bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly the [2Fe‐2S] Cluster in the Cytochrome bc1 Complex from Saccharomyces cerevisiae
Authors: Briones Luis Alberto Sanchez , Pérez Alma Laura Díaz , Alcántar Lorena Martínez , García Jesús Campos
Published: April 2020

Objective

Participation of the Fe‐S‐IBG subsystem in the assembly of the [2Fe‐2S] cluster into the bc1 complex.

Methods

Mutants of S. cerevisiae: Δiba57, Δisa1, Δgrx5, and Δrip1 were grown in YPD medium at 30°C. Mitochondria were isolated by differential centrifugation according to Auchére et al. (2008). Respiratory complex and aconitase activities were evaluated according to Gomez et al. (2014). Mitochondria were treated with digitonin to evaluate the formation of respiratory supercomplexes by electrophoresis in Polyacrylamide Blue Native Gels (BN‐PAGE), and after 2‐dimensional SDS‐PAGE analysis according to Schagger et al. (2000). Detection of the Rip1p subunit was performed by Western blot using the anti‐Rip1p monoclonal antibody at 1:20,000 dilution and developed with anti‐mouse IgG HRP‐conjugate.

The iron‐sulfur clusters [Fe‐S] are inorganic cofactors contained in enzymes involved in multiple cellular processes. In eukaryotes, [Fe‐S] synthesis mainly occurs in the mitochondrial matrix by the biogenesis of Iron Sulfur Cluster system (ISC). This system involves two steps: the novo assembly of the [Fe–S] clusters on scaffold proteins, and the transference of the [Fe–S] cluster to target apo‐proteins. These steps involve the participation of several proteins, which perform specific reactions. The ISC subsystem Fe‐S‐IBG conformed by the Grx5p, Isa1p, Isa2p, and Iba57p proteins is essential for the synthesis of the [4Fe‐4S] clusters. Yeast mutants in the Fe‐S‐IBG subsystem showed deficiency in respiration and excessive ROS generation was found, similarly to the mutant Δrip1, which gene encodes for the Rieske subunit of cytochrome bc1. In this work, the activity of the cytochrome bc1 in mitochondria from the Δiba57,Δisa1, Δgrx5, and Δrip1 mutants strains were determined. Results showed that the cytochrome bc1 complex in mitochondria from the Δiba57,Δisa1, and Δrip1 mutants was dysfunctional; whereas in the Δgrx5, the cytochrome bc1 activity was partially decreased in comparison with the WT strain. BN‐PAGE analysis shows that the respiratory complex lost the capacity to form supercomplexes in mitochondria from the Δiba57 and Δisa1 mutants, Immunodetection assays showed that Rip1p was absent in the iba57Δ and Δisa1 mutants; whereas the Δgrx5 mutant shows an unaltered pattern profile of the supercomplexes bands in BN‐PAGE, without the loss of Rieske protein. In conclusion, results indicate that the Isa1p and Iba57p proteins are involved in the insertion of the [2Fe‐2S] cluster on the Rip1p apoprotein, suggesting that the Fe‐S‐IBG subsystem is not exclusive for the maturation/assembly of proteins with [4Fe‐4S] clusters, but also is involved in [2Fe‐2S] clusters assembly such as occurred in the Rieske subunit.
Characterization of skeletal muscle fiber types and electrocytes in the tail of the electric fish Apteronotus albifrons before and after regeneration following tail amputation.
Authors: Singh Anita Ler , Mills Kayley , Unguez Graciela
Published: April 2020

In most groups of electric fish, cells that make up the electric organ (EO), or electrocytes, derive from skeletal muscle cells that suppress many muscle properties. This muscle‐to‐electrocyte conversion is repeated during tail regeneration in adult fish after tail amputation. One of few exceptions to the adult muscle‐derived EO is the Apteronotus albifrons. In A. albifrons, the first electrocytes that form during the larval stage do derive from muscle. However, an EO that is solely composed of the terminal endings of electromotor neurons subsequently replaces this larval EO. We hypothesize that following tail amputation, regeneration of the adult electric organ includes the process of formation of the myogenically derived EO as it occurs during larval stage of development.

Adult fish were obtained commercially and housed individually in 15 to 20‐gallon aerated aquaria maintained at 25–28°C and fed three times weekly. Ten fish were anesthetized using 2‐phenoxy ethanol (1:1,500 in tank water), and the distal segment of the tail was transected at the caudal end of the anal fin, then returned to their tanks and monitored until they fully recovered from anesthesia. The cut control tail segments were mounted on cork, frozen in liquid nitrogen‐cooled isopentane, and stored at −80°C. These fish were re‐anesthetized 2 weeks after amputation and their regeneration blastema were removed and frozen as done with control tails. Longitudinal and cross‐sections (20μm‐thick) were mounted on glass slides and processed histologically for analysis of general cell type morphology. Immunolabeling studies used antibodies against markers known to detect different muscle fiber types, axon projections, myogenic stem cells, and nerve endings. These approaches will allow us to determine the muscle fiber type composition of the tail region that contains the adult EO in A. albifrons and the fiber type transition that occurs during regeneration. Our preliminary data shows that 1) type I and type II adult muscle fibers surround the EO with type II being more predominant in number, and 2) muscle proliferation is abundant along the entire periphery of the regeration blastema. Further immunolabeling studies are underway to complete testing our hypothesis.

Support or Funding Information

Funded in part by the NIH MARC Program Grant #GM07667‐41
Overexpression of catalase targeted to mitochondria improves neurovascular coupling responses in aged mice.
Authors: Csiszar Anna , Yabluchanskiy Andriy , Ungvari Anna , Ungvari Zoltan , Tarantini Stefano
Published: April 2020

Moment‐to‐moment adjustment of cerebral blood flow (CBF) to neuronal activity via the homeostatic mechanism known as neurovascular coupling (NVC) has an essential role in maintenance of normal brain function. In advanced age cerebromicrovascular endothelial dysfunction impairs NVC responses, which contribute to age‐related cognitive decline. Recently, we have shown that pharmacological treatments that attenuate mitochondrial production of reactive oxygen species (ROS) provide significant neurovascular protection, improving NVC responses in aged mice. Transgenic mice that overexpress human catalase localized to the mitochondria (mCAT) are protected from age‐related mitochondrial oxidative stress and exhibit a longevity phenotype associated with resistance to several age‐related pathologies. The present study was designed to test the hypothesis that mitochondria‐targeted overexpression of catalase also confers protection against age‐related impairment of NVC responses. To achieve this goal, NVC responses were assessed in aged (24 months old) mCAT mice and compared with those in age‐matched wild‐type mice and young control mice by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that mitochondrial overexpression of catalase resulted in improved NVC in aged mice due to preserved NO‐mediated (L‐NAME inhibitable) component of the response. Thus, our present and previous findings demonstrate that interventions that boost mitochondrial antioxidative defenses confer significant cerebromicrovascular protective effects, which preserve NVC responses in aged mice. Our findings provide additional proof‐of‐concept for the potential use of mitochondria‐targeted antioxidants as therapy for prevention of vascular cognitive impairment associated with aging.

Support or Funding Information

This work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE).
Two‐pore channel 2 (TPC2) Role in Melanoma
Authors: Hanbashi Ali , D'Amore Antonella , Parrington John
Published: April 2020

Two‐pore channels (TPC), a recently described class of NAADP‐ and PI(3,5)P2–sensitive calcium‐permeable cation channels in the endolysosomal system of cells have a crucial rule in cellular functions and have been linked to various type of diseases including cancer. Here, TPC2 protein stable silencing in melanoma cancer cell lines was developed using CRISPR/Cas9 genome editing technology. TPC2 knockout (KO) in B16 murine melanoma cancer cell was associated with higher viability and migration rates with higher melanin accumulation inside the cells compared to wild type controls. TPC2 KO CHL1 human melanoma cancer cells showed an increased level of proliferation with reduced migration capacity. Both KO cell lines had lower invasion rates and lacked capacity for angiogenesis compared to wild type controls. Proteomic analysis was used to identify novel targets which were affected by genetic modification of TPC2. Thus, the findings of the present study indicated that CRISPR/Cas9 model is an ideal and cost‐effective tool to study the role of TPC2 in melanoma cancer cells which can be further used as a target to understand the biological basis of this disease.

Support or Funding Information

Royal Embassy of Saudi Arabia Cultural Bureau
Chronic oral NaHCO3 treatment and high tubular flow promote insulin resistance in rats
Authors: Mannon Elinor , O'Connor Paul
Published: April 2020

Diabetes is the leading cause of chronic kidney disease (CKD), and worsening insulin resistance likely contributes to CKD progression. Several small clinical trials have indicated that sodium bicarbonate (NaHCO3) slows the decline in GFR in CKD. Chronic ingestion of alkali like NaHCO3 can lead to potassium (K+) wasting by the kidney. While K+ wasting has been linked to the dysregulation of insulin metabolism, no studies have examined the metabolic impacts of chronic NaHCO3. We hypothesized that chronic NaHCO3 would promote K+ wasting and worsen insulin resistance in rats. In order to test our hypothesis, 16 male, 12 wk old Sprague Dawley (SD) rats underwent four weeks of high‐fructose (60%) diet (HFruD) prior to a baseline insulin tolerance test (ITT). Treatment with 0.1M NaHCO3 (Bicarb; n=8) or 0.1M NaCl (Vehicle; n=8) was initiated in drinking water after the baseline ITT and continued along with fructose feeding for another 3 weeks. ITTs were performed again following 1 week and 3 weeks of either bicarb or vehicle drinking. A similar protocol was performed using 18 male, 12 wk old SD rats, with the exception that these animals were maintained on normal rat chow, and following a baseline ITT, were placed on either Bicarb (n=6), Vehicle (n=6), or Tap H2O (n=6). At the conclusion of the protocol, animals were sacrificed, and visceral and subcutaneous fat pads and the spleen and kidneys harvested. In HFruD rats, there were no significant differences in the magnitude of the fall in blood glucose (area under the curve (AUC)) in response to insulin (0.75U/kg) at baseline (Veh 4229 ± 1625 vs Bicarb 3624 ± 1093; unpaired t‐test, p=0.76) or following 1 week of treatment (Veh 7187 ± 1613 vs Bicarb 5151 ± 1121; unpaired t‐test, p=0.32). However, following 3 weeks of treatment, animals that received bicarb demonstrated a significant decrease in the response to insulin, when compared to vehicle treated animals (Veh 5170 ± 690.0 vs Bicarb 1902 ± 846.0; unpaired t‐test, p=0.001). In naïve animals, there were no significant differences in the response to insulin at baseline (Veh 2337 ± 396.2 vs Bicarb 3651 ± 690.7 vs Tap H2O 3349 ± 544.2; One‐way ANOVA, p=0.25) or following three weeks of treatment (Veh 1773 ± 559.5 vs Bicarb 1745 ± 338.3 vs Tap H2O 1320 ± 215.7; One‐way ANOVA, p=0.67). In conclusion, our data show that prolonged NaHCO3 ingestion can significantly impair the response to insulin. As this effect was only present in HFruD fed rats, we speculate that high tubular flow may act synergistically with NaHCO3 to increase K+ wasting or that underlying metabolic dysfunction may be required to observe impairments in insulin sensitivity. As both underlying metabolic dysfunction and high single tubular flow due to reduced nephron mass are present in many CKD patients, these findings could have significant implications for CKD patients who are currently supplemented with NaHCO3. Such patients may require more vigilant blood glucose monitoring.

Support or Funding Information

NIH PO1HL134604 and DK099548
RETHINKING ANATOMY IN A COMPRESSED TIME FRAME: CLINICAL SYMPTOMS, NOT CASE STUDIES, AS THE BASIS FOR INTRODUCTORY INSTRUCTION IN GROSS ANATOMY
Authors: Zill Sasha
Published: April 2020

The goal of this study was to develop effective approaches to train first year medical students in Gross Anatomy/Embryology in a compressed time frame. Pedagogical reorganization was based upon a core of clinical conditions taught in second‐year and Step One board review courses. These conditions were not presented as clinical problems (many students had limited prior training in medical terminology) but lectures and discussions focused upon clinical symptoms, allowing for a direct correlation of structure and function. Instruction in anatomy was extended to prepare for laboratory dissections. Overall methodology was multi‐modal, including 'flipped' and traditional lectures, guided study of prosections/radiographs and small group laboratory review sessions. Some content was reduced: knowledge of muscle actions and innervations was required, not origins and insertions. Performance in anatomy was specifically evaluated by computer‐based written examinations that included a set of questions (36) previously asked over a 7 year period (repetitively or with minor modification) and by regional practical exams. Mean scores in all areas were sustained or numerically improved, despite the compression of instruction duration. There were no significant differences in performance based upon question format (Basic science anatomy, Clinical Vignette) or instructional modality ('flipped' vs traditional lectures). Point biserial data indicated that students needing assistance in learning could still be identified. These results suggest that training in Gross Anatomy can be effectively maintained in a compressed duration by instruction in the context of symptomatology of clinical syndromes and conditions, a format that should also facilitate review for USMLE Step One Board examinations.

Support or Funding Information

WVCTSI grant U54GM104942
Estrogen Protects Against Olanzapine‐induced Hyperglycemia
Authors: Medak Kyle D. , Wright David C.
Published: April 2020

Olanzapine is a second‐generation antipsychotic (SGA) used frequently in the treatment of schizophrenia and a growing list of off‐label conditions. Though effective in reducing psychoses, acute olanzapine treatment causes rapid increases in blood glucose that are mediated by increases in liver glucose output, skeletal muscle insulin resistance, and beta cell dysfunction. We recently reported that female, compared to male, mice are protected against acute olanzapine‐induced hyperglycemia. To date the mechanism by which female sex confers protection against olanzapine‐induced excursions in blood glucose have not been identified. The purpose of this study was to determine if the protective effects of the female sex against acute olanzapine‐induced hyperglycemia are due, in part, to female sex hormones and if this is mediated by GLP1 signalling. Ovariectomized (OVX) C57BL/6J mice or sham‐operated female controls were treated with olanzapine (5 mg/ kg, IP) or vehicle and blood glucose was measured at baseline, 15, 30, 60, 90, and 120 minutes post‐treatment. These experiments were repeated in female OVX mice following 2‐days of estradiol (E2) treatment (80 ug/kg/day; I.P.) compared to OVX or SHAM mice treated with vehicle. OVX exacerbated the effects of olanzapine on blood glucose, while the add back of E2 rescued olanzapine‐induced impairments in glucose homeostasis in OVX mice. Serum GLP1 concentrations were increased in female compared to male mice and thus, we wanted to determine if GLP1 signaling could be involved in the protective effects of female sex. Female mice were co‐treated with olanzapine and the GLP1 antagonist Exendin 9–39 (25 nmol/kg BW). GLP1 antagonism in female mice exacerbated olanzapine‐induced hyperglycemia. Given the evidence of E2 and GLP1 conferring a protective effect against olanzapine induced hyperglycemia in female mice, we co‐treated male mice with the GLP1 receptor agonist liraglutide (0.4 mg/kg BW) and olanzapine (5 mg/kg, IP). GLP1 agonism protected male mice from olanzapine‐induced hyperglycemia. Taken together, we provide evidence that the protection conferred by female sex on the hyperglycemic effects of olanzapine is due, at least in part, to E2 and GLP1 and that the protective effects of female sex can be recapitulated in male mice treated with liraglutide. Future work will be needed to determine if the protection afforded by female sex hormones on olanzapine‐induced hyperglycemia are dependent on GLP1 and the importance of this proposed mechanism in humans to provide insight into new adjunct treatments to offset the metabolic side effects of SGA treatment.
Designing 3D Synthetic Matrices for Studying Interactions Between Bacterial Peptidoglycan and Macrophages in the Context of Immune Misrecognition Diseases
Authors: Wodzanowski Kimberly A. , Kloxin April M. , Grimes Catherine L.
Published: April 2020

The immune system is a complex network comprised of various cellular components that work together to protect the body from damage, with a key function of differentiating between pathogenic bacteria and the 39 trillion commensal bacteria that constitute our microbiome. Misrecognition of commensal bacteria has been linked to many inflammatory bowel disorders in humans yet remains not fully understood and difficult to study. Enabled by recent advances in molecular tools, fragments of the bacterial cell wall peptidoglycan have been identified that bind to pattern recognition receptors within macrophages, which activate macrophages to start a signaling cascade that turns on the immune response. Typically, these interactions are studied using two‐dimensional invasion assays on tissue culture plastic plates; however, the dimensionality and ‘stiffness’ of these traditional culture systems may not be representative of the cell‐microenvironment interactions that exist in vivo. In order to better simulate the native cellular environment, three‐dimensional (3D) cell culture systems can be used to control key biochemical and mechanical properties of the cell microenvironment. Here, we established an approach for the encapsulation and culture of human macrophages in 3D within well‐defined, biomimetic hydrogels decorated with integrin binding peptides that mimic the natural in vivo environment and linked with cell‐degradable sites for cell‐driven migration and remodeling to probe macrophage response to bacterial invasion. With this approach, we have shown invasion with bacteria whose glycans are remodeled and their engulfment through immunostaining and assessed activation of macrophages using RT‐qPCR and ELISA. These studies provide new insights into the interactions between bacteria and immune cells and this new method affords unique opportunities for examining how our immune system recognizes and misrecognizes bacteria toward a better understanding of the mechanisms of immune misrecognition diseases.

Support or Funding Information

NIH CBI Training Grant‐5T32GM008550NIH U01 Common Fund program‐U01CA221230‐01
Group A Streptococcus Prevents NOX‐2 Complex Formation to Limit Oxidative Burst in Macrophages
Authors: Mackey Kialani , Clark Mary , Lee Ryan C. , Yuen Isabelle , Okumura Cheryl Y.M.
Published: April 2020

Group A Streptococcus (GAS) is a Gram positive bacterial pathogen responsible for about 500,000 deaths a year, causing diseases ranging from superficial pharyngitis to acute rheumatic fever. Immune cells such as macrophages and neutrophils generate reactive oxygen species (ROS) to aid in bacterial destruction. Unlike other bacterial species, GAS lacks enzymes that can detoxify ROS. We thus sought to determine whether GAS can prevent ROS production altogether. We found that THP‐1 macrophages infected with GAS indeed produce less ROS as measured by DCF‐DA assays. We next sought to determine whether GAS affect the formation of the NOX‐2 enzyme complex that produces ROS. The NOX‐2 complex is made up of cytosolic and membrane bound subunits that come together when activated by the phagocytosis of a pathogen. Analysis of whole cell lysates by Western blot indicate decreased NOX‐2 complex formation in GAS‐infected cells compared with controls. Furthermore, we analyzed recruitment of the cytosolic components to the phagosome membrane by analyzing crude cell fractions. These results confirmed that GAS infection prevents NOX‐2 complex assembly. Interestingly, our data indicates that GAS can not only prevent the formation of the NOX‐2 complex, but can disassemble the complex in stimulated cells. Our findings suggest that one way GAS evades defenses of our immune system is by preventing the formation of toxic ROS. Inducing ROS production in GAS‐infected cells would therefore provide an alternative to antibiotic treatment for GAS infections.

Support or Funding Information

American Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center
Hormonal Regulation of Glycine Decarboxylase and its Impact on Cellular Physiology
Authors: Jog Ruta , Chen Guohua , Wang Jian , Leff Todd
Published: April 2020

The amino acid glycine is involved in generation of multiple critical metabolites including glutathione, heme, and creatinine. Interestingly, in both humans and rodents, circulating glycine levels are significantly reduced in obesity, glucose intolerance, type II diabetes and non‐alcoholic fatty liver disease. The glycine cleavage system (GCS) is the predominant glycine degradation pathway in humans. The rate‐limiting enzyme of GCS is glycine decarboxylase (GLDC), and loss‐of‐function mutations of GLDC cause hyperglycinemia. Here, we show that GLDC gene expression is upregulated in livers of mouse models of diabetes and diet‐induced obesity as well as in the fasted state in normal animals. In exploring the hormonal signals that mediate these regulatory events we found that both glucagon and insulin stimulated GLDC gene expression. In primary rat hepatocytes, GLDC expression was strongly stimulated by glucagon and cAMP, and mildly with insulin while in a rat hepatoma cell line, insulin strongly stimulated GLDC expression as compared to cAMP. We identified the cAMP‐response element binding protein 1 (Creb1) and insulin responsive transcription factor Sterol regulatory element binding protein 1c (Srebp‐1c) as mediators of the glucagon and insulin stimulatory effect, respectively. By knocking down or overexpressing GLDC in cell lines we observed GLDC expression levels are strongly linked to intracellular glutathione and reactive oxygen species (ROS) levels. Our findings suggest that the hormonal regulation of GLDC may contribute to compensatory changes in glutathione production as a defense against metabolic disease‐associated oxidative stress.

Support or Funding Information

This work is supported by NIH R21AG050741, Grants Boost OVPR Wayne State University and the Michigan Diabetes Research and Training Center.
Diet Restriction Modulates Heat Shock Gene Expression and Impairs Heat Shock Response in Drosophila melanogaster
Authors: Tresser Jason Warren
Published: April 2020

Diet restriction (DR) has been shown to extend life span in adult Drosophila melanogaster in a manner similar to that of calorie reduction in organisms from worms to primates. Long‐lived flies are smaller, have global down regulation of protein levels, and lower fecundity. We hypothesized that flies under diet restriction, although long‐lived under ideal conditions, would be more susceptible to environmental stresses. We fed adult flies control or diet restricted food for seven days and then performed qPCR on various transcripts known to be involved in stress response. We show here that various transcripts levels are modulated in response to diet restriction, particularly, HSP22, HSP70 and HSP83. We show also that diet restricted flies are limited in their ability to upregulate heat shock transcripts when exposed to heat stress and have an increased mortality rate under heat stress conditions.
The effects of algae oil on laying performance, egg quality, adiponectin and hepatic lipogenesis in laying hens
Authors: Liao P.X. , Lin Yuan-Yu
Published: April 2020

The purpose of current study is to explore the effects of algae oil on laying performance, egg quality, fatty liver syndrome and circulatory adiponectin in laying hens. Sixty‐four 26‐week‐old, Hy‐line W36 laying hens were allotted to two groups with 4 replicates, eight hens per replicate. The control group and the treatment group were fed basal diet with 3% soy oil and basal diet with 3% algal oil, respectively, totally for 4 weeks. The egg production rate and total egg weight per replicate were recorded every day. Egg quality, the fatty acids profile in yolk and cholesterol contents were measured every two weeks. Body weight and feed intake were recorded at beginning of experiment and week four. Blood samples were randomly taken from both groups (2 hens per replicate at week zero and four) . The level of macromolecule adiponectin and total adiponectin in plasma were analyzed. At last week of experiment, liver tissues were weighed and got liver biopsy. The results showed that treatment of algae oil did not affect laying performance and egg shell quality, but egg weight was significantly lighter in the treatment group. At week two, the width and height of yolk were significantly lower than the control group. At the result of histological examination, the accumulation of hepatic fat were significantly higher in the treatment group. The treatment of algae oil did not affect the level of macromolecule adiponectin and total adiponectin in plasma. The results showed that replacing soy oil with 3% algal oil in diets could accelerate the processes of fatty liver syndrome in chicken.

Support or Funding Information

Ministry of Science and Technology, Taiwan (106‐2313‐B‐029 ‐003 ‐MY2)
The Biased Kappa Opioid Receptor Agonist Nalfurafine Reduces the Reinforcing Properties of Oxycodone and Enhances Oxycodone‐Induced Spinal Anti‐Nociception
Authors: Setola Vincent , White Allison , Wix Kimberley , Siderovski David Peter
Published: April 2020

We recently described the effect of two G protein‐biased kappa opioid receptor agonists [1 mg ethoxymethyl ether (EOM)‐salvinorin B/kg and 15 μg nalfurafine/kg] on the reinforcing and anti‐nociceptive properties of co‐administered morphine (5 mg/kg) in mice (Kaski et al., JPET, 2019). EOM‐salvinorin B and nalfurafine each reversed the reinforcing properties of co‐administered morphine in the conditioned place preference paradigm, and each potentiated morphine‐induced spinal anti‐nociception in the warm‐water tail withdrawal paradigm. EOM‐salvinorin B had no effect on morphine‐induced supraspinal anti‐nociception in the hot‐plate paradigm; however, nalfurafine potentiated supraspinal anti‐nociception. In the current study, we addressed whether nalfurafine co‐administration (15 μg/kg) had similar beneficial effects on the prescription opioid painkiller oxycodone (2 mg/kg) in mice. Nalfurafine markedly reduced the reinforcing properties of oxycodone in the conditioned place preference paradigm. In addition, nalfurafine potentiated oxycodone‐induced spinal anti‐nociception in the warm‐water tail withdrawal paradigm. Nalfurafine did not affect oxycodone‐induced supraspinal anti‐nociception in the hot‐plate paradigm. Because nalfurafine has a decade‐long record of safety, tolerability, and long‐lasting efficacy (as an anti‐pruritic agent) in Japan, it is an attractive anti‐addictive, dose‐sparing adjuvant to conventional opioid painkillers that have shown tremendous addiction liabilities.

Support or Funding Information

ANW acknowledges support from the WVU SOM BBS T32 (1 T32 GM132494).
J‐Curve Effects on Basal Autophagy and ER Stress by Sympathetic Activation in the Vascular Endothelium
Authors: Tran Linh , Giles Jennifer , Callaway George , Wauson Eric , Tran Quang-Kim
Published: April 2020

Activation of the sympathetic nervous system is important in the regulation of cardiovascular functions. Excess sympathetic nervous activity, however, is deleterious and is involved in pathological remodeling of cardiovascular tissues. The vascular endothelium is essential for the control of vascular tone, coagulation and metabolism and functions of other cardiovascular cell types. Basal autophagy is critical in maintaining the homeostasis of the endothelium. We hypothesized that long‐term adrenergic stimulation can impair basal autophagy and induce ER stress in the vascular endothelium. In freshly isolated aortic endothelial cells, norepinephrine (10−5 – 10−4 M for 1, 2, 5, 15, 24, 48 hrs) produced J‐curve effects on the autophagic markers, such that expression levels of LC3‐II and p62 are reduced following short‐term NE treatment, but are increased over time and significantly over control value following 48 hrs of treatment. Immunofluorescence showed increases in the numbers of LC3 and p‐62 puncta in cells treated with NE for 48 hrs. NE treatment also produces a J‐curve effect on the expression of ER stress markers calnexin and Ero‐1. Measurement of lysosomal pH indicates reduction in acidification of this compartment in cells treated with NE. Mechanistically, we hypothesized that NE affects basal autophagy via changes in mTOR activity. Indeed, a J‐curve effect was also seen with pS6, an mTOR activity marker, in endothelial cells treated with NE for 1, 2, 5, 15, 24, and 48 hrs. These data suggest that short‐term adrenergic activation stimulates basal autophagy, while long‐term sympathetic activation inhibits basal autophagy and induces ER stress, potentially via altering mTOR activity.

Support or Funding Information

Grant IOER091707 to Quang‐Kim Tran; AHA grant 15SDG25090279 to Eric Wauson
Proenkephalin predicts organ failures, renal replacement therapy, and mortality in sepsis
Authors: Kim Hanah , Hur Mina , Lim Jihyang , Struck Joachim , Bergmann Andreas , Di Somma Salvatore
Published: April 2020

Introduction

Kidney failure frequently occurs in sepsis and is associated with high mortality. Proenkephalin (PENK) is an emerging biomarker for sepsis. We evaluated whether the PENK level could predict severity, organ failure, and 30‐day mortality in septic patients.

Methods

The PENK levels were measured using the sphingotest penKid assay (Sphingotec GmbH, Hennigsdorf, Germany) in a total of 215 septic patients. The PENK levels were analyzed in terms of sepsis severity, vasopressor use, 30‐day mortality, sequential (sepsis‐related) organ failure assessment (SOFA) renal subscore, the Chronic Kidney Disease Epidemiology Collaboration estimated glomerular filtration rate (CKD‐EPI eGFR) categories, and requiring renal replacement therapy (RRT). The number of organ failures, SOFA subscores, requiring RRT, and 30‐day mortality were compared according to the PENK quartiles.

Results

The PENK levels were significantly higher in patients with septic shock, vasopressor use, and non‐survivors than in patients with solitary sepsis, no vasopressor use, and survivors, respectively (all P < 0.01). The PENK levels were significantly associated with SOFA renal subscore and CKD‐EPI eGFR categories (all P < 0.0001). The PENK quartiles were associated with the number of organ failures as well as SOFA renal, cardiovascular, respiratory, and central nervous system subscores (all P < 0.05). The PENK levels were significantly higher in patients who required RRT than in those who did not require RRT (P < 0.0001). High PENK level was also associated with high 30‐day mortality (P < 0.0001).

Conclusions

PENK could be a useful and objective marker to predict severity, organ failure, and 30‐day mortality in septic patients.
INVESTIGATING MOTOR LEARNING IN A LRRK2 MOUSE MODEL OF PARKINSON’S DISEASE
Authors: Eby Allison , Crown Lindsey , Cowen Stephen
Published: April 2020

While most cases of Parkinson’s disease are idiopathic, meaning of unknown origin, in 10% of cases, a genetic link has been identified. Of these, the most common is the G2019S mutation of the leucine‐rich repeating kinase 2 (LRRK2) gene. Investigation into this mutation could prove useful not only for those with LRRK2 Parkinson’s, but for idiopathic Parkinson’s as well. Genetic mouse models of this mutation are important for translational research, but despite the increasingly large number of studies on these animals, few have identified a reliable phenotype. In this study, we measured the average latency to fall during a rotarod‐based motor‐learning task conducted twice a week for two weeks with transgenic (Tg) and non‐transgenic (NTg) mice 6–8 months old. During the second week, half the animals were given a LRRK2 inhibitor, MLi‐2. Although we detected no effect of the drug, results have been mixed for overall motor performance with a trend towards LRRK2 Tg animals performing worse on the task. In this study, we examine whether LRRK2 Tg mice perform similarly to NTg mice for within‐day learning, between‐day learning, and overall latency to fall measures.

Support or Funding Information

Funding from Michael J. Fox Foundation.
Hypoglycemic effect and hepato protective role of Saccharomyces boulardii THT 500101 strain in a murine model of streptozotocin‐induced diabetes
Authors: Cunha Tatiana Sousa , dos Santos Letícia Barssotti , Abreu Isabel Mallosto Emerich , Albuquerque Raquel Cristina Melo Ferreira , Brandão Ana Beatriz Pereira , Ferreira Fabiana Gomes , da Silva Dias Danielle , de Angelis Katia , Salgado Miguel Angel Castillo
Published: April 2020

The chronic hyperglycemia of diabetes mellitus (DM) is associated with long‐term damage and dysfunction of different organs, including the liver. Hyperglycemia induces hepatic mitochondrial dysfunction causing changes that include decreased oxidative phosphorylation, increased oxidative stress, ultra‐structural abnormalities, which in turn worse metabolic health. The disturbance in metabolic homeostasis also modulates gut microbiota by a variety of mechanisms while the maintenance of intestinal microbiota has a significant influence on metabolic state. The aim of this study was to evaluate the hypothesis that streptozotocin‐diabetic mice treated with Saccharomyces boulardii THT 500101 strain (S. boulardii, THT: Probiotics and Starters Cultures, Belgium) present improvement of blood glucose, reduction of oxidative stress and maintenance of hepatocytes structure, concomitant with a beneficial impact in the gut microbiota profile. C57BL/6 male mice were randomly assigned into four groups: Control (C), Control + Probiotic (CP), Diabetes (D), and Diabetes + Probiotic (DP) (n = 9/group). Diabetic mice treated with S. boulardii presented a reduction of blood glucose (~30%) when compared to D group (DP = 251.9 ± 36.40 mg/dl vs. D = 362.9 ± 21.26 mg/dl, p<0.05). D group presented a higher level of carbonylated proteins compared with C and CP groups (C = 3.26 ± 0.42, CP = 3.00 ± 0.37 vs. D = 3.62 ± 0.33 nmol/mg), and probiotics reduced hepatic protein damage in DP (DP = 3.29 ± 0.58 nmol/mg). Diabetes reduced the activity of antioxidant enzymes superoxide dismutase (SOD) (D = 8.00 ± 0.12 vs. C = 8.49 ± 0.10, CP = 8.44 ± 0.05 USOD/mg, p<0.05) and glutathione peroxidase (GPx) (D = 73.50 ± 9.50 vs. C = 83.00 ± 4.00, CP = 81.00 ± 7.25 nmol/min/mg, p<0.05) and S. boulardii increased the activity of both enzymes in DP group as compared with D (SOD: DP =8.20 ± 0.14 USOD/mg and GPx: DP = 5.08 ± 2.29 nmol/mg, p<0.05). Signs of severe hydropic degeneration were noticed in hepatocytes from D group and to a lesser extent in hepatocytes from DP group. S. boulardii treatment was associated with increased proportion in Bacteroidetes, Firmicutes and Deferribacteres, and a decreased proportion of Proteobacteria and Verrucomicrobia phylum in DP group, as compared with D. Thus, the data presented here show up a novel potential therapeutic role of S. boulardii for glycemic control and attenuation of diabetes‐induced liver injury.

Support or Funding Information

FAPESP 2016/24059‐2
Empagliflozin Prevents Renal Dysfunction and Inhibits Proximal Tubule NHE3 Activity in Nondiabetic Heart Failure Rats
Authors: Junior Flavio Araujo Borges , dos Santos Danubia Silva , Crajoinas Renato Oliveira , Wisnivesky Aline Cavalcantti T. , Benetti Acaris , Rios Thiago Matheus Santos , Antonio Ednei Luiz , Tucci Paulo , Girardi Adriana Castello Costa
Published: April 2020

Sodium‐glucose cotransporter 2 (SGLT2) inhibitors improve glycemic control by suppressing glucose reuptake in the renal proximal tubule (PT). Primarily designed as antidiabetic agents, these drugs also reduce cardiovascular death and hospitalization for heart failure (HF), regardless of the presence or absence of type 2 diabetes. However, the mechanisms underlying the cardioprotective effects of SGLT2 inhibitors remain unknown. Given the importance of the cardiorenal axis in HF care, this study aimed to test the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) is capable of exerting renoprotective effects in the setting of nondiabetic HF. To this end, male Wistar rats were subjected to ligation of the anterior descending coronary artery or sham operation. Four weeks after surgery, rats were fed normal chow, with or without EMPA, for an additional 4 weeks. Compared with sham‐groups, HF rats treated with EMPA had similar serum BNP levels, whereas cardiac dysfunction deteriorated it further in HF rats (Sham: 0.5 ± 0.1; Sham+EMPA: 0.5 ± 0.1; HF: 2.1 ± 0.3; HF+EMPA: 0.9 ± 0.1 ng/mL). Additionally, HF rats exhibited an increase in lung (Sham: 3.2 ± 1.5; Sham+EMPA: 31.8 ± 1.1; HF: 44.7 ± 3.3; HF+EMPA: 32.7 ± 1.7 mg/mm) and right ventricle (Sham: 6.3 ± 0.2; Sham+EMPA: 5.2 ± 0.2; HF: 8.9 ± 1.0; HF+EMPA: 6.4 ± 0.3 mg/mm) mass. Renal function was impaired in HF rats as evidenced by low glomerular filtration rate (GFR) and high levels of plasma urea and urinary protein excretion. However, EMPA treatment restored these parameters to sham rats levels [(GFR, Sham: 6.5 ± 0.2; Sham+EMPA: 7.7 ± 0.6; HF: 4.5 ± 0.4; HF+EMPA: 7.7 ± 0.4 mL/min/kg) (Plasma urea, Sham: 37 ± 1; Sham+EMPA: 38 ± 2; HF: 48 ± 2; HF+EMPA: 39 ± 1 mg/dL) (Urine protein to creatinine ratio, Sham: 1.2 ± 0.1; Sham+EMPA: 1.0 ± 0.1; HF: 1.8 ± 0.1; HF+EMPA: 1.3 ± 0.1)]. Furthermore, EMPA prevented reduction of hematocrit in HF rats (Sham: 48.7 ± 0.3; Sham+EMPA: 49.5 ± 0.7; HF: 45.8 ± 0.5; HF+EMPA: 49.7 ± 0.4 %). In vivo stationary microperfusion showed that EMPA reduced Na+/H+ exchanger isoform 3 (NHE3) activity in HF rats, similar to sham‐groups (Sham: 2.05 ± 0.04; Sham+EMPA: 1.40 ± 0.05; HF: 4.36 ± 0.08; HF+EMPA: 1.54 ± 0.05 nmol/cm2). Collectively, these findings demonstrate that SGLT2 inhibition ameliorates renal dysfunction and improves the volemic status of nondiabetic HF rats.

Support or Funding Information

Supported by FAPESP and CAPES.
The Effect of the Virus (MdSGHV) on the Ultrastructure of the Salivary Glands of Musca domestica L
Authors: Palacios Daniela Molina , Stoffolano John
Published: April 2020

The salivary glands of insects play a key role in the replication cycle and vectoring of viral pathogens. Consequently, Musca domestica L, and the Salivary Gland Hypertrophy Virus (MdSGHV) serve as a model to study insect vectoring of viruses. A better understanding of the structural aspects of this virus, and the tissues it invades, will help obtain a better picture of the pathological impact that the virus has on adult flies. Furthermore, since the virus inhibits mating and egg development in the adult fly, its study can lead to development of novel techniques to control this world‐wide pest for food safety improvement. Using scanning and transmission electron microscopic techniques (SEM, TEM), researchers have shown the effects of infection by MdSGHV on the salivary glands; however, the exact location among the glands where the infection was found is unclear (i.e., given that the salivary glands extend throughout the entire adult body). For this reason, this study intends to do a comprehensive examination of the ultrastructural effects of the hypertrophy virus on the salivary glands. Here, we report the clear hypertrophy present throughout the salivary glands in their entirety, as well as enveloped virus particles in the cytoplasm of infected cells. Moreover, the virus provokes an erosion of the cuticular lining that separates the epithelial cells from the lumen of the internal duct.
Impact of GPER, Sex, and Age on Arterial Stiffness and Fibrotic Gene Expression
Authors: Kilanowski-Doroh Isabella M. , Ogola Benard O. , Harris Nicholas R. , Gentry Kaylee , Satou Ryousuke , Lindsey Sarah H.
Published: April 2020

Arterial stiffness is associated with cardiovascular risk independent of blood pressure. Previously, we showed that pulse pressure, a measure of arterial stiffness, is higher in female mice with global deletion of G protein‐coupled estrogen receptor (GPER) despite similar blood pressure. Additionally, we found that the GPER agonist, G1, provides protection from arterial remodeling. Therefore, the current study assessed the impact of GPER, sex, and age on arterial stiffness and aortic expression of four profibrotic genes: decorin (DCN), endothelin 1 (EDN1), type III collagen α1 (COL3A1), and connective tissue growth factor (CTGF). We hypothesized that global GPER knockout (KO) would increase fibrotic gene expression in females more than males, would be exacerbated with age, and would correlate with increased arterial stiffness.

Methods

Pulse wave velocity (PWV) was obtained in the carotid artery of 20 week‐old and 52 week‐old male and female wildtype and GPER KO mice (n = 3/group) using a Vevo 1100 high resolution ultrasound. RNA was isolated from aortas using a Qiagen RNeasy Mini Kit, and gene expression was quantified using droplet digital polymerase chain reaction (ddPCR). Statistical analysis was performed using three‐way ANOVA and Pearson r Correlation.

Results

In females, GPER deletion increased expression of all four profibrotic genes in 20 week‐old mice, but this relationship was reversed in 52 week‐old mice. In males, GPER deletion was associated with lower expression of fibrotic genes in both age groups. PWV significantly correlated with increased expression of CTGF (r = 0.66, p = 0.019) and EDN1 (r = 0.61, p = 0.036). In addition, the expression of CTGF, COL3A1, EDN1, and DCN were positively correlated (p < 0.005). Interestingly, expression of both ERα and GPER were increased with aging in female mice (p < 0.05).

Conclusion

Increased PWV was accompanied by upregulation of four profibrotic genes in the aorta. Adult female GPER KO mice had increased stiffness and higher expression of fibrotic genes. However, GPER deletion was associated with lower PWV and fibrotic gene expression in middle‐aged mice. Therefore, the protective effect of GPER in the vasculature was reversed with aging, despite the fact that aging upregulated estrogen receptor expression. Future studies will determine the mechanism by which GPER influences the expression of fibrotic genes and arterial stiffness.

Support or Funding Information

NIH HL133619
What Neutrons Read in Tea Leaves: A High‐Pressure, Small‐Angle Neutron Scattering Study
Authors: Spruill Racheal
Published: April 2020

Green tea polyphenols contribute to its taste, aroma, and health benefits such as reduced risk of degenerative diseases and cancer. 85% of polyphenols in green tea are catechins, mostly Epigallocatechin gallate (EGCG), a potential alternative to synthetic food additives. EGCG has been shown to prevent the growth of bacteria responsible for food spoilage while showing antioxidant activity in food systems. Polyphenols are however susceptible to light, high extraction temperatures, and alkaline conditions, which cause their degradation during extraction and food processing. Several approaches have been used to improve polyphenol yields, stability, and production costs, including various solvent free extraction techniques, modification of the structure of EGCG or association with protein‐based carriers. Bovine serum albumin (BSA), found in blood serum and milk, functions as a transporter of endogenous and exogenous compounds in the circulatory system and has been shown to bind and stabilize polyphenols. The nature of the interactions is thought to be mostly hydrophobic. The underlying mechanisms of BSA‐polyphenol interactions, as well as their stability, are central to the development of effective carriers as delivery systems and to the engineering of improved food processing. While high‐pressure (HP) is increasingly used in sterilization and food processing, HP is also a non‐invasive tool for the study of protein‐polyphenol interactions. This project combines neutron scattering and HP techniques to investigate the structure and stability of complexes of BSA with polyphenols, particularly EGCG. Small‐angle neutron scattering (SANS) is a non‐destructive technique that can be used to probe a broad range of interactions under various concentrations, temperature, pH and pressure conditions. The results highlight how neutron scattering can contribute new insights into polyphenol bioavailability in biological and food‐processing environments.

Support or Funding Information

Support for Racheal Spruill was provided by the Center for High Resolution Neutron Scattering, a partnership between the National Institute of Standards and Technology and the National Science Foundation under Agreement No. DMR‐1508249.