bluuebunny/crossref_metadata_2025 huggingface dataset:
I say assertive, you say bossy; I say leader, you say bi***: Gender bias in academia
Authors: O'Loughlin Valerie
Published: June 2021

Despite progressive changes in the workforce, a double‐standard still remains when it comes to assessment of competency and leadership in male and female faculty, especially in the STEM fields. The same behavior that is perceived as assertive in a male may be described as bossy in a female. This double‐standard has implications for female faculty in all aspects of their career, from negotiating the new hire to achieving full professorship.

This session will begin with a brief overview of research that examines gender bias in the workforce and academia. The presenter will then discuss her personal experiences in navigating these issues, as she transitioned from a new faculty member to a leader in her mid‐career (including past‐president of the Human Anatomy and Physiology Society). For the presenter, increased maturity (a.k.a. – age) fostered increased comfort as a leader and a decreased need for external approval. Cultivating strong mentor networks can facilitate leadership development and recognition of one's self worth, despite existing gender bias. The presenter will provide advice to all about empowering yourself in all career stages.
Relationship of percentage of adequacy and distribution of macronutrients in the diet of the mother‐child dyad of schoolchildren of Arandas, Jalisco, Mexico
Authors: Miranda Ríos Lizette , Vasquez‐Garibay Edgar , Romero‐Velarde Enrique , Nuño‐Cosío Maria , Campos‐Barrera Liliana
Published: June 2021

In order to explore the relationship between the percentage of adequacy in energy and protein and the percentage of energy distribution of lipids, carbohydrates and sugars in the diet of the mother‐child dyad of schoolchildren, it was carried out a cross‐sectional study of randomly selected schoolchildren of public elementary schools. 128 24h dietary recall surveys on working days and 49 on weekends were obtained. Correlation and determination coefficients were determined.

Table 1. Relationship of percentage of adequacy and distribution of macronutrients in the diet of the mother‐child dyad of schoiolchildren of Arandas, Jalisco, México

Variable
Male
Female

r
r2
p
r
r2
p

Percentage of adequacy

Energy
WD1
0.420
0.177
<0.001
0.214
0.046
0.124

WE2
0.443
0.196
0.039
0.165
0.027
0.500

Protein
WD
0.085
0.007
0.486
0.233
0.054
0.090

WE
0.368
0.136
0.064
0.358
0.128
0.133

Percentage of distribution

Lipids
WD
0.115
0.013
0.345
0.517
0.267
<0.001

WE
0.436
0.190
0.023
0.619
0.383
0.005

Carbohydrates
WD
0.096
0.009
0.434
0.337
0.113
0.013

WE
0.262
0.069
0.187
0.622
0.386
0.004

Sugar
WD
0.387
0.150
0.002
0.117
0.014
0.415

WE
0.586
0.344
0.003
0.301
0.091
0.258

1WD: Working day (n=128); 2WE: Weekends (n=49).

The adequacy in energy consumption and the proportion of sugars correlated on dyads mother‐child of males; the proportion of lipids and carbohydrates correlated on mother‐child dyads of girls. The correlations were more significant for weekends in all nutrients.
Effects of Cow Milk versus Extensive Protein Hydrolysate Formulas on Infant Cognitive Development
Authors: Mennella Julie , Trabulsi Jillian , Papas Mia
Published: June 2021

There is a paucity of research on how feeding different types of currently available infant formulas impacts developmental outcomes other than growth. To this end, the present study examined motor and cognitive development in healthy infants who fed formulas that differed substantially in free amino acid, but shared similar fat, content. The infants (n=80) were randomized to a control group who were fed only cow milk formula (CMF) during the first 8.5 months or to an experimental group which fed extensive protein hydrolysate formula (ePHF) for > 1 month during early life. Mullen assessments of fine and gross motor control, receptive and expressive language, visual reception, and a general intelligence (early learning composite) score were measured at monthly intervals from 1.5 to 8.5 months. Generalized estimating equations (GEE) that included diet group, time, and a diet group‐by‐time interaction were used to account for repeated measurements over time by examining whether the slopes of the line created by the Mullen scores over time differed for the two diet groups. We found that between the ages of 1.5 and 4.5 months, ePHF infants averaged visual reception scores 2.9 points higher (95% CI: 1.0, 4.9) and general intelligence scores 2.5 points higher (95% CI: 0.7, 4.4) than the CMF group, after adjusting for baseline maternal BMI. These differences were not apparent after the introduction of solid foods (5.5‐8.5 months). This is the first study to suggest that the form of protein in infant formula may impact cognitive development during early infancy and further supports the contention that formula‐fed infants are not a homogenous group. Supported by NIH grants R01HD37119 and R01HD072307.
Improvement in Provincial Plans for Nutrition through Targeted Technical Assistance and Local Advocacy in Vietnam
Authors: Harris J , Nguyen P , To Q , Hajeebhoy N , Phan L , Vu H , Frongillo E , Lapping K , Menon P
Published: June 2021

Vietnam has been decentralizing nutrition planning to provinces, which could help with local relevance and accountability. Assessment in 2009 found a continuing top‐down approach, limited human capacity, and difficulty in integrating multiple sectors. Alive and Thrive (A&T) provided targeted assistance and capacity‐building for 15 provincial plans for nutrition (PPNs) including: developing provincial nutrition profiles, planning guidelines and tools, organization of national and provincial workshops, and local advocacy for nutrition. We aimed to 1) assess PPN content and quality improvements 2009‐2014 and 2) explain processes through which change occurred, particularly how planning support and local advocacy contributed. Data consisted of interview‐based assessments of provincial planning processes, annual PPN quality, and tracking of A&T involvement. At endline, some provinces produced higher quality plans. Local planning skills improved, but capacity remained insufficient. Awareness of and support for nutrition improved, but some policy and legal environments were contradictory. Objectives were clearer, but use of data for planning remained inconsistent. Provinces became more proactive and creative, but remained constrained by slow approval processes and insufficient funding. Targeted assistance and local advocacy can improve decentralized planning, but success depends on policy and programming contexts and ability to overcome constraints around capacity, investment, data use, and remnants of centralized planning.

Funding: Bill and Melinda Gates Foundation, through Alive &Thrive, managed by FHI360.
Metabolite Biomarkers for Assessing the Integrity of Human Plasma and Serum
Authors: Zhang Shucha , Sheldon Diane , Kristal Bruce
Published: June 2021

Rigorous quality control (QC) of biospecimens, e.g. plasma/serum, is critical to ensure the validity of clinical and/or epidemiological results. Here we focus on assessing biochemical degradation associated with pre‐processing delays. Specifically, we generated degradation standards of serum and plasma with heparin, ethylenediaminetetraacetic acid (EDTA), or citrate after incubating the whole blood samples for 0‐44 hours (11 time points) at 4oC prior to processing. Metabolomic profiles of ~460 samples from 42 human subjects were acquired using high performance liquid chromatography coupled to electrochemical detection. Principal component analysis (PCA) was performed to visualize metabolic variations of the samples. A clear trend of sample degradation during incubation was revealed by PCA score plots of the samples' metabolic profiles. The abundance of individual metabolites was plotted against incubation time. A panel of biomarkers (e.g. purine catabolites) increased significantly in serum and plasma regardless of anticoagulants during incubation. For example, the fold changes of hypoxanthine from 0 to 44 hours are 8±5, 15±8, 19±6, and 10±6 for serum, heparin plasma, EDTA plasma, and citrate plasma, respectively. The increase of hypoxanthine with time is significant with Pearson correlation coefficients of 0.80, 0.91, 0.80, and 0.82, respectively (p‐value < 0.00001 for all sample types). Models built from this study will be useful to assess the degradation level/integrity of human blood plasma and serum. Funded by NIH/NHLBI.
Integrins Cooperate during Mechanosensing
Authors: Fässler Reinhard
Published: June 2021

It is unclear how integrins that bind to the same extracellular matrix protein mediate specific functions. To address this question we analysed β1‐ and αv‐class integrins expressed in pan‐integrin‐null fibroblasts seeded on fibronectin. Our experiments revealed that αv‐class integrins induce low myosin‐II activity and the formation of large focal adhesions, β1‐class integrins induce intermediate myosin‐II activity, formation of small peripheral adhesions and cell protrusions, and co‐expression of both integrin classes leads to highest myosin‐II activation and highest traction force development on stiff FN‐coated substrates. Using quantitative proteomics we identified signalling pathways enabling α5β1 integrins to generate force and αv‐class integrins to establish the structural requirements to transmit force. The findings will be discussed at the meeting.
Engineering Interactions between Growth Factors and the Extracellular Matrix
Authors: Hubbell Jeffrey , Martino Mikael , Briquez Priscilla , Kilarski Withold , Guc Esra , Swartz Melody
Published: June 2021

In natural situations, growth factors such as VEGF and PDGF are present bound to the ECM, yet therapeutic use of such growth factors has focused on application in soluble forms. To explore ECM immobilization of growth factors, we have explored complexation with recombinant variants of EMC proteins such as fibronectin and engineering growth factors fused to a domain that binds strongly to ECM proteins. To explore noncovalent immobilization upon matrices, we have engineered a fibrin‐binding domain of fibronectin, containing the 12th‐14th type III repeat (which was known to bind VEGF‐A). In studies of the 12th‐14th type III repeat, we determined that the growth factor binding activity of this domain was promiscuous. Incorporation of this domain into fibrin, through engineered transglutaminase activity, provides a powerful and generalizable method to retain such growth factors into surgical matrices. Examination of promiscuous growth factor binding to ECM proteins revealed broad binding to not only fibronectin but also fibrinogen, vitronectin, osteopontin, and tenascin C, among others. We examined these affinities to determine that placenta growth factor‐2 (PlGF‐2) binds the mentioned ECM molecules very strongly. We identified the binding domain and engineered it into other growth factors to confer nM affinity to them, demonstrating enhanced efficacy in the context of angiogenesis, skin repair in chronic wound models, and bone repair. In this way, using variant growth factors that have super‐affinity for ECM proteins, either a fibrin matrix or a tissue itself may be turned into a reservoir for growth factor sequestration and presentation.
Effects of Konjac Glucomannan and Inulin Oligosaccharide on the Colonic Mucosa Barrier, Antioxidant Enzyme Gene Expression and Gut‐associated Lymphoid Tissue Immunity in Mice
Authors: Chen Hsiao‐Ling , Wu Wen‐Tzu , Han Yi‐Chun
Published: June 2021

Konjac glucomannan (KGM) and inulin fructo‐oligosaccharide (mean degree of polymerization=8) effectively protect the AOM‐induced DNA damage in the colonocytes which may reduce the colonic tumorigenesis. The aim of this study was to determine how these fibers modulate mucosa barrier function and immunity that could also contributes to the anti‐carcinogenic potential. Six‐week‐old C57BL/6J mice were randomly assigned to the following diet: control AIN‐93 diet (control group) or that supplemented with 2% (w/w) KGM (KGM group) or inulin (Inulin group) for 21 days. After 12‐h fasting, mice were sacrificed and the plasma and colon were collected. Results indicated that the mucosa barriers were intact in all groups. However, inulin group was characteristics of mucin‐rich crypts and longer crypt length compared with the control group. The gene expression of ZO1, a tight junction protein, was enhanced both in the KGM and inulin groups. The gene expressions of colonic TNF‐α and IL‐10 were increased with KGM and inulin, respectively. The inulin supplementation also significantly increased the plasma TNF‐α level compared with the control group, but not the IL‐10 level. In addition, both KGM and inulin increased the gene expression of glutathione transferase pi and glutathione peroxidase. In summary, our study suggested that both konjac glucomannan polysaccharide and inulin oligosaccharide enhanced the mucosa barrier and antioxidant machinery and modulated the gut‐associated lymphoid tissue immunity. This study was supported by the National Science Council Grant MOST‐103‐2320‐B‐040‐013‐MY3, Taipei, Taiwan.
Blueberry Puree Increases HDL‐cholesterol in Rats
Authors: Gillespie Daniel , Gottschall‐Pass Katherine , Sweeney Marva
Published: June 2021

Oxidative stress has come to the forefront as a contributor to a number of diseases, including cancer, neurodegenerative and cardiovascular diseases (CVD). High‐fat diets (HFD) are thought to increase LDL, which can contribute to oxidative stress by becoming oxidized in artery walls, leading to atherosclerotic plaques that promote CVD. Blueberry (BB) polyphenolics are thought to act as antioxidants, which can potentially mitigate oxidative stress and improve CVD outcomes. The objective of our study was to evaluate the effect of a HFD supplemented with BB puree on serum lipids and biomarkers of oxidative stress. Male Wistar‐Kyoto rats (n=7) were fed either a control diet (CON), CON with 5mL/kg BB (CON+BB), 20% lard diet (HFD) or HFD with BB (HFD+BB) for eight weeks. A serum lipid panel was measured in a clinical laboratory analyzer and urinary 8‐isoprostane, a marker of lipid peroxidation, and urinary nitrite, a product of free‐radical reactivity, were measured using Cayman and Promega kits, respectively. HDL in CON group was 0.747±0.014mM and was increased by 10% in CON+BB to 0.822±0.019mM (p=0.002, ANOVA). All other serum lipids, urinary 8‐isoprostane and urinary nitrite were not significantly changed by either HFD or BB enrichment. We have shown that feeding BB with a normal, healthy diet can improve serum HDL, which could potentially improve excretion of cholesterol from artery walls, improving CVD. Funding support provided by NSERC, UPEI internal grants and WBANA.
Photoprotective Effects of Pistachio Antioxidants in a 3‐Dimensional Human Skin Equivalent Tissue Model
Authors: Chen C‐Y , Smith Avi , Blumberg Jeffrey , Garlick Jonathan
Published: June 2021

Reactive oxygen species (ROS) generated principally by irradiation from ultraviolet light (UV) exposure mediate many pathways leading to skin damage and skin aging. Antioxidants provide protection against oxidative injury to the skin through “quenching” ROS. Pistachios contain several antioxidants, including β‐carotene, lutein, anthocyanins, and γ‐tocopherol. Using a validated 3‐dimensional human skin cell equivalent (HSE) tissue, we examined whether pistachio antioxidants could protect the skin against 35 J/cm2 UVA‐induced damage following their uptake from cell culture media (a model reflecting systemic distribution). Lutein and γ‐tocopherol are the predominant lipophilic antioxidants in pistachios. Treatment with 0.1 µM lutein or 1 µM γ‐tocopherol for 48 h prior to UVA exposure appears to exert protection against UVA‐induced morphological changes to both the epithelial and connective tissue compartments of HSE. Overall skin thickness and organization, as well as fibroblast morphology, appears preserved in HSE treated for 48 h with a pistachio methanol extract containing 0.14 µM lutein, 0.59 µM γ‐tocopherol & 0.37 µM cyanidin‐3‐galactose prior to exposure to UVA. However, this protection from individual antioxidants and the pistachio extract was based primarily on the assessment of skin architecture and was not substantiated by tests of the proliferation of keratinocytes and apoptosis of fibroblasts. Additional studies are warranted to elucidate the basis of these discordant results and extend research into the potential protection by pistachios against UVA‐induced morphological changes in human skin.
Effects of Chronic and Acute Grape Consumption on Gene Expression of Inflammatory Mediators in PBMC
Authors: Stevens Allison , Giebler Kathryn , Hu Qian , Ford Sarah , Steinberg Francene
Published: June 2021

Studies have demonstrated that polyphenol‐rich foods offer protection against chronic metabolic diseases caused by inflammation and oxidative stress. We investigated whether consumption of grapes alters expression of inflammatory pathway genes and transcription factors in a chronic and acute setting. In this double‐blind, crossover trial, 20 obese subjects (BMI 37.0 ± 9.9) were randomized to consume either 60 g of freeze‐dried whole grape powder (Grape) daily (297 mg polyphenols) or matched placebo (C) daily (0 mg polyphenols) for 4 weeks separated by a 2‐week washout. Acute study days at the beginning of each intervention consisted of a high fat, high carbohydrate meal challenge (1,036 kcal, 31 g protein, 107 g carbohydrate, 54 g fat) accompanied by Grape or C consumption. Peripheral blood mononuclear cells (PBMCs) were isolated from fasted blood on acute study days and at postprandial time points 1, 3, and 5 hr. Fasted blood PBMCs were isolated at the end of each chronic, 4‐week intervention. Gene expression was assessed using real‐time quantitative PCR. Relative expression levels of target genes were calculated using the comparative CT method following normalization to the endogenous reference gene GAPDH. Expression of TNFα, NF‐κB, and IκBα genes in PBMCs shows no significant differences for Grape vs. C after 4 weeks of chronic consumption. Preliminary data examining expression of the same genes in the acute setting are inconclusive. Initial results indicate that grape consumption does not impact gene expression in inflammatory pathways; however additional genes must be investigated. (Supported by the California Table Grape Commission and USDA CRIS CA‐S*‐NTR‐6316‐H)
Effect of Sun‐Dried Raisins (Vitis vinifera L.) on the In Vitro Composition of Gut Microbiota
Authors: Mandalari Giuseppina , Chan Luisa , Carughi Arianna
Published: June 2021

Our aim was to investigate the effect of sun‐dried raisins on the colonic microbiota using an in vitro batch culture seeded with representative human gut bacteria. A model of gastric and small intestinal digestion was used to simulate the upper gastrointestinal digestion. Samples collected during a time course at 0, 4, 8 and 24h were analyzed for microbial composition and organic acid concentrations by 16S V4 rDNA analysis and high‐performance liquid chromatography (HPLC), respectively.

The most abundant phylum identified across all samples was Firmicutes. Significant differences (P<0.05) in the proportional abundance of Proteobacteria and Bacteroidetes were observed after 8 and 24h fermentation. The shift in the whole microbioma suggested that raisins selectively modulated gut bacteria differently from fructo‐oligosaccharides (FOS). Growth of Bifidobacterium spp. was higher (P<0.05) with raisins and FOS after 4h compared to the negative control. Amongst Clostridiales, the numbers of Blautia producta increased after 24h with raisins compared to the negative control. The highest concentration of acetate, propionate, butyrate and lactic acid in the raisin samples was detected after 24h.

These findings indicate that sun‐dried raisins could beneficially alter the composition of the human gut microbiota, thus improving gastrointestinal health.
Hierarchy of Anti‐Clumping Salts
Authors: Zem G , Tosunyan O , Hambarsoomian A , Lim K , Khatiblou K , Niamat S , Choi E , Monroy C , Chavez M , Karaan N , Basmajian M , Kim S , Kurginyan A , Sorooshiani S , Manzo L , Bahri F , Holverson A , Chacon G , Allatabakhsh N , Irikyan C , Tortorice C , Balyan S , Amer N , Shtivelman S , Vahdati V , Kaur T , Danialian S , Allard A , Jorshari L , Kazmi N , Sarkissian N , Mkhitarian K , Chavez C , Havaei M , Adzhemian A , Takakura M , Huang Y , Haritounian A , Arakelian A , Bohorquez J , Asatryan M , Herrera Y , Mayorga L , Garcia A , Oppenheimer Steven
Published: June 2021

We are identifying reagents that unclump cells in a model yeast system (Saccharomyces cerevisiae) for potential uses in clinical and other applications. Here 7 salts (0.1M concentration range) and no salt controls were stirred with fixed yeast at 20 min intervals over a 60 min time course in 1 ml distilled water droplets on glass microscope slides in 2545 trials by 45 independent investigators counting percentages of unclumped yeast vs clumps at each time point. The 7 salts were chosen because of their relationship to a known anti‐clumping agent magnesium sulfate. The following hierarchy of best to worst unclumping (compared with controls) was established at the 60 min time. Average percent single cells vs controls of all the trials appears in parentheses: sodium sulfate (+20.0), magnesium sulfate (+17.6), potassium sulfate (+9.9), magnesium carbonate (‐1.6), magnesium chloride (‐7.3), calcium carbonate (‐8.4), calcium sulfate (‐13.0). Individual groups of values varied from +59% to ‐41%. T tests were performed to compare results of experimentals vs controls. These results might help identify reagents that can be useful in unclumping cells in the bloodstream such as in anti‐thrombocytic and anti‐cancer applications, in reducing infectivity and biofilm development as well as in industrial applications (support: Sidney Stern Memorial Trust, NIH RISE, NSF Presidential Award 0731633 and Cal State).
Deletion of Sorting Nexin 27 Reverses Epithelial‐Mesenchymal‐Transition in Highly Aggressive Breast Cancer Cells
Authors: Li Kendy , Wu Shaopig , Playford Martin , Sun Jun
Published: June 2021

Sorting Nexin 27 (SNX27) plays critical roles in intracellular trafficking of proteins with functions of cell motility. However, its role in cancer cell motility remains largely unknown. Here, we showed a positive correlation of SNX27 expression with the malignancy of breast cancer cells. Furthermore, we generated a stable SNX27 knockdown clone in a highly aggressive MDA‐MB‐231 cell line. Wound healing showed that SNX27 knockdown significantly decreased the cell motility and proliferation. Morphologically, SNX27 knockdown cells differentiated into round cells, whereas the parental cells had many spikes. 3D cell culture in soft agar showed that the SNX27 knockdown cells formed significantly fewer and smaller colonies than the parental cells. Depletion of SNX27 made cells adhere together and move collectively and decreased cell motility. Western blots and immunostaining showed that SNX27 knockdown led to increased E‐cadherin, β‐catenin, and F‐actin protein expression, which facilitate the adhesion formation and reverse Epithelial‐mesenchymal transition (EMT). SNX27 Knockdown led to β‐catenin translocation to cell membrane. Furthermore, the expression levels of Vimentin and slug, the critical transcription factors of EMT, were significantly diminished in SNX27 knockdown cells. Taken together, our data at the morphological, transnational, and transcriptional levels have demonstrated that SNX27 plays a crucial role in tumor cell motility and differentiation. SNX27 is a potential target for controlling metastasis and inhibiting EMT.
*KL and SW equally contribute to this study.
Renal Effect of Triolein in a Rat Model of Fat Embolism Syndrome
Authors: Hsiung Ingrid , Qin Chang , Poisner Alan , Herndon Betty , Quinn Timothy , Molteni Agostino
Published: June 2021

Fat embolism syndrome (FES) is a lung process following bone fracture, surgery, and liposuction, expressed by inflammation, vasculitis, and fibrosis. A rat model of FES is induced by IV injection of triolein. Besides the lung damage at 48‐96 hours post injection, fat emboli were present in renal glomeruli, with vasoconstriction, glomerular and tubular hemorrhages, and increased renin secretion. Captopril and Losartan protected both organs, suggesting a role of the renin‐angiotensin system in injury pathogenesis. The pulmonary damage worsened at six weeks post injection. A second mild injury (i.p. injection of lipopolysaccharides (LPS)) exacerbated the condition. Renin was present in the lungs at this time.
Reported is the renal damage following triolein at six weeks. Triolein (0.2mL IV) was given to 18 rats and saline to 18 controls. Six weeks post‐injection, each group received i.p. LPS (3mg/kg) n = 9 or saline n = 9. Two days later at necropsy, kidneys were stained for H&E and renin and damaged scored.
Triolein caused severe glomerular damage with basal membrane thickening, crescents, hemorrhages, but no fat globules. Cortical arterial vasculitis with reduced lumen patency was seen in 59% of the triolein‐treated groups vs. 36% for saline (P = 0.003). Renin staining was seen in 32% of glomeruli of rats receiving saline, 53% of LPS‐treated, 54% of triolein‐treated, and 76% of those treated with triolein and LPS.
Therefore, triolein not only damages the lungs, but also the kidneys up to six weeks post‐embolization. Only lungs had fat droplets. The increased glomerular secretion of renin underlines its role in the FES pathogenesis; clinically, the renal damage should also be taken in consideration.
ΔNp63 Regulates HTRA4 Expression During Extravillous Trophoblast Differentiation
Authors: Li Yingchun , Parast Mana , Moretto Zita Matteo , Soncin Francesca
Published: June 2021

During early placentation, trophoblast cell columns help anchor the developing embryo into the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB), distally these cells differentiate into invasive extravillous trophoblast (EVT). In the absence of normal EVT differentiation, pregnancies can terminate early or be complicated by fetal growth restriction. We previously reported that ΔNp63, a p53 family member, is highly expressed in CTB but is lost when these cells further differentiate into EVT. We now further explore the downstream targets of p63 during EVT differentiation using both primary CTB and the JEG3 human trophoblast. Using gene microarrays, we identified HTRA4 as one such p63 target: HTRA4 is a member of the high temperature requirement A family of serine proteases, and have been shown to regulate trophoblast fusogenic activity and invasion. p63‐overexpressing JEG3 cells showed decreased expression of HTRA4, and decreased cell migration; conversely, down‐regulation of p63 increased HTRA4 expression and restored cell migration. In primary human trophoblast, isolated from early placental tissues, we found that HTRA4 is expressed at low levels in p63‐high EGFR+/HLAG‐ CTBs, and at high levels in p63‐low EGFR‐/HLAG+ EVTs. Taken together, these data indicate that EVT differentiation is accompanied by loss of p63 and gain of HTRA4. Our data reveal a potentially novel p63‐HTRA4 axis, involved in trophoblast migration and invasion.
Linking Pharmacology to Clinical Therapeutics: An Interdisciplinary Laboratory Experience in Optimizing Antiplatelet Therapy using Pharmacogenetic Data
Authors: Calinski Diane , Kisor David
Published: June 2021

The goal of this study was to engage students across professional disciplines, including medical, pharmacy, and nursing relative to collection, interpretation and application of pharmacogenetic data. Here focusing on the cytochrome P450 2C19 (CYP2C19) *1 and *2 alleles as it relates to clopidogrel therapy. This interprofessional exercise was based within the Manchester University College of Pharmacy curriculum. Seventy‐four students volunteered to serve as the sample population for this study. Student samples were kept anonymous. Students were instructed on the process of DNA sample collection through data reporting. Individuals collected their own DNA sample via buccal swab. The samples were genotype using standard PCR and gel electrophoresis. The pharmacy students interpreted the results and reported the findings to the nursing and medical students. Then medical students provided a clinical decision relative to antiplatelet therapy utilizing the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for the clopidogrel‐CYP2C19 drug‐gene interaction. Individual results were available for students and the population frequency of the *2 allele in the study population was calculated and compared to previously published data. Students were surveyed regarding their attitudes and understanding of pharmacogenetics prior to and following the laboratory experience. The vast majority of the students, regardless of their discipline, agreed that the laboratory enhanced their understanding of pharmacogenetics as applied in the clinical setting.
Akt signaling in fear memory processing and depression‐like behaviors
Authors: Willock Christine , Franke Thomas
Published: June 2021

Background

The Akt/PKB family of serine/threonine kinases plays a key role in neuronal cell function. Mutations in human AKT genes are found in human schizophrenic subjects, and other neurodevelopmental and psychiatric disorders. Genes encoding the three Akt isoforms (Akt1, Akt2, Akt3) are expressed in the mouse brain suggesting that Akt‐mutant mice are suitable models to understand the consequences of AKT gene dysfunction in the human brain.

Methods

To model the impact of Akt genotypes on fear memory processing and depression‐like behaviors, we used knockout strains for mouse Akt1, Akt2 and Akt3 to examine behavioral and biochemical phenotypes.

Results

Mice deficient for Akt genes had specific behavioral phenotypes during the acquisition and processing of fear memories. To complement our results with a pharmacological approach, we also inhibited Akt with an allosteric inhibitor. Baseline anxiety levels in naive Akt‐mutant mice or wild‐type littermates with inhibited Akt signaling were not altered. Acute stress exposure of Akt‐mutant or inhibitor‐treated mice resulted in depression‐like behavioral responsiveness. Akt knockout mice exhibited an increased susceptibility to chronic stress and decreased responsiveness to antidepressant treatment.

Discussion

Our data in Akt‐mutant mice converge with genetic findings in human subjects to support a critical role for Akt signaling in cognition and mood regulation. Our findings define the physiological contribution of selected Akt kinase isoforms to fear memory processing and the resilience to chronic stress. Our results support the study of Akt isoforms as diagnostic markers and therapeutic targets for treatment of neurodevelopmental and psychiatric disorders.

This work is supported in part by NIH grants DA032280 and OD018339, and AFSP.
Connecting Cell Death and Survival Pathways via the ASK1/IKKβ Interaction
Authors: Rusnak Lauren , Puckett Mary , Du Yuhong , Fu Haian
Published: June 2021

Cells must constantly integrate pro‐death and pro‐survival signals, and respond accordingly. Dysregulation of this signal integration can lead to human disease, which highlights the importance of balancing life and death signaling inside the cell. Previously we reported the novel interaction between apoptosis signal regulating kinase 1 (ASK1) and the inhibitor of κB kinase β (IKKβ). As a key effector of cell stress signaling, ASK1 is tightly regulated. IKKβ is part of the IKK complex which transduces pro‐inflammatory signaling. The interaction between ASK1 and IKKβ creates a junction between cell death and survival pathways. Thus understanding how ASK1 interacts with IKKβ may offer strategies for therapeutic intervention. Truncation studies led to the identification of the binding interface at the carboxyl terminal regions of both ASK1 and IKKβ. This was also confirmed using competition assays showing that the C‐terminal fragment was capable of disrupting the association between the full‐length proteins. Functionally, the binding of IKKβ decreased ASK1‐mediated pro‐apoptotic signaling. The same C‐terminal of IKKβ is also responsible for mediating binding to NF‐kappa‐B essential modulator (NEMO). We found that the ASK1/IKKβ complex did not disrupt the NEMO/IKKβ interaction, raising the possibility that there are two distinct structural determinants governing separate protein‐protein interactions with a very narrow region of IKKβ. With the defined regions of the binding interface, small peptide antagonists will be utilized to validate the importance of the ASK1/IKK axis in the control of cell fate. This work was supported by the National Institutes of Health grant P01 CA116676.
Mechanistic role of PDE4/cAMP in the development of HIV‐PI induced FasL mediated hepatotoxicity
Authors: Donde Hridgandh , Ghare Smita , Barker David , Joshi‐Barve Swati , Barve Shirish , McClain Craig
Published: June 2021

HIV protease inhibitors (HIV‐PIs) have been successfully used in the anti‐retroviral therapy in the treatment of HIV‐1 infection in the past two decades. Hence it is highly relevant to study the underlying mechanisms in HIV‐PIs induced hepatotoxicity that can negatively impact treatment outcomes. Hence, activation of hepatotoxic Fas/FasL signaling in HIV‐PI induced hepatotoxicity was investigated.

Clinically relevant combination of HIV‐PIs [Ritanovir (Rit)+Lopinavir (Lop)] significantly up‐regulated FasL and Fas expression in H4IIE rat hepatoma cell line as well as primary rat hepatocytes. Our recent work showed that PDE4 expression/cAMP metabolism plays a significant role in hepatic inflammation and injury. Hence, we examined the potential role of PDE4 expression in the HIV‐PIs mediated activation of Fas and FasL expression. Commensurate with the increase in FasL and Fas, PDE4 expression was also increased with Rit+Lop treatment. To address the causal role of PDE4 expression, cells were treated with a highly specific PDE4 inhibitor rolipram which markedly inhibited Fas and FasL mRNA and protein expression induced by HIV PIs.

Increased PDE4 decreases cellular cAMP levels and downstream cAMP effector molecules (cAMP‐PKA and ‐EPAC). Hence, effect of H‐89 (PKA inhibitor) and ESI‐09 (EPAC inhibitor) was examined in hepatocytes treated with HIV‐PI and rolipram. Both inhibitors significantly reversed the rolipram protective effect leading to re‐expression of Fas and FasL. Overall, this data strongly support and identify a mechanistic role for PDE4 regulated cAMP‐EPAC/PKA in the development of HIV‐PI induced FasL mediated hepatotoxicity.
Acetaminophen (APAP) hepatocyte toxicity is associated with increased translocation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) to the nucleus.
Authors: Ciriaci Nadia , Vigo Belén , Rigalli Juan , Mottino Aldo , Ruiz María , Ghanem Carolina
Published: June 2021

Previously, we have described that APAP intoxication induces nuclear translocation of GAPDH in rat liver. Recently, GAPDH nuclear traslocation was highlighted as a mediator of cell death.

Aim

To test if GAPDH nuclear translocation is associated with loss of viability of rat hepatocytes exposed to APAP. M&M: Rat isolated hepatocytes were exposed to different doses of APAP (0‐50 mM), for 24 h, in the absence or presence of RAS (10 µM), an inhibitor of GAPDH nuclear translocation. Cell viability was evaluated by the MTT assay and IC50 was calculated. Nuclear and cytosolic GAPDH, cytosolic cytochrome c, and mitochondrial BAX contents were evaluated by WB in hepatocytes treated with APAP (30 mM) and/or RAS (10 µM) for 24 h.

Results

IC50: RAS: 40.0±8.5 mM*; Control: 18.5±0.6 mM. Nuclear/Nuclear+cytosolic GAPDH expression ratio: APAP: 155±29*$; APAP‐RAS: 26±10*; RAS: 42±17*; C: 100±5. Cytosolic cytochrome c: APAP: 320±7.5*$; APAP‐RAS: 31±22; RAS: 19±32; C: 100±59. Mitochondrial BAX: APAP: 468±57*♦; APAP‐RAS: 342±45*♦; RAS: 152±81; C: 100±59. WB data are expressed as % of control. * P<0.05 vs C; $ P<0.05 vs RAS and APAP‐RAS. ♦ P<0.05 vs RAS.

Conclusion

APAP acute intoxication increased GAPDH nuclear translocation in isolated hepatocytes. RAS blockage of such traslocation was associated with prevention in alteration of apoptotic/necroptotic marker (cytochrome C) and consequently with increased survival of the cells to APAP cytotoxicity. The data strongly suggest a link between APAP liver toxicity and GAPDH translocation.
Regulation of the Brain Organic Cation Transporter Plasma Membrane Monoamine Transporter (PMAT) by Alternative Splicing
Authors: Wagner David , Duan Haichuan , Wang Joanne
Published: June 2021

PMAT is a polyspecific organic cation transporter highly expressed at the blood‐cerebrospinal fluid (CSF) barrier and facilitates the removal of monoamine neurotransmitters, organic cation drugs and toxicants from the CSF. We found that human SHSY5Y neuronal cells co‐express the full‐length wild type (wt) PMAT mRNA with two alternatively spliced mRNA species. One spliced variant (PMAT_v1) introduced a premature stop codon before transmembrane domain (TMD) 3 and the other (PMAT_v2) had a frame shift resulting in the deletion of TMD 3‐4. The goal of this study was to determine 1) the function and expression of the spliced variants in normal human tissues; 2) if the spliced variants exhibit a regulatory role on wt PMAT. Variant‐specific PCR indicated the spliced variants were co‐expressed with wt PMAT in all tested human tissues (whole brain, choroid plexus, small intestine and colon) at levels 37‐84% of the wt PMAT. When expressed in HEK293 cells, wt PMAT‐expressing cells showed up to 20‐fold increase in [3H]MPP+ uptake as compared to vector‐transfected cells. In contrast, there was no significant increase in uptake in cells‐expressing PMAT_v1 or PMAT_v2. PMAT_v1 protein was not detected by immunocytochemistry in HEK cells while PMAT_v2 was exclusively expressed in intracellular organelles. Co‐expression of PMAT_v1 or PMAT_v2 with wt PMAT in HEK293 cells did not significantly interfere with the normal activity of wt PMAT. Together our data showed PMAT pre‐mRNA is alternatively spliced in normal human tissues; and the overall PMAT activity in certain human tissues and cells may be regulated by altering the ratio between the functional and the non‐functional mRNA species.

This study was supported by NIH Grants R01GM066233 and T32GM07750.
Pharmacogenomics Studies of PAR4 Regulation in Human Platelets
Authors: Tourdot Benjamin , Yu Johnny , Holinstat Michael
Published: June 2021

Aberrant platelet activation contributes to the mortality associated with coronary heart disease (CHD). Our lab group has recently shown that platelets from black donors were hyper‐responsive to activation of the thrombin receptor Protease‐Activated Receptor‐4 (PAR4) compared to platelets from white donors. The racial difference observed through PAR4 persists in platelets treated ex vivo with dual antiplatelet inhibitors (COX‐1 and P2Y12 receptor inhibitors), a standard of care for many CHD patients. The racial difference in PAR4 signaling is largely attributable to one PAR4 variant more frequently expressed in blacks than whites, PAR4 T120A. Independent of race, T120 is associated with greater PAR4‐mediated platelet reactivity compared to the A120 variant. The role PAR4 variation plays in regulating thrombin‐mediated platelet activation in CHD has not been elucidated to date. We hypothesize that patients homozygous for the T120 will have an increase in PAR4‐mediated platelet reactivity compared to patients homozygous for A120. To investigate this hypothesis, we studied PAR4 signaling in platelets obtained from healthy donors and patients with CHD on dual antiplatelet therapy. Both healthy donors taking either a COX‐1 or P2Y12 inhibitor or cardiac patients on dual antiplatelet therapy, who are homozygous for T120, have an increase in platelet reactivity as measured by aggregation, αIIbβ3 activation and granule secretion compared to individuals who are homozygous for A120. This study reinforces the personalized medicine approach to therapeutic intervention and challenges the one size fits all approach which often leaves at risk populations without adequate protection from thrombotic events and stroke.
The Effect of Pentadecapeptide BPC 157 on System Circulation and Thrombogenesis after Ligation of Vena Cava Inferior
Authors: Vukojevic Jaksa , Siroglavic Marko , Kasnik Katarina , Kralj Tamara , Stancic Duje , Drmic Domagoj , Seiwerth Sven , Sikiric Predrag
Published: June 2021

In this study we analyzed the effect of pentadecapeptide BPC 157 on the system circulation and thrombogenesis after ligation of vena cava inferior. We used female Wistar rats (200g) which underwent vena cava inferior ligation just above the right vena ovarica. Immediately after, controls received 1 ml of saline bath and the BPC 157 rats received 1 ml of BPC 157 (10 µg/kg) bath. The thrombi from the vein were evaluated macroscopically after extraction and the pressure in the abdominal aorta and vena cava was assessed after 1h and 24h. Control group at 1h had the thrombi lenght of 0.83±0.15 cm and the thrombi mass of 0.04±0.01g, and the control group at 24h had length of 1.60 ± 0.43 cm and mass of 0.074 ± 0.006 g, with blood pressure in abdominal aorta of 70.50 ± 2.12 mmHg after 24h and 72.4± 3.1 mmHg after 1h, while the pressure in vena cava inferior was 27± 1.2 mmHg after 1h and after 24h 26.3± 1.4 mmHg. BPC 157 treated group at 1h had the thrombi lenght of 0.4± 0.05cm and the mass of 0.01± 0.004 g, and the 24h BPC 157 group had length of 0.78 ± 0.07cm and mass of 0.032 ± 0.003g, with pressure in abdominal aorta of 98.0 ± 1.40 after 1h and after 24h it was 96.5± 1.8 mmHg, while the pressure in the vena cava inferior was 13± 1.5 mmHg after 1h, and after 24h 15± 0.8 mmHg. As conclusion, this study has shown significant beneficial effect of pentadecapeptide BPC 157 on system circulation and decrease both in thrombi size as well as blood pressure in vena cava inferior. Supported by Grant 108‐1083570‐3635, Croatia.
HuR is Activated in Cardiac Hypertrophy and Regulates β1‐AR mRNA
Authors: Kraynik Stephen , Anthony Sarah , Tranter Michael
Published: June 2021

Heart failure is a worldwide health burden that is often preceded by a long period of increasing hypertrophy, or enlargement, of the heart prior to the loss of cardiac function. This progression of hypertrophy is marked by a decrease in the function and expression of key proteins that regulate cardiac contractility, such as the β1‐adrenergic receptor (β1‐AR). Despite a compensatory increase in sympathetic tone, cardiac contractility and output are substantially impaired, partially through β1‐AR desensitization and decreased receptor expression via mRNA degradation. It is thought that reversing the loss of the β1‐AR mRNA may preserve cardiac function and ameliorate the progression of hypertrophy. Human Antigen‐R (HuR), a known RNA binding protein, has been shown to bind to the 3'‐untranslated region of the β1‐AR mRNA, and we hypothesized that HuR plays a role in the development of cardiac hypertrophy by regulating β1‐AR mRNA stability in cardiomyocytes.

In hypertrophic hearts from mice that were subjected to chronic isoproterenol, we observed a shift in HuR subcellular localization from predominantly nuclear to increasingly cytosolic, an indication of HuR activation. Surprisingly, in hypertrophic hearts from mice that had undergone transaortic constriction (TAC), we observed that HuR activation strongly localized to regions of increased fibrosis. Futhermore, HuR translocation in hypertrophic hearts positively correlated with a near 50% decline in β1‐AR mRNA in both models of hypertrophy. Additionally, we show that in vitro knockdown of HuR by siRNA resulted in increased β1‐AR mRNA. In conclusion, these results suggest a novel role for HuR in the development of cardiac fibrosis in hypertrophy, and have demonstrated that HuR may be mediating β1‐AR mRNA degradation.
Clinical Biopotency of Insulin Analogues: Comparative Assessment in Different Species
Authors: Stricker‐Krongrad Alain , Hanks Chris , liu Jason , Bouchard Guy
Published: June 2021

Objective

The clinical potency of human insulin has classically been evaluated following the U.S. Pharmacopeia (USP) guideline for comparison to an international standard (IS) of activity (IU). However, since insulin analogues cannot be standardized against IS, there is a need for a bioassay to assess clinical specific activity (U). To this extent, we compared the biopotency (U) of different insulin products in different species and used human (recombinant) and pork (natural) insulin as reference standards.

Methods: New Zealand White rabbits were fasted and injected subcutaneously (s.c.) at a dose level of 0.5 U/kg. Diabetic Yucatan miniature swine (Sus scrofa) were injected s.c. at dose level of 0.1 U/kg. Normal insulin suppression tests (nIST) were conducted in primates (Macaca fascicularis) receiving a bolus intravenous (i.v.) infusion of glucose. Insulin products were given at dose level of 0.05 U/kg. Glucose levels were recorded using handheld glucometer devices. The blood glucose kinetic (BGPK), the blood glucose area under the curve (BGAUC) and the slope of the blood glucose clearance rate (kG) were used to assess clinical biopotency.

Results: The BGPK for short‐acting human and pork insulin were similar in the rabbit assay but their respective BGAUCs were different (ratio of 1.2). Accordingly pork insulin was more potent during nIST in the non‐human primate (slope of ‐0.012 vs. ‐0.010; pork and human, respectively).

Conclusion

Our data indicate that the clinical biopotency of insulin products can be assessed using BGAUC and kG, but that only the type 1 diabetic miniature swine can discriminate between differences in clinical biopotency for all aspects of BGPK.
Activation of Aryl Hydrocarbon Receptor Dissociates Fatty Liver from Insulin Resistance by Inducing Fibroblast Growth Factor 21
Authors: Lu Peipei , Yan Jiong , Garbacz Wojciech , Xie Wen
Published: June 2021

Background & Aims

The aryl hydrocarbon receptor (AHR), also known as the dioxin receptor, was originally characterized as a xenobiotic receptor that senses xenotoxicants. Here we investigated the endobiotic and hepatic role of AHR in fatty liver and energy metabolism, and identified the endocrine factor that mediates the metabolic function of AHR.

Methods

Wild type and liver‐specific constitutively activated human AHR transgenic (TG) mice were used to investigate the role of AHR in fatty liver and energy homeostasis. Adenovirus expressing short hairpin RNA targeting the fibroblast growth factor 21 (FGF21) were used to determine the involvement of FGF21 in the metabolic effect of AHR.

Results

We showed that despite their severe fatty liver, the TG mice were protected from diet‐induced obesity and type 2 diabetes. We identified the endocrine hormone FGF21 as a mediator for the metabolic benefit of AHR and established FGF21 as a direct transcriptional target of AHR. Interestingly, the transactivation of FGF21 by AHR contributed to both hepatic steatosis and systemic insulin hypersensitivity, both of which were largely abolished upon FGF21 knockdown.

Conclusions

The AHR‐FGF21 endocrine signaling pathway establishes AHR as a pivotal environmental modifier that integrates signals from chemical exposure in the regulation of lipid and energy metabolism.

Funding

This work was supported in part by the National Institutes of Health grants DK083952 and HD073070 to Wen Xie.