bluuebunny/crossref_metadata_2025 huggingface dataset:
vCCI:Chemokine Interactions: Experimental Biochemistry Meets Computational Prediction
Authors: LiWang Patricia , Guan Wenyan , Stark Lauren , Showalter Laura , Colvin Michael
Published: April 2020

Inflammation plays a major role in many pathologies including asthma, arthritis, atherosclerosis and traumatic brain injury. As such, controlling inflammation is an important goal. While many biological pathways are involved in inflammation, an appealing target is the chemokine pathway. Chemokines are small immune system proteins that mediate chemotaxis of leukocytes bearing cognate chemokine receptors to the site of infection or inflammation. Many viruses have evolved strategies to counter the chemokine system, including the production of chemokine binding proteins. In particular, poxviruses encode vCCI (viral CC chemokine Inhibitor; also called p35), a protein that binds members of the CC class of chemokines. vCCI has been shown to bind many CC chemokines with high affinity and as such vCCI could be a potent tool as part of an anti‐inflammation strategy.

The mechanistic details of how vCCI is able to bind dozens of CC chemokines with nanomolar (or sub‐nanomolar) affinity is still being elucidated. Structures of vCCI alone and in complex have revealed a beta sandwich composed of two beta sheets that binds chemokines using one face of the sandwich in conjunction with a long, highly acidic loop after the second beta strand. Experimental work shows that this highly negatively charged loop in vCCI can act as a lid on the bound chemokine and interacts with positive charges on the chemokine.

A particularly puzzling vCCI‐chemokine interaction is in the area at the edge of the negatively charged loop in vCCI, where a conserved tyrosine (Y80) is close to K48 of the chemokine. It was hypothesized that mutating Y80 to Ala in vCCI would allow more room for the chemokine to bind, but experimentally the opposite has been shown to be the case: the Y80A variant in vCCI has a greatly decreased ability to bind chemokines.

We report a collaborative effort to understand the role of Y80 and the action of the acidic loop in vCCI to bind chemokines. Molecular dynamics simulations suggest that rather than being a hindrance to the function of the loop, the role of Y80 is actually to prop the loop in the “open” conformation so that the chemokine can bind. When Y80 is replaced with Ala in simulations, the loop closes and blocks the chemokine binding site. This intriguing and unexpected hypothesis from in silico work has been tested experimentally using NMR, isothermal titration calorimetry and other biophysical techniques. We therefore report a collaborative set of experiments that includes computation and experimentation to aid in the understanding of the vCCI:chemokine interaction

Support or Funding Information

Support provided by UC Merced summer graduate funding.
Effect of peroxynitrite scavenger on brain microvascular nitrotyrosination and S‐nitrosylation
Authors: Kolli Lahari , Sakamuri Siva Sankara Vara Prasad , Evans Wesley R. , Sure Venkata N. , Albuck Aaron L. , Mostany Ricardo , Busija David W. , Katakam Prasad V.G.
Published: April 2020

Objective

Peroxynitrite, generated from nitric oxide and superoxide, plays a pathological role in both ischemic and neurodegenerative brain injury. Free radical mediated microvascular dysfunction is a key initiator of cerebrovascular and neurological pathologies. Thus, understanding the acute effects of peroxynitrite on brain microvessels will help identify potential therapeutic targets. The objective of the study was to investigate the impact of peroxynitrite scavenger on the nitrotyrosination and S‐nitrosylation of brain microvessels.

Methods

Brain microvessels were isolated from the brains of 12‐week‐old female C57bl6 (WT, n= 4) mice by a combination of filtration (40μm – 300μm) and gradient centrifugation. Microvessels were treated with 2.5 μmol/L of peroxynitrite scavenger (FeTMPyP, Fe (III)tetrakis (1‐methyl‐4‐pyridyl) porphyrin pentachlorideporphyrin pentachloride) for 60 minutes at 37°C. Subsequently, western Blots were then performed to detect the nitrotyrosine residues in the protein lysates from microvessels. In addition, we determined the protein S‐nitrosylation in lysates by biotin‐switch method followed by immunoblotting. Nitrotyrosine residue is an indicator of peroxynitrite generation and protein‐S‐nitrosylation is an indirect measure for nitric oxide production.

Results

Acute treatment with FeTMPyP decreased nitrotyrosination of proteins from microvessels (3.16±0.63 vs 19.79±10.82, p=0.03, n=4 mice) in WT mice. In addition, 2.5 μM of FeTMPyP treatment decreased the protein S‐nitrosylation by 40% (p≤0.05).

Conclusions

Brain microvessels form peroxynitrite at basal levels. Surprisingly, peroxynitrite inhibition decreases protein S‐nitrosylation in the brain microvessels.

Support or Funding Information

Support: Undergraduate Summer Research Fellowship by American Physiological Society (Albuck); and National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834) and National Institute on Aging (Mostany: AG047296).
Novel transcriptome assembly and annotation of the banana slug (Ariolimax dolichophallus)
Authors: Tavares Noah Park , Vennam Sujay , Sweeney Robert Trip , De Luke
Published: April 2020

In this research, we present a new transcriptome assembly of the Santa Cruz wild banana slug (Ariolimax dolichophallus), the species most important to redwood ecology. Its functional annotation, the first of its kind, will be provided freely. RNA‐Seq with Illumina HiSeq technology was used to analyze samples extracted from slug. We used Trinity and Trinotate for transcriptome assembly and annotation. This assembly and annotation greatly enriches the studies of the banana slug, given its is the first study of banana slug exonic expression. This large‐scale gene discovery gives the broadest depth yet to the annotated transcriptome of this important species and should be of value to ongoing genomic studies of banana slug behavior with an emphasis on their roles in redwood ecology and seed dispersion.
Effect of inhibition of the PI3K‐Akt pathway on the Unfolded Protein Response
Authors: Nguyen Anh , Araujo Kelly , Bhandari Deepali
Published: April 2020

The endoplasmic reticulum (ER) is an organelle in eukaryotic cells that plays a vital role in many important cellular functions including proper folding of secretory and membrane proteins. Any disruption of ER function triggers ER stress leading to activation of an evolutionarily conserved signaling program called the Unfolded Protein Response (UPR). UPR signaling initially aids in re‐establishing normal ER functions and promotes cell viability. However, if the homeostasis is not restored in a timely manner, UPR eventually commits the cells to death. Cancer cells are known to be able to evade ER stress‐induced cell death, which helps them acquire resistance to therapy. Our laboratory has previously shown that Protein kinase B aka Akt is activated in ER stressed cancer cells leading to enhanced cell survival. The goal of this project was to test if Akt activation modulates the outcome of UPR signaling. We treated cells with commercially available inhibitors of the PI3K‐Akt pathway and analyzed changes in the expression and/or post‐translational modification of known UPR signaling proteins by immunoblotting. Our data show that the PI3K‐Akt pathway inactivation differentially regulates the outcome of UPR signaling. Our current experiments are focused on studying the effect of PI3K‐Akt inhibition on viability of multiple cancer cell lines exposed to ER stress.

Support or Funding Information

This project is supported by the NIGMS‐NIH grant #SC2GM121246.
Elucidating the role of antibiotics on PAL release from E.coli
Authors: Jackson Niaya A. , Khokhar Maha , Cheng Susan (Shi) , Gallardo Leslie , Michel Lea V.
Published: April 2020

Sepsis can result from a systemic bacterial infection, followed by an over‐exuberant immune response, which leads to widespread inflammation. Severe sepsis can result in organ failure and in severe cases, death. Past studies have proposed a role for bacterial Peptidoglycan‐Associated Lipoprotein (Pal) in the pathogenesis of Gram‐negative bacterial sepsis. In this study, we confirmed that Pal is released from Gram‐negative bacteria, Escherichia coli (E. coli), under certain culture conditions, including in the presence of certain antibiotics. Our preliminary results suggest that different antibiotics have differential effects on Pal’s release from E.coli. Since the majority of sepsis patients are administered antibiotics, our findings may be of great significance to the medical field and the sepsis research community.

Support or Funding Information

RIT FEAD/DRIG and NIH Bootcamp , RIT LSAMP program, Dr. Michael Pichichero Rochester General Hospital Research Institute
Resident bacteria in the small intestine can shape host drug metabolism through innate lymphoid cell‐derived IL‐22
Authors: Flannigan Kyle L. , Erickson Sarah L. , Johnston Michael J. , Jijon Humberto , Gallo Marco , Hirota Simon A.
Published: April 2020

The ability of the intestinal microbiota to influence drug metabolism has been recognized, however the mechanisms through which this occurs remain relatively unexplored. Recent work in germ‐free mice has demonstrated that conventionalization with specific pathogen free (SPF) microbiota influences the expression of cytochrome P450 (CYP) enzymes in the liver and small intestine (SI), two important sites for drug metabolism. Given that CYP enzymes, including CYP3A11 in mice, account for roughly 75% of total drug metabolism, we sought to investigate the role of specific gut‐residing microbes in shaping the expression and activity of these enzymes. Utilizing PCR arrays, we found that colonization of SPF mice with the immunomodulatory bacteria segmented filamentous bacteria (SFB) altered the expression of various CYP enzymes in the SI (but not liver), with Cyp3a11 being one of the most downregulated genes. Further analysis showed that SFB‐induced IL‐22 production by type 3 innate lymphoid cells (ILC3) correlated with reduced SI Cyp3a11 expression. Additionally, SFB colonization had no effect on the expression of Cyp3a11 in the SI of IL‐22−/− mice or RAG1−/− mice in which ILCs were depleted with anti‐Thy1 antibody. Both SFB colonization and administration of IL‐23, to activate IL‐22 from ILC3, increased the ability of the CYP3A11‐metabolized drug glibenclamide to decrease basal blood glucose levels when administered orally. In mouse SI enteroid cultures, IL‐22 dose‐dependently reduced the expression of Cyp3a11 and decreased the ability of SI enteroids to metabolize CYP3A11 specific substrates, effects that were blocked by the STAT3 inhibitor Stattic. Finally, when applied to human ileal organoids, IL‐22 induced robust changes in the transcriptome, with substantial effects on drug metabolism pathways centred around CYP3A. Our data suggest that specific gut‐resident microbes can influence the expression and activity of the drug metabolizing enzyme CYP3A11. This involves the production of IL‐22 by small intestinal ILC3, which down regulates CYP3A11 in the SI epithelium. These findings provide an understanding of how the intestinal microbiota modulates host drug metabolism, and how microbiota‐immune cell crosstalk may influence the efficacy and toxicity of various drugs.

Support or Funding Information

Alberta Innovates, Canadian Association of Gastroenterology, Canadian Institute of Health Research
Evaluating a High‐throughput method of Shearing DNA Using a Bead Mill for ChIP
Authors: Easparro Brandon
Published: April 2020

Chromatin Immunoprecipitation (ChIP) is key to understanding DNA‐protein interactions. This includes the mapping transcriptional factors and histone modifiers. Recent advancements have shown a transition from ChIP‐chip experiments to ChIP‐seq experiments, partly due to a reduction in materials cost, an increase in genomic coverage, as well as a reduction in required starting material. However, preparation of the chromatin is still prepared by either shearing cross‐linked chromatin by sonication or native chromatin micrococcal nuclease digestion. These options require longer incubation times and lower throughput, resulting in overall longer preparation. Bead milling provides an alternative of shearing DNA through the use of high impact beads. Through the use of specific beads, DNA shearing is achievable. Here we report the comparison of bead milling to ultrasonic shearing of DNA extracted from HEK cells for ChIP analysis.

Support or Funding Information

Omni International, Inc

935 Cobb Place Blvd

Kennesaw Ga 30144
“Genetic Predisposition to Type II Diabetes in the Hispanic Population in Relation to SNP rs2070744 on the eNOS Gene”
Authors: Silverio Rachel , Curiel Alejandro , Dominguez Jhonathan , Ramirez Alexa , Calleja-Macias Itzel Evelyn
Published: April 2020

Type 2 diabetes (T2D) is caused by the inability to break down sugar in the blood and ultimately starving the sufferer’s body and causing a cascade of various health issues. Rates of T2D in the U.S stood at 1.5 million newly diagnosed people in 2016 alone, proving the disease is rampant and dangerous. One of the top ethnicities affected is the Hispanic population. A statistic from 2016 revealed that 9.07% of the Hispanic population in the U.S was affected by T2D. With such high rates, the necessity of prevention research is evident. Since there is a broad spectrum of ethnicities that are affected by the disease, research must be done on certain populations to get reliable results. Single nucleotide polymorphisms (SNPs) may be the key to prevention of T2D. SNPs are genetic variations of alleles that have shown correlations to genetic predisposition to T2D in previous research. Using SNPs we aim to find the same correlations. SNP RS2070744 located on the eNOS gene has shown correlation of the T by C change decreasing the likelihood of attaining T2D. Our project’s goal is to add results to solidify the correlation of SNP RS2070744’s role in T2D predisposition. This was done by collecting buccal samples, extracting the DNA, amplification through PCR, gel electrophoresis, digestion, and lastly analysis for correlation. Our results show that the allele T is more prevalent in the Diabetes patients (82%) in relation to the controls (53%). This research has added data to potentially proving the correlation of SNP rs20707044’s role in genetic predisposition to T2D, but further research and data must be collected to make finite conclusions.
Dietary unsaturated fatty acid metabolites modulate life span and health span of C. elegans
Authors: Lee Kin Sing Stephen , Alan Jamie , Kessler Benjamin , Dattmore Devon , Zhang Fan , Sarparastzardroudi Morteza
Published: April 2020

Dietary unsaturated fatty acids are essential to physiology in different organisms. However, their mechanism remains largely unknown. Recent studies showed that the downstream metabolites play a critical role in inflammation, blood pressure regulation, pain perception, carcinogenesis, wound healing, and tissue and neural repair. However, their role in lifespan and healthspan in mammals remain largely unknown. To study the role and associated mechanism on the effect of unsaturated fatty acid metabolites on lifespan and healthspan in organism, we use C. elegans as a model organism because of the following reasons: 1) It is genetically close to humans; 2) the genome is known; 3) genetic modification is relatively easy; 4) imaging C. elegans is relatively easy, and more importantly, 5) part of the unsaturated fatty acid metabolism is conserved between humans and C. elegans. In this presentation, we will present the effect of cytochrome P450 metabolites on lifespan and healthspan on C. elegans and will discuss the potential mechanism behind such effects.

Support or Funding Information

This work was supported by the National Institute of Environmental Health Sciences (NIEHS) R00 ES024806. This work was also supported by National Science Foundation DMS‐1761320.
Intestinal epithelial cell‐specific deletion of Hif1a affects the hepatic energy metabolism in cholestasis induced liver injury
Authors: Luo Beibei , Xiang Dao , Cao Wei , Huang Xiaoqian , Wu Die , Chen Peijie
Published: April 2020

The hypoxia‐inducible factor (HIF) and its prolyl hydroxylases (PHDs) are pivotal in the transcriptional response to oxygen flux. Our previous study found that exercise elevates in vivo HIF‐1α expression in the small intestine and liver. Hepatic HIF‐1α and PHD2 signaling pathways are involved in exercise‐induced protection against cholestasis. Bile acids are synthesized from cholesterol and further metabolized by the intestinal microbiota. The integrity of intestinal barrier and the function intestinal epithelial cell (IEC) are regulated by HIF‐1α. Therefore, we hypothesized that intestinal HIF‐1α and PHD2 are engaged in the hepatic energy metabolism in response to cholestasis. The noninvasive bioluminescence imaging was applied to monitor HIF‐1α expression in ROSA26 ODD‐Luc/+ mouse model (ODD‐Luc) that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF‐1α. We found that in vivo HIF‐1α markedly increases in the small intestine of DDC fed ODD‐Luc mice. Next, the Villin Cre mediated, IEC‐specific deletion of Hif1a (Hif1aVKO) and IEC‐specific deletion of Phd2 (Phd2VKO) mice were generated. We fed Hif1aVKO, Phd2VKO and wild‐type (Hif1afl/fl and Phd2fl/fl) mice (male, 8‐week, n=6–8 per group) with normal chow or 3.5‐diethoxycarbonyl‐1.4‐dihydrocollidine (DDC) for 4 weeks, to induce cholestasis with biliary obstruction and bile duct injury. The liver structure was detected by H&E staining. Bile acid profile was analyzed by high performance liquid chromatography. Expression levels of bile acid metabolism related genes were determined using PCR array. The body weight, liver weight, albumin, alanine aminotransferase, aspartate aminotransferase, total bilirubin, triglycerides, total cholesterol, LDL cholesterol, and HDL cholesterol levels were also measured. All animal experiments were performed in compliance with and approved by the university ethical review board. The data was shown that DDC fed mice were suffered from cholestasis including biliary obstruction and bile duct injury with no group differences. The cholestasis induced symptoms in body weight reduction and liver injury related makers in DDC‐Hif1aVKO were severer. In Hif1aVKO, bile acid synthesisgenes related genes expression were increased. IEC Hif1a deletion affected bile acid profile in liver. The levels of taurocholic acid sodium salt hydrate, glycocholic acid hydrate synthetic and taurodeoxycholic acid sodium salt monohydrate were higher in Hif1aVKO (p<0.05), compared to Hif1afl/fl. Taken together, these results demonstrated that intestinal HIF‐1α signaling is involved in the hepatic energy metabolism in cholestasis induced liver injury.

Support or Funding Information

The National Natural Science Foundation of China (31701040, 31801003 and 31471135), Shanghai Sailing Program (17YF1418000) and Chenguang Program (16CG57).
Prebiotic effects of Jerusalem artichoke prevent hepatic steatosis in young rats submitted to a Western diet
Authors: St-Pierre David H , Lupien-Meilleur Joseph , Ou Ya , Lilya Melbouci , Auclair Nickolas , Marette André , Emile Levy , Denis Roy
Published: April 2020

The gut microbiota is a key mediator of metabolic functions. Alterations in its microbial ecology are closely associated to obesity and ensuing disorders. The present study was designed to investigate the beneficial effects of whole Jerusalem artichoke powder (JA; enriched in inulin prebiotic fibers) in a rodent model of diet‐induced obesity. Young rats (3 weeks old) were submitted to: 1) a Western diet (WD); 2) a WD supplemented with whole JA powder (10 %); or 3) a conventional CHOW diet for 8 weeks. Body weight, food consumption, fecal energy content and glucose disposal were measured. Muscle, liver and fat pads were weighted, intra‐organ fat accumulation was analysed in liver and muscles, and triglycerides and cholesterol levels were quantified in plasma. Fecal short chain fatty acid (SCFA) production and metataxonomic analysis were also performed. As expected, body weight, fat accumulation (intra‐organ and fat pads), plasma triglycerides and cholesterol concentrations were higher (p<0.05) in WD‐fed rats vs. CHOW‐fed rats. Energy intake and fecal energy excretion were not different in WD vs. JA animals. On the other hand, plasma triglyceride levels and intra‐organ fat accumulation were significantly reduced in JA‐supplemented rats. SCFA profiles were modified and the microbial production of propionate was increased in feces of the JA group (p <0.05). Such an upregulation of propionate synthesis was associated to potent changes in gut microbial ecology. Metataxonomic analysis showed that the treatment with JA significantly increased numbers of organisms from the Verrucomicrobiales and Bifidobacteriales orders. Therefore, the present results highlight the promising prebiotic potential of JA. This further supports the importance to consider JA for food‐based interventions against obesity and the onset of metabolic disorders.

Support or Funding Information

CMDO, NSERC, FRQS
The role of calcium regulation in the expression of PERK protein induced by sorafenib in hepatoma cells
Authors: Li Fu-Jian , Liu Ze-feng , Yang Zhou-fan , Liang Yong-han , Zhang Guo
Published: April 2020

Aim

This study aimed to determine whether calcium signal participates in the sorafenib induced ER stress process and has an impact on the action of sorafenib for the hepatoma.

Methods

Liver cancer HepG2 cell lines were maintained in DMEM with 10% fetal bovine serum and subcultured in plates or dishes at different densities for the following designed studies. HepG2 cells were basically treated with different doses of sorafenib (5–200 μM) for different time (0–48h) in the present or absent of trans‐Ned‐19 (a NAADP inhibitor, 100μM ) . The intracellular calcium signal was simultaneously recorded by confocal microscopy, and the expression level of PERK and caspase3 protein was detected by westernblot in HepG2 cells. The cell growth and apoptosis in these cells were analyzed by flow cytometry as well.

Results

Confocal microscopy showed that sorafenib (20–200 μM) induced substantial calcium release in HepG2 cells in a concentration‐dependent manner. Compared with the sorafenib intervention alone, trans‐Ned‐19 significantly attenuated sorafenib‐induced intracellular calcium elevation. Meanwhile, westernbolt assay showed that sorafenib time‐dependently but doses‐independently induced an increment in PERK expression in HepG2 cells. However, in the present of trans‐Ned‐19 was found to significantly enhance this sorafenib‐induced PERK and caspase3 levels, as well as apoptosis rate of HepG2 cells (P<0.05). For further treatment with trans‐Ned‐19 for 24 h, microscopic observation showed that trans‐Ned‐19 significantly inhibited cell growth and caused abnormal cell morphology changes compared with the sorafenib alone group.

Conclusion

Our preliminary data showed that NAADP‐mediated intracellular calcium release was importantly involved in sorafenib‐induced perk protein expression and cell apoptosis. Blocking NAADP‐mediated calcium channels can significantly enhance the efficacy of sorafenib in the hepatoma cells, suggesting that the combination of NAADP analogues and sorafenib might be a potential strategy in the treatment of liver cancer.

Support or Funding Information

Support or Funding InformationSupported by NSFC Grants 81360077, 81860654, 30630145, 81172260 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang
Characterization of PRAP1 During Murine Gestation
Authors: Smith Taylor , Wolfarth Alexandra , Neish Andrew S.
Published: April 2020

The development of Assisted Reproductive Technologies, such as in vitro fertilization (IVF), has allowed many couples struggling with fertility to conceive. However, IVF has a 60% failure rate. The leading cause of IVF failure is understood to be unsuccessful implantation. Improper or defective implantation leads to improper placentation which can affect fetal growth and development. While it is known that implantation is a complex reproductive process dependent on both competent embryos and a receptive uterus, many aspects of implantation remain unclear. Proline Rich Acidic Protein 1 (PRAP1), a 17 kDa protein, is expressed and secreted by the uterine endometrium. In the absence of PRAP1, female mice show defective implantation suggesting that PRAP1 has a role in this process. Nonetheless, PRAP1 remains largely uncharacterized. The objectives of this study are to characterize the role of PRAP1 during gestation and implantation. We hypothesize PRAP1 plays a role in implantation/placentation and overall pregnancy maintenance. Using timed pregnancies of wildtype C47BL/6J mice, uterine tissue was collected for each day of gestation and four days post‐partum, then analyzed by RT‐qPCR, immunofluorescence with a‐PRAP1 antibodies, and H&E staining. We found that Prap1 transcript is highly expressed during gestation compared to non‐ovulating females. Additionally, PRAP1 is downregulated during the window of implantation beginning at day 3.5 and increases on day 5.5. This increase continues and peaks just prior to parturition, and PRAP1 expression decreases 1000x within 4 days post‐partum. Immunofluorescence analysis demonstrated PRAP1 is localized to the endometrial epithelia during early gestation, but localized specifically to the anti‐mesometrial epithelia that interfaces with the amnion during mid‐ and late‐gestation. We have also found that our PRAP1−/− female mice have a slightly reduced fecundability (the ability to achieve a live birth from one cycle’s exposure to the risk of pregnancy) attributable to a reduced rate of implantation. We conclude PRAP1 plays a role in implantation/placentation and overall pregnancy maintenance.
Implications of Systemic Hematocrit on the Radial Distribution of Red Cells in Arterioles
Authors: Johnson Paul C. , Yalcın Ozlem , Jani Vivek P. , Intaglietta Marcos , Cabrales Pedro
Published: April 2020

It is well‐established that the tube hemacocrit (HctT) in blood vessels and glass tubes with an internal diameter less than 200‐microns is significantly lower than feed or discharge Hct (the Fahraeus Effect). Microscopic imaging of the flow stream reveals a decrease in Hct in the vicinity of the wall (the plasma layer, PL) but the mechanism of the effect is unclear. To address this question, we recorded high speed videos (3000 fps) of blood flow in 35‐micron diameter arterioles of the rat cremaster muscle at systemic Hct (HctS) levels of 20% to 72%. We found that the PL is an annulus bordering the wall that occupies 12% to 50% of the flow stream cross section, and that it is inversely related to HctS. In addition, results indicate that the Hct in the core region of the flow stream is not significantly different from HctS. The position of the PL coincides with the region of maximal shear stress in the flow stream, which may strip away the red cell monolayer adjacent to the wall, creating the PL while the monolayer moves toward the faster‐flowing center of the flow stream. In conclusion, while volumetric flow is related to the cross section of the flow stream, the PL is related to the circumference, thus reducing HctT as radius decreases.

Support or Funding Information

This work was supported by the NIH Heart Lung and Blood Institute under Grants R01HL126945 and R01HL138116.
Mineralocorticoid receptor antagonism prevents associated parenchymal arteriole remodeling
Authors: Yen Ting-Chieh , Laimon-Thomson Erinn , Abolibdeh Bana , Jackson William F. , Dorrance Anne M.
Published: April 2020

Obesity, a major risk factor for cerebrovascular injury, is associated with an increase in mineralocorticoid receptor (MR) activation. Obesity induced middle cerebral artery remodeling can be prevented by MR antagonism. However, the role of obesity‐induced MR activation in cerebral parenchymal arteriole (PA) remodeling has not been defined. PAs are responsible for regulating microcirculatory blood flow and play a significant role in cerebrovascular resistance. We tested the hypothesis that obesity induced PA remodeling is remediated by MR antagonism. Three‐week‐old Sprague Dawley rats were randomized to a control or high fat (HF) diet. At 12 weeks of age, 50% of rats in each group began treatment with the MR antagonist, Canrenoic acid (20mg/kg/day; CAN). PA structure in 24 week‐old rats was assessed by pressure myography under calcium‐free and zero flow conditions at 60 mmHg. Data are presented as means ± SEM, n = 3–10 per group. MR antagonism increased systolic blood pressure in HF‐diet only (HF+Vehicle: 125 ± 6 vs HF+CAN: 153 ± 6 mmHg, p<0.01). At the time of euthanasia, HF‐fed rats, both vehicle and CAN treated, had more abdominal fat per body weight than controls (Control+Vehicle: 1.44 ± 0.37 vs HF+Vehicle: 4.31 ± 0.44 % of body weight, p<0.01; Control+CAN: 2.26 ± 0.33 vs HF+CAN: 4.70 ± 0.44 % of body weight, p<0.01). HF‐diet decreased PA outer diameter (Control+Vehicle: 85.00 ± 5.51 vs HF+Vehicle: 49.09 ± 3.55 μm, p<0.01) that was reversed by MR antagonism (HF+Vehicle: 49.09 ± 3.55 vs HF+CAN: 96.00 ± 3.49 μm, p<0.01). HF‐diet decreased PA lumen diameter (Control+Vehicle: 68.67 ± 6.74 vs HF+Vehicle: 34.59 ± 2.72 μm, p=0.028) that was reversed by MR antagonism (HF+Vehicle: 34.59 ± 2.72 vs HF+CAN: 73.17 ± 7.38 μm, p<0.01). HF‐diet tended to decrease PA vessel wall area (Control+Vehicle: 1948 ± 55 vs HF+Vehicle: 973 ± 148 μm2, p=0.103); MR antagonism increased wall area in HF‐fed rats (HF+Vehicle: 973 ± 148 vs HF+CAN: 2027 ± 234 μm2, p=0.016). Vascular remodeling is associated with cerebrovascular injury and cognitive impairment. Our results suggest that inward hypotrophic PA remodeling is MR dependent providing a potential therapeutic target to combat obesity‐induced cerebrovascular disease.

Support or Funding Information

This work was supported by National Institutes of Health (NIH) Grants R01‐HL‐137694‐01 to W. F. Jackson and A. M. Dorrance.
O‐GlcNAc Transferase Regulates Growth Factor Signaling and Extracellular Matrix Composition in Idiopathic Pulmonary Fibrosis
Authors: Vang Shia , Helton Eric Scott , Denson Rebecca , Thannickal Victor J. , Krick Stefanie , Barnes Jarrod
Published: April 2020

Rationale

Idiopathic pulmonary fibrosis (IPF) is a chronic disease that affects the lungs. The scarring and progressive fibrosis, mediated by communication from the intracellular network and the extracellular matrix (ECM), results in impaired lung function causing respiratory failure and mortality. Over the years, IPF has been documented as a metabolic and age‐related disease. Several hallmarks of aging have been studied, including dysregulated nutrient sensing, altered metabolism, and intracellular communication. Interestingly, dysregulated nutrient sensing, via altered glucose utilization, has been shown to drive metabolic reprogramming in myofibroblasts. O‐linked N‐Acetylglucosamine (O‐GlcNAc) transferase (OGT) is a metabolic ‘stress sensor’ and is responsible for the O‐GlcNAc modification of proteins involved in many cellular processes including metabolism, intracellular communication, and growth factor signaling. However, the potential role of OGT/O‐GlcNAc on growth factor signaling, intracellular communication, and ECM changes during transdifferentiation has not been studied.

Methods

Lung tissue from individuals with IPF or controls was subjected to immunohistochemistry (IHC) staining to assess O‐GlcNAc modification. In addition, the role of OGT on transforming growth factor beta 1 (TGFb1) induced fibroblast to myofibroblast transition (FMT) was assessed on primary human lung fibroblasts cultured in the presence or absence of an OGT inhibitor, OSMI‐1. Alpha smooth muscle actin (a‐SMA), type 1 collagen (COL1a1), and Smad3 phosphorylation following TGFb1 stimulation with and without OSMI‐1 was examined by Western blot analysis.

Results

In human lung tissue sections, O‐GlcNAc levels were increased in IPF lung tissue compared to control human lungs as determined by IHC staining. In primary human lung fibroblast, TGFb1 administration resulted an increase in fibroblast transdifferentiation, which was determined by higher COL1a1 and a‐SMA expression. Interestingly, inhibition of OGT by OSMI‐1 following TGFb1 administration reduced expression of COL1a1 and a‐ SMA. Furthermore, OGT inhibition was shown to alter Smad3 phosphorylation upon treatment with TGFb1 (up to 2 hours) in the human lung fibroblasts.

Conclusion

Collectively, these data suggest that the metabolic sensor, OGT, and the O‐GlcNAc protein modification may have a role in regulating the expression of ECM proteins including COL1a1 by altering TGFb1 signaling during FMT. More importantly, the OGT/O‐GlcNAc axis may be directly involved in the pathogenesis of IPF and regulation of cellular transdifferentiation through TGFb1 signaling and overall ECM composition. Determining the mechanistic role of the O‐GlcNAc modification of proteins on these processes is needed, and may help to identify novel therapies for the treatment of IPF or any other fibrotic diseases.
Identify effects of phenytoin and losartan on TGF‐beta using human gingival fibroblast‐1 (HGF‐1) cells and establish HGF‐1 cell culture as a model to study drug‐induced gingival hyperplasia
Authors: Lipovskyte Kamile , Burrows Anna , Kale Vijay
Published: April 2020

Purpose

The main objectives of this study were 1) to evaluate the effects of phenytoin (antiepileptic drug) and losartan (TGF‐beta inhibitor) on cytotoxicity, ATP, and TGF‐beta in human gingival fibroblasts (HGF‐1) cells, 2) to identify whether losartan inhibits gingival overgrowth caused by phenytoin and 3) to establish HGF‐1 cell culture as a model to study druginduced gingival hyperplasia.

Methods

HGF‐1 cells obtained from ATCC were grown to 90% confluency in DMEM media supplemented with PenStrep, and bovine serum (FBS 10%). Cells were incubated in 5% CO2, 37°C and 95% relative humidity. For cytotoxicity, LDH leakage in the medium was measured using the CytoTox 96 Non‐radioactive Cytotoxicity Assay Kit, and for ATP, total % ATP was measured using CellTiter‐Glo Luminescent Cell Viability Assay Kit. For ATP and LDH measurements, cells were treated with various concentrations of phenytoin (0, 10, 25, 50, 100 and 200 μM) and losartan (0, 25, 50, 100, 250 and 500 μM) dissolved in DMSO, and incubated for 24h (30,000 cells/well in 96 well plates), 5% CO2, 37°C and 95% relative humidity. Final DMSO concentration was 0.1%. To identify effects on TGF‐beta, cells were treated with DMSO (0.1% final concentration), Phenytoin (100 μM), Losartan (100 μM), and Phenytoin (100 μM) + Losartan (100 μM). After 24h treatment of cells, TGF beta was measured as per supplier’s protocol using ELISA assay kit (Thermo Fisher, Grand Island, NY). All experiments were carried out as triplicate measurements and mean of triplicates were considered as one experiment (n). Minimum of three repetitions (n = 3) were carried out unless otherwise noted in results.

Results

Preliminary results show that none of the drug concentrations had a cytotoxic effect on HGF‐1 cells. No increase in ATP levels were observed for losartan. However, phenytoin showed greater increase in percent ATP levels when compared to vehicle control. Percent ATP levels for losartan and phenytoin were in the range of 101 ± 4 to 113.5 ± 8.2% and 135 ± 11.1 to 138.1 ± 16.5% respectively. Data from one TGF‐beta experiment showed no increase in TGF‐beta levels with phenytoin treatment, however losartan, and losartan plus phenytoin treatment showed 1.3 and 2‐fold increase in TGF beta levels over untreated cells respectively.

Conclusion

Our study suggests that phenytoin causes cell proliferation as noted from increased ATP levels. Preliminary results form TGF‐beta experiments were inconclusive and further experiments are needed to ascertain effects of losartan and phenytoin on TGF‐beta in HGF‐1 cells and identify whether HGF‐1 cells can be used as a model to study drug‐induced gingival hyperplasia.

Support or Funding Information

This project is supported by College of Pharmacy, Roseman University of Health Sciences, South Jordan, Utah 84095
Thermal Hyperpnea in Humans Changes Following Passive Heat Acclimation
Authors: Etter Clara Vada , Beaudin Andrew E. , Walsh Michael L. , White Matthew D
Published: April 2020

Introduction

Exercise ventilation was shown to increase after a 10‐day, 2 h·day−1 passive heat acclimation (HA) to 50°C and 20 % RH and this study assessed if thermal hyperpnoea for those at rest show a similar adaptation.

Purpose

It was hypothesized that similar to that seen for eccrine sweating and cutaneous blood flow, the core temperature thresholds for human pulmonary ventilation would decrease after heat acclimation. It was also hypothesized that pulmonary ventilation would increase following heat acclimation as it does for an active heat acclimation.

Methods

Six male participants performed two 40°C immersions separated by a 10‐day passive heat acclimation. Heat acclimation included 10 consecutive days with participants’ rectal temperatures maintained between 38.5–39.0°C. The heat acclimation was performed in a climatic chamber at 50°C and 20 % RH where participants remained seated at rest for 2 hr·day−1. A repeated measures ANOVA was employed with factors of Acclimation State (Pre & Post) and delta TES (0 to 1.4°C from rest). The p value was set at 0.05. An ANOVA with oxygen consumption as the covariate was employed to assess for heat acclimation‐induced changes in pulmonary ventilation. This study was approved by the SFU Office of Research Ethics.

Results

Acclimation was confirmed by a decrease in resting esophageal temperature (TES) from 37.70 ± 0.19 (mean ± SD) to 37.31 ± 0.11°C (p=0.001), as well as by significant decreases in the TES thresholds for temple cutaneous blood velocity from 37.30 ± 0.12 to 37.07 ± 0.13°C (p=0.014) and for forehead eccrine sweating from 37.60 ± 0.25 to 37.25 ± 0.22°C (p=0.03). The pulmonary ventilation thresholds were 38.28 ± 0.28 before and 37.84 ± 0.44°C after heat acclimation (p=0.13). After accounting for the covariate of oxygen consumption, a significant main effect of Acclimation State (F=10.05, p=0.025) plus an acclimation state x delta TES interaction for pulmonary ventilation (F=3.6, p=0.008) were explained by a significantly greater pulmonary ventilation following acclimation at delta TES of 0.6, 0.8, 1.0°C (0.018 <p < 0.05).

Conclusion

Following a passive heat acclimation the resting TES and TES thresholds for human thermoregulatory responses were significantly lower and pulmonary ventilation was significantly increased during passive heating as indicative of an increased thermal hyperpnea.

Support or Funding Information Funding Source

This project was supported by Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation.
Novel Mutations in OCTN2 Identified in Newborn Screening Exhibit Reduced Function and Lead to Carnitine Transporter Deficiency
Authors: Koleske Megan , Giacomini Kathleen
Published: April 2020

Newborn screening programs have revolutionized the diagnosis and treatment of genetic disorders. A compelling example is found in SLC22A5 encoding OCTN2, a bi‐directional plasma membrane transporter, which acts in the influx carnitine and efflux of drugs. Newborns with low plasma carnitine levels and biallelic SLC22A5 variants are diagnosed with Carnitine Transporter Deficiency (CTD), a potentially fatal disorder that can be treated with L‐carnitine supplementation. Here, we sought to validate the role of novel OCTN2 variants in CTD via functional characterization of mutant transporters in vitro.

In a recent study (PMID 31364285), three novel OCTN2 missense variants were identified in newborn screening. Following site‐directed mutagenesis and transient transfection in HEK293T cells, we performed isotopic uptake assays using 14C‐carnitine. Sodium phenylbutyrate, a chemical chaperone previously shown to increase function of other OCTN2 variants, was used to treat transfected cells. Welch’s two‐sample t‐test was used to determine significance.

Two novel missense variants, p.Asp165Glu and p.Gly473Val, exhibited substantial reduction of carnitine transport, retaining less than 5% of wildtype transporter function (p = 9.63*10−6 and 1.02*10−5, respectively). In contrast, only moderate reduction was observed for p.Met433Thr, which retained 61% wildtype function (p = 0.021). Sodium phenylbutyrate showed trends (which were not significant) toward increasing the function of all three variants, suggesting that intracellular errors of protein folding, processing, or trafficking may contribute to their reduced function.

Our studies validate the role of three novel variants in CTD, none of which have been associated previously with the disorder. The studies are consistent with the idea that even partial loss‐of‐function alleles may be disease‐causing for CTD. Finally, we suggest that sodium phenylbutyrate represents a potential modulator of OCTN2 function, with further studies warranted to confirm effects on function and membrane localization of OCTN2 variants.

Support or Funding Information

This work is supported by the Chan Zuckerberg Biohub.
A Meta Analysis of Advancements in Prevention and Therapy of Chronic Kidney Disease
Authors: Negere Mulunesh Lee , Einstein George P. , Tulp Orien Lee
Published: April 2020

This meta‐analysis study was focused on estimating the prevalence of chronic kidney disease (CKD) in the USA and Ethiopia to show the relevance of global awareness toward chronic kidney disease, advanced treatment practices, and its eradication in these two Nations. Kidney disease has become a global burden, especially CKD, according to CDC 2019 data. CKD is most common in adults. Evidence shows nine in ten adults with chronic kidney disease do not know if they have the disease or not. The estimated ratio shows from the CDC data indicate that 15% of US adults have CKD. Studies indicate that Diabetes mellitus and hypertension are the leading causes of chronic kidney disease more than other factors or diseases. The accepted common treatments for CKD are medication, dialysis, and transplant. The other modality of treatment now emerging is stem cell therapy, which has been proposed to improve the quality of patent life, replace dialysis, and decrease the frustration behind the transplant donor waiting list. In the present study, an extensive search was done with the use of MedLine, EmBase, PubMed, BIOMED Central, CDC Database, Web of Science, and Scopus databases to identify applicable published studies. Search items included CKD prevalence, diabetic and hypertensive causes of CKD, and other common causes of decreased renal function, advancements in CKD treatment, stem cell therapy, clinical trials, diagnostic criterion to diagnose CKD, including possible blood and urine tests, chronic kidney failure, and Renal insufficiency, including pool risk estimates, fixed and random effects models where applicable. The participants were chosen with inclusive criteria using meta‐analysis. The study group was focused on the participants with both diabetic and hypertensive contributors. The results show there is a high prevalence of CKD among diabetes, and the rates for hypertensive patients were consistently high regardless of the country and/or their socio‐economic status. CKD was significantly associated with older age; prevention of Diabetes mellitus and hypertension is mandatory in programs to reduce or eliminate the prevalence of CKD. While stem cell therapy shows promise to decrease the incidence of kidney transplant rate or as a replacement for dialysis, it has not yet become the universally accepted treatment of choice.

Support or Funding Information

Support from University of Science Arts & Technology, Montserrat and the Einstein Medical Institute USA
Evaluation of CB1 antagonists as antidotes for synthetic cannabinoid toxicity in male and female mice
Authors: Raghav Jimit G. , Van Kralingen Thomas , Kumar Hritik , Makriyannis Alexandros , Desai Rajeev I. , Vemuri Kiran
Published: April 2020

While a plethora of recreational marijuana and cannabis derived products have been legalized across the United States, recent trends also indicate an increase in the number of emergency calls relating to the overdose or accidental exposure to Δ9‐tetrahydrocannabinol (THC) and/or synthetic psychoactive cannabinoids. There are currently no FDA approved medications for treating acute cannabinoid toxicity. To address this public health issue, here we provide key data towards the discovery and development of an effective antidote for treating acute toxicity symptoms that may be associated with cannabis overdose, and by ingestion of certain cannabimimetics such as the potent CB1 receptor agonist JWH‐018. Previously, we established proof‐of‐concept and presented preliminary data showing that the novel CB1 antagonist AM11503 (10 mg/kg, i.v.) effectively reversed the hypothermic effects of an acute dose of JWH‐018 (6 mg/kg, i.p.), effects that were similar to the known CB1 antagonist rimonabant (10 mg/kg, i.v.). Herein, we extend this line of research in male and female CD‐1 mice by: a) examining whether AM11503 induces rimonabant‐like effects on observable behavior; b) assessing the ability of AM11503 to prevent JWH‐018‐mediated tremors and convulsions; and c) comparing the ability of AM11503 and rimonabant to precipitate somatic signs of withdrawal in JWH‐018‐dependent mice. Results show that rimonabant (3.2–32 mg/kg, i.v.) produced a dose‐related increase in scratching and grooming in both male and female mice compared to controls, whereas AM11503 (3.2–32 mg/kg, i.v.) did not alter stereotypic behavior. In interaction studies, administration of 10 mg/kg (i.v.) AM11503 fully blocked tremors and convulsions induced by “suprapharmacological” doses of JWH‐018 (18 mg/kg, i.p.). Finally, rimonabant (10 mg/kg, i.v.) induced significant somatic signs of withdrawal, i.e., head twitches and paw tremors, in male and female mice that were repeatedly exposed to JWH‐018 (3 mg/kg, i.p. once per day for 6 days) compared to vehicle‐treated subjects. In contrast, AM11503 (10 mg/kg, i.v.) did not induce somatic signs of withdrawal in JWH‐018‐dependent mice and effects were similar to vehicle‐treated mice. In conjunction, the inability of AM11503 to produce rimonabant‐like stereotypic behaviors and precipitate somatic signs of withdrawal yet prevent CB1 agonist‐induced acute toxicity allows for the identification and development of safer CB1 antagonist‐based therapies for nullifying synthetic cannabinoid toxicity.

Support or Funding Information

Funding Information: Work supported by NIDA/NIH grants R21DA045882 and R41DA044048