bluuebunny/crossref_metadata_2025 huggingface dataset:
ROLE OF DNA METHYLAION IN THE PROMOTER REGION OF CLOCK GENES IN THE REGULATION OF LIVER DISEASE
Authors: Aljobaily Nouf , Han Yuyan
Published: April 2020

Background and aim

Obesity is becoming a major global health concern, as it is associated with many diseases, such as Non‐Alcoholic Fatty Liver Disease (NAFLD). NAFLD is associated with diabetes, metabolic syndrome and, and is known to be multifactorial. Meaning that it is affected by multiple external and environmental factors. The progression of NAFLD to Non‐alcoholic Steatohepatitis (NASH), liver carcinoma and further to liver failure, makes it very crucial that we pay attention to the disease at its earlier stages. Further, NAFLD is affected by the circadian rhythm, which is a 24‐hour cycle that controls biological activities of all cells. In the liver, this rhythmic cycle is called liver clock and is very sensitive to nutritional intake, physical activity and sleeping patterns, and it is controlled by Clock genes. The expression of clock genes can be controlled by DNA methylation, which is a modification that acts as a switch to turn genes on and off. Therefore, this study aimed to study the methylation status of clock genes and clock‐controlled genes at the promoter region.

Methods

C57BL/6 mice were treated with high‐fat diet (HFD) or control (CTR) for 8 wk. Mouse livers were harvested for further analysis. Liver function was evaluated by serum chemistry, fibrosis was measured by IHC and PCR and lipid deposition were evaluated by Oil red O staining. Genomic DNA and total RNA were obtained for measuring the DNA methylation of core clock genes and RNA expression as described above. Methylation‐Specific PCR (MSP) and Sanger sequencing were used to evaluate the change in the gene expression of genes of interest; CLOCK, BMAL‐1 and PPAR‐a in a high‐fat diet group and control‐diet group.

Results

We found increased steatosis and hepatic fibrosis in mice fed a high‐fat diet compared to control. Expression of fibrotic markers (a‐SMA, FN1 and collagen I) was upregulated in mice fed high‐fat diet compared to control. Differential DNA methylation status in promoter regions was observed in different core clock genes in high‐fat‐fed mice.

Conclusion

DNA methylation of core clock genes and clock‐controlled genes may lead to dysregulation of the circadian rhythm during consumption of a high fat diet, thus leading to increased steatosis and liver fibrosis.
Multiplex analysis in a Drosophila gene‐environment model identifies interactions among LRRK2, rotenone and α‐synuclein
Authors: Sarkar Souvarish , Maor Gali , Feany Mel B.
Published: April 2020

Epidemiological studies strongly support a role for environmental factors, particularly pesticide exposure, in the pathogenesis of Parkinson’s disease (PD). Genetic risk factors, including penetrant single gene mutations and risk factors identified from genome‐wide associated studies (GWAS) also contribute to PD risk and progression, but available model systems are limited in their ability to interrogate gene‐environment crosstalk in vivo and at scale. Here we use Drosophila as a model system to understand complex gene‐environment interactions in PD. We developed a multiplex model in Drosophila in which we knocked down GWAS candidate genes in neurons in a robust new α‐synucleinopathy model and exposed the flies to environmental neurotoxicants. We identified an interaction between LRRK2, rotenone and α‐synuclein. Expression of the disease‐causing Lrrk‐G2019S mutant in the presence of rotenone and alpha‐synuclein induced behavioral deficits and mitochondrial dysfunction. Further, super resolution microscopy analysis revealed that the interaction of LRRK2, α‐synuclein and rotenone leads to hyperstabilization of actin cytoskeleton. We have previously shown that LRRK2 has actin severing activity and previous studies have implicated the GTPase domain in regulating actin severing. We next expressed a GTPase domain mutant, Lrrk‐Q1003H, designed to mimic the human protective mutant LRRK2‐R1398H. Interestingly, expression of the LRRK2 protective mutant attenuated behavioral deficits mediated by LRRK2‐rotenone‐synuclein interactions. Further, expression of the protective mutant also attenuated the actin stabilization and mitochondrial deficits. Together, using our novel multiplex model in Drosophila we have identified an interaction between LRRK2, α‐synuclein, and rotenone which is modulated by actin stabilization and mitochondrial dysfunction. Our findings have implication towards development of a personalized approach for drug discovery and lead identification.
Maternal Immune Activation Alters Placental and Fetal Brain Amino Acid Transport in Rats
Authors: McColl Eliza R. , Piquette-Miller Micheline
Published: April 2020

Problem

Maternal immune activation (MIA) during pregnancy is an environmental risk factor for neurodevelopmental disorders such as autism and schizophrenia. However, the exact mechanism through which this occurs is unknown. Due to the important roles that amino acids play in neurodevelopment, altered transport of amino acids across the placenta and throughout the developing fetal brain could play a role in neurodevelopmental disorders. Our objectives were to determine whether virally mediated MIA alters amino acid transporter expression in the placenta and fetal brain, and to explore the underlying mechanism(s) associated with these changes.

Methods

Pregnant rats received poly(I:C) on gestational day 14 and placental and fetal tissues were collected 6, 24, and 48 hours later. Amino acid transporter expression in the placenta and fetal brain was measured using qPCR and immunoblotting. Free amino acid concentrations in the fetal brain were quantified using HPLC. To explore a possible mechanism, the expression and phosphorylation of components of the placental mTOR and AMPK signaling pathways were investigated using immunoblotting.

Results

Poly(I:C) induced the mRNA expression of multiple amino acid transporters in the placenta and fetal brain over all three timepoints. Conversely, poly(I:C) significantly decreased protein expression of ASCT1 and EAAT2 in the placenta, and SNAT5, EAAT1, and GLYT1 in the fetal brain. Changes in transporter expression were accompanied by functional changes in the concentrations of numerous amino acids in the fetal brain. We observed activation of AMPK and inhibition of mTOR signaling in the placentas of poly(I:C)‐treated dams.

Conclusions

MIA induced by poly(I:C) significantly alters placental and fetal brain amino acid transporter expression as well as amino acid concentrations in the fetal brain. Due to the importance of amino acids in neurodevelopment, changes in amino acid transporters could contribute to the development of a neurodevelopmental disorder. AMPK and mTOR have been previously shown to regulate amino acid transporter expression. Since poly(I:C) altered the activation of placental and AMPK and mTOR, these could provide novel treatment targets to alleviate MIA‐induced changes in amino acid transport and investigate the implications of these changes in neurodevelopment.

Support or Funding Information

This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) awarded to Dr. Piquette‐Miller [MOP 13346]. Eliza McColl was a recipient of a CIHR Canadian Graduate Scholarship (Master’s) and an Ontario Graduate Scholarship, and is currently funded by a CIHR Banting and Best Canadian Graduate Scholarship (Doctoral) [GSD‐164238].
Recapitulating GSK3β and Tau interaction in brain injury dynamic model
Authors: Cheng Pei-Wen , Lin Yu-Te , Wu Yi-Chung , Wong Tzyy-Yue
Published: April 2020

Introduction

Living systems experience various form of mechanical stimulations in daily life. Some mechanical stimulation are required for cell survival, others are capable of causing injuries. In vivo, the neuronal system is linked closely to the muscular system. As a result, we respond to pain immediately after physical injury. Proliferating neuron cells can be used to study the effect of mechanical stimulation on neurons. However, cell models developed from neurons have been lacking due to the fact that neuron cells do not or are difficult to proliferate by in vitro culture. In this study, we investigated the effect of stretching on the SH‐SY5Y cells, neuroblastoma cells that proliferate in vitro. The cells retain the dopamine‐β‐hydroxylase activity; thus, it is suitable for neuron mechanistic study.

Method

The culture conditions contain non‐stretched (control) and stretched groups. SH‐SY5Y cells were cultured on stretchable membranes, subjected to 6, 24h of cyclic stretching, 25% elongation at 1Hz.

Results

After 6 and 24h stretching, cells showed decreased red fluorescence for JC‐1 probe which stains the mitochondrial membrane, indicating that cells experienced oxidative stress. Results show that stretching increased b‐amyloid precursor, total Tau protein by western blot. Co‐immunoprecipitation showed that Tau formed complex with GSK3b after 6h stretching.

The observations of change in mitochondrial potential, and formation of Tau complex imply that the neuron cells were under a stress. The stress may be a kind of injury suffered from physical stimulation.

Support or Funding Information

108‐2320‐B‐075B‐002 ‐
Filament Formation Induces a Shape Change and Activation of the Nuclease SgrAI
Authors: Horton Nancy C.
Published: April 2020

SgrAI is allosteric and is both activated as well as undergoes a change in substrate specificity (cleaving a secondary class of DNA sequences) upon binding to its primary recognition sequence in DNA. We have investigated the allosteric activation of SgrAI and this unusual expansion of DNA sequence specificity using structural, biophysical, and enzyme kinetic methods.
1–
2

A variety of biophysical techniques (native gel electrophoresis, analytical ultracentrifugation, ion mobility native mass spectrometry, x‐ray crystallography and electron microscopy) to investigate complexes formed by SgrAI. Single turnover DNA cleavage assays were used to investigate the reaction mechanism of SgrAI, and global modeling to create a computation model of the full reaction pathway including rate constants for each important step. The biophysical characterizations revealed that SgrAI forms run‐on oligomers or filaments when activated, and that these filaments are induced when SgrAI binds to its primary recognition sequence. SgrAI binds tightly to secondary site sequences, which differ from primary by a single base pair, but fails to cleave them and does not induce filament formation. However, SgrAI bound to the secondary site sequences will join filaments formed from SgrAI bound to primary site sequences. Protein‐protein and protein‐DNA contacts between SgrAI/DNA complexes in a filament stabilize a different conformation of SgrAI, hypothesized to be activated due to a reorganization of critical residues in the active site. DNA cleavage kinetics reveal activation of DNA cleavage activity by SgrAI of 200–1000 fold for primary and secondary site sequences upon filament formation. Full global kinetic analysis reveals a slow second order association step of SgrAI addition to a filament, followed by rapid DNA cleavage, then dissociation of SgrAI from the filament, and finally rapid product (i.e. cleaved DNA) release. The slow association step controls SgrAI activity to form filaments preferentially on DNA contiguous with primary site sequences, and incorporation of secondary site sequences into the filament increases the number of DNA cleavages on the contiguous DNA. Hence, this system allows for rapid cleavage of invading phage DNA (with unprotected primary sites).

The SgrAI system uses formation of a filamentous form to stabilize an activated conformation. DNA sequence and slow second order association kinetics control where and when this filament is formed in a way that is tuned for optimal performance of its biological role.

Support or Funding Information

This work was supported by the National Science Foundation under Grant No. MCB‐1410355 (to N.H.).
Connecting the microbiome to host metabolites: understanding how the microbiome controls immune activity in birds
Authors: Koci Matthew , Ballou Anne , Wei Xubiao , Zhang Lulu , Liew Zie Qing , Ali Rizwana
Published: April 2020

The gastrointestinal (GI) microbiome plays an important role in the development and function not only of the GI, but also the immune system. Products such as probiotics represent promising treatments for improving animal health through the GI, but their modes of action are largely still unknown. Previous research in our laboratory has demonstrated supplementation with a lactic acid bacteria probiotic product alters host energy partitioning in immune tissues, including increased ATP production and consumption in circulating leukocytes; and was associated with a more rapid antibody response to antigen. To better understand the communication between probiotics and the immune system our laboratory has focused on characterizing how supplementation affects the microbiome and host systems. We hypothesized that the changes previously observed in immune tissue activity and energy metabolism were regulated by a probiotic‐stimulated factor in the serum. We examined the ability of serum isolated from probiotic‐fed animals to augment ATP production in vitro. Chicken lymphocyte cell lines were cultured for 4 days in media supplemented with serum from treated and control birds. Cells cultured in serum from probiotic‐fed birds had higher levels of ATP (P < 0.05) compared to controls. Transcriptomic analysis of these cells suggest an increase in genes associated with cell survival and differentiation, and signaling via TGF‐β, IFN‐γ, IL‐7, and IL‐1β. Analysis of the metabolites in serum and digesta from these animals identified several metabolite changes correlate with the change in ATP levels in lymphocytes and putative changes in pro‐ and anti‐inflammatory cytokine production.
Glycolysis enzymes coalesce in G bodies in hypoxia nucleated by RNA
Authors: Fuller Gregory , Freeberg Mallory , Han Ting , Kim John
Published: April 2020

Cells deprived of oxygen rely on glycolysis alone for energy production, generating far less energy per molecule of glucose than aerobic respiration. While modulating levels of glycolysis enzymes and their activity can increase the rate of glycolysis, spatial reorganization of pathway components may also contribute to enhancing rates of glycolysis. We have demonstrated that in S. cerevisiae, glycolysis enzymes, including the rate limiting phosphofructokinase (PFK), coalesce into single non‐membrane‐bound cytoplasmic foci called “glycolytic (G) bodies
1” in hypoxia. Cells lacking the AMP activated protein kinase, SNF1, fail to form single foci and are enriched for upstream glycolysis metabolites, while being depleted for the downstream metabolite, acetyl CoA. Furthermore, cells preconditioned in hypoxia (thereby forming G bodies) deplete glucose from media at a faster rate than those cultured in normoxia (lacking G bodies). Thus, the presence of G bodies is correlated with greater rates of glycolysis. However, we know very little about how G bodies form. Many glycolysis enzymes moonlight as RNA binding proteins despite lacking known RNA binding domains, and proteomic analysis of purified G bodies revealed an enrichment of RNA binding proteins, suggesting that G bodies are ribonucleoprotein (RNP) granules. Tethering PFK to nonspecific endoribonucleases to degrade RNA at sites of G body formation resulted in complete loss of G body formation, indicating that RNA nucleates nascent G bodies. Induction of PFK‐RNase in cells with existing G bodies leads to fragmentation into multiple foci, indicating that RNA is required to maintain G body integrity. Consistent with RNA nucleating G body formation, RNAs bound to glycolysis enzymes in normoxic conditions identified by PAR‐CLIP strongly overlapped with RNAs that copurify with G bodies in hypoxia. Many RNP granules form via phase separation of protein and RNA. Although G bodies are distinct from stress granules, G bodies exhibit similar physical properties to stress granules, which are thought to form via phase separation. Proteomic analysis revealed similar protein components as stress granules including HSP70 chaperones. In addition, we have shown that G bodies in mating yeast cells have the ability to fuse and do so during their biogenesis. Furthermore, FRAP revealed that G body components exchange with the cytoplasm. G body fusion, however, takes multiple minutes to occur and FRAP recovery rates occur with half times of greater than 10 minutes, indicating that G bodies behave more akin to gels than a liquid‐liquid phase condensate. Taken together, we propose that G bodies are novel RNP granules that function to enhance rates of glycolysis in response to energy limiting hypoxic stress. Future studies will clarify the mechanisms of G body function and further test their physiological roles in cells.

Support or Funding Information

This work was funded by NIH grant (GM129301‐01).
CD248 plays important role in profibrotic phenotype switching of macrophages and tissue fibrosis
Authors: Pai Chen-Hsueh P. , Lin Shu-Rung , Liu Chia-Hao , Pan Szu-Yu , Hsu Hao , Chen Yi-Ting , Tsai Yu-Fei , Yu I-Shing , Lin Shuei-Liong , Lin Shu-Wha
Published: April 2020

In human chronic kidney disease, upregulated CD248 in myofibroblasts is linked to poor renal survival. Although the role of macrophages and myofibroblasts in organ fibrosis have been extensively studied, how macrophages and myofibroblasts crosstalk to promote organ fibrosis is not clear. In this study we showed that Cd248 was upregulated in myofibroblasts in murine models of renal and peritoneal fibrosis. Cd248 knockout (Cd248−/−) could attenuate both renal and peritoneal fibrosis. By parabiosis of both the GFP reporter mice and Cd248−/− mice, we showed that decrease of fibrosis was related to a decrease in macrophage infiltration. Moreover, decreases of chemokine (C‐C motif) ligand 17 and Ccl22 was also found in Cd248−/− or galectin‐3‐deficient macrophages demonstrated that CD248 and galectin‐3 played a similar role in profibrotic phenotype switching of macrophages in injured organs. We also showed that CD248 interacted specifically with macrophage galectin‐3 and promoted myofibroblasts collagen synthesis. Mice with DNA vaccination targeting CD248 showed decreased fibrosis. We propose a novel interaction between CD248 on myofibroblasts and galectin‐3 on macrophages is a key step in mediating kidney fibrosis.

Support or Funding Information

This study is supported by Taiwan MOST (108‐2319‐B‐002‐002 and 108‐2320‐B‐002‐051‐MY3), National Core Facility for Biopharmaceuticals, and the Mrs. Hsiu‐Chin Lee Kidney Research Foundation.
Is Hyperoxic Hyperventilation Caused by Reduced Carbon Dioxide Washout or Disturbed Brain Redox Homeostasis?
Authors: Fernandes Igor A. , Mattos João D. , Monique Campos O. , Rocha Marcos P. , Mansur Daniel E. , Rocha Helena M. , Garcia Vinícius P. , Alvares Thiago S. , Secher Niels H. , Nóbrega Antonio C. L.
Published: April 2020

Under oxygen‐enriched conditions, humans present a biphasic ventilatory response that is initially characterized by reduced ventilation (V̇E) as hyperoxia (HX) inhibits the peripheral chemoreceptors. This short‐lived response precedes a paradoxical increase in V̇E that has been termed hyperoxic hyperventilation. Proposed but untested mechanisms for this paradoxical response include central chemoreceptor stimulation via either an increased cerebral partial pressure of carbon dioxide (CO2), which would result from reductions in cerebral blood flow (CBF) and brain CO2–[H+] washout, or (2) an HX‐induced exaggerated production of reactive oxygen species (ROS). To determine the mechanism behind the paradoxical increase in V̇E during hyperoxemia, we submitted 8 healthy young men (24±1 yrs) to 10‐min trials of poikilocapnic HX under (1) saline or (2) ascorbic acid (AA) infusion. V̇E, arterial (a) and internal right jugular venous (Jv) PO2, PCO2, pH, oxidant (8‐isoprostane), and antioxidant (AA) markers, as well as volumetric CBF (Doppler ultrasound) responses, were quantified at the last minute of each trial. Under saline infusion, HX evoked an increase in PaO2 followed by a hyperventilatory response (+3.0 ± 1.2 L/min, p = 0.003) and arterial hypocapnia (−2.4 ± 0.9 mmHg, p = 0.03). HX also evoked a small but significant reduction in CBF (−219.6 ± 36.8 mL/min, p = 0.001) that was followed by unchanged PjvCO2 (−0.1 ± 0.7 mmHg, p = 0.619) and pH (0.001 ± 0.005, p = 0.957). HX under saline infusion also provoked an increase in arterial 8‐isoprostane (18.6 ± 7.5 pg/mL, p = 0.04). On the other hand, intravenous AA augmented arterial antioxidant markers (+584.1 ± 64.6, p = 0.0001 vs. saline) and blunted not only the increase in arterial 8‐isoprostane (−10.7 ± 9.5 pg/mL, p = 0.204) but also the HX‐induced hyperventilation (+0.87 ± 0.64 L/min, p = 0.232) and the arterial hypocapnia (−1.7 ± 0.5 mmHg, p = 0.04 vs. saline) observed during infusion of saline. HX under AA infusion also blunted CBF hypoperfusion (−41.8 ± 26.3 mL/min, p = 0.001 vs. saline) that, in turn, was followed by increases in PjvO2 (+5.3 ± 0.7 mmHg, p = 0.006 vs. saline), PjvCO2 (+1.1 ± 0.50 mmHg, p = 0.03 vs. saline) and a reduction in pH (−0.012 ± 0.004, p = 0.01) that may be explained by the Haldane effect. These findings suggest that hyperoxic hyperventilation is not a consequence of an increased cerebral CO2 partial pressure and a reduced brain CO2–[H+] washout but mediated by central chemoreceptor stimulation likely evoked by excessive production of ROS.

Support or Funding Information

Support: FaperRJ, CNPq, CAPES
Mapping Purinosome Protein‐Protein Interactions Using In Vivo Photocrosslinking
Authors: He Jingxuan , Zou Ling-Nan , Anand Ganesh S. , Benkovic Stephen J.
Published: April 2020

Efficient de novo purine biosynthesis in higher eukaryotes is hypothesized to depend on the existence of a multienzyme complex — the purinosome. However, there is as yet no compelling identification of specific protein‐protein interactions between purine biosynthetic enzymes. Similarly, functionally important interactions between purine biosynthetic enzymes and other proteins have yet to be elucidated. Inspired by the highly symmetric oligomeric structure of PAICS, a de novo purine biosynthetic enzyme, we identified several putative interaction surfaces. Using genetically encoded unnatural amino acid incorporation, we inserted photocrosslinking amino acid analogs into these surfaces at specific residues. Upon UV irradiation, we were able to detect multiple crosslinked products between PAICS and other proteins, formed in their native context inside living cells. Distinct sites of photocrosslinker incorporation yielded distinct products; this suggests they are the result of specific protein‐protein interactions. Using mass spectrometry, we are in the process of identifying PAICS’s crosslinked partners, as well as their interaction interface with PAICS. We will also extend this approach to study other pathway enzymes as we seek to identify functionally important protein‐protein interactions in de novo purine biosynthesis.

Support or Funding Information

The National Institutes of Health (GM024129 to S.J.B.)
Influence of Oxidative stress and NF‐κβ/CRP/Bcl‐2 Signaling on Gentamicin Induced Renal Toxicology and the ameliorative effect of chloroform extract of Abrus precatorius in male Wistar rats
Authors: Falayi Olufunke Olubunmi , Ogunpolu Blessing Seun , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Yakubu Momoh Audu , Adedapo Adeolu Alex
Published: April 2020

The kidneys play an important role in attenuating the toxicity of various drugs and chemicals. Gentamicin, a widely used effective aminoglycoside is one of such drugs, however, dose and time‐dependent nephrotoxicity limits its clinical use. This study evaluates the influence of oxidative stress, and inflammatory transcription factors in the pathogenesis of Gentamicin induced‐renal damage and the ameliorative potential of chloroform stem extract of Abrus precatorius. Twenty‐four male Wistar rats were divided into five equal groups. Group I was the negative control group while II was the positive control group which received gentamicin 100 mg/kg intra‐peritoneally daily for 6 days. Groups III and IV were pretreated with 100 and 200 mg/kg extract, respectively, for the first 3 days and then concurrently with gentamicin 100 mg/kg for 6 days. Blood samples, kidneys and kidney homogenates were collected for haematological, biochemical, histopathological and immunohistochemical analyses. Results revealed no significant hematological changes across the groups, Gentamicin caused significant increases in serum creatinine, urea, xanthine oxidase, nitric oxide, and myeloperoxidase levels. Gentamicin toxicity also resulted in increases in levels of renal advanced oxidative protein products, protein carbonyl, hydrogen peroxide, malondialdehyde levels. Furthermore, depletion in the activity of superoxide dismutase, glutathione S‐transferase, and contents of reduced glutathione, protein thiol, and non‐protein thiol. Gentamicin also caused focal areas of inflammation, fatty degeneration, congestion of vessels, tubular necrosis and glomerular atrophy in the kidney tissue. Levels of expressions of inflammatory mediators such as CRP and NF‐κβ levels were increased, alongside a decrease in Bcl‐2, an anti‐apoptotic protein. Gentamicin induces renal damage through the generation of oxidative stress, up‐regulation of pro‐inflammatory cytokines and down regulation of anti‐apoptotic protein. These were ameliorated with chloroform stem extract of Abrus precatorius.
Endothelial Cell Bulge Occludes the Vasa Vasorum in the Cerebellar Vascular Hyalinosis Lesions
Authors: Shults Nataliia V. , Marcocci Lucia , Rybka Vladyslava , Suzuki Yuichiro , Gychka Sergiy G. , Gavrish Alexander S.
Published: April 2020

Background

The remodeling of the cerebral vascular wall contributes to the occurrence of ischemic stroke in systemic hypertension patients, representing a leading cause of death in the U.S. and worldwide. Cerebral vascular remodeling in systemic hypertension is characterized by progressive degradation of structural components of each layer of the vessel wall with the subsequent transformation to hyaline material leading to the thickening vascular wall and lumen narrowing. The vasa vasorum as a network of small blood vessels supplying the blood to the vascular wall tissues contributes to the damage vascular structure and results in the formation of hyalinosis. The occlusion of vasa vasorum contributes to development of cerebral vascular hyalinization in systemic hypertension. However, mechanisms of occlusion of vasa vasorum that contribute to the brain vascular remodeling events have not been well defined. Lack of such knowledge interferes with the development of therapeutic strategies to reduce the occurrence of ischemic stroke.

Methods & Results

Light microscopy and transmission electron microscopy (TEM) of hyalinized cerebral vessels from human patients who suffered from systemic hypertension and died by ischemic stroke visualized the occlusion of the vasa vasorum due to the formation of the “bulge” structure that protrude from the endothelial cells. Treatment of microvascular endothelial cell with adrenaline (a major biogenic amine that are increased in the blood due to disbalance of neurohumoral homeostasis in systemic hypertension) resulted in a cytoskeletal rearrangement that contributes to the formation of the bulge‐like structure in the vasa vasorum of the brain vessels with hyalinosis lesion. The adrenaline activated clathrin‐mediated endocytosis of endothelial cells that in turn elicited SNX9/N‐WASP‐dependent disruption of cytoskeleton. siRNA knockdown of SNX9 inhibited endocytosis and prevented the adrenaline‐induced bulge formation in human microvascular endothelial cells. TEM visualized the adrenaline‐induced an increase endocytosis of the endothelial cells. Immunofluorescence staining showed the actin filament structural reorganization and TEM confirmed myofilament damage within endothelial cells. Adrenaline treatment induced post‐translational modifications of N‐WASp as a regulator of actin polymerization.

Conclusions

This is the first demonstration that adrenaline contributes to the occlusion of vasa vasorum in the hyalinized cerebral vascular wall that will allow to the development new strategies that can be used to prevent ischemic stroke.

Support or Funding Information

Supported by NIH
Capsaicin changes the pattern of brain rhythms in sleeping rat
Authors: Tian Yuhua , Du Guangxu , Jin Jingyu , Yang Zhihong , Hu Fang , Lu Wei , Wang KeWei , Zheng Jie
Published: April 2020

The heat and capsaicin sensor TRPV1 ion channels were originally discovered from sensory neurons in dorsal root ganglia, and later found in many other tissues and organs. However, whether TRPV1 channels are present in brain regions other than hypothalamus has been a subject of debate. Here we addressed this issue with an unbiased functional test, by recording electroencephalogram (EEG) to examine whether capsaicin injection directly into the rat lateral ventricle could alter brain electrical activity. We observed that EEG during the sleep stage could be significantly perturbed by capsaicin, whereas EEG during the awake stage did not show a detectable change. Our results are consistent with TRPV1 expression in selective brain regions whose activities are dominative during the sleep stage.

Support or Funding Information

This work is supported by funding from the Qingdao Postdoctoral Research Project to YHT.
The Howl of Rancho La Brea: the functional morphology of Pleistocene canid hyoids
Authors: Flores Mary Deanna , Eldridge Emma , Elminowski Erin , Dickinson Edwin , Hartstone-Rose Adam
Published: April 2020

The Rancho La Brea (RLB) fossils housed at the La Brea Tar Pits and Museum in Los Angeles, California represent one of the richest collections of carnivoran fossils in the world. The collection is particularly well known for exceptional preservation, including that of of rare and understudied bones. Among these, the RLB collection houses hyoids from several extinct species, including relatively large samples of specimens from the dire wolf (Canis dirus) and the coyote (Canis latrans). In this study, we compare fossilized hyoid elements from these two fossil samples to modern specimens: the gray wolf (C. lupus), red wolf (C. rufus), and modern coyote (C. latrans). Seven measurements were taken on each long bone of the hyoid apparatus and eight measurements of the u‐shaped basihyoid. Principal component analysis was performed to determine statistical differences between species and the drivers of those morphological differences. For most of the bones, the majority of the variation was driven by size. Red wolves, modern coyotes, and RLB coyotes could not be differentiated from each other based on any of the hyoid elements, but dire wolves could be clearly differentiated from all other canids for all elements. The hyoid apparatus of C. dirus is larger and more robust than that of C. lupus, its closest modern analogue. As larger hyoids have been associated with a lower vocal frequency, this distinction would likely result in the vocalizations of C. dirus occurring at a lower frequency than those produced by C. lupus.

Support or Funding Information

North Carolina State University Office of Undergraduate Research Grant
Capsaicin enhances the pro‐apoptotic activity of Camptothecin in human small cell lung cancer
Authors: Dasgupta Piyali , Friedman Jamie R , Perry Haley E , Colclough Kate W , Stevenson Cathryn D , Hurley John D , Akers Austin T , Nolan Nick A , Brown Kathleen C , Chen Yi Charlie , Denning Krista L
Published: April 2020

Small cell lung cancer (SCLC) is characterized by early dissemination, aggressive clinical course and a dismal survival rate. The first line therapy for SCLC patients is cisplatin‐based combination chemotherapy along with radiation. Initially, cisplatin shows excellent results causing remission in 60–80% of the SCLC patients but within a year the tumor inevitably relapses and this relapsed tumor is resistant to cisplatin. A subset of SCLC tumors are unresponsive to cisplatin from the outset. These patients are said to have platinum‐refractory SCLC tumors. Patients with platinum resistant or refractory disease have very limited options, as the only standard chemotherapy with an FDA‐approved drug, camptothecin (and its derivations), has an objective response rate of approximately 3% and little or no survival benefit. Such sobering statistics define the arena where there is an urgent need for drugs which enhance the pro‐apoptotic activity of camptothecin in human SCLC. The present study tests the hypothesis that capsaicin can sensitize human SCLC cells to the apoptotic effects of camptothecin. Caspase‐3 activity assays reveal that the combination of camptothecin and capsaicin displayed greater apoptotic activity than the drugs used alone. We verified these results using a second apoptosis assay, namely the Cell Death ELISA and obtained similar results. Chou‐Talalay isobologram analysis showed the interaction between camptothecin and capsaicin is synergistic. Chicken chorioallantoic membrane (CAM) assays confirmed the combinatorial apoptotic effects of capsaicin and camptothecin in human SCLC. The co‐operative effects of capsaicin and camptothecin were also confirmed in human SCLCs xenografted on athymic mice. The results of our experiments pave the way to novel combination therapies for human small cell lung cancer.

Support or Funding Information

Support or Funding Information Funding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by a Center for Natural Products pilot grant from Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH).
Cigarette Smoke Upregulates Phospholipase A□‐Mediated Metabolic Pathway Expression in the Bladder: A Potential Promoter of Tumorigenesis
Authors: Kispert Shannon , Nix Jessica , Beggs Hadley , McHowat Jane
Published: April 2020

Cigarette smoking remains the leading cause of preventable death and disease in the United States. Despite known risks, 42.1 million Americans continue to smoke, making research into tobacco‐related pathologies relevant and necessary. Tobacco smoking remains the best‐established risk factor for bladder cancer, and is implicated in primary tumor growth and metastasis. In a recently published study, we exposed bladder cancer cells to cigarette smoke extract and observed a significant increase in platelet‐activating factor (PAF) accumulation and expression of the PAF‐receptor. PAF is a downstream metabolite of phospholipase A□ (PLA□)‐catalyzed membrane phospholipid hydrolysis that results in the release of lysophospholipids and arachidonic acid. Arachidonic acid and its metabolites have been shown to be involved in inflammation and tumor progression. Immunohistological analysis of bladder cancer biopsies from smokers indicated increased PAF and PAF‐receptor expression in the tumor region when compared to normal tissue. PAF accumulation suggests an upregulation of PLA2 activity and arachidonic acid release. Accordingly, we examined the immunohistological expression of enzymes and metabolites involved in eicosanoid generation, including cyclooxygenase‐2 (COX‐2), prostaglandin E‐synthase (PGES), prostaglandin E□ (PGE□), and 15‐prostaglandin hydrogenase (15‐PGDH) in the bladder wall of mice exposed to room air and cigarette smoke. After 6 months’ exposure to cigarette smoke (48 min/day, 5 days/week), we observed increased expression of metabolic enzymes (COX‐2, PGES) involved in the production of PGE2, and a corresponding decrease in 15‐PGDH (responsible for its degradation) in the bladder wall. Immunohistological analysis of the bladder wall revealed a 2‐fold increase in expression of PGE□. This was associated with a 25% increase in COX‐2 expression and a 40% decrease in 15‐PGDH, suggesting that PGE2 accumulation is a result of increased production and decreased degradation. Our studies show for the first‐time cigarette smoke‐induced dysregulation of a PLA□‐mediated metabolic pathway which could contribute to tumorigenesis and progression. These results may provide a potential therapeutic target for cigarette smoked‐induced bladder cancer and progression.
Circulating Irisin Level as an Early Biomarker of Decline of Metabolic Health
Authors: Romanelli Massimiliano Marco Corsi , Vianello Elena , Sitzia Clementina , Benedini Stefano , Gorini Silvia , Rampoldi Benedetta , Rigolini Roberta , Tacchini Lorenza , Ambrogi Federico , Dozio Elena
Published: April 2020

A decline in metabolic health may take place before observing an alteration in the levels of the traditional metabolic markers. New indicators of metabolic derangement are therefore compelling. Irisin is a myokine which exerts important metabolic functions: it increases energy expenditure, improves insulin resistance, reduces glucose level and advanced glycation end products (AGE)‐induced inflammation. The optimal level for irisin in human and its potential role as a metabolic biomarker have not been fully established yet.

To this aim, we quantified plasma irisin level in 106 healthy subjects (age 43 ± 11.72 years, 58 males/48 females) and we performed a cluster analysis to evaluate the association between irisin and subject characteristics [age, total cholesterol, LDL, HDL, tryglicerides, NEFA (non‐esterified free fatty acids), glucose, HbA1c (glycated hemoglobin), insulin, GA (glycated albumin), uric acid, creatinine, irisin, sRAGE (total soluble receptor for AGE), esRAGE (endogenously secreted receptor for AGE), cRAGE (membrane‐cleaved receptor for AGE) and cRAGE/esRAGE ratio]. ELISA tests were used for the quantification of parameters different from routine biochemical markers. Glycated albumin (GA) was meausured by the enzymatic method Lucica®GA‐L. The study was conducted in accordance with the Declaration of Helsinki.

Irisin median level was 7.89 ng/ml. Although the mean levels of the traditional metabolic blood markers were within the ranges of normality, subjects in cluster 1 displayed a metabolic profile shifted toward the highest reference values. In fact, we observed higher concentrations of total cholesterol, LDL, triglycerides, NEFA, glucose, HbA1c, insulin, uric acid and creatinine. The reduced levels of the anti‐AGE molecules sRAGE and esRAGE, the increased levels of cRAGE/esRAGE (a surrogate marker of inflammation) and GA (an AGE product), and a greater percentage of subjects with visceral obesity in cluster 1 confirmed an increased metabolic stress and a pro‐inflammatory profile in this group. Irisin concentration was higher in this group too.

The traditional metabolic blood tests seem to be not enough to early identify a metabolic decline. The increase of irisin level may reflect a metabolic derangement at the beginning of its development. The role of irisin as an early biomarker of decline in metabolic health seems an interest topic to be further explored.

Support or Funding Information

The study was supported by funding from Fondazione E. A. Fiera Internazionale di Milano to Università degli Studi di Milano and Ricerca Corrente funding from Italian Ministry of Health to IRCCS Policlinico San Donato.
The Metabolic Woes of the Alveolar Macrophage in HIV
Authors: Yeligar Samantha M. , Auld Sara C. , Staitieh Bashar S.
Published: April 2020

Objective

Despite anti‐retroviral therapy, people with human immunodeficiency virus (HIV) are still at high risk for respiratory infections and non‐infectious pulmonary diseases. Alveolar macrophages (AM), which act as a viral reservoir, are critical immune effectors in the lung that phagocytize and clear pathogens. We have previously shown that oxidative stress impairs AM phagocytic function. Therefore, we hypothesized that HIV causes AM immune dysfunction by increasing oxidative stress by (1) up‐regulating NADPH oxidases (Noxes), which are the primary sources of cellular oxidative stress in the AM, and (2) causing mitochondrial derangements and consequent increases in mitochondria‐derived oxidative stress.

Methods

AM were isolated from the bronchoalveolar lavage fluid of HIV transgenic rats that express the HIV proteins, trans‐activator of transcription and glycoprotein‐120, or littermate control rats. AM were also isolated from the bronchoalveolar lavage fluid of people with HIV or otherwise healthy control subjects. mRNA and protein levels of Noxes were assessed by qRT‐PCR and cytoimmunostaining, respectively. Mitochondrial oxygen consumption rate and bioenergetics were measured by Cell Mito Stress Test assay using an extracellular flux analyzer. AM phagocytosis was evaluated by pHrodo‐labelled S. aureus internalization.

Results

Compared with controls, AM from HIV transgenic rats and people with HIV exhibited: (a) increased mRNA levels of Nox1, Nox2, and Nox4; (b) increased protein expression of Nox1, Nox2, and Nox4; (c) decreased mitochondrial respiration and bioenergetics; and (d) decreased AM phagocytic function.

Conclusions

HIV increases cellular sources of oxidative stress via upregulation of Noxes and mitochondrial sources of oxidative stress via derangements in mitochondrial function, which may contribute to impaired AM phagocytosis. Therapeutic targeting of oxidative stress pathways could provide a novel approach to improving lung immune defense in people with HIV.

Support or Funding Information

Research Funding Sources: NIAAA R00AA021803 and R01AA026086 to SMY, NIAID K23AI134182 to SCA, and NIAAA K08AA024512 to BSS.
Differential inhibition of target gene expression by human microRNAs
Authors: Zeng Yan , Li Peng , Chen Yi , Juma Conslata , Yang Chengyong , Zhang Xiaoxiao
Published: April 2020

microRNA (miRNA) target genes are commonly computationally predicted, yet how dependable predictions are remains unclear, as most predicted target genes have not been rigorously tested experimentally. In addition, the degree to which a miRNA differentially inhibits the expression of its targets is underappreciated. We selected human miR‐1, miR‐122, and miR‐124 as representatives to investigate the reliability of miRNA target predictions and to examine how miRNAs suppress their target genes. We first constructed target gene reporter libraries for the miRNAs based on three prediction programs: TargetScan, miRanda, and PicTar, and used reporter assays in cell cultures to directly evaluate whether as well as how strongly a predicted target gene is repressed by its cognate miRNA. We found that all three programs have approximately 72–85% success rates in predicting genuine miRNA targets, and that miRNAs inhibit different targets to various degrees. We then performed statistical and correlation analyses to examine how well parameters offered by the online programs and features in target mRNAs might predict the degrees of repression by miRNAs. To show that endogenous miRNAs act through the same mechanism, we correlated our reporter assay results with publically available mRNA expression data in vivo. Overexpression of miR‐1, miR‐122, or miR‐124 further led to differential reduction of target mRNA levels in cell cultures. Our studies systematically investigated hundreds of miRNA target genes, shed light onto the performance of miRNA target gene prediction programs, and suggested a new mechanism by which differential target repression by miRNAs regulates gene expression in vivo.

Support or Funding Information

This work was supported in part by the National Natural Science Foundation of China [grant number 31570843].
ALGX‐XC20, a Novel Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist Attenuates Joint Pain and Articular Cartilage Damage in an Osteoarthritis Rat Model
Authors: Desclaux Scarlett , Sriwatananukulkit Orada , Buated Wannipat , Himakhun Wanwisa , Herz Jeffery M. , Hemstapat Ruedee
Published: April 2020

Currently, there are no approved disease‐modifying drugs for osteoarthritis (OA). OA patients depend upon analgesic drugs such as nonsteroidal anti‐inflammatory drugs (NSAIDs) and opioid analgesics for pain management. Despite existing therapies, pain relief in OA remains largely inadequate. Recently, the functional role for TRPA1 receptor‐channels which are expressed on nociceptor sensory nerves and other non‐neuronal cells within the joint has been identified, although studies of the role of TRPA1 in the pathogenesis of OA pain are limited. TRPA1 represents a promising target for the development of novel therapeutic agent for managing OA pain. This study investigated the effects of ALGX‐XC20, a highly selective and potent TRPA1 antagonist with a long in‐vivo half‐life on pain behaviors and cartilage damage in a rat model of monosodium iodoacetate (MIA)‐induced OA. The analgesic effect of a single dose (30 mg/kg; oral) of ALGX‐XC20 was evaluated for a period of 14 days post‐OA induction and compared to celecoxib (30 mg/kg; oral), a selective cyclooxygenase‐2 (COX‐2) inhibitor by using pain‐related behavior assessments, including hindlimb weight distribution and von Frey tests. The histological scoring of cartilage damage was also determined according to the Mankin grading system. The MIA‐induced OA rat group displayed a large reduction in the weight bearing distribution on the ipsilateral hindlimb (p<0.01–0.05) and hind paw mechanical withdrawal thresholds compared to saline control group, which was maintained for 14 days. The MIA‐evoked reduction in weight bearing distribution was significantly reversed (~50%) in the MIA‐induced group treated with ALGX‐XC20 compared to the MIA‐treated group for the 2‐week duration, while the antiallodynic effect of ALGX‐XC20 was less pronounced. In addition, histopathology studies showed that ALGX‐XC20 was also effective in reducing the severe cartilage damage in the MIA‐injected knee joint as shown by a significant improvement in the Mankin score in the ALGX‐XC20 treated group at 14 days (p<0.05). The magnitude of the effect on spontaneous weight bearing behavior due to ALGX‐XC20 treatment is closely comparable to the effects observed for the MIA‐celecoxib group and reported for TRPA1‐KO animals. The positive analgesic effects in this OA model due to pharmacological inhibition of TRPA1 by ALGX‐XC20 occur without any adverse behavioral effects. These in vivo data suggest that ALGX‐XC20 may be used as a novel therapeutic analgesic/anti‐inflammatory drug for the treatment of chronic arthritis, such as OA pain. This TRPA1 antagonist drug may also represent a new class of disease‐modifying drug by providing a chondroprotective effect on articular cartilage.
Cell Toxicity Studies of Tremetone Isolated From Rayless Goldenrod (Isocoma pluriflora)
Authors: Green Benedict , Lee Stephen , Davis Zane , Welch Kevin
Published: April 2020

This research compared the cytotoxic actions of the benzofuran ketone tremetone in B16 murine melanoma cells, TE‐671 human rhabdomyosarcoma cells, and SH‐SY5Y human neuroblastoma cells using a MTT assay, with and without exposure to rat liver microsomes (RLM). Tremetone is of interest because it is a putative toxin in white snakeroot and rayless goldenrod. Identifying the toxic principal(s) of white snakeroot is important because in addition to poisoning livestock by causing “trembles,” it also causes “milk sickness” in humans that drink the milk from dairy animals that have eaten these toxic plants. Tremetone was not cytotoxic in B16 cells. In TE‐671 and SH‐SY5Y cells, concentration‐dependent tremetone cytotoxicity occurred without microsomal activation. In SH‐SY5Y cells the best fit values were; tremetone IC50 = 490 μM, tremetone and RLM IC50 = 505 μM. In TE‐671 cells the best fit values were; tremetone IC50 = 2.5 μM, tremetone and RLM IC50 = 2.2 μM. These results suggest that in B16 cells, tremetone is not cytotoxic. The results from experiments with SH‐SY5Y and TE‐671 cells suggest that tremetone does not require microsomal activation to be cytotoxic in a concentration‐dependent manner. This study demonstrates that tremetone is cytotoxic and plays a role in the disease caused by white snakeroot and rayless goldenrod.

Support or Funding Information

This research was supported by USDA.