bluuebunny/crossref_metadata_2025 huggingface dataset:
Carotid Arterial Stiffness Predicts White Matter Lesion Volume in Older Adults
Authors: Balestrini Christopher S. , Al‐Khazraji Baraa K. , Lingum Navena R. , Vording Jennifer L. , Suskin Neville , Shoemaker J. Kevin
Published: June 2021

Markers of arterial wall stiffness are used commonly as indicators of cardiovascular risk. Stiff conduit vasculature transmits pulse waves with increased velocities, which may lead to end‐organ microvascular damage. However, the association of peripheral vascular stiffness to the brain's vulnerability for vascular impairment has not been established. In the brain, this microvascular damage could include breakdown of the blood‐brain barrier causing leakage of fluid through the cerebrovascular endothelial layer. Fluid‐attenuated inversion recovery (FLAIR) imaging allows the visualization of fluid‐abnormalities in subcortical tissue, which can be quantified as white matter lesions (WML). These WML have been linked to cognitive impairment, and labelled as risk factors for stroke, dementia, and cardiovascular mortality. This research tested the hypothesis that peripheral arterial stiffness predicts levels of WML volume. We examined normotensive controls (CTRL; n = 12; 6 males; age: 42–69 years) as well as normotensive ischemic heart disease patients (IHD; n = 17; 14 males; age: 40–75 years). Common carotid artery diameters were measured during systole and diastole (B‐mode ultrasound images with ECG‐gating), and time‐aligned corresponding blood pressure values were used for measures of arterial strain (strain), arterial stiffness index (β), carotid distensibility, carotid compliance, and arterial elastic modulus. All measures were taken in the supine posture. Magnetic resonance FLAIR imaging at T2 was used to assess periventricular WML volume (WMLv) using SPM software. The IHD and CTRL cohorts had similar WMLv (4.4 ± 3.2 vs. 3.2 ± 3.5 mL; p = 0.33). Also, all participants were normotensive. Therefore, the data were pooled (n = 29) to examine the association between WMLv and peripheral arterial stiffness. Following multiple linear regressions while adjusting for age and BMI, WMLv was correlated with arterial stiffness index (r2 = 0.39, p = 0.001), strain (r2 = 0.37, p = 0.002), carotid distensibility (r2 = 0.36, p = 0.004), carotid compliance (r2 = 0.26, p = 0.009), and absolute change in carotid diameter across one cardiac cycle (r2 = 0.24, p = 0.01). Regardless of the age, BMI, and participant cohort, structural periventricular WMLv correlated with markers of arterial stiffness. These findings support the emerging hypothesis that systemic arterial stiffness affects damage in the microvasculature of the brain. Further, the resulting subcortical structural damage is apparent in older populations regardless of ageing and vascular pathology.

Support or Funding Information

Supported by the Canadian Institute of Health Research (201503MOP‐342412‐MOV‐CEEA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ZEB1 REGULATES MULTIPLE ASPECTS OF ENDOTHELIAL QUIESCENCE DURING VASCULAR REMODELLING
Authors: Bates David O. , Fernandes Cristiano S. , Usman Kiren Yacqub , Bourne Joshua , Collier Pamela , Ali Shahzeb , Kaira Spandan , Gregory Scott , Pang Vincent , Allen Claire , Grabowska Anna M. , Denning Chris , Benest Andrew V.
Published: June 2021

Generation of new blood vessels is essential for vascular remodeling in exercise to provide greater blood flow to developing muscles. As blood vessels grow, the sprouting endothelial tips need to recognise each other, and move from a motile, angiogenic phenotype to a maturing, quiescent phenotype,, form tubes and allow blood flow to occur and regulate their development. They grow in response to cytokine signalling such as VEGF and other angiogenic factors, but they mature as a consequence of cell contact and flow. As they do so, they change their metabolic state from growing, glycolytic, to quiescent oxidative metabolism. The mechanisms behind the transcriptional control of this phenotype are unknown, but recent work has demonstrated the dynamic interplay between ‘quiescence’ factors (such as FOXO1 expression) and expression of glycolytic enzymes such as PFKFB3 through c‐MYC. We investigated potential drivers of this process and identified Zeb1 (a transcription factor with known functions in cancer progression) to be expressed by quiescent (non‐angiogenic) endothelial cells and reduced in angiogenic endothelial cells in the developing murine retina. In vitro analyses demonstrates that Zeb1 is expressed by confluent monolayers of Human Vein Umbilical Cord Endothelial cells (HUVECs) but is downregulated in actively growing cells. Zeb1 knockdown promotes an angiogenic EC phenotype: including enhanced PFKFB3, c‐myc and VEGFR2 and reduced FOXO1, Tie2 and VE‐cadherin expression. Consistently, Zeb1 knockdown EC were metabolically more active (both through oxidative and glycolytic pathways). Furthermore, loss of Zeb1 promoted EC migration and proliferation in vitro. In vivo shRNA knockdown of Zeb1 led to altered vessel formation and hypercellularity. In conclusion, Zeb1 appears to be linked to key quiescence regulating signaling pathways and furthermore regulates aspects of these pathways. Zeb1 expression appears to be a critical component for endothelial cells to generate functional vasculatures that can support enhanced muscle blood flow, by its control of FOXO1, PFKFB3 and Myc.

Support or Funding Information

This work was supported by the British Heart Foundation (grant PG/11/67/29067), the University of Nottingham Cancer Research Priority Area, and the Medical Research Council Newton Fund (MR/N01247X/1)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the Expression of TRPV4 in the Choroid Plexus Epithelia as a Prospective Component in the Development of Hydrocephalus in the Gas8GT Juvenile Mouse Model
Authors: Hochstetler Alexandra Elizabeth , Whitehouse Logan , Antonellis Patrick , Berbari Nicolas F. , Blazer‐Yost Bonnie L.
Published: June 2021

Maintenance of homeostatic cerebrospinal fluid (CSF) secretion and absorption is essential for basic neurologic function. The choroid plexus epithelia, which is thought to produce the majority of the CSF, are among the most secretory of all epithelia. However, the control of this secretory process is poorly described. In diseased states such as hydrocephalus, where this homeostasis is disrupted, patients often experience symptoms such as cognitive impairment, motor/stability issues, and incontinence, among others. Current treatment for hydrocephalus is limited to surgically invasive shunting procedures which often fail and need revising – particularly in pediatric cases. In order to study the mechanism of this disease, a knock out mouse line of the Growth Arrest Specific 8 (Gas8) allele was generated. Loss of function of the Gas8 allele induces ciliopathy similar to Primary Ciliary Dyskinesia (PCD) in humans, with one of the symptoms being severe perinatal hydrocephalus. The disease progression in the mutant (−/−) mice is from postnatal day 0 (P0) to postnatal day 12–16 (P12–16), which makes this an excellent model to study pediatric/juvenile hydrocephalus. It is believed that one of the mechanisms of pediatric hydrocephalus is the overproduction of CSF by the choroid plexus (CP) epithelial cells that line the ventricles of the brain. The protein of interest to this research is the non‐specific cation channel, Transient Receptor Potential Vanilloid 4 (TRPV4), which has been shown to be activated by multiple stimuli including osmotic and pressure changes as well as by prostanoid metabolites. Activation of TRPV4 results in Ca2+ influx through the channel resulting in changes in intracellular signaling including the secondary activation of Ca2+‐stimulated ion transporters. Consistent with other studies, TRPV4 is localized to the apical membrane of the CP epithelial tissue in the Wild Type Gas8 mice. Preliminary data on the juvenile Gas8 mice indicate that TRPV4 is overexpressed in CP epithelia, ependymal cells and in the sub‐ventricular zone in the mutants as their hydrocephalus progresses as compared to the wild type (+/+) and heterozygous (+/−) animals. This suggests that antagonistic compounds of the TRPV4 channel have the potential to reduce hydrocephalus in the Gas8 model. Successfully targeting the molecular mechanisms for hydrocephalus in rodent models can provide a promising base for preclinical studies aimed at developing pharmaceutical agents to treat this disease.

Support or Funding Information

Funding: Grants from the Hydrocephalus Association and the Department of Defense Office of the Congressionally Directed Medical Research Programs (CDMRP).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Whole‐genome DNA hydroxymethylation among monozygotic twins discordant for cardiovascular death: the prospective National Heart, Lung, and Blood Institute (NHLBI) Twin Study
Authors: Dai Jun , Leung Ming , Krasnow Ruth , Reed Terry , El‐Rifai Wael
Published: June 2021

Objectives

Epigenetic modifications are related to cardiovascular disease. It is unknown whether DNA hydroxymethylation is associated with the risk for cardiovascular death that is independent of genes and common environment. This study aims to address this issue.

Methods

19 male monozygotic twin pairs middle‐aged in 1969–1973 and discordant for cardiovascular death through December 31, 2014 were included from the prospective National Heart, Lung, and Blood Institute Twin Study. Buffy coat leukocyte DNA samples were collected in 1986–87. Genome‐wide DNA hydroxymethylation levels were quantified using the hMe‐Seal‐seq. The log2 transformed reads were used for analysis of differentially hydroxymethylated regions (DhMRs) across the genome. Benjamini‐Hochberg false discovery rate (q value) of 0.05 was considered as the significance level. The bioinformatic tools Enrichr with Gene Ontology (GO) and the Cytoscape software package were used to analyze biological functions.

Results

There were 73,311 statistically significant DhMRs after controlling for age and blood cell subtypes (q<0.05 for all DhMRs). These DhMRs were located on all 22 autosomal chromosomes, and both the X and Y chromosomes. Among them, 27% of DhMRs were hypo‐hydroxymethlyated, had a log2 fold change ranging from −15.1 to −0.18, and were within/overlapped with 3′‐UTR (1.2%), 5′‐UTR (0.1%), exon (2.1%), intergenic regions (35.8%), intron (55.6%), non‐coding regions (0.6%), promoter transcription start sites (TSS) (2.4%), transcription terminal site (TTS) (2.1%); while 73% DhMRs were hyper‐hydroxymethylated, had a log2 fold change ranging from 0.14 to 15.7, and were within/overlapped with 3′‐UTR (0.9%), 5′‐UTR (0.1%), exon (1.7%), intergenic regions (49.2%), intron (44.9%), non‐coding regions (0.6%), promoter transcription start sites (TSS) (1.4%), and transcription terminal site (TTS) (1.2%). Using either all DhMRs with q value <0.05, a multi‐dimensional scaling plot demonstrated the complete distinction of case twins from control twins along dimension 1 but not dimension 2. A total of 12 significant GO biological processes (p‐value < 0.01) were identified. Cytoscape clustered the GO terms in these processes into 4 groups: target of rapamycin (TOR) signaling, metabolic process, cholesterol transport, and intracellular transport.

Conclusions

Differentially hydroxymethylated regions are prospectively related to cardiovascular death independent of genes and common environment.

Support or Funding Information

NHLBI 1R21HL127368‐01 and Des Moines University Faculty Research Fund to Dr. Dai

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression Profile of G Protein‐Coupled Receptor 37L1 in mouse
Authors: Ma Xiaobo , Armando Ines , Jose Pedro A. , Konkalmatt Prasad
Published: June 2021

The orphan G protein‐coupled receptor GPR37L1, also known as ETBR‐LP2, has strong homology with the endothelin‐B receptor (ETBR). However, GPR37L1 does not interact with endothelins. GPR37L1 is implicated to participate in brain development and the regulation of arterial blood pressure. GPR37L1 has been shown to be expressed in several regions in the brain, however its expression profile and the function in peripheral organs is poorly understood. We studied the expression profile of GPR37L1 in the brain, heart, kidney, liver, lung, spleen, stomach, pancreas, skeletal muscle, testis, and ovaries by RT‐PCR and immunoblot analyses and further used immunohistochemical analyses on tissue sections to identify the location of GPR37L1 expression within the tissue. Immunoblot analyses revealed the predicted 50 kDa and 40 kDa isoforms in most organs, and a ~35 kDa isoform mainly in the stomach and to a lesser extent in the brain, and a diffuse band of 45 kDa isoform mainly in the testis. The expression of GPR37L1 was highest in the brain, heart, stomach, pancreas, and skeletal muscle while GPR37L1 protein expression was lower in the kidney, liver, lung, spleen, testis, and ovary. The brain and pancreas expressed predominantly a 50 kDa isoform, while heart and skeletal muscle expressed mainly a 40 kDa isoform of GPR37L1. The kidney, liver, lung and ovary expressed almost equivalent levels of 50 kDa and 40 kDa isoforms of GPR37L1. The spleen expressed mainly a 40 kDa isoform of GPR37L1. RT‐PCR analyses confirmed the relative levels of GPR37L1 expression in the organs analyzed. Immunohistochemical analyses revealed the expression of GPR37L1, in the granule cell layer of the cerebellum, apical membrane of proximal tubule cells in the kidney, and airway epithelial cells in the lung. Further, RT‐PCR of proximal tubule and collecting duct cells obtained by laser capture micro‐dissection of mouse kidney sections, confirmed that the GPR37L1 is expressed in renal the proximal tubule but not in the collecting duct. These data show that GPR37L1 is expressed in the specific cell types within the organs and may play a key role in the physiology of the respective organs.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enterocyte Lipid Droplet and Mitochondrial Interaction in Zebrafish (Danio rerio) Larvae During High Fat Diet Absorption
Authors: Walters James William , Compton Gregory , Andrews Lacey
Published: June 2021

Diets associated with the development of dyslipidemias are typically high in triglycerides and cholesterol. These lipids are readily absorbed by intestinal enterocytes and there processed and packaged for delivery to the whole organism. Much remains unknown about the energetic mechanisms regulating dietary lipid processing within intestinal enterocytes. The purpose of this study is to better elucidate the dynamic interaction of lipid droplets and mitochondria during lipid absorption in the zebrafish model. Previous work in our lab has shown that larvae fed a high fat diet undergo lipid droplet biogenesis. We hypothesized that an increase in the size of the lipid droplets will cause mitochondria biogenesis or fusion while increasing apposition with lipid droplets. To test this prediction, zebrafish 6 day post fertilization larvae were fed a high fat diet of chicken egg yolk for one hour, rinsed, and fixed for TEM to create a 10 hour time course. Larvae were then sectioned, and imaged via TEM for each hour post feeding. Numbers of mitochondria decreased (43±7, 0 hours vs. 30±7, 3 hours) early upon feeding while area the total number of mitochondria increased (21±14 μm vs. 28±9 μm). Apposition between lipid droplets and mitochondria increased (from 0 vs. 5±1) during the same time frame and decrease by hour 10. Our results indicate that mitochondrial/lipid droplet apposition events correlate with lipid droplet processing or maturation. Future studies will attempt to further validate the zebrafish enterocyte model by comparison of other vertebrates. This work was supported by WV‐INBRE Grant# 2P20GM1.343_14 P1500699 to the West Virginia IDeA Network for Biomedical Research Excellence, NIH Grant P20GM103434

Support or Funding Information

This work was supported by WV‐INBRE Grant# 2P20GM1.343_14 P1500699 to the West Virginia IDeA Network for Biomedical Research Excellence, NIH Grant P20GM103434

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The DASH Diet Produces a Natriuretic Effect and Interacts with the Renin‐Angiotensin‐Aldosterone System
Authors: Maris Stephen A. , Brown Stacey , Conlin Paul R. , Williams Jonathan S.
Published: June 2021

The DASH diet is widely recommended to lower blood pressure, but its mechanisms of action are unclear. Lines of evidence suggest an interaction with the Renin‐Angiotensin‐Aldosterone system (RAAS), including ACE‐inhibitor or natriuretic effects. The DASH‐Diet in Isolated Systolic Hypertension (DASH‐DISH) trial is a randomized, controlled, cross‐over, study to test RAAS‐related mechanisms underlying the DASH diet effect. Following a one‐week run‐in diet on a Control diet (CON), subjects consumed a DASH and a CON diet for 4 weeks each in a randomized sequence. Calorie content was controlled to maintain weight and sodium was set at about 3 g daily. After each diet, subjects were admitted to a clinical research center where hormonal and hemodynamic assessments were obtained supine at baseline and in response to a RAAS inhibitor—captopril 25mg (CAP). This created four study groups; 1) DASH, 2) DASH+CAP, 3) CON, and 4) CON+CAP. Each group was also studied for responsiveness to a RAAS stimulator—a graded angiotensin II infusion (1ng and 3ng/kg × 45mins each). Primary outcomes were renal blood flow (RBF), mean arterial pressure (MAP), plasma renin activity (PRA), and aldosterone (ALDO). A repeated measure ANOVA was computed for primary outcomes with a Bonferroni adjustment and a statistical significance set at .0125. A total of 44 (19 female) hypertensive subjects (age 55.7±13.6 yrs, BMI 29.7±5.2 kg/cm2, BP 149/82±8/6 mmHg) completed the study protocol. DASH diet showed a trend towards lowered MAP vs CON (96.1±11.7 mmHg vs 98.8±14.1 mmHg, p = 0.07) and DASH+CAP significantly enhanced effects on MAP vs CON+CAP (83.2±10.7 mmHg vs 87.8±14.3 mmHg, respectively, p < 0.01). There were no differences in baseline RBF between the DASH and CON (p = 0.17). However, DASH+CAP increased RBF more than CON+CAP (486.46±149.38 cc/min vs 451.21±171.11 cc/min, p < 0.001). There were no baseline differences in PRA between the DASH and CON (p = 0.92) but DASH+CAP significantly increased PRA vs CON+CAP (1.52±1.78 cc/ml/min vs. 0.89±1.17 cc/ml/min; p < 0.001). DASH showed greater ALDO sensitivity to AngII infusion when compared to CON (17.40±7.74 ng/ml vs 13.84±6.21 ng/ml, p < 0.05) as did DASH+CAP vs CON+CAP (15.06±5.32 ng/ml vs 13.08±5.89 ng/ml, p < 0.05). These results suggest that the DASH diet produces a natriuretic effect in lowering blood pressure and stimulates the RAAS. The DASH diet might be particularly helpful in lowering BP in populations who benefit from a diuretic/natriuretic response and/or are concurrently prescribed RAAS antagonists.

Support or Funding Information

NIH NHLBI: R01 HL77234, NIH NHLBI: K23 HL084236

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutations in the zebrafish ortholog of HCFC1 reveal a critical function in neural precursor function
Authors: Reyes Joel Fernando , Castro Victoria Lynn , Reyes‐Nava Nayeli G. , Quintana Anita M.
Published: June 2021

Mutations in the HCFC1 transcriptional co‐factor cause cblX syndrome, an X‐linked disorder characterized by defects in cobalamin metabolism, intractable epilepsy, microcephaly, intellectual disability, movement disorders, and craniofacial abnormalities. Intractable epilepsy and neurological impairment are two of most severe phenotypes associated with cblX syndrome, however we do not understand the cellular and molecular mechanisms underlying these phenotypes in vivo. The human and mouse orthologs of HCFC1 are located on the X‐chromosome and deletion of the murine HCFC1 gene is subject to compensatory mechanisms, which have limited our ability to understand the function of HCFC1 during early brain development. To circumvent the limitations associated with rodent model systems, we created a stable germline mutation in the zebrafish HCFC1 ortholog, hcfc1a, using CRISPR/Cas9 genome editing. We identified three independent germline alleles in the hcfc1a gene, two of which result in a premature stop codon and a third that results in substitution of two amino acids within the kelch protein interaction domain. In vitro evidence suggests that perturbations in HCFC1 expression affect the number and differentiation of neural precursors. Thus, we analyzed the number of neural precursors and post‐mitotic neurons in hcfc1a mutant embryos. Mutation of hcfc1a resulted in an age dependent statistically significant increase in the number of neural precursors in specific brain regions. In addition, cell cycle analyses demonstrated a critical function of hcfc1a in the transition from S‐phase to G2/M phase. Together, our results provide evidence that HCFC1 plays an important role in the production, maintenance, proliferation, and/or differentiation of neural precursors during early vertebrate development. These data suggest a potential mechanism by which mutations in HCFC1 cause neurological deficits.

Support or Funding Information

Research reported in this poster was supported by the National Institute Of General Medical Science of the National Institutes of Health under the linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Regulators of Melanogenesis Identified by Genome‐wide Transcriptome Analysis
Authors: Yadav shalini , Kirty Kritika , Shukla Mudita , Natarajan Vivek , Gadgil Chetan , Gokhale Rajesh , Natarajan Krishnamurthy
Published: June 2021

Skin pigmentation occurs through an intricate network of pathways involving several signaling and regulatory molecules. The homeostatic mechanisms controlling melanogenesis is of great importance since prolonged activation of melanin synthesis could have disastrous consequences to the skin. We used the small molecules forskolin and IBMX, two activators of cAMP levels, to induce melanogenesis in B16 melanoma cells. Treatment with forskolin and IBMX led to increased tyrosinase protein levels and enzyme activity, hyper‐dendricity, transcriptional upregulation of melanin biosynthetic pathway genes and melanin accumulation. Electron microscopic imaging showed that the two small molecules enhanced the maturation of melanosomes. To identify genome‐wide transcriptomic changes, we carried out a time‐course microarray analysis of B16 melanoma cells treated with forskolin and IBMX. Microarray data analysis showed that a large number of genes are commonly regulated by forskolin and IBMX. Hierarchical clustering analysis showed a temporal pattern of expression of ~1620 genes. DAVID pathway analysis yielded several genes involved in melanosome biogenesis, cell cycle, inflammation and small solute transporters enriched during melanogenesis. Moreover, the hyper‐pigmenting cells contained elevated ROS level consistent with the melanogenesis itself being a pro‐oxidative process indicating a connection between melanogenesis and oxidative stress in melanocytes. A large number of differentially expressed genes belonged to the SLC small molecule transporter family genes. siRNA‐mediated silencing of two of the novel transporter genes led to impaired pigmentation indicating a key role of the SLC genes. Moreover, we identified several differentially expressed genes encoding chromatin modulators and transcription factor genes including MITF, a master regulator of melanocyte lineage. Microarray profiling of B16 cells upon silencing with siMITF identified a large number of MITF‐independent genes. Together our study provided an integrated view of the transcriptome involved in melanogenesis that could facilitate the understanding of the pathogenesis of pigmentation disorders.

Support or Funding Information

Genotypic Technology for microarray hybridization CSIR‐NMITLI, DST‐PURSE and DBT for funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Bio‐analytical Methodology and Physico‐chemical Characterization of Novel Compound RED
Authors: Sarkar Arindam Basu , Blanco Marsha , Reddy E Shyam P.
Published: June 2021

Objective

This study was carried out to develop the analytical methodology and limited characterization of the novel compound codenamed RED. The structure of the novel molecule is being withheld to protect the intellectual rights and patent issues involved.

Introduction

The molecule under investigation is a novel molecule that is custom manufactured for research purpose only. It is believed to have anti‐cancer activity and thus is a subject of interest and investigation.

Method

The UV‐Visble absorption spectra of the compound is established. A reverse phase liquid chromatographic method is developed to separate RED from its close structural analogs code named RmBr, RpM and RPh using a Xorbax Eclipse XDB‐C18 as stationary phase. The mobile phase is a combination of acetonitrile and water used in a continuously changing concentration gradient. Standard curve was prepared between concentrations of 0.5–10 mcg/ml with RmBr as internal standard; limit of detection (LOD) and quantitation (LOQ) was established. Statistical analysis of the data was carried out. The solubility of the compound in water, water containing 5% DMSO and water containing 5% DMSO and 5% Cremaphor was established by vortexing and ultra‐centrifugation. The stability profile of the compound in water and water containing 5% DMSO and 5% Cremaphor is established in glass and polypropylene container.

Results

Within the specified range the standard curve yielded a slope of 0.1631 (±0.0045) and an intercept of 0.0035 (±0.0054) and good linearity (R2≥0.999). RED solubilities are found to be 0.34, 0.7 and above 5 mcg/ml in water, 5% DMSO and 5% DMSO+5% Cremaphor ( a commonly used bio‐compatible solubilizing agent) respectively. The compound is found to be stable (≥90% of original content) for more than 7, 9 and 7 days in water, 5% DMSO and 5% DMSO+5% Cremaphor respectively when preserved in room temperature in glass containers. However, the compound Red rapidly degenerates when kept in room temperature in polypropylene bottles. The shelf life (>90% of original) of Red in polypropylene containers in room temperature is less than a day. The stability studies were carried out in both transparent and amber colored glass bottles, the compound was found to stable up to 7 days. The stability is not dependent on light within the time period of investigation.

Conclusion

The project is successful in establishing an analytical methodology for RED that is repeatable, robust, accurate and precise in the range. The solubility and stability of the compound in common aqueous solvents have been established. More work is required achieve a complete physico‐chemical characterization of compound.

Support or Funding Information

*This work has been financially supported by University of Findlay, College of Pharmacy funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes of urinary proteins in DDC‐induced chronic pancreatitis rat model
Authors: Zhang Linpei , Gao Youhe
Published: June 2021

Biomarkers are changes associated with the disease. Without homeostasis mechanisms, theoretically urine can be better and earlier biomarker source than blood especially for chronic diseases. Chronic pancreatitis is a chronic inflammatory disease characterized by pancreatic fibrosis as a major pathological change. If diagnosed earlier, effective preventive measures may be used. In this study, we test if early changes can be detected in rat model of chronic pancreatitis induced by intraperitoneal injection of sodium diethyldithiocarbamate. Urine samples before injection and 2 weeks, 3 weeks and 4 weeks after injection were collected. Urinary proteins from three rats were profiled by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS). Compared with before injection, the urinary patterns have changed significantly in all three rats. At week 2, fifteen differential proteins were identified when there was no obvious pathological changes yet. Twelve of these differential proteins were altered at other two time points and five proteins had previously been associated with chronic pancreatitis. At week 3, twenty‐nine differential proteins were identified, when inflammatory cytokines infiltration, acinar disruption, and slight fibrosis occurred. Significant fibrosis was detected at week 4 and thirty‐eight differential proteins were identified. At the stages of week 3 and week 4, fourteen urinary proteins had been reported to deferentially expressed in the serum or pancreatic tissue of chronic pancreatitis patients and other animal model studies. These findings demonstrate that changes caused by chronic pancreatitis can be reflected early in urinary proteins. Interesting clues can be found with only small number of modeled animals for further validation.

Support or Funding Information

This work was supported by the National Key Research and Development Program of China (2016YFC1306300), Beijing Natural Science Foundation (7173264, 7172076), and funds from Beijing Normal University (11100704, 10300‐310421102).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of FZD5 as Genetic Vulnerability in RNF43 Mutant Cancer
Authors: Steinhart Zachary , Pavlovic Zvezdan , Chandrashekhar Megha , Mascall Keith , Hart Traver , Wang Xiaowei , Zhang Xiaoyu , Brown Kevin R. , Adams Jarrett , Pan James , Sidhu Sachdev , Moffat Jason , Angers Stephane
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and novel effective treatments are greatly needed. Recent research revealed that a subset of PDAC cell lines is dependent on the Wnt cell‐signaling pathway for cell proliferation and survival. Wnt‐dependent PDAC all contain mutations in the gene encoding RNF43. RNF43 acts as a negative regulator of the Wnt pathway through ubiquitination and degradation of the Frizzled (FZD) family of Wnt receptors. RNF43 mutation status may therefore serve as a suitable biomarker for targeted therapy against the Wnt pathway in PDAC. In order to find novel genetic vulnerabilities in RNF43 mutant PDAC, we used genome‐wide CRISPR/Cas9 knockout screens in RNF43 mutant and wild‐type PDAC cell lines. Comparison of essential genes between RNF43 mutant and wild‐type groups was used to find context‐dependent essential genes for the RNF43 mutant genotype. This revealed the unexpected unique requirement of FZD5 (one of ten FZD homologs in humans) as a common genetic vulnerability in RNF43 mutant PDAC lines. This finding was validated through cell proliferation assays in vitro in both established cell lines and a primary patient derived cell line containing RNF43 mutation. Monoclonal antibodies derived against FZD5 were found to be remarkably effective as a single agent therapy both in vitro and in vivo, in subcutaneous and orthotopic xenograft models. Anti‐FZD5 biologics represent a novel potential targeted therapy in RNF43 mutant pancreatic cancers.

Support or Funding Information

This work was supported from grants funded by the Canadian Institutes for Health Research to S.A. (CIHR‐273548) and J.M. (CIHR‐342551) and by the Ontario Research Fund to S.S. J.M. holds a Canada Research Chair in Functional Genomics of Cancer and S.A. holds a Canada Research Chair in Functional Architecture of Signal Transduction. Z.S. is supported by a scholarship from the Centre for Pharmaceutical Oncology at the Leslie Dan Faculty of Pharmacy, University of Toronto.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) in Non‐small Cell Lung Cancer
Authors: Kooiker Amanda J. , Provost Joseph J. , Wallert Mark
Published: June 2021

Lung cancer is a debilitating disease with 222,000 new cases diagnosed each year and causing 156,000 deaths each year in the United States. Approximately 80% of lung cancers are categorized as non‐small cell lung cancer (NSCLC). Of these NSCLCs, 50% are comprised of adenocarcinomas, or tumors that originated within glandular epithelium, while 30% are due to squamous cell carcinomas, or a cancer that began at in a squamous epithelial lining. The Na+‐H+ Exchanger Isoform 1 (NHE1) acts as a key regulator in cellular proliferation, migration, and metastasis in a variety of solid tumors. NHE1 exchanges 1 extracellular Na+ ion for 1 intracellular H+ ion driving changes in both intracellular and extracellular pH in these developing tumors. To evaluate the role of NHE1 in NSCLC, we employed three cells types: 1) NCI‐H1299 a carcinoma cell line, 2) NCI‐H520 a squamous cell carcinoma cell line, and 3) NCI‐H23 and adenocarcinoma cell line. For each cell type, changing the media conditions from 0.5% serum to 10% serum increased the rate of cell proliferation 2 to 3 fold. This increased proliferation was blocked when the NHE1 inhibitors EIPA (ethylisopropylamiloride) or cariporide were present. Similarly, the change in serum concentration increase cell migrations rates in an NHE1‐dependent manner. To further evaluate the role of NHE1 in these cells, western blot analysis was completed to evaluate NHE1 expression in both low and normal serum conditions for each cell type. We also employed CRISPR‐Cas9 technology to develop an NHE1‐knockout (NHE1‐KO) cell line for each cell type. The rates of cell proliferation and migration where then evaluated in the NHE1‐KO cells as compared to NHE1 expressing cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The aggressive nature of prostate cancer of African Americans is correlated with massive down‐regulation of many immunoregulatory genes of microenvironment
Authors: Rahmatpanah Farah , Zi Xiaolin , Sawyers Anne , Agrawal Anshu , Lilly Michael , McClelland Michael , Mercola Dan
Published: June 2021

Background

The overall survival of prostate cancer for African Americans (AAs) is significantly worse than for European‐Americans (EA) which cannot be attributed to social‐economic status and may be related to racial‐genetic differences. We previously applied expression analysis (U133A) and observed significantly decreased expression of AA patient compared to EA patients especially in the tumor‐adjacent stroma. We tested the hypothesis that gene express in the stroma AA patients was significantly decreased compared to EA.

Methods

We developed primary cultures of turmor‐adjacent carcinoma‐associated fibroblasts (CAFs) of AA and EA tumors and matching distant contralateral fibroblasts (dCAFs), ~100 lines in all. We examined secretion of immunomodulators from the CAFs and dCAFs from prostate cancer of AA and EA tumors and distant stroma (dCAFs) as controls and applied RNAseq of the same cells as well as archived FFPE (formal‐fixed paraffin‐embedded) blocks of AA and EA patients.

Results

All 30 measured secreted immunomodulators were lower for AA CAFs. In contrast, few if any differences were seen between dCAFs of AA and EA. In particular Interferon‐alpha2 (IFN‐a2) is significantly reduced which is in keeping with the reduced immunomodulatory and Interferon‐induced genes (ISGs) observed for AA patients. The widespread reduction in immunomodulatory chemokines and cytokines observed in AA CAF supernatants suggests a state of significant immunosuppression as compared to the EA CAFs. The higher expression levels of chemokines Eotaxin, IP‐10, MIP1‐a, MIP‐1b, and MCP‐1, in the EA stroma may attract and activate immune cells to the tumor microenvironment thereby improving prognosis. Higher levels of several of inflammatory cytokines such as IL‐17, IL‐12, IL‐1a, IL‐1b in EA patients also may help activate immune cells leading to greater inflammation and immune responses in EA patients. The RNAseq data of AA and EA CAFs and for the FFPE tissue of AA and EA patients supported the decreased expression of the immunomodulatory genes in the stroma of AA patients compared to EA patients.

Conclusion

The detection of many statistically significant differences among ISGs and other immunomodulatory genes between the races is supported by RNAseq analysis of FFPE stroma tissue and by in vitro analysis of primary tumor‐adjacent stroma indicating that differences in immunomodulatory mechanisms may be a factor in the poor prognosis of AA prostate cancer. The primary lines of AA and EA CAFs and dCAFs may be a valid system to test role of these factors.

Support or Funding Information

DOD/CDMRP W81XWH‐15‐1‐0696

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative analysis of RORA expression in brain tissue from multiple sclerosis and normal individuals:a pilot study
Authors: Schroeder Stephanie C. , Heath Leah
Published: June 2021

The key to understanding the autoimmune disease multiple sclerosis (MS) lies in a deeper analysis of the genes that play a role in the pathology of the disease. A new gene of interest encodes the retinoic acid receptor‐related orphan receptor alpha (RORA). This receptor is expressed in several tissues and plays a critical role in regulating inflammatory response, neuronal cell development, bone metabolism, and has been proposed to promote the differentiation of Th17 cells. Th17 cells play a role in initiating autoimmune central nervous system inflammation, which is the cause of this debilitating disease. To better understand the significance of RORA in MS, the focus of this study was to observe the expression of this receptor and determine its role in susceptibility of multiple sclerosis. We did so by isolating RNA from 10 brain samples, 5 from multiple sclerosis patients and 5 from healthy controls. We then subjected the RNA to quantitative RT‐PCR and normalized the samples to control RNAs TBP and GAPDH. We found that there was a significant increase in expression of RORA (P<0.05) in brain samples from MS patients compared with healthy controls. This novel finding could be used as diagnostic criteria for susceptibility of multiple sclerosis and could potentially be a target for new treatments.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Human Cerebral Organoids to Study the Role of the Microbiome in Neuroinflammation
Authors: DiUbaldi Gianluca , Toth Charles
Published: June 2021

All cells within the central nervous system (CNS), including neurons and glial cells, respond to inflammatory mediators. It is integral to understand how these inflammatory pathways are activated within the CNS by both endogenous and exogenous factors. Diverse neuro‐immune and neuro‐psychiatric disorders are correlated with or modulated by changes of microbiome or microbiota‐derived metabolites. To study such a complex system, it is best to develop a model that best recapitulates in vivo biology. As a model of the CNS, cortical spheroids, derived from human pluripotent stem cells, were used to study inflammatory responses since they are a physiologically relevant 3D co‐culture of neurons and glial cells. For this experiment, spheroids were treated for 24 hours with the inflammatory mediators poly (I:C) (10 mg/mL) and indoxyl‐3‐sulfate (I3S) (50 mM). Poly (I:C) is a Toll‐like receptor agonist (TLR3), and I3S is a metabolite from tryptophan generated by the resident microbiome and an aryl hydrocarbon receptor agonist. The degree of neuroinflammation were analyzed using immunofluorescence of iNOS co‐localization with neurons and astrocytes in organoid sections. qPCR was also used to analyze the expression levels of TNF alpha and iNOS. Metabolic remodeling of treated spheroids was determined by using a Seahorse XFe96 Analyzer to show changes in metabolic pathways in the treated cells. These inflammatory responses have known biological links to many neurodegenerative disorders such as Huntington's and ALS, so the use of human cerebral organoids to examine these responses is pertinent to disease research and drug discovery.

Support or Funding Information

The authors would like to thank the Walsh Fund from Providence College and alumni Dr. John Mullen, Dr. Herbert Brennan and Roderick Lichtenfels for their generous research support.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biological and Health Aspects of Edible Wild Omani Plants in the Primary Prevention of Oxidative Stress‐mediated Colon Cancer
Authors: Waly Mostafa I. , Guizani Nejib , Rahman Mohammad Shafiur , Al‐Attabi Zaher
Published: June 2021

Background

Epidemiological, clinical trials and observational studies continue to support the notion that colon cancer is a nutritionally modified disease in which oxidative stress is a cofactor. Oxidative stress is involved in the pathogenesis of colon cancer. Although edible wild Omani plants (Portulaca oleracea, Pteropyrum scoparium, and Oxalis corniculata) are rich in phytonutrients (polyphenols and flavonoids), yet their medicinal use in relation to cancer was not well studied.

Objective

This study aimed to identify the antioxidant potential properties of three different edible wild Omani plants (Portulaca oleracea, Pteropyrum scoparium, and Oxalis corniculata) against colon cancer pathogenesis in rats.

Methods

A colon specific carcinogen, azoxymethane, was used to induce cancer in rat colon. The animals were allocated into 8 groups (10 rats/group) as follow; control group and azoxymethane‐injected group. Meanwhile the other 6 groups were rats fed one of the three plants extracts in the presence or absence of azoxymethane. After 12 weeks, all rats were sacrificed and colon tissues were dissected and used for aberrant crypt foci enumeration as well as for biochemical analysis of oxidative stress markers.

Results

It was observed that, the assayed three plants extracts had a protective role against colon cancer by abrogating the azoxymethane‐induced colonic carcinogenesis and oxidative stress.

Conclusion

Edible wild Omani plants act as a dietary bioactive agents and combat oxidative stress‐mediated colon cancer disease. It is recommended to be utilized in the daily food chain of the first degree relatives of colon cancer patients. This nutrition intervention approach might be effective in the primary prevention of colon cancer.

Support or Funding Information

This research was supported by Internal Grant Funding (IG/AGR/FOOD/17/1), College of Agricultural and Marine Sciences, Sultan Qaboos University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Neuronal Fate in C. elegans
Authors: Chalfie Martin
Published: June 2021

Neuronal differentiation is not simply the turning on of a developmental switch that results in the production of cell‐specific expression and features. We have used the six touch receptor neurons (TRNs) in C. elegans to identify and study the factors needed to initiate and maintain the differentiation of a specific type of neuron. We have identified transcription factors that specify, maintain, and restrict cell fate and others transcription factors, including Hox proteins, that reduce stochastic variability and ensure differentiation. The first class of transcription factors includes selectors, which direct the production of cell‐characteristic proteins, but also transcription factors that restrict and allow the selectors to act. We are calling the second class of transcription factors, which allow maximal function of selectors, guarantors. Our most recent studies are directed at understanding how different subtypes of TRNs are specified. One particular differences between the cells is that some are monopolar cells whereas others are bipolar cells.

Support or Funding Information

This work was supported by grants GM30997 and GM122522 from the National Institutes of Health

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glucocorticoid Receptor‐Dependent Gene Expression in the Central Amygdala of Alcohol‐Dependent Animals
Authors: McGinn Mary Adrienne , Edwards Kim , Edwards Scott
Published: June 2021

Alcohol Use Disorder (AUD) is a chronic, relapsing psychiatric disorder that is characterized by the development of motivational symptoms (e.g., escalation of alcohol intake) and the parallel emergence of negative emotional states (e.g., dysphoria, anxiety, pain). Consequently, the transition from recreational, limited alcohol consumption to uncontrolled, escalated intake is proposed to involve a transition from positive to negative reinforcement mechanisms. Our previous research indicates that alcohol dependence is associated with potentiated glucocorticoid signaling in brain regions that mediate stress and nociception, namely the central amygdala (CeA). Glucocorticoid receptor (GR) antagonism with mifepristone has demonstrated promising efficacy in reducing alcohol consumption in humans with AUD and rodent models of alcohol dependence. However, mifepristone's mechanism of action remains unclear. Given the role of GR as a transcription factor, we hypothesized that potentiated GR activity increases the expression of downstream neuropeptide system genes, and mifepristone normalizes these transcriptional effects. We tested our hypothesis in a well‐ established rodent model of AUD that utilizes chronic intermittent ethanol vapor exposure (CIEV). One day prior to the start of vapor exposure, animals were implanted with either placebo or 30d chronic release mifepristone pellets. After the 4‐week treatment regimen, we utilized a fluorescent bead‐based multiplex assay to determine brain region‐specific changes in mRNA levels of expected GR‐regulated genes. Results were analyzed via two‐way ANOVA, with alcohol vapor exposure (modeling alcohol dependence) and mifepristone treatment as factors. We found that within the CeA, alcohol dependence was associated with increased mRNA coding for the nociceptin peptide as well as its receptor, opioid receptor‐like 1 (Orl1), while mifepristone treatment significantly attenuated this effect. The nociceptin system is a part of the endogenous opioid class of signaling molecules, and previous research has demonstrated a role for nociceptin in both chronic pain and the manifestation of negative affect during alcohol withdrawal. Our findings suggest that the therapeutic benefits of mifepristone could be associated with a blunting of the nociceptin system in the central amygdala.

Support or Funding Information

Our research is supported by the following grants from the National Institute on Alcohol Abuse and Alcoholism: F31AA025812 (MAM) and R00AA020839 (SE).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.