bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular basis for import cargo discrimination by importin α isoforms (793.2)
Authors: Pumroy Ruth , Cingolani Gino
Published: June 2021

The human genome encodes seven isoforms of importin α grouped into three subfamilies, α1, α2 and α3. All isoforms have an N‐terminal auto‐inhibitory Importin β binding (IBB)‐domain and a C‐terminal helical core that associates with cargos bearing a Nuclear Localization Signal (NLS‐cargos). Although all importin α isoforms can associate with classical NLS‐cargos, certain cargos are selectively imported by specific isoforms. This specificity is not seen in vitro, as NLS‐cargos bind different ΔIBB isoforms with comparable affinity. To identify rules for importin α selection, we have determined high‐resolution crystal structures of representative isoforms from each family of importin α in complex with a model NLS‐cargo. In parallel, structure‐based mutagenesis and an in vitro binding assay were used to examine the degree of IBB‐dependent auto‐inhibition among different isoforms. This work led us to three findings: (i) all isoforms share an identical NLS‐binding groove across the inner surface of ARM 2‐8. (ii) Although the NLS‐binding groove is invariant, importin α isoforms present different curvature, which positions NLS‐cargos with subtly different binding energetics. (iii) Importin α isoforms are differentially auto‐inhibited by their IBB‐domains. We propose that these three factors determine NLS‐cargo discrimination and explain the specificity for importin α isoforms observed in living cells.

Grant Funding Source: Supported by T32 GM 100836‐2
BRAF inhibitor resistance is mediated by N‐Ras upregulation in melanoma (796.3)
Authors: Ahn Jun‐Ho , Lee Michael
Published: June 2021

The clinical benefit of BRAF inhibitors therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying acquired resistance to BRAF inhibitor by comparing the signaling pathway in the parental A375P cells and resistant sub‐line (A375P/Mdr). We demonstrate that MAPK re‐activation is not attributed to the mechanism of resistance to UAI‐201 in A375P/Mdr cells. Relative quantitative analysis with the 2‐ΔΔCt method revealed that BRAF inhibitor resistance observed in A375P/Mdr cells was not mediated through the overexpression of MDR proteins. We found that expression of N‐Ras was up‐regulated in BRAF inhibitor‐resistant A375P/Mdr cells compared to A375P cells. Actually, siRNA‐mediated N‐Ras knockdown partially conferred UAI‐201 sensitivity to A375P/Mdr cells, implying that acquired resistance to UAI‐201 developed by N‐Ras up‐regulation. Notably, the flow cytometric analysis revealed that UAI‐201 caused a significant accumulation of cells in G0/G1 phase in N‐Ras knock‐downed A375P/Mdr cells. However, PDGFRβ knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI‐201, although the remarkable increase in PDGFRβ was observed in A375P cells. Thus, the discovery of newly acquired genetic alterations that mediate resistance following exposure to BRAF targeted therapy would logically lead to sequential treatment strategies.
Yeast phospholipase C is required for normal glucose metabolism, acetyl‐CoA homeostasis, and global histone acetylation (800.1)
Authors: Zhang Tiantian , Galdieri Luciano , Vancura Ales
Published: June 2021

Phospholipase C (Plc1p) is required for the initial step of inositol polyphosphates (InsPs) synthesis. Plc1p is also required for normal level of histone acetylation; plc1∆ cells that do not synthesize any InsPs display decreased acetylation of bulk histones, global hypoacetylation of chromatin histones, and altered transcriptional regulation. In accordance with the role of Plc1p in supporting histone acetylation, plc1∆ mutation is synthetically lethal with mutations in several subunits of SAGA and NuA4 histone acetyltransferase (HAT) complexes. Conversely, the growth rate, sensitivity to multiple stresses, and the transcriptional defects of plc1∆ cells are partially suppressed by deletion of histone deacetylase HDA1. The histone hypoacetylation in plc1Δ cells is due to the defect in Grr1p/proteasome‐dependent degradation of repressor Mth1p, and consequently lower expression of the HXT genes and reduced conversion of glucose to acetyl‐CoA, a substrate for HATs. Our current experiments test three possible mechanisms whereby InsPs affect Mth1p degradation: (i) InsPs are required for Mth1p phosphorylation prior to degradation, (ii) InsPs are required for Grr1p activity, and (iii) InsPs are required for the activity of the proteasome.

Grant Funding Source: Supported by NIH
Understanding the role of myosin‐10 at the mitotic spindle (801.3)
Authors: Hine Ken , Sandquist Joshua
Published: June 2021

The proper formation and function of mitotic spindles are essential for any organism. Disruption of myosin‐10 (myo10), an actin‐based motor protein, has been shown to perturb mitotic spindle structure and function. Such an important role for a myosin at the mitotic spindle is surprising since spindles are microtubule‐based structures. It thus stands to reason that myo10 either directly or indirectly interacts with spindle microtubules. In support of a direct interaction, a microtubule‐binding domain (MTB) has been identified in the C‐terminal tail of Myo10, suggesting that myo10 can interact with both cytoskeletal polymers. Here we use a dominant negative approach to investigate the function of the MTB in myo10’s spindle function. In brief, mRNA encoding various fragments of the myo10 C‐terminal tail with either intact or altered MTB were injected into Xenopus laevis embryos, and the embryos were subsequently analyzed for protein expression and spindle phenotypes. Results indicate that protein fragments containing intact MTB function as dominant negatives and cause spindle phenotypes similar to myo10 depletion, while fragments that either excluded or contained a mutated MTB did not. This suggests that the MTB of myo10 plays a crucial role in myo10’s function at the mitotic spindle.
Molecular basis of the activation of the Rabex‐5 GEF activity by Rabaptin‐5 in endocytosis (802.27)
Authors: Zhang Zhe , Zhang Tianlong , Yu Fang , Chen Jiangye , Ding Jianping
Published: June 2021

Rabex‐5 and Rabaptin‐5 function together to activate small GTPase Rab5 and further promote early endosomal fusion in endocytosis. The Rabex‐5 GEF activity is autoinhibited by the Rabex‐5 CC domain (Rabex‐5CC) and activated by the Rabaptin‐5 C2‐1 domain (Rabaptin‐5C21) with yet unknown mechanism. We report here the crystal structures of Rabex‐5CC, the Rabex‐5CC‐Rabaptin‐5C212 complex, and the Rabex‐5Δ‐Rabaptin‐5C212 complex. Our structural and biological data together show that Rabex‐5CC assumes an amphipathic α‐helix which may bind weakly to a nonpolar surface groove of the GEF domain to occlude the substrate‐binding site, resulting in autoinhibition. The linker between the GEF and CC domains plays a role in the autoinhibition probably by modulating the relative conformation of the two domains. Binding of Rabaptin‐5C212 to Rabex‐5 displaces Rabex‐5CC, yielding a largely exposed substrate‐binding site and thus releasing the GEF activity. These results reveal the molecular mechanism of the regulation of the Rabex‐5 GEF activity.

Grant Funding Source: Supported by grants from the National Natural Science Foundation of China (31230017 and 31000327)
Cdk1‐dependent phosphorylation of leukemia‐associated RhoGEF during mitosis (802.30)
Authors: Helms Michelle , Grabocka Elda , Martz Matthew , Suzuki Nobuchika , Wedegaertner Philip
Published: June 2021

Rho GTPases are crucial players in the regulation of actin cytoskeleton‐dependent processes such as mitosis. Our laboratory has discovered a novel role and localization of the leukemia‐associated Rho guanine‐nucleotide exchange factor (LARG), a regulator of G protein signaling (RGS)‐RhoGEF, during mitosis. Rho and LARG have been implicated in diseases such as cancer and heart disease, thus highlighting the importance for further research. The purpose of this study is to determine the molecular mechanism and role of mitotic‐dependent phosphorylation of LARG. Using mitotic‐shift assays and kinase inhibitors, we report that LARG undergoes a mitotic‐dependent and Cyclin‐dependent kinase 1 (Cdk1) inhibitor‐sensitive phosphorylation. Cell synchronization followed by Western blot also identified the temporal feasibility of Cdk1 as a potential kinase. Furthermore, using in vitro kinase assays, we show that LARG can be directly phosphorylated by Cdk1. Using both N‐ and C‐terminal deletion and multi‐site phosphorylation mutants, we demonstrate that the mitotic‐dependent shift of LARG relies on phosphorylation occurring in both termini. Using custom phosphospecific antibodies, we confirm that two sites that are phosphorylated during mitosis and also in a Cdk1‐dependent manner. We conclude that Cdk1 phosphorylates LARG during mitosis, and phosphorylation of LARG may play a role in LARG activity and function.

Grant Funding Source: Supported by NIH GM062884
Proteomic analysis of chronic morphine treated SH‐SY5Y human neuroblastoma cells (803.10)
Authors: Randell Kesa , Taka Equar , Goodman Carl
Published: June 2021

The development of opioid tolerance is a barrier to its clinical use. Continuous exposure to opioids, like morphine, induces several molecular and cellular changes at the receptor and signal transduction pathway. Chronic opioid treatment of differentiated SH‐SY5Y human neuroblastoma cells has been shown to be an excellent in vitro model for studying cellular changes observed after opioid exposure. The present study was conducted to gain a better understanding of alterations in protein expression induced after opioid treatment, using mass spectrometry based proteomics, which in recent years has had a major impact on investigating the molecular and cellular mechanism of protein‐protein interactions. Cells were treated with morphine (10 μM), naloxone (10 μM), or 1 hr naloxone (10 μM) pre‐treatment followed by morphine (10 μM) for 24 hours, harvested, and separated by 2‐D gel electrophoresis. Analysis of these gels revealed alterations in protein expression induced by morphine treatment. Proteins with the most significant changes following drug treatment were identified using MALDI‐TOF. Calmodulin, alpha‐enolase, stathmin, nucleoside diphosphate kinase A, and nucleophosmin were identified. These results form the basis for the analysis of differential protein expression in SH‐SY5Y cells. The proteins detected will further be investigated to determine their role in morphine tolerance.

Grant Funding Source: NIH‐NIMHD G12MD007582
Regulator of G protein signaling 6 promotes anxiety and depression by attenuating serotonin‐mediated activation of the 5‐HT1A receptor‐adenylyl cyclase axis (803.12)
Authors: Stewart Adele , Maity Biswanath , Wunsch Amanda , Meng Fantao , Wu Qi , Wemmie John , Fisher Rory
Published: June 2021

The limited efficacy, delayed onset of action, and side effects restrict the clinical utility of selective serotonin (5‐HT) reuptake inhibitors (SSRIs), which remain first line therapies in the treatment of mood disorders. Regulator of G protein Signaling (RGS) proteins have been implicated as key regulators of 5‐HT1ARs, believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS protein involved remains unknown. Here, we identify RGS6 as the critical negative regulator of 5‐HT1AR‐dependent anxiolytic and antidepressant actions. RGS6‐/‐ mice exhibit spontaneous anxiolytic and antidepressant behavior, a phenotype reversible by 5‐HT1AR blockade. Effects of the SSRI fluvoxamine and 5‐HT1AR agonist 8‐OH‐DPAT were also potentiated in RGS6+/‐ mice. The phenotype of RGS6‐/‐ mice was associated with decreased phospho‐CREB and reduced PKA activity implicating enhanced Gαi‐dependent adenylyl cyclase (AC) inhibition as a causative factor in RGS6‐/‐ animal behavior. In fact, direct activation of AC with forskolin reversed the antidepressant phenotype of RGS6‐/‐ mice. Thus, by inhibiting 5‐HT activation of postsynaptic 5‐HT1ARs, RGS6 exerts powerful anxiogenic and pro‐depressant actions indicating that RGS6 inhibition may represent a viable means to enhance the efficacy of serotonergic drugs through selective potentiation of 5‐HT1AR‐mediated inhibition of AC.

Grant Funding Source: Supported by NIH CA161882, 1R01MH085724‐01, 5 R01 HL113863‐01
Multiple palmitoyl acyltransferases modify DAT palmitoylation (803.5)
Authors: Rastedt Danielle , Foster James , Vaughan Roxanne
Published: June 2021

The dopamine transporter (DAT) mediates the reuptake of synaptic dopamine, thus regulating dopaminergic neurotransmission. DAT undergoes palmitoylation in which a saturated 16‐carbon palmitate group is linked to cysteine via a thioester bond. Palmitoylation regulates diverse aspects of neuronal protein trafficking and function, but its role in DAT function is poorly understood. Palmitoylation is a reversible process in which the protein undergoes cycles of palmitoylation and depalmitoylation catalyzed by palmitoyl acyltransferases (PATs) and palmitoyl‐protein thioesterases (PPTs). The 23 member family of DHHC (Asp‐His‐His‐Cys) proteins, identified as PATs, remains poorly characterized and the identities of the PATs and PPTs involved in the regulation of DAT palmitoylation are unknown. To address this question, we co‐expressed each PAT individually with DAT and assessed DAT palmitoylation, total expression, and transport activity. Preliminary results indicate that specific neuronal‐linked enzymes DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17 increase DAT palmitoylation, while several other DHHC enzymes have no effect. Furthermore, DHHC enzymes found to increase DAT palmitoylation also increase total DAT levels, while treatment with the PAT inhibitor, 2‐bromopalmitate, led to DAT degradation. These findings suggest that palmitoylation enhances total DAT levels, opposing its degradation.

Grant Funding Source: Supported by DA 13147, DA 031991, EPS 0184442
Shared lunch intake: implications of food sharing in double fortified salt intervention trial (804.30)
Authors: Venkatramanan Sudha , Marquis Grace , Haas Jere
Published: June 2021

Shared lunch intake: implications of food sharing in double fortified salt intervention trial Sudha Venkatramanan1, Grace S. Marquis1, Jere D. Haas2 1McGill University, Canada, 2Cornell University, NY Treatment contamination can be a challenge in randomized control trials. Sharing foods during lunch is a common social practice in the tea garden culture and it has a significant role in creating a communal bond. The objective of this analysis was to assess the amount of treatment that was diluted due to contamination. The double masked study randomized 248 women to either double fortified salt (DFS) or iodized salt (control) for 10 mo. Information on the amount of food shared between the women in two different ethnic groups and total amount consumed was measured by weighing the lunch intake. The women were 18‐55 y old, Adivasi or Nepali, not pregnant or lactating, and full‐time experienced tea pickers. The endline lunch intake data from 206 women was used to estimate the amount of treatment received from sharing and the effect of treatment on the energy and nutrient intakes at lunch. Average shared foods received from other members and given away to others were 91.6 and 93.6 g, respectively. Food sharing resulted in both the control and treatment groups benefitting from DFS. The proportion of treatment received through the lunch by the IS and DFS groups were 40% and 54 %, respectively. A significant association of ethnicity (P <0.0001) on the total food and macronutrient intakes at lunch was observed. The results conclude that sharing lunches resulted in the contamination of the control group, somewhat diminishing the expected group difference in iron intake.

Grant Funding Source: Supported by the Mathile Institute for the Advancement of Human Nutrition and the Micronutrient Init
“Amarrarnos Las Tripas” (tie up your gut): perspectives on food insecurity from Mexican‐origin seniors living in colonias along the Texas‐Mexico border (805.3)
Authors: Munoz Linda , Sharkey Joseph , Dean Wesley
Published: June 2021

Latinos are the largest and fastest growing minority group in the United States, with persons of Mexican‐origin as the largest subgroup. Accompanying an increased number of Mexican‐origin seniors are persistent poverty and high rates of food insecurity, especially among Mexican‐origin seniors who live in the burgeoning colonias along the Texas‐Mexico border. This study described the perceptions of food security among Spanish‐speaking Mexican‐origin seniors under conditions of material hardship and structural vulnerability in colonia areas of the Lower Rio Grande Valley of Texas. In 2012, 95 Mexican‐origin seniors (22 men and 73 women) were recruited by promtoras de salud (community health workers) from clusters of colonias in four geographic areas of Hidalgo County (TX) to participate in focus group discussions conducted in Spanish. A promotora, trained as a moderator, conducted the focus groups using a semi‐structured interview guide developed with the assistance of promotoras. Discussions were audio recorded and underwent a 6‐stage process for transcription and translation into English. The mean age of participants was 65 y, 87 were born in Mexico, and 29% completed < 9 years of education).The results identified six major themes: stretching, most necessary, that’s good enough, amarrarnos las tripas (tie up your gut, which implies bear with the hunger), situación difícil (difficult situation), and not natural. This study shows that vulnerable Mexican‐origin seniors in the rural colonias are experiencing food insecurity and hunger. The voices of these marginalized people also stress the need for policy changes and action on federal, state, and the local community level to reduce food insecurity by providing more economic resources, expanding access to food and nutrition programs, and integrating culturally sensitive interventions and services.

Grant Funding Source: Supported by AARP Foundation and CDC's PRC Healthy Aging Research Network
Self‐efficacy for healthy eating of Hispanics participating in a lifestyle intervention (806.3)
Authors: Duarte‐Gardea Maria , Burgos Ximena , Balcazar Hector , Braham Sandra
Published: June 2021

The increasing epidemic of cardiovascular disease (CVD) in US requires effective interventions to reduce its multiple risk factors. Our study focuses on an intervention for Hispanics, a population of high prevalence for CVD and the largest minority group in US since 2009. OBJECTIVE: To asses self‐efficacy to eat healthier of participants engaging in an intervention led by Community Health Workers (CHW). The intervention is part of a CBPR named HEART that uses an ecological approach to demonstrate the efficacy of CHW at reducing CVD risk in Hispanics. METHODS: 421 low‐income Hispanics ages 46 ± 12 years engaged in a 16‐week culturally appropriate intervention consisting of a menu of nutrition and physical activities led by CHW at partner agencies. Demographic and anthropometric measures and self‐efficacy using a Likert scale were recorded at baseline and four months after the intervention. General linear mixed model analysis for repeated measures was conducted to test for time effect. RESULTS: 85% of participants were female and overweight or obese. Differences in average scores between baseline and follow up indicate that the intervention was effective at increasing self‐efficacy for healthy eating across six domains (e.g., can prepare with less salt, can cook using less fat) and at engaging in physical activity of 30 min 3 times per week (× = 8.41 ± 1.54 vs x = 9.1 ± 1.12: p = .0000). The mean difference in body mass index was also significantly different after the intervention (× = 31.3 ± 6.47 vs × = 30.7 ± 6.78: p = .0000). CONCLUSION: A culturally appropriate intervention led by CHW appears to be a promising approach for low‐income Hispanics to positively impact self‐efficacy for heart healthy eating and for reducing overweight and obesity, a risk factor for CVD.

Grant Funding Source: National Institute on Minority Health and Health Disparities
Biophysics and dynamics of bioinspired stem cell microenvironments (81.1)
Authors: Healy Kevin
Published: June 2021

Highly regulated signals in the stem cell microenvironment, such as growth factor presentation and concentration, matrix stiffness, and ligand adhesion density have been implicated in modulating stem cell proliferation and maturation. Therefore, it is desirable to have independent control over both the biochemical and mechanical cues presented to cells to analyze their relative and combined effects on stem cell function. Accordingly, we have developed synthetic hydrogels to assess the effects of adhesion ligand presentation, MMP labile crosslinkers, and material moduli spanning physiologically relevant ranges (10 to 10,000 Pa) on stem cell function. Employing these soft materials, we have demonstrated that the mechanical and biochemical properties of a stem cell microenvironment can be tuned to regulate the self‐renewal and differentiation of different types of stem cells including human embryonic, neural, and mesenchymal stem cells. We hypothesize that injectable bioinspired hydrogels, designed with a range of mechanical and biological properties, will yield material‐dependent improvements during either in situ tissue regeneration or matrix assisted stem cell transplantation.
“Don’t eat so much:” how parental comments about a girl’s weight influence her as an adult (811.20)
Authors: Latimer Lara , Pope Lizzy , Wansink Brian
Published: June 2021

Objective: How do the comments that a parent makes about a girl’s weight or eating behavior influence them long after they have left home? Methods: 501 women aged 20‐35 completed a survey assessing what comments they recalled their parents making about their eating habits and their weight as a young girl. These responses were correlated with their current BMI, eating habits, and satisfaction with their weight.

Results: Regression analyses indicated that women’s BMIs were positively associated with parental comments about the women’s weight (p<0.001) and how much they ate (p<0.01). Moreover, young women’s weight satisfaction was negatively impacted by parental comments about the women’s weight (p<0.01). Importantly, all of these findings were significant even after accounting for their parents’ concern about their own weight.

Conclusions: A parent’s comment about their daughter’s weight or eating habits may have a lingering influence long after it is spoken. A parent’s negative comments about eating habits are related to their daughter having a higher BMI and more dissatisfaction with her weight as an adult.
Effects of omega‐3 and omega‐6 fatty acids on the ethograms of rodents simulating PTSD‐like aspects (814.7)
Authors: Chakraborty Nabarum , Melige Monique
Published: June 2021

The influences of unsaturated fatty acids in various clinical and patho‐physiological paradigms are documented.A balanced composition of omega‐3 and omega‐6 fatty acids (n‐3 and n‐6 FA) is beneficial for the daily diet, but the Western diet is biased towards n‐6 FA.The optimum balance between these two fatty acid types is yet to be established.

Probing the suitable dietary compositions purposed for post‐traumatic stress disorder (PTSD) amelioration, groups of C57BL/6Jmice were fed three different diet types. The standard lab diet (diet c) containeda highn‐6 FA/n‐3 FAratio, while the two other customized diets had high(diet 1) and equivalent (diet 2)ratios of n‐3/n‐6 FAs, respectively.

The rodents were subjected to an aggressor‐induced social stress established to elicit PTSD‐like behavioral and histo‐pathological symptoms.The subject mice were co‐habited with aggressors for 6 h/d for 10 days with 3x/d random direct exposures. A situation reminder study assayed their behavioral shiftsas a function of time delay. The behavioral traits simulating PTSD among the mice consuming diet cwerecriticallyaugmented during the delay period. Diets 1 and 2 facilitated development of stress resistance,but these impacts varied among the different traits of behaviors, such as fear, anxiety, and social withdrawal.
Development and characterization of Hephaestin knockdown Caco‐2 cells (818.13)
Authors: Doguer Caglar , Gulec Sukru , Collins James
Published: June 2021

Intestinal transport of inorganic iron has been intensively studied. Ferric iron is reduced, possibly by duodenal cytochrome b (Dcytb), and transported into cells by divalent metal‐ion transport 1 (Dmt1). Ferrous iron is exported from enterocytes via ferroportin 1 (Fpn1) and is then oxidized prior to binding to transferrin. The efflux step, involving the export‐oxidation functional couple, is the rate‐limiting step in iron absorption. Although an iron oxidase has been discovered (Heph), it is likely that other oxidases also exist. The current study was thus designed to assess the necessity of Heph for iron transport using the Caco‐2 cell model. Caco‐2 cells, which differentiate in post‐confluent culture, express an inducible, vectorial iron transport system, faithfully recapitulating mammalian iron absorption. Heph was effectively silenced in Caco‐2 cells using shRNA technology. A control cell line was also established with negative‐control shRNA transfected cells. Heph KD cells grew normally and were visually indistinguishable from control cells. Gene expression studies showed that Dcytb (~55%) and Dmt1 (~25%) mRNA expression was decreased, while Fpn1 expression was unaffected. Heph protein levels were significantly reduced (<90%) in cytosolic and membrane fractions of the KD cells. Ferrous iron oxidation measurements, using a transferrin‐coupled assay, showed that Heph KD had very little effect on membrane and cytosolic ferroxidase (FOX) activity. Additional studies showed for the first time that Heph was detected in early and late endosomes isolated from Caco‐2 cells, fractions in which Rab7 was also detected (an endosomal marker). This investigation has thus provided further evidence that Heph is not the sole FOX in mammalian intestinal epithelial cells.

Grant Funding Source: Supported by NIH Grant, 2R01DK074867‐07A1
Aligned polymer nanofibers for tissue engineering (82.3)
Authors: Zhang Miqin
Published: June 2021

The ability to produce aligned poly‐blend nanofibers may open new avenues in the development of scaffolds for tissue engineering and regenerative medicine. An area of particular interest is functional restoration of damaged or diseased nerves, muscles and tendons where the aligned fibers serve to support cell adhesion and proliferation, and guide tissue growth in the direction of fiber orientation. The poly‐blend nanofibers represent an emerging class of biomimetic nanostructures that can act as proxies of the native tissue, while providing topographical and biochemical cues that promote healing. These poly‐blend nanofibers are prepared using mixtures of synthetically and naturally derived polymers that can behave cooperatively to demonstrate unique combinations of mechanical, biochemical and structural properties. In this talk, I will discuss design criteria and properties of aligned polymer‐blend nanofibers for use in tissue engineering, various methods of making them, and their applications in nerve, muscle, and tendon tissue regeneration.
Alterations in the transsulfuration reactions in rats acutely challenged with ethanol (820.5)
Authors: Kwon Do , Lee Yoo , Jun Doo , Kim Young
Published: June 2021

It is well known that ethanol intake decreases hepatic glutathione (GSH) level. We examined the changes in GSH synthesis in rats treated with ethanol acutely. Ethanol treatment rapidly depleted hepatic and renal GSH, which persisted in liver, but recovered in kidney in 24 h. Cysteine, the essential substrate for GSH synthesis, was also decreased both in liver and kidney, suggesting that the reduction of cysteine availability was associated with the ethanol‐induced depletion of GSH. In ethanol‐treated rats, however, cysteine dioxygenase (CDO) was up‐regulated in liver as shown by induction of CDO gene and protein expression as well as its activity. Taurine and hypotaurine levels were elevated markedly in liver. Plasma taurine level was also increased. It is therefore suggested that the increment in metabolic conversion of cysteine to taurine would further aggravate the reduction of cysteine availability for GSH synthesis. In contrast, expression of glutamate cysteine ligase, the rate‐limiting enzyme in GSH synthesis, was unaffected by ethanol. Taken together, the results indicate that ethanol treatment reduces GSH by decreasing the availability of cysteine, in which an enhancement of cysteine catabolism to taurine via up‐regulation of CDO plays a critical role. The physiological significance of this finding is being investigated.
Fatty acid profiles of undifferentiated and differentiated white and brown adipose cell lines supplemented with alpha‐linolenic acid (821.5)
Authors: Qin Xia , Park Hui Gyu , Zhang Jiyao , Lawrence Peter , Liu Guowen , Kothapalli Kumar , Brenna James Thomas
Published: June 2021

Introduction. Adipose tissue is a complex endocrine organ which coordinates several crucial biological functions including fatty acid metabolism, glucose metabolism, energy homeostasis, and immune function. White adipose tissue (WAT) stores energy, while brown adipose tissue (BAT) dissipates energy.

Our aim was to characterize fatty acid profiles of the undifferentiated and differentiated WAT and BAT transformed cells.

Methods. Mouse WAT and BAT cells were grown in regular media to confluence, and differentiation was induced. At days 10, 12 and 14, cell were supplemented with 50 μM of albumin bound alpha‐linolenic acid (ALA). Cells with no ALA supplementation served as controls. Twenty‐four hours after supplementation, fatty acid profiles of the cells were analyzed by GC and structural mass spectrometry.

Results. ALA supplementation caused increase in cellular eicosapentaenoic acid (EPA, 20:5n‐3) with no change in docosahexaenoic acid (DHA, 22:6n‐3) levels in undifferentiated and differentiated WAT cells. Differentiated WAT cells accumulated more EPA. However, differentiated BAT cells accumulate significant amounts of DHA when compared to undifferentiated cells.

Conclusion. When provided with ALA, WAT cells accumulate EPA with no change in DHA; BAT cells accumulate EPA and DHA.

Grant Funding Source: NIH R01AT007003
Human breast cancer cells stably expressed FADS2 synthesize sapienic acid (16:1n‐10) from palmitic acid (16:0) (821.8)
Authors: Park Hui Gyu , Kothapalli Kumar , Park Woo Jung , Qin Xia , Lawrence Peter , Brenna J.
Published: June 2021

Introduction. Sapienic acid, 16:1n‐10, is synthesized in sebocytes and is the most abundant unsaturated fatty on human skin. Its biosynthesis from palmitic acid (16:0) has not been demonstrated apart from skin glands. MCF‐7 human breast cancer cells have no detectable 6‐desaturase activity despite detectable FADS2 expression.

Our aim was to characterize the fatty acid profile of MCF‐7 cells stably expressing FADS2 grown in MEM‐α media containing 16:0, 18:2n‐6, 18:3n‐3, and long chain PUFA.

Methods. Cells stably expressing FADS2, FADS1, and empty vector were created by using pcDNA3.1 expression vector system. At confluence cells were dosed with 100 μM of albumin‐bound 16:0 fatty acid. Cells were harvested after 24 hours, FAME were analyzed by GC and structures confirmed by CACI‐MS/MS.

Results. FADS2 MCF‐7 cells preferentially biosynthesize 16:1n‐10 which is more abundant than 16:1n‐9. The two fatty acids have nearly identical retention times under our standard GC conditions. No synthesis of 16:1n‐10 was observed for FADS1 or vector‐only cells.

Conclusion. Human cells stably expressing FADS2 synthesize 16:1n‐10 even in the presence of alternative FADS2 substrates 18:2n‐6 and 18:3n‐3. Sapienic acid detected in human plasma may be the product of biosynthesis in internal organs.

Grant Funding Source: NIH (R01AT007003)
Mutagenic and apoptotic effects of white mold (Sclerotinia sclerotiorum) in vivo and in vitro (824.6)
Authors: Roque Aline , Kim Hyemee , Azevedo Luciana , Barrocas Ellen , Machado Jose , Trevisan Jerusa , dos Santos Marcelo Henrique , Mertens‐Talcott Susanne
Published: June 2021

Various common foods, including beans, soybeans, peas, and potatoes, are often contaminated by soil fungus called white mold, Sclerotinia sclerotiorum. To data, little information is available regarding the toxicological effects. This work aimed to determine the apoptotic and mutagenic potential of aqueous extract of Sclerotinia sclerotiorum (AESS) in vivo and in vitro. Chemical analysis showed that the extract was rich in oxygenated monoterpenes, such as camphor. Swiss mice were fed 10, 100 and 1000mg/kg bodyweight of AESS for 15 days. The comet assay was performed in peripheral blood cells and the number of micronucleated polychromatic erythrocytes (MNPCEs) was determined in bone marrow cells. In addition, HT‐29 and CCD‐18 cell lines were treated with 0.005, 0.01 and 0.02ppm of AESS for performing cell viability assay and RT‐PCR. In results, the extract had mutagenic effects in the micronucleus test, by increasing the number of MNPCEs up to 439%. However, the extract did not show any DNA‐damage in the comet assay. In vitro, the extract demonstrated pro‐apoptotic effects through modulation of gene expressions associated with p53‐mediated apoptosis pathway in both cancer and non‐cancer colon cells within a low concentration range. In conclusion, the AESS did not cause DNA damage in the comet assay, but potential mutagenic and cytotoxic activities within a low concentration range should be further investigated.
Effect of plant‐based polyunsaturated fatty acids on characteristics of fatty liver disease in laying hens (829.27)
Authors: Cain James , Hales Dale , Small Carrie , Banz William , Davis Jeremy
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) and fatty liver hemorrhagic syndrome (FLHS) are common problems related to human and poultry health. Omega‐3 polyunsaturated fatty acids (n‐3 PUFA) are known to modify liver disease development and progression. Flaxseed is the most commonly consumed plant‐based source of n‐3 PUFA, but it remains unclear how different components of the flaxseed food matrix affects characteristics of liver disease. The objective of this study was to determine the effect of feeding different plant‐based sources of PUFA on the development of liver disease in laying hens. Leghorn laying hens aged 1 yr were provided high‐fat (23 en%) diets supplemented with differing sources of fatty acids‐‐corn (CORN), soybean (SOY), and flaxseed (FXO) oils. Defatted (DFM) and whole flaxseed (WFX) meal were provided to additional groups in order to elucidate the components of the flaxseed whole food matrix account for different outcomes. Plasma and tissues were collected and analyzed for metabolic and morphologic parameters, histological grading, and fatty acid profiles. Results indicate WFX supplementation was associated with decreased body weight (p< 0.05) compared to all other groups. All treatment groups displayed significant steatosis, with WFX showing reduced severity compared to CORN, SOY, and DFM (p< 0.05). No treatment consistently resulted in advanced liver disease characterized by hepatocellular injury or inflammation. Taken together, our data suggest flaxseed based diets modestly ameliorate characteristics of fatty liver disease in lay hens, and that flaxseed is most effective when consumed as part of its whole food matrix.
Method development for investigating probiotic effects on fat storage in a Caenorhabditis elegans model (829.33)
Authors: Shay Mack , Al‐Tameemi Marwa , Wright Saxon , McLean Robert , Vattem Dhiraj , Maitin Vatsala
Published: June 2021

Gut microbiota via an ability to influence fat synthesis and storage has emerged as an important factor in host adiposity, and a target for its dietary modulation. To examine the role of specific gut bacteria in modulating fat stores and associated mechanisms, we need simple, inexpensive yet powerful animal models. An attractive choice is the nematode C. elegans, in which many pathways of fat storage and energy use are conserved. Based on our previous observations that a probiotic Bifidobacterium longum can reduce triglyceride storage in vitro, our objective was to use the C. elegans model to investigate this in vivo. Our initial attempts at growing C. elegans on B. longum as a food source rather than E. coli OP50 were met with several challenges: reluctance of C. elegans to consume B. longum and preference for OP50, carry‐over of OP50 on the worm surface and in the gut, and non‐comparable growth of C. elegans on B. longum and OP50. By varying the type of medium (solid/liquid) and its composition, increasing B. longum concentration and inhibition of OP50 growth by an antibiotic, we devised a successful method to grow C. elegans on B. longum for over one generation, detect B. longum in the C. elegans gut at numbers comparable to the human small intestine (with no detectable E. coli OP50) and study its impact on triglyceride storage. The method may be adapted to other probiotic bacteria to serve as a screening tool with the scope of investigating detailed mechanisms of action.
Dietary p‐coumaric acid modulates hepatic gene expression involved with lipid metabolism in rats (829.6)
Authors: Kishida Kunihiro , Suzuki Masaya , Ihara Hayato
Published: June 2021

p‐Coumaric acid (pCA) is a phenolic acid that is ubiquitous in edible plant. pCA has been reported to reduce plasma and hepatic lipids in rats. However the detailed mechanism remains to be elucidated. The aim of this study was to investigate how pCA affected hepatic gene expression involved with lipid metabolism in rats. Male SD rats were fed a western diet (high‐fat, high‐cholesterol, and high‐sucrose) containing 0.2% pCA for 35 days. Dietary pCA attenuated hepatic triacylglycerol and cholesterol accumulation although serum lipid profile was not changed. The hepatic expression of lipogenic genes, acetyl‐CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), malic enzyme (ME), stearoyl‐CoA desaturase 1 (SCD1) was significantly up‐regulated, while their transcription factors, sterol regulatory element binding protein 1c (SREBP1c) and carbohydrate‐responsive element binding protein (ChREBP) were not changed. As for cholesterol metabolism, cholesterol 7α‐hydroxylase (CYP7A1) was down‐regulated, and low density lipoprotein receptor (LDLr) and its transcription factor, sterol regulatory element binding protein 2 (SREBP2) were up‐regulated significantly. These results suggest that pCA improved hepatic lipid accumulation and the variations in mRNA expression might occur as a result of decreased triacylglycerol and cholesterol levels in liver.
An omega‐3 epoxide of docosahexaenoic acid lowers blood pressure in angiotensin II‐dependent hypertension (832.5)
Authors: Ulu Arzu , Lee Kin Sing Stephen , Miyabe Christina , Yang Jun , Hammock Bruce , Dong Hua , Hammock Bruce
Published: June 2021

Mediators of anti‐hypertensive actions of docosahexaenoic acid (DHA) are largely unknown. The omega‐3 epoxide of DHA, 19, 20‐EDP (epoxydocosapentaenoic acid) is metabolized by soluble epoxide hydrolase (sEH), which also metabolizes the anti‐inflammatory and anti‐hypertensive arachidonic acid (ARA) epoxides, EETs (epoxyeicosatrienoic acids). Based in part on plasma levels of EDPs following a DHA‐rich diet, we hypothesized that 19, 20‐EDP contributes to the anti‐hypertensive actions of DHA in angiotensin‐II dependent hypertension. Treatment individually with 19, 20‐EDP, and a potent sEH inhibitor (sEHI) TPPU (1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea) significantly lowered blood pressure (BP) as compared to angiotensin‐II infused animals. The largest reduction in BP was obtained with the combination of 19, 20‐EDP and TPPU, which was more efficacious than the combination of 14, 15‐EET and TPPU. Oxylipin profiling revealed that 19, 20‐EDP and 14, 15‐EET infusion affected mostly metabolites of the P450 pathway but also renal levels of prostaglandin‐E2. Our findings suggest that 19, 20‐ EDP is a mediator of the anti‐hypertensive and anti‐inflammatory effects of DHA in angiotensin‐II dependent hypertension. It appears that 19, 20‐ EDP requires metabolic stabilization with a sEHI to be most effective in lowering BP, although both TPPU and 19, 20‐ EDP are so effective on their own that demonstrating additive or synergistic interactions is difficult.

Grant Funding Source: Supported by NIEHS R01 ES002710, NIEHS Superfund Research Program grant P42 ES004699
Ligand binding in PECAM‐1‐dependent cell motility (832.8)
Authors: Abraham Valsamma , Parambath Andrew , DeLisser Horace
Published: June 2021

Although PECAM‐1 is able to mediate homophilic or heterophilic ligand binding, the roles of these interactions in PECAM‐1‐dependent cell motility have not been determined. Mutants of mouse PECAM‐1 (MP) in which homophilic (MPΔHom) or heterophilic (MPΔHet) binding are disabled by targeted mutations in the extracellular domain of the molecule were generated and expressed in REN cells (an endothelial cell surrogate). The resulting cells, along with wild type controls, were assessed for localization at intercellular junctions and wound‐induced tyrosine phosphorylation (required for PECAM‐1 dependent cell motility), cell migration and filopodia formation. Our findings are summarized in the table.

Construct
Localization to Intercellular Junctions
Wound‐Induced Tyrosine Phosphorylation
Increased Wound‐Induced Migration
Increased Filopodia Formation

Wild type
Yes
Yes
Yes
Yes

MPÎ”Hom
No
Yes
No
No

MPÎ”Het
Yes
Yes
No
No

As expected, disabling homophilic (but not heterophilic) adhesion prevented the localization of PECAM‐1 at intercellular junctions. Further, despite preservation of tyrosine phosphorylation, loss of either homophilic or heterophilic binding completely eliminated the increases in wound‐induced migration and filopodia formation resulting from the expression of PECAM‐1. These data therefore suggest that in the setting of PECAM‐1‐dependent cell motility, ligand interactions occur downstream of PECAM‐1 tyrosine phosphorylation and that both homophilic and heterophilic binding are necessary for the increase in cell motility and filopodia formation mediated by PECAM‐1. Thus during PECAM‐1‐dependent cell motility, PECAM‐1‐PECAM‐1 interactions in the membrane, as well as PECAM‐1‐matrix interactions may be active.

Grant Funding Source: Supported by VA Merit Award
Role of adducins in endothelial barrier formation (832.9)
Authors: Kugelmann Daniela , Radeva Mariya , Waschke Jens
Published: June 2021

Adducins tightly regulate actin dynamics which is critical for endothelial barrier function. Adducins were reported to regulate epithelial junctional remodeling by controlling the assembly of actin filaments at areas of cell‐cell contacts. Here we investigated the role of adducins for endothelial barrier regulation by using microvascular human dermal and myocardial murine endothelial cells. Transendothelial electrical resistance (TER) measurements revealed that siRNA‐mediated adducin depletion impaired endothelial barrier formation. To further test whether the peripheral localization of adducins is functionally linked with the integrity of endothelial adherens junctions, junctional remodeling was induced by a Ca2+ switch‐assay. Ca2+‐depletion disturbed both linear VE‐cadherin and adducin location along cell junctions, whereas the localization of both proteins was restored as result of Ca2+‐repletion. By using TER measurements we verified that endothelial barrier properties and junction reorganization can be effectively modulated by altering Ca2+ concentration. Thus, Ca2+‐depletion drastically reduced TER whereas Ca2+‐repletion led to recovery of endothelial barrier properties reflected in increased TER. Taken together, our results indicate that adducins maybe involved in remodeling of endothelial junctions and thereby contribute to endothelial barrier regulation.
Comparative studies of purified poloxamer 188 using clot‐based and viscoelastic measurements of coagulation (833.5)
Authors: Hoppensteadt Debra , Iqbal Omer , Abro Schuharazad , Emanuele Marty , Fareed Jawed
Published: June 2021

Poloxamer 188 (P188; Mast Therapeutics, Inc.)is a surface‐active, non‐ionic block copolymer which binds to hydrophobic surfaces on damaged cells improving membrane hydration and lowering adhesion and viscosity. We compared the effect of this agent using various clot based, chromogenic and viscoelastic measurements of blood coagulation. Whole blood activated clotting time ACT) studies were carried out in healthy individuals (n=10) at a concentration range of 1.872‐15.0mg/mL. TEG analysis was carried out on TEG 5000 (Haemoscope Corp, Niles, IL) at concentrations of 0‐0.45 mg/mL. The effect of P188 on normal plasma clotting parameters, such as PT and aPTT, was measured at a concentration range of 0‐10 mg/mL. The effect of P188 on thrombin‐induced clot formation was investigated using a fibrinokinetic method. The effect of P188 on thrombin generation was measured using the fluormetric method (Technoclone, Vienna, Austria). The anti‐protease effects of P188 were studied using chromogenic substrate methods using isolated biochemical systems. At concentrations up to 10 mg/mL, P188 did not produce any modification of ACT or the standard clotting assays. In the TEG analysis, P188 produced a concentration dependent hypocoagulant effect in as evidenced by increased angle and shortening of maximum amplitude (MA). In fibrinokinetic studies, P188 produced an increase in the fibrin clot density and rate of fibrin polymerization. The marked discordance between TEG and other coagulation tests suggest that P188’s effect on viscosity and adhesive interactions result in an artifact in TEG analysis and an incorrect indication of a hypocoagulant effect.
Pulmonary histopathology of a case of fat embolism (FE) in a patient and its similarity to an animal model of FE (834.11)
Authors: Ajemba Ogugua , Zia Hamid , Lankachandra Kamani , Singh Gurmukh , Poisner Alan , Herndon Betty , Molteni Agostino
Published: June 2021

BACKGROUND: FE, predominantly a respiratory disease, is thought to be the result of fat globules entering the bloodstream from tissue that has been disrupted by trauma but scarce information about the histopathology is available from clinical reports on pregnancy and other FE ‐ induced conditions.

CASE REPORT: A 29‐year‐old Caucasian female, obese, with a complex medical history, underwent Cesarean section at 35 weeks gestation due to preeclampsia. Postoperatively, 53 hours after surgery, she was found dead. At necropsy, lungs showed edema, mononuclear and histiocytic cell infiltrate and scattered fat emboli (Oil Red O stain) in the lumen of small arteries and in air spaces. Early interstitial fibrosis mostly in the adventitia of the same vessels, peribronchial musculature and lobular septa was seen with trichrome stain, while smooth muscle actin was present in the media of medium size vessels, suggesting incipient fibrosis.

CONCLUSION: The histopathological findings in lungs of this patient concur with the earlier reported findings in lungs of an animal model of fat embolism produced by i.v. triolein (J. Orthopedic Res, 28, 191‐197, 2010). Such histologic alterations may underscore the pulmonary pathobiology after pregnancy or other FE causes.