bluuebunny/crossref_metadata_2025 huggingface dataset:
Interethnic Diversity and Association of STAT6 Genetic Variants With Schistosomiasis in West Africa
Authors: Liou Gialeigh , Seamans Brooke , Adedokun Samuel , Ojurongbe Olusola , Thomas Bolaji
Published: June 2021

Published reports suggests a major role for signal transducer and activator of transcription (STAT6) polymorphisms in driving susceptibility and response to several diseases, including urinary schistosomiasis, showing an association with disease burden. We have shown previously that Th2 cytokine genes (IL‐4, IL‐10 and IL‐13) are differentially regulated in urinary schistosomiasis, with significant interethnic delineation across sub‐Saharan Africa. Therefore, we analyzed the genetic frequency of the STAT6 gene (rs3024974), located in the promoter region, in interethnic and disease groups, as well as evaluate association with urinary schistosomiasis and other disease covariates. Blood and urine samples collected from 114 urinary schistosomiasis patients, recruited from Nigeria were used for DNA extraction and egg counts respectively. Additional genomic DNA samples extracted from 225, 284 and 159 uninfected Africans, African Americans and Caucasians respectively served as uninfected controls. We genotyped these samples for comparative analysis among and between groups, utilizing a polymerase chain reaction‐restriction fragment length polymorphism assay. The prevalence of rs3024974 wild type genotype (CC) was 70.7%, 82.4% and 81.1% for Africans, African Americans and Caucasians respectively, and significantly different between groups (chi square=12.28; p=0.001). Mutant variants (rs302497TT) showed frequencies of 4.9%, 0.4% and 3.2% between groups (chi square=13.53; p=0.003), with similar results obtained for the minor allele, rs302497T (17.1% versus 8.8% versus 11.0% for Africans, African Americans and Caucasians respectively; p=1.42E‐05). Among the schistosome‐infected group, the frequencies of the rs3024974 genotypes CC and CT genotypes were 75.4% and 24.6%; none of the subjects had the TT genotype. The frequencies of the minor allele were 87.7% (rs3024974C) and 12.3% (rs3024974T), with subjects carrying the mutant genotype TT significantly protected from infection (p=0.02), while both CC and CT genotypes show an association with schistosomiasis. Despite minor differences in covariates between infected and uninfected subjects, we found no association between STAT6 (rs3024974) genetic variants with age, gender, packed cell volume, temperature or egg burden. The absence of the mutant allele among infected subjects reveal the necessity of the STAT6 promoter gene in driving susceptibility to, and association with schistosomiasis in Nigeria. Additionally, our results show that its genetic variants are insignificant for driving disease covariates.

Support or Funding Information

Laboratory Support and Faculty Development Award, College of Health Sciences and Technology, Rochester Institute of Technology

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional Fusions of Putative G‐protein Coupled‐Receptors from Hookworm (Ancylostoma ceylanicum) Expressed in Caenorhabditis elegans
Authors: Erickson Patti T. , Ratnappan Ramesh , Bernot James , Haile Meseret , Hawdon John M.
Published: June 2021

Many parasites, including hookworms of the genus Ancylostoma, are specialists and require host‐specific signals to resume development upon infection. Although little is known about the nature of these signaling molecules, evolutionary conservation between hookworms and the model nematode C. elegans suggests that hookworm host‐signal receptors are likely to be G‐protein coupled receptors (GPCRs) that are expressed in amphid neurons. Based on bioinformatic analysis of developmental stage‐specific RNA‐Seq data from A. caninum and A. ceylanicum, predicted hookworm GPCRs with increased expression levels during the infective larval stage 3 (iL3) were identified.

Approximately 1500 bp of genomic sequence upstream of the predicted start site for the putative A. ceylanicum GPCRs were fused to green fluorescent protein (GFP) coding sequence to generate promoter‐GFP transcriptional fusion constructs, which were microinjected into C. elegans. Of nine hookworm promoter fusions tested, four drove differential GFP expression in C. elegans tissues, including the digestive tract, pharynx, and a neuron pair. Preliminary analysis identified the neurons as interneurons, potentially involved in locomotion responses to chemosensory input in C. elegans. The putative GPCR encoded by the neuronally expressed gene is upregulated in both A. ceylanicum and A. caninum, suggesting that it may play a general role in hookworm development during host infection. These experiments indicate that hookworm promoters can be expressed in C. elegans, providing a surrogate system for the study of hookworm gene expression patterns.

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 Role of Acylated Homoserine Lactone Among Predator‐Prey Interactions of Arthrobotrys oligospora and Caenorhabditis elegans
Authors: Jauregui Mario Richard , Rocha Destany , Ochoa Roxanna
Published: June 2021

The necessity for biological control agents has increased over the past decade. Many pathogens become resistant to drugs and chemicals, pesticides are harming the environment and genetically‐modified pest‐resistant crops are a problem (Mankau, 1880). It has been previously established that Acylated Homoserine Lactone (AHL) is essential for bacterial cell to cell communication known as quorum sensing. AHL is part of the Lux –R family of enzymes used to transcribe and translate bacterial pheromones (Tsai, Winans, 2011). Studies have shown that the fungal organism, Arthrobotrys oligospora, has a homologous AHL gene in its genome. The parasitic nematode, Caenorhabditis elegans, has been shown to move towards synthetic AHLs. Therefore, we hypothesize AHL acts as an attractant leading C. elegans to A. oligospora, where the fungus traps and kills the nematodes. The entrapment of C. elegans has been observed for many years; however, the mechanism is not understood. A biosensor will be used to detect AHL production in various fungus‐nematode interactions.

Support or Funding Information

The project was supported in part by grants from the Department of Education (Award #PO31S150199), Southern California Edison and through Vanguard University's SURP Program generously supported by the Office of the Provost and the Institute for Faculty Development.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multi‐Targeted Inhibition of an Essential Bacterial Enzyme
Authors: Soares da Costa Tatiana P. , Gardi Chamodi K. , Christoff Rebecca , Sutton J. Mark , Abbott Belinda M. , Perugini Matthew A.
Published: June 2021

The cell wall of Gram‐negative bacteria consists of peptidoglycan chains linked together by oligopeptidic sequences comprised of the amino acids L‐Ala, D‐Ala, D‐Glu and meso‐diaminopimelate (DAP). Meso‐DAP is synthesized via the DAP pathway that also yields the basic amino acid, L‐lysine. Gene knock‐out studies show that enzymes functioning in the DAP pathway are essential to bacteria, including dihydrodipicolinate synthase (DHDPS). DHDPS is an allosteric enzyme that catalyzes the first‐committed and rate‐limiting step in DAP biosynthesis. It forms a homo‐tetrameric structure that gives rise to at least two ‘druggable’ sites, namely (a) the active site and (b) the allosteric site, which binds lysine to mediate a feedback inhibition response. Given its essentiality to bacteria and absence in humans, DHDPS represents a valid but as yet unchartered target for antimicrobial development. Recently, we have developed two classes of small molecule inhibitors that target the DHDPS active site and allosteric site using a contemporary multi‐disciplinary workflow spanning biophysics, biochemistry, medicinal chemistry, microbiology and structural biology. Inhibition studies in combination with biophysical techniques have demonstrated that these compounds are broad‐spectrum inhibitors of bacterial DHDPS in vitro, representing the most potent DHDPS inhibitors discovered to date. Using viability and time‐kill assays, these inhibitors have been shown to be bactericidal against both drug‐sensitive and drug‐resistant strains of Gram‐negative bacteria (MIC = 8 – 64 μg/ml), including Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli, but were non‐toxic against cultured human cells at >1024 μg/ml. Importantly, these compounds have been shown to synergize with FDA‐approved classes of antibiotics, including β‐lactams, fluoroquinolones, rifampicin and aminoglycosides. This study illustrates the potential for DHDPS inhibitors to be developed into a new class of antimicrobials with excellent potential to be combined with current antibiotics to yield innovative multi‐targeted formulations to minimize the emergence of resistance.

Support or Funding Information

Australian National and Health Medical Research Council (NHMRC)and CASS Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Functionalization of Azetidines
Authors: Gianatassio Ryan , Kadish Dora
Published: June 2021

While azetidines have always been known to be structurally strained and difficult to synthesize, a new reaction has been invented that shortens their preparation time significantly. Out of the top 200 marketed drugs, only two have azetidine‐based functional groups. Thus, the untapped prevalence of azetidines in drug discovery was the one of the driving forces to pursue this project. The proposed synthesis allows for the regioselective preparation of functionalized azetidines. Previous syntheses of azetidines are generally 3–5 steps, while this proposed synthesis can be performed in one‐pot and is operationally simple. It is envisioned that this method will be adopted medicinal chemists to expand chemical space and improve structure‐activity relationships (SAR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation Of Phosphoserine Aminotransferase 1 And Its Role In Breast Cancer Progression
Authors: Clem Brian , Metcalf Stephanie , Kruer Traci , Klinge Carolyn
Published: June 2021

Metastasis and endocrine resistance are two factors that complicate therapeutic intervention in breast cancer patients and lead to poorer overall survival. Metastasis is known to be responsible for 90% of cancer related deaths, and is especially prevalent in triple negative breast cancer (TNBC); while endocrine resistance can affect up to 50% of patients diagnosed with estrogen receptor positive breast cancer (ER+BC). Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second step within de novo serine biosynthesis and increased expression of enzymes in this pathway have been linked to progression of breast cancer and poor clinical outcomes. Within our preliminary retrospective analysis of human breast cancer patients, we identified an inverse association with elevated transcript levels and poorer distant metastasis free survival, which, coupled to a previously reported correlation of PSAT1 with response to endocrine therapy in patients with ER+BC, we postulate that PSAT1 contributes to breast cancer progression through promotion of metastasis and/or endocrine resistance. To initially determine relevance for PSAT1, immunohistochemistry was performed to determine PSAT1 expression in human breast cancer patients. We found that PSAT1 expression is increased through breast cancer progression, with highest levels observed within metastatic conditions. To investigate the metastatic contribution of PSAT1, we silenced PSAT1 expression within the triple negative breast cancer cell line (TNBC), MDA‐MB‐231. While suppression of PSAT1 had no effect on proliferation, there was a significant decrease in the motility and invasion of these cells. In addition, decreased PSAT1 substantially inhibited tumor nodule formation following tail‐vein injections of MDA‐MB‐231 cells in vivo. To investigate PSAT1's role in endocrine resistance, we used parental MCF‐7 cells and an endocrine‐resistant derivative cell lines and found that resistant cells exhibited higher PSAT1 expression compared to parental MCF‐7 cells. Lastly, suppression of PSAT1 trended to sensitize the resistant cells to 4‐hydroxytamoxifen treatment. Taken together, these data indicate that PSAT1 may contribute to the progression of human breast cancer via either metastasis or endocrine resistance or both and could potentially serve as a viable target for new therapies.

Support or Funding Information

Supported by a Research Scholar Grant, RSG‐13‐139‐01 from the American Cancer Society

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selenium and Sex: Competition Between Brain and Testes for Selenium Results in Male‐ Specific Consequences in Mice and Men
Authors: Berry Marla J. , Pitts Matthew , Kremer Penny , Hashimoto Ann , Seale Lucia , Ogawa‐Wong Ashley , Torres Daniel
Published: June 2021

Selenium (Se) is essential for normal neurological development and male fertility in mammals, setting the stage for competition between brain and testes when Se is limiting or its distribution is perturbed. Se supplementation studies in humans, and genetic knockout (KO) of selenium transport and recycling proteins in mice, unveiled the surprising finding that males exhibit more severe consequences than females when selenium homeostasis is altered. Se is also critical for thyroid hormone action, with implications for many downstream effects on endocrine and metabolic pathways. In one Se supplementation study in humans, an increased risk of type 2 diabetes (T2D) was reported in men with high baseline Se. However, other studies found no correlation between increased Se and T2D susceptibility, and the discrepancies in these human trials are controversial. The objective of our current studies is to elucidate the mechanisms underlying the sex specific differences in phenotype observed following disruption of the Se transport and recycling pathways in mice, the effects of varying dietary Se in combination with genetic KO, and the implications for human dietary Se requirements. Mice with KO of the Se recycling enzyme, selenocysteine lyase (Scly), exhibit metabolic syndrome that is more pronounced in males. Double KO (DKO) of Scly and the Se transport protein, Selenoprotein P (Sepp1) resulted in low viability and severe neurological deficits, including seizures. These traits are also more pronounced in males. Strikingly, while female DKO mice exhibit milder neurological deficits, they develop hallmarks of metabolic syndrome not seen in male DKO mice. Pre‐pubescent castration of male mice reversed the DKO neurological phenotype and partially attenuated the Scly KO metabolic phenotype. Assessment of brain and testes Se levels in these mice suggests that competition for available Se between these two tissues is a significant contributor to the observed phenotypes. However, preliminary data from testosterone replacement studies indicate that there are also hormone‐dependent mechanisms at play. These studies unveil novel insights into the complex mechanisms involved in Se distribution and utilization, and may be informative regarding sex specific Se requirements in humans.

Support or Funding Information

Supported by grants NIH NIMHD G12MD007601, and NIH NIDDK R01DK047320 to MJB.

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 Role of Sphingosine Kinase 2 in Chronic Alcohol‐Induced Liver Injury and Disease
Authors: Kwong Eric K. , Li Xiaojiaoyang , Liu Runping , Wang Xuan , Hylemon Phillip B. , Zhou Huiping
Published: June 2021

Introduction

Alcoholic liver disease (ALD) is one of the most common liver diseases worldwide. Currently, there are no effective therapeutics against ALD and liver transplantation remains the only curative option for end stage liver disease. Hepatic steatosis is a characteristic hallmark of alcohol‐induced hepatocyte injury and we have previously reported that sphingosine kinase 2 (SphK2) deficient mice on an alcohol diet develop overt fatty liver compared to wild type in the chronic and binge drinking NIAAA mouse model. In addition, there was significant downregulation of hepatic lipid genes and upregulation of pro‐inflammatory mediators in SphK2 deficient mouse livers. However, there are currently no effective mouse models that can recapitulate ALD progression and our aim is to determine whether SphK2 deficient mice on a chronic alcohol diet can produce advanced ALD pathology.

Methods

Mouse primary hepatocytes derived from wild type or SphK2 knockout (KO) mice were treated with different concentrations of ethanol (0–200 mM) for various time points (0–24h). For in vivo studies, C57BL/6J wild type and SphK2 KO mice (10–14‐week‐old, male and female) were fed a Lieber‐DeCarli 5% ethanol diet for 60 days. Cultured hepatocytes and liver tissue were harvested for RNA and protein using standard laboratory protocols. Quantitative RT‐PCR and western blot analysis were used to determine relative mRNA levels and protein levels, respectively. Hematoxylin and eosin (H&E) staining was performed on liver tissue for pathological analysis using standard laboratory protocols. Immunofluorescence was performed by staining frozen liver sections with anti‐CD11b antibody conjugated with FITC.

Results

Ethanol increased the mRNA and protein expression levels of SphK2 in mouse primary hepatocytes (MPH). H&E staining revealed that SphK2 KO mice on an 8‐week alcohol diet produced extensive steatosis and steatohepatitis compared to wild type mice and the NIAAA binge model. In addition, liver enzyme assay levels of ALT, AST and ALP are significantly increased in SphK2 KO mice suggesting increased liver injury and inflammation. The mRNA expression levels of hepatic lipid genes and bile acid synthetic gene mediators (SIRT1, CYP7A1, CYP7B1 and FXR) are downregulated and pro‐inflammatory mediators (IL‐1β, F4/80 and TNFα) are upregulated in SphK2 KO mice compared to wild type. Immunofluorescent staining of CD11b reveals increased macrophage infiltration in SphK2 KO mouse livers.

Conclusion

SphK2 is involved in hepatic lipid metabolism and plays an important role in ameliorating alcohol‐induced hepatic injury. Our results suggest that SphK2 KO mice on a chronic alcohol diet exacerbate alcohol‐induced liver injury and promotes ALD progression.

Support or Funding Information

NIDDK R01NIAAA F30

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Bias in Cytokines Transported by High‐Density Lipoproteins in Patients with Coronary Artery Disease
Authors: Creasy Kate Townsend , Stock Eveline , Pullinger Clive R. , Malloy Mary J. , Kane John
Published: June 2021

Inflammation plays an important role in the induction of Coronary Artery Disease (CAD) and the progression to deadly coronary syndromes. Cytokines are key mediators of inflammation, both in pro‐ and anti‐inflammatory capacities. We have recently discovered that cytokines are transported by high‐density lipoproteins (HDL), suggesting a novel mechanism contributing to the anti‐inflammatory effects of HDL. We examined the HDL‐associated cytokine profiles of male and female patients with CAD to better understand the differences in coronary disease presentation and therapeutic response between men and women.

Methods

Patients with confirmed CAD, defined by history of myocardial infarction, coronary artery bypass surgery, or angiography, with controlled serum lipid levels were included in the study. Native HDL species were isolated from plasma samples by selected‐affinity immunosorption. Plasma and isolated HDL samples were assayed for cytokine content by a multiplex array detecting the quantity of 35 cytokines.

Results

The majority of cytokines assayed were measurable in HDL samples. The distribution of HDL‐associated cytokines varied significantly between males and females. Female HDL samples indicate a much greater degree of remodeling of cytokine cargo than males. Cluster analysis of cytokine expression patterns suggests a difference in inflammatory pathway activation and response in females with CAD.

Conclusions

The cytokines transported by HDL in CAD patients differs greatly between men and women. The pattern of HDL‐associated cytokines provide new insights into the inflammatory processes contributing to CAD in a sex‐dependent manner. The amount of cytokine‐HDL remodeling is exhibited to a greater degree in females; the physiological relevance of this phenomenon is unknown. We aim to elucidate the mechanism of HDL transport of these cytokines and the clinical implications of sex differences in inflammatory response in CAD in future studies.

Support or Funding Information

This work is supported by the Read Family Foundation and the Campini Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation Of Cellulase Activity Of Fungi Isolates For Bioethanol Production By Using Cassava Bagasse
Authors: Igwo‐Ezikpe Miriam Nwanna , Okunowo Wahab Oluwanisola , Ayanshina Oluwamuyiwa
Published: June 2021

The continual energy demand worldwide and the progressive demising of oil reserves motivate the search for alternative energy resources especially from renewable sources such as agrowaste biomass. This study investigated the optimization of cellulase activity for bioethanol production through modulation of the cellulase activity of three fungi isolated from cassava bagasse. Fungi were isolated from cassava bagasse and genetically identified using 16S rDNA. The isolates were screened for cellulase enzyme production by filter paper and carboxymethylcellulase (CMC) assay. Physiochemical factors (Temperature, moisture content, nitrogen source, pH, substrate and inoculum concentration) which affect the cellulase activity were evaluated. Three fungi isolates were identified as Trichoderma Spp, Aspergillus spp and Rhizopus spp. The exoglucanase and endoglucanase had optimum activity at 25°C and 35°C, moisture content effect at 40 ml, pH 7, inoculum concentration of 10 – 20 ml and substrate concentration of 10–15g. Peptone greatly enhanced enzyme production better than urea and ammonium sulfate at day 3 incubation for both the exoglucanase and endoglucanase. Present study established the potential of these fungi isolates from agrowaste as good cellulolytic enzyme for potential use in bioethanol production.

Support or Funding Information

TETFUND Research Grant Award, 2015

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ARB treatment ameliorates triacylglycerol accumulation during insulin‐resistant conditions in the liver of OLETF rats
Authors: Godoy‐Lugo Jose Arquimides , Lee Debby , Thorwald Max Andrew , Nakano Daisuke , Nishiyama Akira , Hui David , Ortiz Rudy M.
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is a growing concern globally, and is associated with obesity, hyperlipemia, hypertension, insulin resistance, and diabetes; all traits linked to metabolic syndrome, which affects 20% of the US population. While angiotensin receptor blockers (ARB) effectively ameliorate the hypertension associated with metabolic disorders, its potential benefits on other aspects of metabolism during such conditions are not fully appreciated. ARB treatment may reduce overproduction and accumulation of hepatic triacylglycerols (TG), but it remains unknown whether ARB treatment has favorable effects in fatty livers associated with insulin resistance. To evaluate the benefits of the ARB treatment on lipid‐related genes, three groups (n=4–6) of 25‐week‐old rats were used: (1) LETO (lean strain‐control), (2) insulin resistant OLETF, and 3) OLETF + ARB (10 mg olmesartan/kg/d × 8 wks). An oral glucose tolerance test (oGTT) was performed to mimic a high glucose challenge. Liver and plasma were collected at T0, T30, and T60 min of the oGTT. We measured gene expression of hepatic PPARα, SREBP1c, FASN, and CPT1 along with TG and cholesterol levels in liver and plasma. PPARα expression was 1.6‐fold lower at T0 in OLETF+ARB compared with OLETF, but no changes were detected for other genes evaluated. Hepatic TG content was 1.5‐, 3.4‐ and 1.5‐fold lower at T0, T30, and T60, respectively, in OLETF+ARB compared to OLETF, along with 1.2‐fold lower plasma TG at T30. These changes suggest that AT1 signaling contributes to stabilizing lipid metabolism especially during a glucose challenge, and contributes to maintaining lower hepatic TG content that translates to reduced plasma TG during a glucose challenge.

Support or Funding Information

M. Thorwald was supported by NIH NCMHD9T37MD001480. R.M. Ortiz was partially supported by NIH NHLBIK02HL103787. Research was partially funded by NIH NHLBIR01HL091767.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingosine 1 Phosphate Regulates Store‐Operated Calcium Entry through binding to STIM1
Authors: El‐Shewy Hesham M. , Parnham Stuart , Fedarovich Dzmitry , Bullesbach Erika , Luttrell Louis M.
Published: June 2021

A growing body of evidence suggests that sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) pathway contributes to vascular dysfunction through dysregulation of Ca2+ mobilization. We demonstrated recently that intracellular S1P triggers store‐operated Ca2+ (SOC) entry and independent of G protein‐coupled S1P receptors suggesting SOC as intracellular target for S1P. However, the mechanism by which S1P modulates SOC entry is not well defined. SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. While STIM function as Ca2+ sensors within the ER, the Orai proteins constitute the pore‐forming unit of SOC in PM. We first investigated the role of SK1/S1P on STIM1/Orai1 assembly by confocal microscopy. Pretreating transiently transfected HEK293 cells with SK1 specific inhibitor inhibited angiotensin‐induced colocalization of GFP.STIM1 and mCherry Orai1. Similarly, using BRET assay, inhibition of SK1 decreased NET BRET values of STIM1‐FlAsH and Orai1‐Rluc in transiently transfected HEK293 cells stimulated with GPCR agonists. Our molecular modeling simulation predicted direct binding of S1P to EF/SAM1 domain of STIM1 molecule. This is initially assessed by in vitro binding assay revealed binding of biotinylated S1P to STIM1 in the membrane fractions of vascular smooth muscle cell lysates. We then used circular dichroism spectroscopy (CD) to measure the thermostability of the EF/SAM1 protein in the presence or absence of S1P. Addition of S1P to EF/SAM1 induced a consistent left shift indicative of lower melting temperature (Tm) compared to vehicle control. To further validate direct binding, we prepared 15N‐labeled EF/SAM1 for the NMR spectroscopy. A five point titration series with S1P caused perturbations of residue specific chemical shift due to binding interactions between EF/SAM1 and S1P using 2D TROSY (Transverse Relaxation Optimized Spectroscopy). Collectively, our data provide evidences of intracellular role for S1P in regulation of SOC through direct binding to STIM1.

Support or Funding Information

American Heart Association 17GRNT33700222 (HME)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the Function of Apolipoprotein A‐I Using Chimera Proteins
Authors: Patel Nairuti , Weers Paul M. M.
Published: June 2021

Human apolipoprotein A‐I (apoA‐I) is a 243 residue, 28 kDa protein that consists of an N‐terminal (NT) helix bundle domain and a less structured C‐terminal (CT) domain. ApoA‐I is the major protein component in high‐density lipoprotein (HDL) and is known to play an important role in reverse cholesterol transport. HDL promotes the removal of excess cholesterol from peripheral tissues to the liver. The CT domain of apoA‐I has a high affinity for phospholipids and initiates lipid binding, whereas the NT domain harbors the site of lecithin‐cholesterol acyltransferase activity. A recent study showed that removal of residues 231–243 led to a decrease in lipid binding affinity. Other studies have suggested that segments in the NT domain, residues 1–43 and 44–65, initiate lipid binding. Thus, conflicting data exists regarding the location of high‐affinity lipid binding in apoA‐I. A chimera protein was recently designed in which the CT domain of apoA‐I was attached to insect apolipophorin (apoLp‐III). The insect apolipoprotein acquired apoA‐I like properties, including high affinity lipid binding. Thus, this system allows for the identification of apoA‐I segments that have a high affinity for lipid surfaces. To identify potential lipid binding regions within apoA‐I, three chimeric constructs were designed using L. migratoria apoLp‐III. Residues 1–43 or 44–65 of apoA‐I were attached to the NT end of apoLp‐III. In addition, residues 231–243 of apoA‐I were attached to the CT end of apoLp‐III. The three DNA constructs were inserted into the pET‐20b (+) expression vector through restriction enzymes HindIII and NdeI. Site‐directed mutagenesis was conducted to introduce two cysteines into apoLp‐III at positions Thr‐20 and Ala‐149. The two cysteines lock the protein in an inactive state under oxidizing conditions to prevent lipid binding. This enables to study the lipid binding properties of the attached apoA‐I segments in the chimera proteins. Proteins are currently being expressed in Escherichia coli BL21(DE3) pLysS cells and will be purified through nickel‐affinity and size‐exclusion chromatography. The lipid binding properties will be assessed by the ability to solubilize phospholipid vesicles.

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 Roles of the Juxtamembrane Cysteine and Glutamine Residues in Mucin 1 (MUC1) Dimerization
Authors: Li Edwin , Herrera Roberto , Cani Ksandros , Freeman Christina
Published: June 2021

Mucin 1 (MUC1) is an O‐glycosylated transmembrane protein involved in forming protective mucous barriers on epithelial cells that line the mucosal surfaces of different tissues including breast, stomach, pancreas, and lung. When this protein is overexpressed, MUC1 dimerizes through the formation of disulfide bonds. A MUC1 fragment is then cleaved and transported to the nucleus, where it regulates apoptotic genes that allow for tumorigenesis. The CQC motif, located in the juxtamembrane domain of MUC1, has been shown to be important for dimerization to occur. However, the importance of the position of the CQC motif and the roles of the glutamine and each cysteine residues remain unclear. To answer these questions, we have been using the ToxR assay to measure beta‐galactosidase activity as a reporter of transmembrane dimerization. In this assay, we use a chimeric protein that contains the transmembrane domain and the juxtamembrane CQC motif of MUC1. To investigate the relevance of the position of the cysteines with respect to each other within the CQC motif, we have inserted an alanine before the glutamine (CAQC). To determine the role of the glutamine residue in dimerization, we have exchanged it for an alanine (CAC). Furthermore, to study the contribution of each cysteine, we have substituted the cysteine residues with alanine (CQA, AQC, and AQA). By providing additional information about the role of the CQC motif in dimerization we will further our knowledge about the mechanism of the MUC1 pathway that is involved in cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
COQ9 Membrane Association and Its Role in Coenzyme Q Biosynthesis
Authors: Von Bank Helaina C. , Lohman Danielle C. , Aydin Deniz , Smith Robert , Bingman Craig , Peraro Matteo Dal , Pagliarini David J.
Published: June 2021

Coenzyme Q is a ubiquitous and hydrophobic lipid essential in cellular respiration. The de novo eukaryotic synthesis of coenzyme Q involves at least nine different proteins (COQ1–9), several of which have undiscovered functions. Many of these proteins are peripherally associated with the inner mitochondrial membrane as part of the biosynthetic complex Q. How these proteins bind the membrane and interact with coenzyme Q lipid intermediates remains unresolved. Filling these gaps in knowledge provides potential for improving the treatment of diseases associated with Coenzyme Q deficiency, such as Parkinson's disease and cardiomyopathy. Recently, we discovered that one member of complex Q, COQ9, can associate with mitochondrial membrane lipids. Our biochemical, crystallographic, and computational data reveal that a structurally unique amphipathic helix of COQ9 drives membrane association. Collectively, this work provides mechanistic insight into how a lipid‐binding protein associates with biological membranes and has important implications toward understanding its role in coenzyme Q biosynthesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myocardin‐Related Transcription Factor Plays a Role in Myofibroblast Transdifferentiation of Retinal Pigment Epithelial Cells
Authors: Ueda Shunichiro , McDonald Kevin , Tamiya Shigeo
Published: June 2021

Retinal pigmented epithelial (RPE) cells have been implicated in fibrotic scarring of the retina. Past studies have shown transforming growth factor (TGF)‐beta plays a critical role in this process by inducing myofibroblast transdifferentiation. Myocardin‐related transcription factor (MRTF) has been implicated to play a key role in TGF‐beta initiated signaling in several fibrotic diseases, and thus, we examined its role in an in vitro model using small molecule inhibitors.

Primary cultured porcine RPE cells were cultured for 3 days in media containing TGF‐beta2 (10ng/ml) in the presence or absence of the following inhibitors: TGF‐beta receptor inhibitor SB431542 (10 micro M), MRTF signaling inhibitors CCG100602 and CCG203971 (25 microM), and CCG1423 (10 microM). Staining of actin cytoskeleton and alpha‐smooth muscle actin (SMA) were performed using phalloidin and anti‐alpha‐SMA antibody, respectively. For the collagen matrix contraction assay, an in vitro model for fibrosis, cells were cultured three days on type 1 collagen gels, which were subsequently released from 24 well plates and photographed to evaluate contraction by measuring gel size. These same gels with attached cells were then lysed by RIPA buffer and used for Western blot analysis of alpha‐SMA, and tropomyosin1.

TGF‐beta treatment induced myofibroblast transdifferentiation as evidenced by expression of alpha‐SMA incorporated prominent stress fibers and the associated significant increase in contractility. This phenotypic change was completely blocked by the TGF‐beta receptor inhibitor SB431542. Similarly, MRTF inhibitors CCG100602, CCG203671, and CCG1423 prevented cytoskeletal changes as well as the enhanced collagen matrix contraction induced by TGF‐beta. Interestingly, MRTF inhibitors did not significantly alter alpha‐SMA expression, but reduced expression of tropomyosin 1, a MRTF target that has been implicated to play a role in alpha‐SMA incorporation into stress fiber.

These data strongly implicate that MRTF plays a key role in myofibroblast transdifferentiation of RPE cells downstream of TGF‐beta receptor activation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pericyte Cd248 Promotes Fibrosis Through Induction Of Pro‐Fibrotic Macrophages During Kidney Injury
Authors: Pai Chen‐Hsueh , Tsai Yu‐Fei , Yen Ching‐Tzu , Lin Shu‐Rung , Lin Shuei‐Liong , Lin Shu‐Wha
Published: June 2021

CD248, also known as endosialin or tumor endothelial marker 1, is a type I transmembrane glycoprotein that is expressed in stromal cells of normal kidneys and uterus after birth. Using lacZ knock‐in (KI/+) mice we have shown that Cd248 expression decreases in most organs but increases and persists in postnatal kidneys, specifically in glomerular mesangial cells and pericytes/perivascular fibroblasts. In human chronic kidney disease, upregulated expression of CD248 is shown in myofibroblasts, which is negatively associated with renal survival. Although limited experimental data suggest that CD248 is involved in the cross‐talk with the post‐receptor signalling and cell proliferation of fibroblasts activated by platelet‐derived growth factors, the role of CD248 in kidney fibrosis is still unclear. Many studies including ours have identified CD248+ pericytes as the major source of precursors of scar‐producing myofibroblasts during progressive kidney fibrosis. Our data showed that expression of Cd248 was upregulated in the fibrotic kidney myofibroblasts of mouse model induced by unilateral ureteral obstruction (UUO). Cd248 knockout mice (Cd248−/−) showed less fibrosis and cell numbers of macrophages in the UUO kidneys. Moreover, UUO kidney macrophages expressed lower levels of chemokinde (C‐C motif) ligand 17 (Ccl17) and Ccl22 in Cd248−/− mice, suggesting CD248 deficiency led to a decrease of pro‐fibrotic macrophages. In vitro functional polarization assay of macrophages induced by co‐culture with wild‐type or CD248‐deficient myofibroblasts confirmed that CD248 played a role in the differentiation of pro‐fibrotic macrophages. Our results suggest that pericyte CD248 promotes fibrosis through induction of pro‐fibrotic macrophages during kidney injury.

Support or Funding Information

This study is supported by MOST (105‐2314‐B‐002 ‐096 ‐MY3 and 106‐2319‐B‐002‐001) and the Mrs. Hsiu‐Chin Lee Kidney Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Frutapin can interact with TLR4 and increase IL‐6 production by fribroblasts
Authors: Moreira Renato Azevedo , Sousa Felipe Domingos , Silva Ayrles Fernanda Brandão , Xu Shiwen , Owen James S. , Abraham David J. , Monteiro‐Moreira Ana Cristina Oliveira
Published: June 2021

Objective

Frutapin (FTP) is a glucose/mannose‐binding lectin derived from Artocarpus incisa L. seeds which is a wide spread plant, common in pan‐tropical regions, popularly known as “fruta‐pão” (breadfruit) and it is consumed cooked, by the local Brazilian population. FTP is the second most abundant lectin in A. incisa seed extracts, but proved difficult to purify with very low yields and contamination with Frutalin, a multiple binding lectin, frustrating its characterization. Herein, we assess the effect of rFTP, obtained by heterologous production in Escherichia coli, on fibroblast biology.

Methods

rFTP was successfully isolated in its biologically active form using SUMO‐tag strategy. Human skin fibroblasts (HSF) were used to perform cell viability and migration assay. Mitomycin C was used as an anti‐mitotic agent to assess whether FTP induces migration in scratch assay. The ability of rFTP to interact with TLR4 was evaluated using HEK‐Blue™‐hTLR4 cells. The effect of rFTP on IL6 levels was estimated by ELISA. Also, the effect of this lectin in HSF protein expression was evaluated by western blot.

Results

rFTP did not show cytotoxicity in a concentration range of 0.001 – 1mg/mL. HSF were cultured with media supplemented with rFTP to perform migration assay. By 24 h, samples treated with FTP had completely repaired the scratch, migrating across the entire denuded area effecting efficient wound closure. rFTP was able to potently stimulate TLR4 pathway in a manner similar to that observed with LPS. Also, rFTP stimulation increased reaching a plateau after five hours. High levels of IL6 produced by HSF were observed when compared to untreated cells (p<0.0001). The protein expression levels of pERK 1/2 and MyD88 were significant higher when compared to untreated cells during 24 h of treatment with rFTP concentration (p<0.05).

Conclusions

In conclusion, this study showed that rFTP was non‐cytotoxic to human fibroblasts. This lectin contributes substantially to cell migration and increase IL6 production by these cells, also stimulating TLR4 receptor. Given that fibroblasts play an important role in tissue repair, rFTP may represent a potential therapeutic biomolecule for wound healing and other related skin diseases.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MAST CELL NUMBERS OF RAT LUNGS IN AN ACUTE MODEL OF FAT EMBOLISM ARE REDUCED BY ALISKIREN AND LOSARTAN BUT NOT BY CAPTOPRIL
Authors: Colson Jordan , Arif Dauod , Voelker Dayne , Spaedy Abigail , Hamidpour Soheila , Poisner Alan , Pour Mohammad , Monaghan Paula , Khalafi Farnaz , Molteni Agostino
Published: June 2021

Introduction

Fat embolism (FE) induced by iv injection of triolein (T) causes vasculitis, septal inflammation and fibrosis in rat lungs at 48 hrs that is mitigated by two drugs acting on the renin‐angiotensin system (RAS): captopril and losartan (1). The histopathological changes found at this time are also ameliorated by treatment with the renin inhibitor aliskiren as the associated increase in mast cell numbers (2). This study examines whether captopril and losartan also prevent the increase in mast cell number at this time.

Methods

Sprague‐Dawley rats (280–300g) were treated with 0.2 ml iv (T) n=31 or saline n=7. One hour later 0.2 ml of saline (n=7) ip, aliskiren (50 mg/kg n=6), (100 mg/kg n=6), captopril (150 mg/kg n=6) or losartan (10 mg/kg n=6) were given. Saline treated rat (n=7) receive ip, one hour later, the same dose of saline. 48 hours later all the rats were necropsied after isofluorane anesthesia, lungs fixed in 10% formalin and H&E stained for morphology, Masson trichrome for fibrosis and CD 117 for mast cells. Two pathologists unaware of slides identity took at random 10 photographs of all the slides at 400× and separately counted the mast cells.

Results

As previously reported, 48 hours after triolein the lungs showed severe histopathological damage that was prevented by all three of the RAS drugs (1–3) and was associated with an increase in the number of mast cells (3). Aliskiren treatment reduced their numbers confirming previous findings (3,4). A significant reduction was also induced by losartan but not by captopril despite its protective effects on lung histopathology.

Conclusions

Mast cells may be involved in the pathological damage found after FE as suggested by their increase in number in the lungs after T and their reduction during protective treatment with aliskiren. Aliskiren and losartan also reduced their number. However, the lack of effect of captopril on mast cell number at this acute time period may suggest that some other pathways are involved in the time course of mast cell recruitment, such as angiotensin (1–7).

Support or Funding Information

Supported by the Catherine T Geldmacher Foundation, St. Louis, MO

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gas6/AXL Modulates Increased Inflammatory Cytokines During Preeclampsia in Rats
Authors: Hall Parker D. , Caskey Bryson , Hirschi Kelsey M. , Reynolds Paul R. , Arroyo Juan A.
Published: June 2021

Preeclampsia (PE) is a complicated obstetric complication characterized by increased blood pressure and the production of inflammatory cytokines. The growth arrest‐specific 6 (Gas6) protein is known to induce dynamic cellular responses and is elevated in PE. Gas6 binds to the AXL tyrosine kinase receptor and AXL‐mediated signaling is implicated in proliferation and migratory mechanisms in several tissues. Our laboratory utilized Gas6 to induce preeclamptic conditions in pregnant rats. Our objective was to determine the role of Gas6 and AXL signaling in the release of cytokines during the development of PE in the rat. Briefly, pregnant rats were divided into 3 groups that received daily ip injections of PBS, Gas6, or Gas6 + R428 (an AXL inhibitor). Treatment was done from embryonic day 7.5 (E7.5) to E18.5, at which time they were euthanized and serum was harvested for Multiplex cytokine determination. Fetal and placental weights were recorded. Blood pressure and proteinuria were determined. We observed: increased proteinuria and elevated blood pressure in Gas6 treated animals, but each were inhibited by R428; a 12‐fold increase of serum interferon gamma was reduced by AXL inhibition; a 12‐fold increase in serum IL1‐alpha was inhibited by AXL inhibition; a 45‐fold increase in serum IL‐2 was reduced by AXL inhibition; a 10‐fold increase in serum IL1‐beta was reduced by AXL inhibition; a 29‐fold increase in serum IL4 was decreased by AXL inhibition; a 13‐fold increase in serum IL5 was reduced by AXL inhibition; an 24‐fold increase in serum IL6 was reduced by AXL inhibition; and a 20‐fold increase in IL12p70 was reduced by AXL inhibition. These data suggest roles for Gas6/AXL in cytokine elaboration coincident with PE and offer possible treatment avenues that may alleviate PE symptoms.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).

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