bluuebunny/crossref_metadata_2025 huggingface dataset:
Anatomical and histological observations on a musk ox brain (Ovibos moschatus)
Authors: Ackermans Nicole L.
Published: April 2020

Musk oxen are large tundra‐dwelling bovids that, when in rut, can strike heads at speeds around 30 mph. The wide base of their large horns armor their skull against such impacts, although it is unknown if they sustain brain trauma. There is no literature describing concussion, or other adverse repercussions of severe cranial contact in musk oxen. Our research explores the brain and skull anatomy of combative bovids, specifically focusing on whether these animals sustain brain injury after head‐butting, and if not, what features most likely provide protection. Achieving a better understanding of how these extreme animals may avoid brain injury will provide insight to develop strategies for the reduction of traumatic brain injury in humans.

An archived brain of an adult male musk ox was used in this preliminary study. It had been collected after humane euthanasia and preserved in formalin following a goring injury in the wild where it had been observed clashing heads with another musk ox bull in the moments before its death. The brain was MR scanned and examined histologically for evidence of traumatic brain injury. The right hemisphere was fixed in paraformaldehyde (4%, buffered) and prepared using a variety of exploratory histological protocols, to investigate abnormalities in neurons, microglia, astrocytes, and blood vessels. Specifically, Tau protein, a biomarker found in the cerebrospinal fluid and in neurodegenerative lesions, was used to detect any brain trauma‐related cellular consequence of chronic or acute head clashing. Preliminary histological results indicate no apparent abnormalities. A high‐resolution 7‐T MRI scan additionally revealed no abnormal neuropathological changes. This brain being a single sample does not allow us to make sweeping assumptions. However, these preliminary investigations lead us to believe that musk oxen do not appear to suffer from chronic or acute brain trauma after head clashing. A more in‐depth exploration with a larger sample size is proposed to better understand the combination of anatomical structures contributing to brain protection in these animals.

Support or Funding Information

NLA is a fellow of the Swiss National Science Foundation
GLP‐1 Induces a Shift in Primary Carbon Metabolites in Late‐fasted Northern Elephant Seals
Authors: Dhillon Jaapna , Viscarra Jose A. , Fiehn Oliver , Ortiz Rudy M.
Published: April 2020

Insulin resistance has been suggested to be an adaptive response to food deprivation since it promotes the preservation of glucose and oxidation of fat. However, the insulin resistance‐like conditions that fasting mammals experience may result from reduced pancreatic sensitivity to glucose/capacity to secrete insulin. Glucagon‐like peptide 1 (GLP‐1) is a gastrointestinal hormone that enhances postprandial glucose‐stimulated insulin secretion. To better assess the effects of GLP‐1 on the metabolism of primary carbon metabolites associated with late‐fasting induced insulin resistance, we dose‐dependently infused late‐fasted seals (naturally adapted to prolonged fasting) with low (10 pM/kg; n=3) or high (100 pM/kg; n=4) GLP‐1 immediately following a glucose bolus (0.5g/kg), using glucose without GLP‐1 as control (n=5). The plasma metabolome was measured from samples collected at 5 time points just prior to and during the infusions and areas under the curve (AUC) were calculated.

The median AUC fold changes between the GLP‐1‐treated (low and high doses combined) and control group were analyzed using ChemRICH which is a chemical similarity enrichment analysis software for metabolomics datasets that uses medical subject headings and Tanimoto substructure chemical similarity coefficients to cluster metabolites into non‐overlapping chemical groups. Statistically significant p‐values for clusters were obtained by self‐contained Kolmogorov–Smirnov tests. ChemRich mapped 149 of the identified metabolites to 23 nonoverlapping chemical classes, of which 2 were found to be significantly different between the GLP‐1‐treated and control group (false discovery rate adjusted P value < 0.05). Treatment with GLP‐1 infusions was associated with altered Kreb Cycle activity represented by increased levels of tricarboxylic acids, and reduced levels of carbocyclic acids (benzoic acid being a key compound). Hence, GLP‐1 infusion induced a shift in primary carbon metabolites in a model of fasting‐induced insulin resistance.

Support or Funding Information

This work was supported by grants: NIH NHLBI HL091767, NIH NHLBI HL091767‐S1, NIH NHLBI K02HL103787, NIH NHLBI R01HL09176, and NIH U24 DK097154.
Structure of sympathetic input to interscapular brown adipose tissue in the mouse
Authors: Muenzberg-Gruening Heike , Huesing Clara , Gummadi Sanjeev , Zhang Rui , Lee Nathan , Qualls-Creekmore Emily , Yu Sangho , Morrison Chistopher D. , Burk David , Berthoud Hans Rudolph , Neuhuber Winfried
Published: April 2020

Interscapular brown adipose tissue (iBAT) is under sympathetic and sensory nerve control. We recently identified the stellate and thoracic sympathetic chain ganglia T1‐T5 as the main origins of sympathetic input to iBAT in adult male and female mice. Previous studies reported that iBAT receives sympathetic innervation from five intercostal nerves; even though intercostal nerves (which originate from ventral rami) are typically known to provide innervation to lateral, ventral body tissues. Furthermore, we were unable to find literature with detailed anatomical studies. Here, we used a reporter mouse model that expresses tdTomato in tyrosine hydroxylase expressing neurons (TH‐tomato mice) in all sympathetic nerve fibers to facilitate dissection and imaging of sympathetic nerves to iBAT. Our data clarifies that iBAT receives sympathetic input via dorsal rami, which combine sensory and sympathetic fibers. This is consistent with the role of dorsal rami to innervate dorsal body structures, while intercostal nerves (traveling along the ribs) provide innervation to lateral and ventral body structures in mammals. Furthermore, we performed whole mount and paraffin embedded anatomical studies of TH (as surrogate for sympathetic nerves) to understand the anatomy and structure of sympathetic iBAT innervation. We found that fine sympathetic nerve fibers branch off the dorsal rami emanating from T1‐T4 to enter the BAT pad, and from there further branch to form axonal varicosities. Meanwhile, the dorsal rami continue to innervate vasculature and skin. We further established a novel tool to quantify axonal varicosities across entire adipose tissue depots using the IMARIS spot detection tool. This allowed us to visualize the extensive variation of axonal varicose density within the entire iBAT depot with dense varicosities adjacent to brown adipocytes, but sparse varicosities adjacent to white adipocytes. We also analyzed the axonal varicosities in inguinal white adipose tissue and found denser varicosities associated with beige islands that were significantly increased after 7day cold exposure (10°C). Finally, we use UCP1‐reporter mice and whole mount iBAT staining of TH to verify that dense axonal varicosities overlap with UCP1‐positive tissue, while UCP1‐negative tissue shows sparse axonal varicosities. The dynamic and anatomically distinct regulation of sympathetic axonal varicose density is likely due to distinct neurotrophic interaction of brown and white adipocytes with sympathetic neurons. Our data clarify the structure of sympathetic innervation to the iBAT in the mouse and highlights the benefits to study endorgans like entire adipose depots in an anatomically defined manner.

Support or Funding Information

Grant support: NIH 4OT2 OD23864‐01
Protective Role Of Estrogen Against Excessive Erythrocytosis In Monge’s Disease
Authors: Azad Priti , Patel Gargi , Bermutez Daniella , Villafuerte Francisco , Haddad Gabriel
Published: April 2020

Rationale

Chronic mountain sickness (CMS), or Monge’s disease, is an advancing and debilitating syndrome caused by chronic (years) exposure to high altitude hypoxia, as experienced by people living in the Andean region. Excessive erythrocytosis (EE) is one of the critical traits of CMS and this excessive pathobiologic response has deleterious effects since a high hematocrit would increase blood viscosity and reduce blood flow to hypoxia‐sensitive organs (brain and heart), often resulting in myocardial infarction or stroke in young adults. Within the population of Cerro de Pasco in the Andes, CMS prevalence is vastly different between males and females, being rare in females. Of interest is that there is a sharp increase in CMS incidence in females after menopause. This suggests the role of gender and sex hormones in altering disease manifestation.

Methods

In order to understand the molecular basis for polycythemia of high altitude, we have generated a disease in‐the dish‐model by re‐programming fibroblasts and native CD34+ve cells from CMS and non‐CMS subjects and converting them to RBC. Using this in‐vitro platform, we are now studying the interactions between genetic factors and sex hormones (Testosterone, Estrogen and Progesterone) and their effect on RBC production in the CMS and non‐CMS subjects at high altitude.

Results

While we found that testosterone increased RBC production somewhat, estrogen in physiologic concentrations (10nM), in sharp contrast, reduced the CD235a (Glycophorin A‐marker of mature RBC) remarkably (from 65.4% in the untreated CMS sample to 11% in the treated CMS) and eliminated the CMS EE phenotype. There was also stage‐specificity since we found that the impact was most severe when estrogen was present at the EB stage. This strongly suggests that estrogen has a protective role against the polycythemia phenotype. To probe further, we studied the effect of estrogen (10nM) on GATA1, a critical link with erythropoiesis. There was a sharp 5 fold decrease in GATA1 (p<0.01) and its target genes such as Alas2 (14 fold, p<0.001) in erythroid cells. This decreased expression resulted in marked reduction (>50%) in RBC production, demonstrating that estrogen constrains RBC production in females.

Conclusion

Our results demonstrate that estrogen inhibits GATA1 expression and prevents excessive erythropoiesis and Monge’s disease in females at high altitude. We believe that these studies not only shed light on role of sex hormones on hypoxia‐induced excessive erythropoiesis but have a broader application in their regulation of erythropoiesis and related blood disorders.
Complex Dental Structure and Wear Biomechanics in Non‐Avian Dinosaurs and Pleistocene Mammals
Authors: Erickson Gregory , Hunt Tyler , Kuhn-Hendricks Stephen , Pathak Siddhartha , Varma Soumya , Babuska Tomas , Grejtak Tomas , Krick Brandon
Published: April 2020

Reptiles rarely approached the biomechanical sophistication for feeding or dietary diversity seen in mammals. Their teeth are typically non‐occluding, semi‐conical structures with simplistic parallel‐crystallite enamel surrounding an orthodentine core. Conversely, most mammals possess multi‐cusped teeth that are drawn across one another during mastication and self‐wear to their functional morphology. The most complex dental architectures are seen in herbivorous mammals. Their teeth are composed of up to four constituents (incl. prismatic enamel) that strategically wear creating coarse grinding or slicing surfaces. These allow them to comminute tough and/or abrasive plants and liberate nutrients inaccessible to other animals. Non‐avian dinosaurs stand out among reptiles in that precise, mammal‐like dental occlusion and self‐wearing teeth evolved on at least four occasions. These topographies enabled access to myriad floral types and facilitated their respective ecological diversifications. My research group and colleagues showed that the teeth of these dinosaurs are histologically more complex than formerly appreciated. Using cutting‐edge material science and tribological engineering indentation techniques we discovered that wear and fracture relevant material properties are preserved in the fossil dental tissues of dinosaurs and Pleistocene mammals. This led to development of a 3D Archard’s wear model from which we determined how horse‐like occlusal surfaces for the grinding of plant matter occurred in hadrosaurids, and fuller‐like slicing surfaces developed and functioned in ceratopsians. In current research we are studying how the grinding dentitions of mammoths similarly formed through self‐wear. Notably our paleontologically‐inspired wear model is seeing broad industrial applications.

Support or Funding Information

NSF CMMI (Civil, Mechanical and Manufacturing Innovation) Eager and AFOSR (Air Force Office of Scientific Research) to GME, SP and BAK
Modulation of the repetitive element transcriptome by healthy aging interventions
Authors: Wahl Devin , Cavalier Alyssa , LaRocca Thomas
Published: April 2020

Growing evidence indicates that non‐coding repetitive elements (RE) may play an important role in aging. For example, recent studies have shown that age‐related RE transcript accumulation may cause pro‐inflammatory signaling and be detrimental to cardiometabolic health. However, RE are often ignored in transcriptome studies on aging. Moreover, their role in well‐known healthy aging interventions has not been comprehensively characterized, and the link between RE transcript accumulation and lifespan is unknown. To investigate this, we have analyzed RE transcript expression in multiple RNA‐seq datasets on aging, and in transcriptome data on mice subjected to several of the most robust and well‐studied lifespan increasing interventions, including: calorie restriction (CR), acarbose, Rapamycin, Protandim, and 17‐□ estradiol. We find that aging is associated with a general accumulation of RE transcripts in multiple tissues, but that CR significantly reduces the expression of >60% of RE transcripts in the liver and brain (p<0.05 vs. controls). We also observe that RE transcript levels are modulated significantly by Rapamycin, acarbose, and Protandim. Overlap analyses indicate that certain RE transcripts are affected by multiple interventions, suggesting that these RE may play a particularly important role in aging and healthy aging interventions, and we are investigating correlations among these RE transcripts and transcriptome signatures of adverse aging processes including inflammation. Collectively, our results suggest that the accumulation of RE transcripts may play an important role in aging and represent a novel target for increasing lifespan.

Support or Funding Information

R21: AG060302
S‐Palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of GC‐1 in Cardiomyocytes
Authors: Tzimas Christos , Russo Ilaria , Dun Wen , Yilmaz Sevde , Rajan Sudarsan , Tsai Emily J.
Published: April 2020

Introduction

The nitric oxide (NO) receptor soluble guanylyl cyclase (GC‐1) was first identified as a cytosolic enzyme but is now known to localize to various cell membrane microdomains. We reported that in normal hearts, GC‐1 localizes to caveolae, where it exhibits enhanced NO‐responsiveness. In failing hearts, caveolae‐localization of GC‐1 is disrupted and GC‐1 outside of caveolae is oxidized. How GC‐1 dynamically localizes to and away from caveolae is unknown. We hypothesized that S‐palmitoylation, a reversible, enzyme‐catalyzed, post‐translational modification that enhances membrane affinity of protein substrates, mediates caveolae‐localization and modulates redox regulation of GC‐1.

Methods

To examine S‐palmitoylation of GC‐1 in the failing heart, we subjected adult male C57BL/6 mice to transverse aortic constriction (TAC) vs thoracotomy (Sham) and performed modified acyl‐biotin exchange (ABE) assays. Expression of palmitoyl‐transferases ZDHHC1, ‐16, and ‐24 were also assessed in mouse hearts at the transcript and protein levels. Using computational bioinformatics, we identified 5 conserved cysteines of potential S‐palmitoylation in the GC‐1α subunit. We created cysteine‐serine mutants of GC‐1α at each and all of the 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). We expressed the mutants and WT GC‐1α in neonatal rat cardiomyocytes (NRCM) via adenoviral mediated gene transfer and compared their palmitoylation status by ABE. We also assessed caveolae‐localization of GC‐1α (CS* vs WT) by confocal microscopy and its oxidation by redox Western.

Results

In TAC hearts, S‐palmitoylation of GC‐1α was diminished by 50% compared to Sham (p=0.02). Likewise, ZDHHC16 was also downregulated in TAC by 31% (p=0.01) at the transcript level and by 56% (p=0.03) at the protein level, relative to Sham. In NRCMs, S‐palmitoylation of CS* was profoundly diminished (6.9±2.0 % of WT, p<0.001), while that of C15S, C176S, and C497S were each modestly diminished (23.8±8.4%, p=0.03; 23.2±6.6%, p=0.01; 22.8±6.0%, p=0.01, respectively of WT). S‐palmitoylation of C3S and C595S were similar to WT. Confocal microscopy of NRCMs revealed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed a trend towards increased cysteine oxidation of CS* vs WT upon H2O2 treatment (1.5‐fold increase, p=0.05).

Conclusions

Our data suggest that S‐palmitoylation of GC‐1α at C15, C176, and C497 mediates GC‐1 caveolae‐localization and may reciprocally regulate cysteine post‐translational modifications of GC‐1.

Support or Funding Information

5R01HL138528 to EJT.
Effects of vitamin C, and B3, separately, on the apoptotic rate of metastatic breast cancer cells treated with etoposide by green fluorescent assay
Authors: Tunc Malik , Witherow Scott
Published: April 2020

Apoptosis is one of the nine hallmarks of all cancer and has been the focus of many chemotherapeutic treatments. Indeed, cells that have escaped programmed death mechanisms are extremely difficult to treat. In recent years, researchers have studied the effects of micronutrient supplementation in complementation with drugs such as cisplatin or etoposide, which are aimed to control cell cycle progression and to initiate apoptosis. The triple‐negative breast cancer cell line, MDA‐MB‐231, does not express human epidermal growth factor receptors, estrogen, or progesterone, and is highly aggressive. This study aimed to determine the effect of niacin or L‐ascorbic acid (vitamin B3 & C) supplementation on the apoptotic rate of MDA‐MB‐231 cells treated with etoposide. Relative apoptotic levels were assessed using the Cell‐Event™ Caspase‐3/7 Green Detection Reagent with a TECAN plate reader and statistical analyses were performed in RStudio. The data suggests vitamin B3 and C both increase the relative apoptotic rate of MDA‐MB‐231 cells treated with etoposide. Furthermore, the concentrations of the two antioxidants were at the lower end of the recommended spectrum, indicating that even a relatively small quantity may augment the efficacy of the drug. Our findings demonstrated an increase in cell death following etoposide treatment when supplementing with either vitamin C or B3, but the molecular mechanism underlying this observation is not yet understood.
Securinine protects vascular dysfunction targeting atherosclerosis in vascular endothelial cells, smooth muscle cells, and apolipoprotein E deficient mice
Authors: Lee Yun Jung , Yoon Jung Joo , Han Byung Hyuk , Lee Hyeon Kyoung , Yang Sun Ryo , Lee Ho Sub , Kang Dae Gill
Published: April 2020

Atherosclerosis is a chronic vascular disease and characterized by accumulation within the intima of inflammatory cells, smooth muscle cells, lipid, and connective tissue. Here, we suggest securinine as an anti‐atherosclerotic agent to attenuate vascular dysfunction and lesion. To identify natural agents that commonly reverse advanced atherosclerotic plaque to early atherosclerotic plaque, significantly regulated genes were analyzed in silico. The differentially expressed genes from 9 intimal thickening and 8 fibrous cap atheroma tissue which were collected from GEO data were assessed by the connectivity map. Natural candidate securinine, a main compound from Securinega suffruticosa, was selected and administrated 1, 5 mg/kg/day in apolipoprotein‐E‐deficient (ApoE KO) mice for 18 weeks. Securinine significantly showed lowered blood pressure and improvement of metabolic parameters with hyperlipidemia. The impairment in vasorelaxation was remarkably decreased by treatment with securinine. H&E staining revealed that treatment with securinine reduced atherosclerotic lesions. Securinine suppressed the expression of adhesion molecules and matrix metalloproteinase‐2/‐9 in both ApoE KO and vascular endothelial cells (HUVEC). In HUVEC pretreatment with securinine significantly inhibited ROS generation and NF‐κB activation. Growth curve assays using the real‐time cell analyzer showed that securinine significantly decreased TNF‐α‐induced aortic smooth muscle cell proliferation and migration in a dose‐dependent manner. Thus securinine may be a potential natural candidate for the treatment of atherosclerosis because it attenuates vascular inflammation and dysfunction as well as vascular lesion.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2017R1A5A2015805)(2017R1D1A3B03029089) (2019R1A2C1085650).
Effect of Histamine‐receptor Antagonism on VO2Peak Improvements to Exercise Training
Authors: Sieck Dylan Charles , Mangum Joshua E. , Ely Matthew R. , Francisco Mike E. , Larson Emily A. , Kaiser Brendan W. , Minson Christopher T. , Halliwill John R.
Published: April 2020

One of the physiological changes following moderate intensity aerobic exercise is a transient reduction in blood pressure, termed post‐exercise hypotension (PEH), which is exercise intensity and duration dependent. This short‐term reduction in blood pressure is dependent upon skeletal muscle histamine production and release, which causes increased conductance via local activation of histamine H1 and H2 receptors. PEH is abolished when antihistamines are consumed prior to exercise. Additionally, histamine‐receptor antagonism during a single bout of exercise modifies expression of the transcriptome related to inflammation, angiogenesis, and metabolism. Due to the large role histamine plays in post‐exercise recovery, the scope of histamine receptor activation in adaptations to aerobic exercise training needs to be determined. Therefore, the purpose of this study was to examine histamine’s role in promoting adaptation to exercise. It is hypothesized that blocking histamine’s actions will attenuate adaptations that occur from aerobic exercise training. 16 young, healthy, recreationally active individuals (5M, 11F) volunteered for this study. All volunteers completed 6 weeks of exercise training (3–4 times a week), for a total of 21 exercise sessions. Exercise training combined 18 sessions of continuous exercise (1 hour at 60% VO2peak) and 3 sessions of high intensity interval training (30 min between 30 and 90% VO2peak). Volunteers were randomly placed into a placebo or histamine antagonism (540 mg fexofenadine; a H1‐receptor antagonist, and 300 mg of ranitidine; a H2‐receptor antagonist) group, and consumed medication 1 hour prior to each exercise session. Data was collected 4 times throughout the study with pre and post exercise training measures, as well as after every 2 weeks of exercise training. Measurements of VO2peak and cycle ergometry work rate were obtained. Exercise training resulted in increased absolute VO2peak of all subjects (9.4±1.5%; P<0.01); however, no difference was seen between groups (10.9±1.8% control vs 7.9±2.4% blockade; P=0.35). Additionally, exercise training resulted in increased cycle ergometry work rate of all subjects (11.2±1.6%; P<0.01); however, there was a trend for blunted improvement with blockade (7.7±2.4%) vs control (14.6±2.8%; P=0.08). In conclusion, blocking histamine’s actions had no effect on VO2 adaptations to exercise training. Additional information about factors related to O2 delivery and consumption are needed to understand the relationship between histamine and cardiovascular adaptation to exercise training.

Support or Funding Information

Support provided by: The Eugene & Clarissa Evonuk Memorial Graduate Fellowship
High‐intensity interval training decreases muscle sympathetic nerve activity and improves peripheral vascular function in patients with heart failure with reduced ejection fraction
Authors: Sales Allan Kluser , Azevedo Luciene F. , Oliveira Thiago O. , Nunes Alves Maria Janieire N. , Rodrigues Amanda G. , Oliveira Patricia A. , Jordao Camila P. , Andrade Ana , Urias Ursula , Guimaraes Guilherme V. , Bocchi Edimar A. , Grunewald Zachary I. , Martinez-Lemus Luis A. , Padilla Jaume , Negrao Carlos Eduardo
Published: April 2020

Background

Moderate intensity continuous exercise (MICT) has been shown to reduce muscle sympathetic nerve activity (MSNA) in heart failure patients with reduced ejection fraction (HFrEF). However, the effects of high‐intensity interval training (HIIT) on MSNA in HFrEF patients are unknown. We hypothesized that reductions in MSNA would be greater following HIIT than MICT and correspond with improvements in peripheral vascular function.

Methods

HFrEF patients (30 ‐ 65 years), left ventricular ejection fraction ≤ 40%, Functional Classes II–III, were randomized into HIIT, MICT or no training (NT) three times/week for 12 weeks. MSNA was assessed by microneurography. Brachial artery flow‐mediated dilation (FMD), blood flow and vascular conductance were assessed by ultrasonography, blood pressure (BP) and heart rate (HR) by plethysmography and peak oxygen uptake (V̇O2peak) by a cardiopulmonary exercise test.

Results

Both, HIIT and MICT, led to reductions in MSNA (frequency and incidence burst) relative to NT (p<0.05). However, the reduction in MSNA was more pronounced following HIIT than MICT (p<0.05). Increases in brachial artery FMD, resting blood flow and conductance were also greater following HIIT than MICT (p<0.05), while V̇O2peak increased similarly following HIIT and MICT relative to NT. No changes in BP or HR were observed in either exercise condition. Further analysis showed a negative correlation between changes in MSNA and FMD following the interventions (r=−0.60, p=0.005).

Conclusion

Our findings indicate that 12 weeks of HIIT is superior to MICT in reducing sympathetic overactivity and improving vascular function in patients with HFrEF, despite similar increases in exercise capacity.

Support or Funding Information

FAPESP Grants (2014/11671‐6 and 2017/25613‐6)
Sensitive and Comprehensive Lipid Mediator Analysis using Advanced Scheduled MRM with Polarity Switching and QTRAP Enhanced Product Ion Scanning
Authors: Norris Paul , Pearson Mackenzie Jo , Kapil Santosh , Ubhi Baljit
Published: April 2020

Using a QTRAP® 6500+ system coupled with an ExionLC™ System, we developed a targeted panel of 88 compounds for comprehensive profiling of lipid mediators and pathway markers. A Kinetex® Polar C18 column was used for LC based separation of lipid mediators, epimers and other isoelemental structures within a 20 min run time. Advanced scheduled sMRM was used to optimize scanning windows and dwell weighting. The negative mode lipid mediator panel includes pro‐inflammatory prostaglandins, leukotrienes and their primary metabolites, specialized pro‐resolving mediators (SPM) including resolvins, protectins, maresins, and lipoxins, biomarkers of ROS and NOS, and fatty acid and mono‐hydroxy fatty acid precursors. The positive mode panel includes cysteinyl leukotrienes, PAF, and maresin and protectin conjugates in tissue regeneration (MCTRs and PCTRs).

Concentration curves were generated by injecting 3 replicate injections of 8 different concentrations of each lipid mediator standard. Excellent linearity is observed with r2 of at least 0.997 for all analytes. Excellent sensitivity was obtained for all lipid mediator species with LLOQ between 0.05 and 1 ng/mL and CV < 30%. The unique qualitative/quantitative QTRAP platform also enabled MRM triggered EPI experiments which provided high sensitivity MS/MS data for confirmation of low level analytes. Baseline separation of LXA4 and 15‐epi‐LXA4 well as RvD1 and 17‐epi‐RvD1 enables the differentiation of enzymatic pathway utilization for pro‐resolving lipid mediator production.
MS/MSALL with SelexION®: A High‐throughput Lipidomic Solution for Untargeted Profiling
Authors: Pearson Mackenzie Jo , Kapil Santosh , Norris Paul , Ubhi Baljit , Dumlao Darren
Published: April 2020

Global lipid profiling is a growing area of interest and is a primary means to measure the changes in lipid molecular species as a function of human disease or drug treatment. One of the most challenging aspects of lipid analysis by mass spectrometry is isobaric overlap, or molecules having the same or nearly the same mass. With direct infusion, or ‘Shotgun’ approaches, the user is able to rapidly profile biological samples. On the Sciex 6600+ TripleTOF®, the MSMSALL method on this system generates data by collecting product ions of all precursor masses. Using this approach, thousands of lipids can be represented in a profile snapshot and allows for retrospective analysis of acquired data. However the issue of isobaric overlap is not addressed.

Isobaric overlap is a complex problem in lipidomic workflows and can make positive identification or quantification of a lipid species very difficult. The issue of isobaric overlap is often approached with liquid chromatography separation. However, lengthy sample preparation, method development, and lengthy chromatographic run times for adequate separation can narrow the scope of the lipidome and diminish throughput. Differential ion mobility separation (DMS) is a relatively new ion mobility technology that uses a molecule’s dipole moment to isolate it rather than shape and size. The SelexION® device is installed at atmosphere, so the addition of chemical modifiers can be applied to the system to assist in additional separation. In this study, 1‐propanol is used as the chemical modifier to separate the six phospholipid subclasses while cardiolipin’s ‐2H charge state is used to separate this class without the assistance of chemical modifier. Unique compensation voltages are then found with standards and assigned to each lipid class to ensure lipid class isolation. In negative ion mode, all seven classes can be acquired in just under twelve minutes of total run time. This allows for untargeted, high‐throughput acquisition of the lipidome while simultaneously acquiring MS/MS spectra for lipid species identification.

LipidView software is used to analyze the data collected from the SelexION® runs in a high throughput fashion. All samples from each class can be loaded and a lipid class can be assigned along with its corresponding COV. Since much of the isobaric overlap has been removed from the sample a positive identification and quantification can be made.
A surface‐exposed, network substitution results in a gain‐of‐function in tryptophan synthase6.5.0
Authors: D’Amico Rebecca N. , O’Rourke Kathleen F. , Bosken Yuliana , Murray Alec M. , Chang Chia-en A. , Boehr David D.
Published: April 2020

Tryptophan synthase (TS) is a α2β2 dimeric enzyme that catalyzes the final two reactions in the production of tryptophan. The product of the alpha subunit (αTS), indole, is channeled to the beta subunit (βTS) where it is then condensed with serine to form tryptophan. TS is a valuable model system for understanding enzyme allostery, and specifically enzyme‐enzyme communication. We previously identified amino acid interaction networks in αTS using NMR‐based methods. We proposed that these network residues play an important role in catalysis and communication between the two subunits. Based on previous studies, αTS residue Ala198 was identified as a residue of interest. Ala198 is a surface‐exposed, network residue that is dynamic on the ms‐μs timescale, according to our previous NMR studies. To better understand the networks, we characterized the A198W variant by performing cell‐based assays, kinetic assays, molecular dynamics (MD) simulations, and NMR experiments. We found that E.coli cells expressing the A198W variant had a faster growth rate in minimal medium compared to cells expressing the wild‐type αTS. Our kinetic studies on purified TS indicated that the A198W variant had little effect on αTS activity, but increased the activity of the TS complex (i.e. from initial indole‐3‐glycerol phosphate substrate to final tryptophan product), perhaps due to an enhancement of indole channeling. MD simulations indicated that the A198W substitution led to structural dynamic changes that propagated throughout αTS, including several key residues at the α/β binding interface. The MD simulations were consistent with the indole channel being more open, more often, which could help explain the increase in TS activity in vitro and in vivo. NMR experiments also indicated that the A198W substitution induced structural dynamic changes throughout αTS, consistent with the MD simulations. Despite Ala198 being distal from both the active site of αTS (27Å) and the α/β binding interface (30Å), substitutions at this position result in changes to catalytic efficiency and the TS complex dynamics. These findings underscore the importance of network residues on enzyme dynamics, allostery, and subunit communication. Engineering of surface‐exposed network residues represents one way of controlling enzyme activity.

6.5.0

Support or Funding Information

6.5.06.5.0
A Chaperon‐Like Protein HYPK Binds Arl4 Small GTPases to Modulate Cell Migration
Authors: Lee Fang-Jen Scott , Lin Ming-Chieh
Published: April 2020

ADP‐ribosylation factor (Arf)‐like 4 proteins are a group of small GTPases that control actin remodeling and promote cell migration. Their expression is finely tuned during development and only detectable in specific tissues. Dysregulation of Arl4s is linked with several cancers but little investigation reveals how they are regulated. Here we identified huntingtin yeast two‐hybrid protein K (HYPK) as a novel interacting partner of Arl4A and Arl4D, but not Arl4C. HYPK, primarily known as huntingtin (Htt) interacting protein, is reported to reduce aggregates and apoptosis induced by Htt and exhibited chaperone‐like activity. We show that HYPK interacts with Arl4A/D and modulates Arl4A/D‐dependent cellular phenotypes and cell migration ability. Arl4A/D does not appear to involve in HYPK‐mediated reduction of Htt aggregation. Residues 115–129 within C‐terminal conserved domain, possessing chaperon‐like activity of HYPK, is required and sufficient to interact with Arl4A/D. Losing of HYPK nullifies Arl4A‐induced cell migration, and only WT, but not Arl4A‐binding‐defective HYPK mutant, can rescue this phenotype. In addition, Arl4A exhibits reduced plasma membrane signals and its ability to maintain trans‐Golgi integrity in HYPK knockdown cells. Taken together, we infer that HYPK may serve as an upstream modulator of Arl4A/D to confer function of Arl4 small GTPases.

Support or Funding Information

This work was supported by grants from the National Health Research Institutes (NHRI) in Taiwan (NHRI‐EX106‐10601B1)
The Novel Roles of Dusp6 in Gut Barrier Modulation and Microbiome Shaping
Authors: KAO CHENG-YUAN , Chang Cherng-Shyang , Liao Yi-Chu , Huang Chih-Ting , Lin Chiao-Mei , Ruan Jhen-Wei , Liao Yu-Chieh
Published: April 2020

Gut microbiome has been associated with many human disorders and conditions such as allergy, obesity, inflammatory bowel disease and various types of cancers. Many of these diseases have become important health issues worldwide and effective strategies for their treatment and/or prevention are highly desired. We previously have found that dual‐specificity phosphatase 6 (Dusp6)‐deficient mice had reduced body weight gain and alleviated metabolic disorders. Notably, 16S rRNA gene sequencing revealed that the gut/fecal microbiota in Dusp6‐deficient mice was more resistant to high‐fat‐diet‐ induced dysbiosis compared with wild‐type mice. The RNA‐seq gut transcriptome analysis has shown several potential mechanisms driving the microbiota alteration in Dusp6‐deficient mice. We also found that Dusp6 knockout mice compared with wild‐type littermate mice were more resistant to dextran sulfate sodium (DSS)‐induced colitis and the mechanisms of the Dusp6 deficiency in shaping gut microbiome and barrier integrity were investigated. In summary, these findings indicate that Dusp6‐deficiency is a novel host genetic factor that could modulate gut barrier and inhibiting DUSP6 might be a novel strategy to maintain gut microbiome eubiosis.

Support or Funding Information

This work was supported by the following grants: IM‐108‐PP‐04 from NHRI and 106‐2628‐B‐ 400‐001‐MY3, 106‐2923‐B‐400‐001‐MY3, 108‐2321‐B‐400‐011‐ from MOST.
Investigation of HMGB1 binding interactions with cell clearance molecules
Authors: Wade Elizabeth , Troisi Maria , Broome Payson , Halsell Jonah , Alverado Alice , Desani Alexis , O’Murphy Fiona , Fairweather Samantha , Bell Anthony Jerome
Published: April 2020

High Mobility Group Box 1 (HMGB1) is a highly abundant DNA‐binding protein. HMGB1 was initially classified as an architectural nuclear protein that facilitates transcription factor binding, chromatin remodeling as well as genetic recombination and repair. It is now clear that HMGB1 also functions in the extracellular matrix (ECM) as a proinflammatory cytokine. More specifically, HMGB1 acts as a damage‐associated molecular pattern molecule. In this capacity, HMGB1 binds immune receptors (i.e. CXR4,RAGE and TLRs) to promote leukocyte migration and amplify proinflammatory gene expression. Ideally, HMGB1 would operate in the ECM to expunge infectious pathogens. However, this does not seem to always be the case. Studies show that the presence of HMGB1 in the ECM is also linked with diseases associated with defective cell clearance, such as lupus (SLE) and cystic fibrosis (CF). The investigation of HMGB1 in cell clearance is an understudied area of research. We hypothesize that HMGB1 exacerbates SLE and CF by binding to cell clearance molecules in the ECM to prevent the proper removal of apoptotic cells. In SLE, failed cell clearance can lead to breaking immune tolerance. In CF, chronic tissue damage is mediated by neutrophils that are not effectively engulfed by macrophages. We will evaluate HMGB1 binding interactions with four categories of cell clearance molecules: i)“eat‐me” signals, ii)“find‐me” signals, iii) phagocyte receptors and iv)adaptor molecules and opsonins. We will use circular dichroism (CD), far‐western blots and surface plasmon resonance to investigate HMGB1 binding interactions with each class of cell clearance biomolecule.

Support or Funding Information

University of San Diego
The Role of Nicotinic Receptors in the Attenuation of Autism‐related Behaviors in a Murine BTBR T+tf/J Autistic Model
Authors: AlSharari Shakir , Aldhalaan Hesham , Mahmood Hafiz , Alqasem Mashael , Alshammari Musaad
Published: April 2020

The nicotinic receptors are distributed throughout the central and peripheral nervous system. Postmortem studies have reported that some nicotinic receptor subtypes are altered in the brains of autistic people. Recent studies have demonstrated the importance of nicotinic acetylcholine receptors (nAChRs) in the autistic behavior of BTBR T+tf/J mice. This study was undertaken to examine the behavioral effects of pharmacologically targeted nAChRs using nicotine and mecamylamine (MEC) in a BTBR T+tf/J mouse model of autism. It was conducted a panel of behavioral tests relating to autism. The behavioral tests included the three‐chamber social interaction, self‐grooming, marble‐burying, locomotor activity (LA), and rota‐rod (RR) test. It was examined the effect of various oral doses of nicotine over a period of two weeks in BTBR T+tf/J mice. The results indicated that the chronic administration of nicotine modulated sociability and repetitive behavior in BTBR T+tf/J mice while no effects in C57BL/6J mice. Furthermore, the non‐selective nAChR antagonist (MEC) reduced sociability and increased the repetitive behaviors in BTBR T+tf/J mice. Overall, the findings indicate that the pharmacological modulation of nicotinic receptors is involved in modulating core behavioral phenotypes in the BTBR T+tf/J mouse model.
Group A Streptococci Shift the Balance of Muscular Mitochondrial Dynamics
Authors: Tsai Pei-Jane , Hsu Chih-Yu , Lai Wei-Ting , Wang Yi-Cian , Tsai Yau-Sheng
Published: April 2020

The most severe form of invasive group A Streptococcus (GAS) disease is necrotizing fasciitis described as rapidly muscle destruction. Muscles are responsible for energetic functions which are mostly provided by proper mitochondrial function. Mitochondria are the highly dynamic organelles, constantly undergoing fusion and fission. Pathogen aim at preserving the bacterial replication niche by targeting to mitochondria and inducing cell death. This prompted us to specifically analyze the effect on muscular mitochondria upon GAS infection. By using electron microscopic analysis of GAS‐infected muscle tissue, the muscle is strongly damaged and the mitochondria are fragmented and structurally abnormal. Further, we found GAS alters the morphology of cell mitochondria, inducing fragmentation of the mitochondria network which accompanied by decreasing the levels of fusion protein in mouse muscle cell, C2C12. Furthermore, our data demonstrate that mitochondrial function is not strictly dependent on morphology. Our results reveal that disruption of fusion induces more profound defects on lacking of fission during GAS infection, and imply that while fusion is required throughout life, fission is more important later in life likely to combat bacterial infection. Overall, our study provides important new insights into the central role of mitochondrial dynamics in innate immunity.

Support or Funding Information

MOST 108‐2320‐B‐006‐013
Exercise Training Combined with Hydrochlorothiazide is More Efficient than Antihypertensive Treatment Alone to Promote Cardiofunctional Benefits in a Model of Hypertension and Menopause
Authors: Ferreira Maycon , Silva Michel , Silva Gabriel , Dias Danielle , Bernardes Nathalia , Irigoyen Maria-Cláudia , De Angelis Kátia
Published: April 2020

The largest proportion of hypertensive women are in postmenopause and this hypertension is poor controlled. Experimental model has shown that exercise training (ET) is effective to reduce arterial pressure (AP) and to mitigate of autonomic dysfunction. Following current guidelines for hypertension treatment, the association of antihypertensives therapy (AT) with ET is strongly recommended. Here, we investigate the effects of the ET plus AT on functional and hemodynamic parameters in an experimental model of hypertension and menopause. Female spontaneously hypertensive rats (150–180g, 3 months old) were ovariectomized and randomly assigned into 3 groups (4–6 animals/group): ovariectomized (OVX) sedentary (OS), OVX sedentary treated with hydrochlorothiazide (OSH) and OVX trained treated with hydrochlorothiazide (OTH). Ovarian hormone deprivation was performed through bilateral ovariectomy. Rats underwent the maximal running test and 1‐repetition maximum (RM) test in the first, fourth and eighth weeks. Hydrochlorothiazide (30 mg/kg) was dissolved in the drinking water. Noninvasive arterial pressure (AP) was assessment by the tail cuff plethysmography. Concurrent ET (40–60% maximal capacity) was performed on a motor treadmill (aerobic training) and on a ladder adapted to rats (resistance training), 3 days/wk for 8 weeks. Carotid artery and jugular vein catheterization were performed and the systolic (SAP), diastolic (DAP) and mean AP (MAP), as well as heart rate (HR) were recorded. OTH presented a higher aerobic capacity compared to OS group (group effect, p<0.05). In addition, trained and treated with antihypertensive therapy showed a higher load resistance capacity in relation to OS and OSH groups (group effect, p<0.05). There was an interaction effect between time and group in both aerobic capacity (p<0.05) and load resistance capacity (p<0.05). Similarly, interaction effect was observed for noninvasive SAP (p<0.05), and OTH presented a lower value compared to OS group (group effect, p<0.05). In addition, DAP and MAP were lower in the OSH compared to OS group, as well as the OTH group showed lower HR in relation to OS group. No differences were observed for DAP and MAP between OSH and OTH groups. To date, our results suggest that exercise training associated with hydrochlorothiazide treatment induce additional cardiofunctional benefits than antihypertensive treatment alone.

Support or Funding Information

CAPES‐DS and FAPESP.
Expression of AIF, PARP and microRNAs miR‐145, miR‐210, miR‐486 associated with apoptosis in the corpus cavernosum of rats subjected to chronic alcoholism model
Authors: Fazan Valéria Paula Sassoli , Assis Cirino Mucio L. , Schiavoni Vagner S. , Lizarte Neto Fermino S. , Melo de Carvalho Camila A. , Novais Paulo Cezar , da Cunha Tirapelli Daniela Pretti , Molina Carlos Alberto F. , Júnior Silvio Tucci , Tirapelli Luis Feranado
Published: April 2020

Introduction

Since penile erection is dependent on competent vascularization, substances that are known to damage vessels and their functions can affect it. An example is excessive alcohol consumption. Chronic and excessive alcohol consumption has a cytotoxic effect that harms health in general, but especially in events that are dependent on the integrity of vascularization such as penile erection. Previous studies from our group described some consequences of using ethanol on penile erection. Nevertheless, the molecular mechanisms surrounding microRNAs, apoptosis process and their relationship with erectile dysfunction associated with alcohol consumption are still poorly understood.

Objectives

To evaluate the mechanism of apoptosis by the expression of AIF and PARP, as well as their regulatory microRNAs: miR‐145, miR‐210 and miR‐486, in the corpus cavernosum of rats submitted to a semivoluntary alcoholism model.

Material and methods

24 Wistar rats were divided into two groups: control (C) and treated with 20% ethanol (A) for seven weeks. The corpus cavernosum samples were prepared for immunohistochemical analysis of AIF and PARP protein expression, and gene expression for miR‐145, miR‐210, miR‐486 microRNAs in cavernous tissue was quantified by real time PCR.

Results

The immunohistochemical analysis showed little nuclear positive labeling for the protein PARP and AIF in the corpus cavernosum of both control and ethanol treated animals. The analysis of miR‐145, ‐210 and ‐486 microRNA expression in the 12 animals studied showed no significant difference between the control and alcoholized groups.

Conclusions

The expression of AIF and PARP and their regulatory microRNAs involved in apoptotic process (miR‐145, miR‐210 and miR‐486) were not altered in the corpus cavernosum of rats submitted to the semivoluntary alcoholism.

Support or Funding Information

FAPESP, CNPq, CAPES and FAEPA