1 is known to produce the antioxidant, cysteamine, from the vitamin B5 precursor, pantetheine. High levels of this protein may represent an adaptation or response to diving in marine mammals. If this is true, then elevated levels of circulating Vanin‐1 may be a state shared amongst, but unique to, diving marine mammals. To test this hypothesis, sera from a diving pinniped (N=6), California sea lion (Zalophus californianus), were assayed for Vanin‐1 concentration by liquid chromatography/tandem mass spectrometry using parallel reaction monitoring. Commercially purchased sera from 9 terrestrial species belonging to five different mammalian orders: Rodentia (Muridae), Primates (Hominidae), Cetartiodactyla (Suidae and Bovidae), Perissodactyla (Equidae), and Carnivora (Canidae) were also monitored via product ion scan for a tryptic peptide homologous to the sea lion Vanin‐1 peptide. The presence of the monitored Vanin‐1 peptide was detected in 5/6 sea lion samples as defined by a lower limit of detection of 3.7 μg/mL, which was calculated by 3.3σ of the lowest standard used in an external calibration curve. Only 3/6 serum samples had measureable concentrations above the lower limit of quantification of 5 μg/mL, which was calculated by 10σ of the lowest standard. Of those samples above the lower limit of quantification the mean ± standard deviation of Vanin‐1 concentration was 5.7 μg/mL ±0.4. Monitoring of serum Vanin‐1 by product ion scan in terrestrial mammals revealed a signal that was well above the lower limit of quantification in sheep, cow, goat, donkey, and horse, but not detected in pig or dog. Product ion scan data in these species were consistent with proteomic rank data. These findings indicate that a high circulating Vanin‐1 phenotype is not unique to diving marine mammals. Data suggest that an elevated Vanin‐1 phenotype is shared amongst some members of the clade Ungulata, which includes the orders Perissodactyla and Artiodactyla, within which fall cetaceans.

Support or Funding Information

Research was supported in part by the Office of Naval Research grant N000141410361, National Science Foundation grant OCE‐1335657, and the John H. Prescott Marine Mammal Rescue Assistance Grant Program.
Activation of Caspase‐8/BID Pathway in Dexamethasone‐Induced Apoptosis in a Human Lens Epithelial Cell Line
Authors: Ali Mohammed S
Published: June 2021

Background

Prolonged glucocorticoid steroid usage is capable of inducing changes to the transcription of genes in lens epithelial cells and is associated with the development of several ocular diseases, including cataract formation. Mechanisms of cataract formation, however, are not well known, although apoptosis has been implicated. We therefore investigated glucocorticoid dexamethasone‐induced apoptosis in a human lensepithelial cell line.

Methods

Human lens epithelial cells were exposed to dexamethasone and apoptosis was evaluated using flow cytometry for AnnexinV expression, chromogenic substrates for Caspase activity measurements, Western blotting for Bax andBCL‐2 expression as well as PARP cleavage, and agarose gel electrophoresis for detection of DNA fragmentation. Mitochondrial pathway activity was determined by measuring the levels of Cytochrome C release, Bax translocation to the mitochondria, and BID cleavage.

Results

A significant induction of early apoptosis was observed by the AnnexinV/PI dual staining flow cytometric analyses. Increased expression ratios of pro‐apoptotic protein Bax toanti‐apoptotic protein BCL‐2 following dexamethasone exposure demonstrated apro‐apoptotic tendency. Sequential activation of caspase‐8 and caspase‐3, and BID cleavage with Cytochrome C extrusion from mitochondria, indicated an extrinsic apoptotic pathway. Nuclear changes of DNA fragmentation and PARP cleavage, both indicative of late stage apoptosis, were detected by electrophoresis only after dexamethasone exposure, despite lack of increased late stage apoptotic detection using Flow cytometry.

Conclusion

Results suggest that Dexamethasone exposure induces an extrinsic apoptotic pathway in a human lensepithelial cell line which follows along the activation of caspase‐8/BIDpathway and supports the hypothesis that apoptosis may be an important mechanism involved in steroid‐induced cataract formation.

Support or Funding Information

Intramural Funding from the Department of Defense
Characterization of Saccharomyces cerevisiae Coenzyme Q Biosynthetic Protein Coq11
Authors: Bradley Michelle C. , Awad Agape M. , Allan Christopher M. , Johnson Jarrett S. , Shirasaki Dyna I. , Wang Charles , Blaby‐Haas Crysten E. , Loo Joseph A. , Clarke Catherine F.
Published: June 2021

Coenzyme Q, also known as ubiquinone or Q, is a redox‐active lipid component of the electron transport chain that functions in cellular energy metabolism. Due to its redox capabilities reduced Q (or QH2) also serves as an important lipid‐soluble antioxidant effective in alleviating lipid peroxidative damage. In Saccharomyces cerevisiae, thirteen known mitochondrial proteins, Coq1‐Coq11, Yah1, and Arh1, drive Q biosynthesis. Many of these Coq polypeptides required for Q production are localized to the matrix side of the mitochondrial inner membrane where they form a high molecular weight, multi‐subunit complex known as the ‘CoQ‐synthome’. Absence of individual Coq proteins causes severe defects in Q biosynthesis, and such coq null mutants lose the ability to respire. However, the functional roles of some Coq polypeptides as well as several steps in the Q biosynthetic pathway remain unknown. Here, we present the identification of a novel mitochondrial protein that associates with the CoQ‐synthome encoded by the open reading frame YLR290C, which we renamed Coq11. Co‐immunoprecipitation and lipid analyses demonstrate that Coq11 interacts with other Coq polypeptides and with Q and Q‐intermediates, and is required for efficient de novo Q biosynthesis. We have begun work towards understanding the functional relationship between Coq11 and its human homolog, NDUFA9. Taken together, this project will provide powerful insights into Q biosynthesis and regulation towards the objective of designing effective therapeutics for diseases caused by Q deficiencies. This research was supported by NSF MCB‐1330803 and the Ruth L. Kirschstein National Research Service Award GM007185.

Support or Funding Information

NSF MCB‐1330803 and the Ruth L. Kirschstein National Research Service Award GM007185
Iodine Status and Its Determinants among Adolescent School Girls in Rural Tanzania
Authors: Bakar Saidah Mohamed , Lee Kyung Won , Song Won O.
Published: June 2021

Iodine deficiency is still one of the most serious public health problems in developing countries in the world. The high rates of adolescent pregnancies in Tanzania exacerbate the vicious cycle of maternal child malnutrition including iodine deficiency. It has been well‐documented that maintaining adequate iodine supply during pregnancy is essential for maternal healthy thyroid function and fetal neurological development. Increased iodine requirements for pregnancy and a high demand for nutrients for growth in adolescent make them vulnerable to inadequate iodine status. However, few studies have reported determinants of iodine status in rural areas of Tanzania. We aimed to assess iodine status and identify its determinants among adolescent schoolgirls in rural Tanzania. Four secondary day schools in Iringa region in Tanzania were randomly selected for this cross‐sectional study during the lean season (January–March) in 2016. A total of 208 adolescent girls aged 12–19 years were surveyed with a questionnaire on general and socioeconomic characteristics of themselves and their caregivers; dietary intakes; and use of iodized salt in cooking at home, and single spot urine samples. Multivariable logistic regressions were performed to identify determinants of iodine status, while controlling for covariates. Based on UICs from urine samples, iodine status of the adolescent girls was adequate (median UIC: 227.1 μg/L) and the prevalence estimate of UIC <50 μg/L was 16.4% among adolescent girls in rural Tanzania. Adolescent girls in the lowest tertile of UIC tended to live in a smaller household and have their caregivers having a low level of education and working as blue‐collar employees (peasant farmers) than those in the highest tertile of UIC. Adolescent girls living in semi‐urban were more likely to be at risks for UIC <50 μg/L (adjusted odds ratio [AOR]=16.32, 95% confidence interval [CI]: 4.92–54.11, P <0.01), compared to those living in rural area. Adolescent girls who have caregivers working as peasant farmers had a lower risk of UIC <50 μg/L, than those who have caregivers working as a white‐collar employee such as educator, health professionals, and business person (AOR=0.17, 95% CI:0.03–0.97, P <0.05). The present study provides the evidences that socioeconomic status of caregivers was important determinants of adolescent girls' iodine status. Therefore, nutrition education for adolescents' caregiver should be emphasized to combat nutritional inadequacies. Further investigations longitudinally designed with large samples are warranted to identify risk factors of iodine deficiency among adolescent girls of childbearing age in Tanzania.

Support or Funding Information

This study was conducted by the generous financial support of a USAID‐funded Food Security Project, Innovative Agricultural Research Initiative (iAGRI) in Tanzania.
Effects of Hepcidin on Zinc Homeostasis in HepG2 cells
Authors: Kelley Alyssa M. , Hennigar Stephen R. , McClung James P.
Published: June 2021

Hepcidin, a peptide hormone produced by theliver in response to iron loading and inflammation, regulates body iron storesby inhibiting iron transporter function. Recently, hepcidin was shown toinhibit zinc efflux in intestinal epithelial cells. The objective of this study was to determine whether hepcidin affects hepatic zinc homeostasis. Human HepG2cells were treated ± 1μM hepcidin for 24 h. Labile zinc pools were labeled with the zinc‐specific fluoroionophore FluoZin‐3‐AM (Kd 15 nM) and visualized and quantified using confocal microscopy and a fluorescent platereader. Expression of metallothionein, ZIP14, and ZnT1 was determined by Western blot. Intracellular zinc in control cells was localized to vesiclesdispersed throughout the cytoplasm; however, the number of vesicles and intensity of FluoZin‐3 fluorescence decreased by 38% in cells treated with hepcidin for 24 h (34.0 ± 11.3 and 21.0 ± 11.2 RFU/OD 562 nm; P<0.05). Metallothionein expression increased in cells treated with hepcidin as compared to control cells(P<0.05). ZIP14 expression remained unchanged (P>0.05), whereas hepcidin increased ZnT1 expression (P<0.05) as compared to control cells. These data suggest that hepcidin may affect zinc homeostasis in liver cells. Understanding the mechanism and role of hepcidin in zinc homeostasis may provide insight in to the hypozincemia associated with inflammation and infection.

Support or Funding Information

The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. Support provided by USAMRMC.
Prolonged high altitude exposure exacerbates fat‐free mass and fat mass loss during negative energy balance regardless of dietary protein intake
Authors: Berryman Claire E. , Karl J. Philip , Cole Renee E. , Kenefick Robert W. , Margolis Lee M. , Carbone John W. , Ferrando Arny A. , Lieberman Harris R. , Young Andrew J. , Pasiakos Stefan M.
Published: June 2021

Protein intake above the recommended dietary allowance attenuates fat‐free mass (FFM) loss during moderate negative energy balance (~40% energy deficit) at sea level (SL). Sustained periods of strenuous physical activity at high altitude (HA) may exacerbate FFM loss, especially during energy deficit. However, increased dietary protein intake has not been evaluated as a countermeasure to FFM loss at HA. This study aimed to determine whether consuming dietary protein at amounts consistent with current recommendations for elevated physical activity (2.0 g protein/kg) attenuates FFM loss during prolonged energy deficit at HA. Seventeen healthy males (mean ± SD, 23.4 ± 5.6y, 81.9± 13.9 kg) participated in a 2‐phase, 43‐d study. During phase 1 (SL), participants were instructed to consume a weight maintenance diet with standard protein intake(1.0 g protein/kg, STD) for 21 d. During phase 2, participants resided at HA (4300 m, Pikes Peak, CO) for 22 d and were randomly assigned to either a STD or high (2.0 g protein/kg, HIGH) protein diet designed to elicit a 40% energy deficit (30% dietary restriction and 10% increase in physical activity, estimated energy deficit was 1074 ± 122 kcal/d based on the total energy requirements necessary to maintain body mass during phase 1) and~3 kg total body mass loss. Diet and physical activity were highly controlled. Total body mass, FFM, and fat mass (FM) were assessed by dual energy x‐ray absorptiometry and resting metabolic rate (RMR) by indirect calorimetry. Volunteers were weight stable during SL and lost −7.9 ± 1.9 kg(P<0.01)during HA, regardless of protein group (P>0.05). Decrements in FFM (−4.0 ± 3.3 vs. −3.2 ± 1.5 kg) and FM (−3.3 ± 1.8 vs. −3.9 ± 0.8 kg) were not statistically different between STD and HIGH (P>0.05). The overall energy deficit was 2306 ± 444kcal/d when calculated based on changes in FFM and FM. The additional energy deficit was attributable, in part, to an increase in RMR, which peaked at 58%above SL on HA day 1 and remained ≥ 25% higher than SL throughout HA(P<0.05), accounting for 27% (634 ± 273 kcal/d) of the total energy deficit at HA. The remaining 598 kcal/d (26% of total deficit) could not be accounted for by estimated energy expenditure of prescribed daily physical activity and measured energy intake, suggesting the energy cost of physical activity may be greater at HA than SL. Prolonged HA exposure exacerbated the catabolic effects of negative energy balance. In this context, consuming a higher versus standard protein diet was equally ineffective at sparing FFM with weight loss. The opinions or assertions contained herein are the private views of the author(s)and are not to be construed as official or reflecting the views of the Army or the Department of Defense.

Support or Funding Information

Supported by USAMRMC.
Expression of Sodium‐Dependent Cation‐Chloride Cotransporters in Adult and Larval Osmoregulatory Tissues of Aedes aegypti Mosquitoes
Authors: Jamil Taylor L. , Chen Jiayu , Crow John C. , Kerkhoff Willa G. , Akuma Daniel C. , McQuiston Alexander , Piermarini Peter M. , Gillen Christopher M
Published: June 2021

Yellow fever mosquitoes, Aedes aegypti, face diverse salt and water challenges. Larvae lose salt to the freshwater environment and gain water osmotically, whereas adults tend to lose water by evaporation. Moreover, adult females take in large quantities of salt and water during a blood meal. We hypothesize that sodium‐dependent cation‐chloride cotransporters (CCCs) contribute to the salt and water balance of Aedes aegypti by participating in epithelial salt secretion and absorption. Sequence analysis revealed three genes (AAEL006180, AAEL009888, and AAEL009886) in Aedes aegypti with similarity to vertebrate Na‐K‐Cl cotransporters. We performed multiple sequence alignment and phylogenetic analysis of the proteins encoded by the Aedes aegypti genes and orthologs in Culex and Anopheles mosquitoes, Drosophila, and other arthropods. The protein encoded by AAEL006180 groups in a clade with the human secretory Na‐K‐Cl cotransporter and a Drosophila cotransporter known to be involved in salt secretion. In contrast, AAEL009888 and AAEL009886 fall into a clade that includes sequences from other insects but not vertebrates. Moreover, both AAEL009888 and AAEL009886 have orthologs in other mosquito genera but other insects only have one ortholog, indicating that AAEL009888 and AAEL009886 probably diverged early in the evolution of mosquitoes. Amino acids that differ among the paralogs include residues known to influence ion and inhibitor binding affinities. Using qPCR, we quantified the relative gene expression of the Aedes aegypti paralogs with at least two isoform‐specific primer sets for each gene. AAEL009886 transcript is expressed at significantly greater levels in larvae compared to adults and at significantly greater levels in anal papillae compared to Malpighian tubules, suggesting that AAEL009886 may be specialized for ion absorption in larvae. In contrast, AAEL006180 and AAEL009888 transcripts are expressed at approximately the same levels in adults and larvae. To evaluate the physiological role of the proteins encoded by AAEL006180 and AAEL009888, we attempted to reduce expression of each transporter using dsRNA. Larvae exposed to dsRNA corresponding to either transporter had up to five‐fold increases in hemolymph ammonium levels compared to negative controls as well as smaller changes in other hemolymph cations. Our results point towards physiological roles for AAEL006180 and AAEL009888 in osmoregulation and ammonia balance of larval mosquitoes.

Support or Funding Information

NSF‐IOS‐1557230
Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies
Authors: Zepeda Samantha K. , Villarreal Michael A. , Biediger Nicole M. , Bonner Natalie A. , Miller Jennifer N. , Ricard Benjamin J. , García Dana M. , Lewis Karen A.
Published: June 2021

Antibodies are produced by the immune system as a defense against foreign proteins due to their ability to bind with these molecules with a high degree of specificity. The highly specific nature of antibody/antigen interaction has made them of great use in a variety of applications that identify the presence of the protein antigen, including western blotting and immunohistochemistry. Zebrafish (Danio rerio) is a versatile model genetic organism that has been pivotal for numerous studies of vertebrate physiology. Historically, there were very few antibodies developed specifically against zebrafish proteins. Therefore, many studies used antibodies against human protein homologs to identify zebrafish proteins. However, the evolutionary ancestor of zebrafish experienced a whole genome duplication event. As a result, the zebrafish genome expresses several paralogs of many genes that have only a single homolog in most other vertebrates. This presents an interesting problem: can the antibodies raised against human proteins be relied upon to identify and discriminate between the different paralogs of zebrafish genes? To test the epitope conservation between human and zebrafish homologs, we tested a commercial antibody against the human melatonin receptor protein 1 (MTNR) for cross‐reactivity with putative zebrafish epitopes. Zebrafish express six paralogs of this protein: Mtnr1Aa, Mtnr1Ab, Mtnr1A‐like, Mtnr1Ba, Mtnr1Bb, and Mtnr1C. The putative epitope binding regions of these proteins display a variety of evolutionary conservation. These peptide sequences were cloned into a bacterial expression vector between an N‐terminal SUMO solubility tag and a C‐terminal 6xHis affinity purification tag. Following expression in Rosetta (DE3) E. coli, the fusion proteins were purified via nickel affinity column. Western blotting was carried out with anti‐His and anti‐human MTNR1 probes.

The zebrafish Mtnr1A subtype demonstrated cross‐reactivity with anti‐human MTNR1, In contrast, the Mtrn1B and Mtnr1C subtypes were not recognized at all. Interestingly, the Mtnr1C protein contains only conservative differences in amino acid sequence from the human MTNR1. These data conclusively show that the proteins being detected in immunohistochemical assays with this antibody are of the Mtrn1A subtype. Additionally, this project demonstrates the importance of validating antibody specificity through experimental means to ensure that the protein(s) of interest is indeed being detected.

Support or Funding Information

National Science Foundation (DBI‐0821252)
Mating yeast cells concentrate the pheromone receptor and its G protein as polarized crescents at the default polarity site that then track to the eventual chemotropic site
Authors: Wang Xin , Stone David E.
Published: June 2021

Chemotropism and chemotaxis are fundamental processes required for a broad range of biological phenomena. The mating process of budding yeast (Saccharomyces cerevisiae) is, to date, the best‐studied example of chemotropism. In mating mixtures, haploid yeast cells can interpret a shallow pheromone gradient, chemotrope toward the closest mating partner, and fuse to form a diploid zygote. The molecular machinery required for polarized growth has been well characterized. However, how yeast cells accurately position the polarity machinery towards the source of pheromone is unclear. It is well known that the pheromone receptor and its cognate G protein are uniformly distributed on the plasma membrane of vegetative cells, and that they polarize in response to pheromone. In our published model of yeast gradient sensing, inhibition of receptor phosphorylation by Gβg results in differential phosphorylation of the receptor across the cell surface, and consequently, lesser internalization of the receptor and G protein on the up‐gradient side of the cell. A key question is how the uniformly distributed surface receptor competes for a limiting amount of G protein. Here we show that in mating cells, the initially uniform receptor and G protein first localize as polarized crescents at the default polarity site. The receptor and G protein crescents then track along the plasma membrane until they reach the region of highest pheromone concentration, centered around the position at which the cell ultimately shmoos towards its partner. We also show that polarization of Gβ to the default polarity site is independent of receptor phosphorylation and polarization, whereas Gβ tracking from the default site to the eventual chemotropic site does not occur if receptor phosphorylation and redistribution are blocked. These observations suggest a new mechanism that localizes the receptor with its much less abundant G protein. In our revised model, we propose that mating cells that are arrested in G1 concentrate Gβg at the default polarity site, likely through its interaction with Far1‐Bem1‐Cdc24‐Cdc42. The polarized Gβg then protects the receptor from being phosphorylated and internalized, thereby triggering local accumulation of the receptor and G protein. Because the pheromone gradient is mirrored by a gradient of signaling activation within the receptor/G‐protein crescent, there are higher proportions of active‐unphosphorylated receptors and active G protein closer to the pheromone source. The peak of signaling activity incrementally moves up the pheromone gradient, as unprotected receptors are phosphorylated and co‐internalized with G proteins at the back, while vesicles containing nascent receptors and G proteins preferentially dock where the receptor is most abundant.

Support or Funding Information

NSF
Involvement of SNX27‐retromer in ASCT2 trafficking and glutamine uptake
Authors: Yang Zhe , Follett Jordan , Kerr Markus , Clairfeuille Thomas , Collins Brett , Teasdale Rohan
Published: June 2021

Mammalian retromer is composed of cargo‐recognition trimer comprised of Vps26, Vps29 and Vps35 and membrane recruiting subcomplex formed by sorting nexin (SNXs), which has been shown to play a critical role in sorting various receptors (cargoes) from the endosome to the TGN. Recently, several studies have been focused on retromer in complex with SNX27 and its function in retrieval of receptors from the endosome to the plasma membrane. Here we investigate functional consequence of SNX27‐retromer‐mediated trafficking of one such cargo, ASCT2.

ASCT2 is a major glutamine transporter, allowing the uptake of glutamine into the cells as one of the major fuel sources and facilitating amino acid stimulated mTOR activation, and ASCT2 overexpression has been observed in several human cancers. We here identify ASCT2 as an SNX27‐retromer cargo mediated by SNX27 PDZ domain, as shown by co‐immunoprecipitation that a SNX27 truncated construct, lacking of PDZ domain, abolished the binding, despite their co‐localization was unperturbed. Using both biochemical (cell surface biotinylation) and subcellular localisation methodologies, we further demonstrate that ASCT2 mainly resides at the cell surface, and this plasma membrane localisation is significantly lost upon CRISPER‐mediated SNX27 knockout (KO). SNX27 KO leads to the missorting of ASCT2 to the degradation pathways, which can be partially reversed by the presence of chloroquine, an inhibitor for vacuolar type H+‐ATPase, or MG132, a proteasome inhibitor. ASCT2 reduction in SNX27 KO cells leads to the decreased glutamine uptake as measured by L‐H3‐glutamine incorporation, associated with the decreased cell proliferation rate and the increased cell cycle arrest. Furthermore, SNX27 KO cells showed the reduced activation of amino acid‐stimulated mTOR pathway, yet the enhanced autophagy activation as measured by LC3 levels. Taken together, this data indicates a role of SNX27‐retromer in glutamine homeostasis via the modulation of ASCT2 sorting.

Support or Funding Information

Australian Research Council Grant DP160101573
Inhibition of Na,K ATPase partial reactions by organic amines
Authors: Stanley Kevin , Artigas Pablo , Gatto Craig
Published: June 2021

The Na,K‐ATPase (NKA) transports 3 Na ions out of the cell and 2 K ions into the cell for every one ATP molecule hydrolyzed. Two of the three ion‐binding sites within the protein reciprocally bind two K or two Na ions (shared sites), while the other site exclusively binds Na). Three modes of transport can be studied with two electrode voltage clamp (TEVC) in Xenopus oocytes to evaluate NKA function and its partial reactions involving external ion binding: 1) outward pump current (IP) induced by forward NKA cycling when extracellular K is added, 2) transient charge movement (QNa) induced by voltage‐dependent binding and release of Na, rate limited by the NKA conformational change and 3) uncoupled proton import at negative voltages without external Na or K the pump transports H down their electrochemical gradient (IH). Tetrapropylammonium (TPA) and ethylenediamine (EDA), are known competitive inhibitors of extracellular K binding and their mechanisms of action are not fully understood (particularly for EDA). We used TEVC to evaluate the effect of TPA and EDA on IP, QNa and IH. TPA acted as a competitive inhibitor of K binding by reducing its apparent affinity for IP activation in a voltage‐independent manner IC50 of 8.78 ± 0.12 mM. EDA also acted as a K competitor, but its IC50 changed from 8.96 ± 0.48 mM at −100 mV to 15.92 ± 1.84 mM a 0 mV. When evaluating QNa, the position of the charge voltage curve (Q‐V), parameterized by the curve's center voltage (V1/2) related to the apparent affinity for external Na (more negative V1/2 indicating a reduction of Na affinity and viceversa), was −54.39 ± 1.65 mV in the absence of amines, −120.88 ± 2.15 mV in the presence of 20 mM TPA and −10.49 ± 6.13 mV in the presence of 15 mM EDA. While TPA did not significantly affect the total charge moved (related to the number of pumps performing the Na binding reactions) EDA reduced it by 53.6 ± 0.3%. This indicates that TPA acts as a prototypical competitive inhibitor of external Na reducing Na apparent affinity, but EDA does not. Finally, TPA inhibited IH (~70% maximal inhibition) at both pH 7.6 and pH 6.0, while EDA had no effect on IH at pH 7.6, but increased IH (by 2 fold) at pH 6.0 at −180 mV. Our results are consistent with the larger TPA acting as a large extracellular access‐channel blocker therefore competing with Na, K and H without reaching the ion‐binding sites, while EDA reaches the shared sites domain, probably becoming occluded there, therefore making NKAs unavailable to induce QNa. The complex effects of EDA on IH may reflect the complex nature of its occlusion there and are still under evaluation.

Support or Funding Information

Work supported by NIH GM 061583 to CG and NSF MCB‐1243842 to PA.
Characterization of Anthocyanins in Sweetpotato leaves
Authors: Su Xiaoyu , Griffin Jason , Chen Xi , Xu Jingwen , Wang Weiqun
Published: June 2021

As phytochemical‐enriched edible greens, sweetpotato (Ipomoea batatas L.) leaves have become popular due to potential health benefits. However, the phytochemical contents in sweetpotato leaves and their subsequent change over harvest stages and growth condition are mostly unknown. In this study we determined the anthocyanin profile and content in leaves of four sweetpotato cultivars (Bonita, Beauregard, Murasaki, and P40). Fourteen anthocyanins, including one newly discovered, were identified by HPLC‐MS/MS. The most abundant was cyanidin 3‐caffeoyl‐p‐hydroxybenzoyl sophoroside‐5‐glucoside, which comprised up to 20% of the total anthocyanins. Of the young leaves (1st and 2nd slip cuttings), Bonita contained the highest anthocyanin content followed by P40. Of the mature leaves (vine stage), Beauregard had the greatest anthocyanin (391 mg/kg DW) and total phenolic (52 GAE/g DW) content. It should be noted that the lowest anthocyanin and total phenolic content of shoots were found in P40, while tubers of P40 contain the highest content of each. Furthermore, the increase in leaf anthocyanin content over the growth stages that was observed in three of the cultivars was absent in P40. No significant difference of anthocyanin content was found in Beauregard leaves grown in the high tunnels or open field.

Support or Funding Information

supported by USDA Cooperative KS511‐1001903
Effective delivery of social‐behavioral change communication through a care group model in a supplementary feeding program: a descriptive analysis
Authors: Wilner Lauren , Suri Devika , Langlois Breanne K , Walton Shelley , Rogers Beatrice
Published: June 2021

Objective

A USAID/FFP‐Funded study implemented by Tufts University (Food Aid Quality Review) in Southern Malawi found that intensified social‐behavioral change communication (SBCC) in combination with an augmented oil ration resulted in an increase in proper preparation of Corn‐Soy Blend (CSB) porridge such that the full intended nutritional content reached the target population of children with moderate acute malnutrition. This analysis describes the flow of key SBCC messages delivered through a care‐group model during an intervention aimed at increasing the oil in porridge prepared by caregivers of beneficiary children, as well as the flow of these key messages among sub‐groups in this intervention – health care workers, care group volunteers, and caregivers of beneficiary children.

Methods

The intervention provided a supplementary food ration of CSB and oil and used a care group model in which healthcare workers were trained to deliver SBCC to care group volunteers who then delivered messages to caregivers of beneficiary children. Healthcare workers also delivered messages to caregivers directly. Interviews and focus group discussions were conducted with all 3 sub‐groups in order to determine the exchange of key messages about ingredient use, storage, and purpose, which were analyzed descriptively. The analysis tracked the flow of information between the groups, and ultimately to the beneficiary caregiver.

Results

Analysis of SBCC flow and information exchange showed that 100% of caregivers reported learning about the amounts of oil and CSB to use while preparing porridge, and over 90% of caregivers, healthcare workers, and care group volunteers reported talking about it. Information flowed to caregivers along multiple lines of communication, and the reported exchange of this information was high among all groups. Focus groups confirmed an effective flow of communication amongst these 3 sub‐groups.

Conclusion

Key SBCC messages were provided and received according to healthcare workers, care group volunteers and caregivers; caregivers received information from multiple sources, contributing to the overall effectiveness of the intervention. While increasing the ration likely contributed to increasing the oil content in prepared CSB porridge, this analysis found that the SBCC was also an important component of the intervention.

Support or Funding Information

This research was made possible by the generous support of the American people through the United States Agency for International Development (USAID). The contents are the responsibility of Tufts University under the terms of Contract AFP‐C‐00‐09‐00016‐00 and do not necessarily reflect the views of USAID or the United States Government.
East Feliciana Parish LA Has Limited Availability of Nutritional Food Sources Which May be Associated with Obesity and Limit Individuals' Ability to Meet Dietary Recommendations
Authors: Alexis Torrey D , O'Neil Carol E , Cater Melissa , Holston Denise
Published: June 2021

Low incomes, rural residence, high obesity rates, and limited healthy food options can increase the risk of chronic disease. Rural East Feliciana Parish (county), Louisiana (EFP) has an area of ~543 sq miles, a population of ~19,000, a per capita income of $21,370, and an obesity prevalence of 39%. With this high rate of obesity and low income, it is important to assess more fully the food environment to determine the availability of healthy food choices. The goal of this study was to assess the food sources (stores and restaurants) of EFP for the availability of healthy options using the validated Nutrition Environment Measures Survey (NEMS) Reduced Item Audit Tool. The NEMS assesses store nutrition environments such as the availability of healthier and less‐healthy options, including: milk, fresh fruit and vegetables, beverages, and whole grain bread. Restaurant assessment includes food indicators including: availability of fruit and vegetables without added sauce, whole grain bread, and beverages. EFP had 25 sites of food availability: two full‐service grocery stores (included in the descriptive analysis, but not in the statistical analysis), eight convenience stores, and six variety stores; there were five fast food, three fast casual, and one “sit down” restaurants. Data were compared using the Mann‐Whitney U test; p<0.05 (two tailed). Eight stores (50.0%) offered non‐ or low‐fat milk, and six stores (37.5%) offered whole wheat or whole grain bread. Fifteen stores offered whole fruit (13%) or 100% fruit juice (87.5%), and thirteen stores (81.3%) offered vegetable options. There were no significant differences between convenience and variety stores regarding availability of non‐ or low‐fat milk, whole or non‐whole grain bread, fresh fruit or 100% fruit juice, or vegetables. Only one restaurant (11.1%) offered non‐ or low‐fat milk, and only two restaurants (22.2%) offered whole wheat or whole grain bread. Four restaurants (44.4%) offered fruit or fruit juice options, and five restaurants (55.6%) offered healthy vegetable or salad options. Healthy food option scores for fast food restaurants did not differ significantly from fast casual restaurants, suggesting that restaurants which may be perceived as offering more healthy foods did not. There was also no significant difference between fast food and fast casual restaurants regarding nutrition information provided on menus or restaurant website or healthy eating signage inside the restaurant. This study suggested that EFP has a limited availability of nutritional food sources making it difficult for individuals to meet dietary recommendations.

Support or Funding Information

Supported by the LSU College of Agriculture, the LSU AgCenter, and USDA Hatch Project LAB 94209.
Predictors of Cardiovascular Health in Older Adults: a Regression Tree Analysis
Authors: Anderson Amy , Sahyoun Nadine , Moshfegh Alanna , Loh Wei‐Yin
Published: June 2021

Background

Cardiovascular health is central to overall health. Studies on predictors of cardiovascular health in older adults have not fully considered potential interactions among predictor variables.

Objective

The purpose of this study was to determine the relationships of lifestyle behaviors and body composition with cardiovascular health in older adults while accounting for potential interactions among lifestyle behaviors, measures of body composition and socio demographic characteristics.

Methods

The Health, Aging and Body Composition (Health ABC)Study is a prospective cohort study of 3,075 adults aged 70 to 79 at baseline. The Generalized, Unbiased, Interaction Detection and Estimation (GUIDE) algorithm for regression tree analysis was used to predict measures of cardiovascular health, including high‐density lipoprotein (HDL) cholesterol, interleukin‐6 (IL‐6), homeostasis model assessment of insulin resistance (HOMA‐IR), and systolic blood pressure (SBP), of Health ABC participants based on their lifestyle behaviors, measures of body composition and socio demographic characteristics.

Results

Participants with the highest mean HDL cholesterol had trunk lean mass ≤21.6 kg and abdominal visceral fat ≤96.2 cm2, while those with the lowest HDL cholesterol had trunk lean mass >27.8 kg. Persons with the lowest IL‐6 had a sagittal diameter ≤24.7 cm (or had missing values for sagittal diameter) and flossed their teeth at least 3 times per week, while those with the highest IL‐6 had a sagittal diameter >24.7 cm. Persons with the lowest HOMA‐IR had a BMI ≤27.4 kg/m2 (or had missing values for BMI), while those with the highest HOMA‐IR had a BMI >27.4 kg/m2 and a sagittal diameter >27.1 cm. Participants with the lowest SBP were those who were not on (or had missing data on) blood pressure medication, were from the Memphis clinical site and were white, while those with the highest SBP were those on high blood pressure medication from the Pittsburgh site.

Conclusion

This study provides further evidence that abdominal adiposity is strongly linked to cardiovascular risk in older adults and supports the use of sagittal diameter as an indicator of cardiovascular health. It also suggests a strong inverse relationship between lean body mass and HDL cholesterol, which warrants further investigation.

Support or Funding Information

This research is supported by USDA‐ARS Specific Cooperative Agreement 8040‐53000‐018‐08. This study is also supported in part by National Institute on Aging (NIA) Contracts N01‐AG‐6‐2101;N01‐AG‐6‐2103; N01‐AG‐6‐2106; NIA grant R01‐AG028050, NINR grant R01‐NR012459, and by the Intramural Research Program of the NIH, NIA.