bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of Caspase‐8/BID Pathway in Dexamethasone‐Induced Apoptosis in a Human Lens Epithelial Cell Line
Authors: Ali Mohammed S 
Published: June 2021

BackgroundProlonged 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.MethodsHuman 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.ResultsA 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.ConclusionResults 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 InformationIntramural 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 InformationNSF 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 InformationThis 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 InformationThe 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 InformationSupported 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 InformationNSF‐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 InformationNational 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 InformationNSF
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 InformationAustralian Research Council Grant DP160101573