bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic Effects of Finasteride on Human Leydig Cells
Authors: Ihentuge Churchill , Csoka Antonei
Published: April 2020

Finasteride is a synthetic 4‐azasteroid, used in the treatment of both androgenetic alopecia and benign prostatic hyperplasia. In some men, its use is associated with persistent erectile dysfunction, ejaculatory dysfunction, loss of libido, altered sperm counts and infertility ‐ even after cessation of the drug. This condition has been termed “Post‐finasteride Syndrome” (PFS). We want to understand the underlying biochemical mechanisms that govern the development of this condition. Therefore in this experiment we tested the effects of exposure of finasteride on human Leydig cells, the main hormone‐producing cell of the testis, to identify epigenetic changes in the cells that could be at least partially responsible for PFS. We cultured human Leydig cells in the presence of 0.5 μM finasteride for 14 days and performed whole‐genome DNA methylation analysis using the NimbleGen Human DNA Methylation 3×720K Promoter Plus CpG Island Array and Ingenuity Pathway Analysis. Here we describe initial identification of specific genes and pathways involved that may ultimately lead to treatments to reverse PFS.

Support or Funding Information

NIH/NIA (IR25AG047843‐01) to ABC
Involvement of ER stress‐mediated sEH Activation in Myocardial Ischemia‐Reperfusion Injury
Authors: Xue Hong-Mei , Sun Wen-Tao , Chen Huan-Xin , Wang Jun , He Guo-Wei , Yang Qin
Published: April 2020

Objectives

Previous studies have suggested the role of endoplasmic reticulum (ER) stress in myocardial ischemia/reperfusion (I/R) injury and inhibition of soluble epoxide hydrolase (sEH) was found to be protective against I/R‐induced injury. Whether ER stress mediates myocardial I/R injury via regulating sEH remains uninvestigated. The present study aimed to understand the role of ER stress‐sEH signaling in cardiac and coronary function in an in vitro rat model of myocardial I/R.

Methods

Isolated rat hearts were randomly divided into four groups including control, I/R, and I/R groups pretreated with either the IRE1α inhibitor GSK2850163 or the sEH inhibitor dicyclohexylurea (DCU). The hearts were mounted on a Langendorff apparatus and I/R groups were subjected to 30‐min global ischemia followed by 60‐min reperfusion. GSK2850163 and DCU were perfused for 15 min prior to the onset of ischemia. Left ventricular function was monitored during the experiment. Branches of the left anterior descending artery were isolated after I/R for study of endothelium‐dependent vasorelaxation in a myograph. The protein expression of IRE1, phosphorylated IRE1 (p‐IRE1), and sEH was determined by western blotting.

Results

Left ventricular end diastolic pressure (LVEDP) was increased and the maximum velocity of contraction (+dp/dtmax) and relaxation (‐dp/dtmax) of the left ventricle was decreased during I/R, which could be partially reversed by GSK2850163 or DCU pretreatment. Relaxant response of coronary artery to acetylcholine was also improved by GSK2850163 or DCU. Inhibition of IRE1 phosphorylation by GSK2850163 significantly downregulated the protein expression of sEH.

Conclusions

Activation of sEH induced by the IRE1 branch of ER stress is involved in I/R‐induced cardiac dysfunction, in which impairment of coronary dilator function is an important contributing factor.

Support or Funding Information

Supported by NSFC 81870227 & 81870288, Tianjin Science and Technology Commission (18PTZWHZ00060) & Binhai New Area Health Bureau (2018BWKZ005), and Non‐profit Central Research Institute Fund of Chinese Academy of Medical Sciences 2018TX31002 & 2019XK310001.
Do Changes in the Composition of Bone Marrow Adipose Tissue Affect the Development of Osteoarthritis?
Authors: Shepherd Rebecca F , Allison Kathryn , Rhodes Bryan , Kerns Jemma G , Taylor Adam M
Published: April 2020

There are three types of adipocytes in the human body, classified as white, brown and beige. Bone marrow adipose tissue (BMAT) is often described as having a heterogeneous appearance and has been suggested to be a distinct class of ‘yellow’ adipocytes. However, recent studies have also identified beige‐like adipocytes present in the bone marrow cavity, and they appear to have a relationship with the formation of bone like structures found in patients with osteoarthropathies, termed trabecular excrescences. The BMAT in osteoarthritic (OA) patients has an increased number of adipocytes compared to healthy and osteoporotic bone marrow. The objective of this study was to identify changes in BMAT composition between non‐osteoarthritic and osteoarthritic (OA) bone samples.

Human joint samples from 28 patients (14 non‐OA, 14 OA) were obtained with informed consent from patients undergoing joint surgery at University Hospitals of Morecambe Bay NHS Trust (Lancaster, UK). North of Scotland NHS Research Ethics Committee (UK) approved this research. Samples were prepared for routine histological analysis and flow cytometry. Flow cytometric analysis gated for adipocytes by size and lipid content. Markers for adiponectin (Acrp30), uncoupling protein 1 (UCP1) and myogenic factor 5 (Myf5), were used to distinguish between white, beige and brown adipocytes. To corroborate the results, sections of joint tissues underwent immunohistochemical analysis. The anatomical location of the adipocytes within the bone marrow cavity was also studied.

The data analysis showed an approximate 9:1 ratio of white to beige adipocytes found within BMAT, with only trace amounts of brown adipocytes identified. Statistical analysis to account for disease, age and biological sex demonstrated no significant difference in the adipocytes present between control and OA samples. However, male sex and ageing were shown to be factors for an increase in beige adipocytes. The histological study confirmed the presence of beige adipocytes and showed that, when present the beige adipocytes were found closer to the bone surface than the centre of the bone marrow adipose tissue.

Previous studies have argued that BMAT is a distinct class of adipocyte. This study has demonstrated the presence of beige adipocytes within the BMAT of patients with and without OA. The methods used give a quantitative method of identifying different types of bone marrow adipocytes. Understanding changes in the anatomical composition of BMAT and the role of adipocytes in skeletal health may prove important in understanding the progression of age associated bone disorders.

Support or Funding Information

This work was funded by the Anatomical Society and sponsored by the American Association for Anatomy.
Mathematical Modeling of In Vitro‐In Vivo Atypical Kinetics of Diazepam
Authors: Wang Zeyuan , Nagar Swati , Korzekwa Ken
Published: April 2020

Cytochrome P450 (CYP) and uridine 5'‐diphospho‐glucuronosyltransferase (UGT) enzymes are responsible for metabolism of most marketed drugs. Due to the large binding sites of certain CYPs and UGTs, CYP and UGT substrates tend to show non ‐ Michaelis‐Menten kinetics (atypical kinetics). Although several in vitro data provide evidence for the existence of atypical kinetics, this characteristic is seldom observed with in vivo data. The purpose of this project is to characterize the atypical kinetics of diazepam and its sequential metabolism in in vitro and in vivo systems. Numerical methods will be used to obtain kinetic parameters, which will be used for in vitro‐in vivo correlations (IVIVC).

Diazepam, which exhibits atypical kinetics in vitro, and its primary metabolites (temazepam, nordiazepam), and secondary metabolite (oxazepam) were characterized with in vitro studies with pooled male Sprague Dawley rat liver microsome (MRLM), pooled female Sprague Dawley (FRLM), and purified rat cyp isoforms (cyp3a1, cyp3a2, and cyp2c11) separately. Rat microsomal partition and rat plasma protein binding assays were conducted via equilibrium dialysis to determine the free fraction of the drug. Two substrate binding (ESS) models provided better fits than one substrate (ES) models, as determined from the resulting AICc values. Preliminary in vitro data from MRLM and cyp3a1 showed obvious sigmoidal saturation curves for temazepam formation. Only cyp3a1 show significant sigmoidal curves for nordiazepam formation. For cyp3a1, Km and Vmax values for temazepam and nordiazepam calculated with the EES model are significantly different from those with the ES model. The intrinsic clearance (CLint) values calculated from the MRLM and cyp3a1 ESS models is not constant within the observed range of diazepam plasma levels of rats. No obvious sigmoidicity of diazepam saturation curves was observed for cyp3a2 (male‐specific) and cyp2c11 (male‐specific) isoforms, which are other cyp isoforms responsible for diazepam metabolism in male rats. In order to characterize in vitro‐in vivo correlations for diazepam, in vivo PK studies with female and male rats are currently underway.

Support or Funding Information

This research was supported by NIGMS grants: R01GM114369 and R01GM104178.
Agreement between ACSM’s equation and cardiopulmonary exercise test in determine maximal oxygen uptake in individuals with metabolic syndrome
Authors: Ceolin-Nascimento Reginaldo , Cepeda Felipe Xerez , DeMoura José Roberto , Camargo Fernanda Cristina Ferreira , Fortes-Queiroz Lucas , Ferreira Fabiana Gonçalves , Hussid Maria Fernanda , Consolim-Colombo Fernanda M. , Correia Marilia Almeida , Trombetta Ivani Credidio
Published: April 2020

Purpose

Low functional capacity is associated with cardiovascular risk factors. Cardiopulmonary exercise test (CPET) is the gold standard evaluation to determine functional capacity through maximal oxygen uptake (VO2max) measurement. However, the high financial cost, the material and the time spent on this method difficult its use in a large number of clinicals. In this sense, it is possible to determine VO2max via the equivalence formulas. The aim of this study was to verify the agreement between indirect calculation of VO2max using American College of Sports Medicine (ACSM) equation and the directly measure of maximal oxygen uptake in metabolic syndrome (MetS) patients.

Methods

Sixty non‐diabetic and without medication patients with MetS (ATP‐III) were divided in 2 groups, according to gender: Male (n=32; 46±9 y; 31.2±3.0 kg/m2); and Female (n=28; 49±9 y; 32.7±3.1 kg/m2). VO2max was measured during CPET in a cycle ergometer with ramp protocol and estimated by ACSM’s equation [VO2= 7.0+ (1.8 x work rate)/body mass].

Results

All patients included achieved respiratory exchange rate (RER=VCO2/VO2) >1.10 at maximal exercise. In Male group, despite correlation between methods (R=0.78; P=0.0001), VO2max estimated by ACSM’s equation was higher than the VO2max of the direct measurement (28.7±3.1 vs. 26.9±6.0 ml/kg/min; P=0.049). Similarly, in Female group there was correlation (R=0.79; P=0.0001), however VO2max estimated by ACSM’s equation was higher than direct measured VO2max (20.3±3.3 vs. 18.8±3.3 ml/kg/min; P=0.0008). In both genders, Bland‐Altman plot analysis suggested a lack of agreement between predicted and direct VO2max.

Conclusion

In bout groups, Male and Female, the ACSM’s equation overestimates the VO2max. It seems that ACSM’s equation is not capable of accurately predicting VO2max in men and women with MetS.
Anatomy Tour at the Mall; an Outreacha Activity for the Public by Nursing and Physical Therapy Students
Authors: Sola Maryvi Gonzalez , George Michael , Woodson Cassandra , Junious Victoria , Jose Aleena , Rosario Martin G.
Published: April 2020

Introduction

Anatomical kinesthetic education is one of the best active learning techniques to educate clinicians to avoid having limited exposure to clinical training. An outreach entitled ‘Anatomy Tour at the Mall’ allows students to practice while interacting with the public who could benefit from these anatomical educational activities.

Aims

1. Have the nursing and physical therapy (PT) students give participants a pre‐test, 2. engage with the public by teaching them anatomy and execute non‐invasive free tests including vital signs, balance and visual tests, and 3. giving them a post‐test and survey. The hypothesis is this activity will allow the students to assimilate knowledge by practicing their skills while edifying the public, which will likewise learn significantly by participating.

Methods

A 10‐question pre‐test (1 point each) was given at the beginning to the public after signing a consent form (IRB approved). Then, participants will pass by 6 stations about the following systems: respiratory, nervous, special senses, musculoskeletal, cardiovascular and human organs, managed by both nursing and PT students. Finally, a 10‐question post‐test (1 point each) and a survey were given to the participants and students.

Results

A total of 77 male and female individuals participated and completed the pre‐test (average 5.42), but only 57 completed the post‐test (average 7.24, 0.00 t‐test, 1.28
st.dev) achieving higher scores in all the stations except for the musculoskeletal. Also, 64 participants rated the Anatomy tour with A, 6 B, and 1 C. Finally, a 1–5 Likert scale of the Anatomy tour being ‘worthy of their time’ and ‘completely beneficial activity’ (both 1 being ‘strongly disagree’ and 5 being ‘completely agree’), the median was 5. The student’s survey revealed that out of 28 students, 15 rated the activity with A, 11 B, and 2 C, 24 students said this activity was beneficial for their future careers and 25 would consider participating again. Additional data is currently being collected and yet to be analyzed.

Discussion and Conclusion

Both the students and the public agreed on this activity is beneficial and constructive for their education. The overall post‐test indicates the public learned about the aforementioned anatomical stations.

Support or Funding Information

Faculty Scholarship Award by West Coast University
Chronic Kidney Disease Impact on Total Joint Arthroplasty Outcomes: A National Inpatient Sample Based Study
Authors: DiMagno Allyson N , Hussein Inaya Hajj , Sayeed Zain , El-Othmani Mouhanad M
Published: April 2020

INTRODUCTION

In the United States, chronic kidney disease (CKD) affects roughly 11% of the population or 19.2 million people. As the prevalence of CKD and demand for total joint arthroplasty (TJA) continue to rise, it is critical to assess the impact of CKD on post‐operative clinical and economic outcomes.

METHODS

Discharge data from 2006–2011 National Inpatient Sample (NIS) was used for this study. A total of 851,150 TJA patients were divided into 3 cohorts: Group 1 included no CKD, CKD Stage I, and CKD Stage II; Group 2 included CKD Stage III and Stage IV; Group 3 included CKD Stage V. Inverse Probability of Treatment Weighting (IPTW)/Propensity Score Weighting was used to predict outcome variables as a function of age, sex, and Elixhauser comorbidities. Patients were compared against group I for in‐hospital postoperative outcomes.

RESULTS

Stage III/IV CKD patients undergoing primary TJA had higher odds of any complication (odds ratio [OR], 2.63; P<0.0001), longer length of stay, and higher total charge (length of stay [LOS], 4.34 vs 3.48 days; total charge, $56,003 vs $46,115; P<0.0001) when compared to patients with no CKD/Stage I or II. Similarly, Stage V CKD patients undergoing primary TJA had higher odds of any complication (OR, 1.64, P<0.0001), longer LOS, and higher total charges (LOS, 5.81 vs 3.48 days; total charge, $59,869 vs $46,115) than their counterparts with no CKD/Stage I or II CKD.

DISCUSSION

Our results indicate that Stage III, IV, or V CKD, compared with those with no CKD, Stage I or II patients are at a greater risk for postoperative complications and consume more resources following TJA.
Crosstalk between brown adipocytes and macrophages
Authors: Zurkovic Jelena , Sanyal Abhishek , Mass Elvira , Schlitzer Andreas , Pfeifer Alexander
Published: April 2020

There are two types of adipose tissue (AT): white adipose tissue (WAT), which is involved in energy storage, and brown adipose tissue (BAT) that dissipates energy in the form of heat (non‐shivering‐thermogenesis). AT acts as an endocrine organ that secretes hormones or cytokines known as adipokines. Dysregulation of secretion of adipokines, caused by adipocyte hypertrophy and dysfunction, is directly linked to chronic inflammation. Inflammation of WAT during obesity leads to increased expression and secretion of pro‐inflammatory cytokines such as TNF‐α, IL‐6, MCP‐1 (Villarroya et al., 2018). This increase in adipocyte‐derived pro‐inflammatory cytokines causes a dramatic shift in the immune cell population, especially in macrophages, and leads to metaflammation. On the other hand, adipose tissue‐resident macrophages (ATMs) play a very important role in innervation and energy expenditure of BAT (Wolf et al., 2017). All these facts are the reason why we investigate signal pathways as well as secreted molecules that play a role in the communication between brown adipocytes and ATM. We are focusing on the crosstalk between brown adipocytes and macrophages. We used three in vitro models: direct co‐cultivation, transwell system and conditioned medium. In our experiments, we used two different types of macrophages: fetal liver derived macrophages (FLDMs) and bone marrow derived macrophages (BMDMs). We found that macrophages regulate expression of adipogenic (e.g. PPARg) as well as thermogenic (e.g. UCP1) markers. Interestingly, we observed that brown adipocytes produce different factors that regulate macrophages and their pro‐ versus anti‐inflammatory phenotype. These data indicate a crosstalk between macrophages and adipocytes in BAT.

Support or Funding Information

Institute of Pharmacology and Toxicology, University of Bonn, 53127 Bonn, Germany Research Training Group 1873, University of Bonn, 53127 Bonn, Germany
Effect of the N‐terminal Region of Arabidopsis thaliana LARP6C in RNA Binding Specificity
Authors: Roberts Julia , Lewis Karen
Published: April 2020

The La‐related proteins (LARPs) are a superfamily of RNA‐binding proteins that are distinguished by a core RNA binding domain called the “La Module”. This family is highly conserved across eukaryotes and exerts diverse functions in RNA processing and function. The genetic model vascular plant, Arabidopsis thaliana (At), has three paralogs of LARP6, denoted “A”, “B”, and “C”. Of these paralogs, AtLARP6B and AtLARP6C are more closely related to each other due to an N‐terminal sequence motif known to associate with other RNA binding proteins, the “PAM2w” motif. Previous work evaluated the RNA binding activity of the isolated AtLARP6C La Module. Recent work on vertebrate LARP4A suggests that N‐terminal PAM2 motifs are important for higher‐order assembly of regulatory complexes. We have generated a set of recombinant constructs to test the role of both the NTR and PAM2w motif in RNA binding activity. These protein variants are stably expressed and have been highly purified for use in electrophoretic mobility shift assays (EMSAs) to measure RNA binding activity. Preliminary work indicates that binding affinity is not significantly impacted by either the NTR or the PAM2w motif, and current studies are focusing on the role that these regions play in ligand specificity. By characterizing the binding specificity of these AtLARP6C proteins to a panel of RNA ligands, we will gain significant insight into the contribution of this domain and motif to ligand binding.
Biological and Pharmacological Profiling of Pentosan Polysulfate (PPS) in Comparison to Heparin and its Relative Neutralization by Protamine Sulfate
Authors: Venkitasubramony Vishnu , Polisky Anna , Siddiqui Fakiha , Kouta Ahmed , Fareed Jawed , Hoppensteadt Debra , Iqbal Omer , Laddu Atul , Jeske Walter
Published: April 2020

Background

Pentosan Polysulfate has traditionally been used to treat patients with Interstitial Cystitis and more recently osteoarthritis, cancer, HIV and prion disease. Such widespread clinical application of PPS and the active search for new effective alternatives to traditional heparin compels a comprehensive investigation of the effect of PPS batches on anticoagulation in comparison to Heparin. The purpose of this study is to create a comprehensive profile and comparison of new PPS batches (L‐15, L‐19, L20 and Ba), old PPS batch (PPS Ba), and Heparin. Furthermore, the feasibility of protamine sulfate as a neutralizing agent to reverse the anticoagulant effects of PPS is investigated.

Materials and Methods

Various batches of PPS at 1 mg/ml were prepared in saline. For whole blood clotting profile, thromboelastography (TEG) was carried from 2.5 – 5.0 ug/ml and activated clotting time (ACT) at 25ug/mL. Clotting profile in whole blood, retrieved plasma and supplementation in normal human pooled plasma (NHP) were carried out at 100 – 0.0 ug/ml by using anticoagulant assays. Amydolytic methods were used to measure anti‐Xa/ IIa effects. Thrombin generation inhibition studies (TGA) were carried out in NHP from 100 ‐ 0.0 ug/ml. Heparin supplementation and protamine sulfate neutralization in NHP from 10 ‐0 ug/mL were compared to PPS batches in a logarithmic scale.

Results

All drugs produced a concentration dependent effect. Similar effects in anticoagulation assays were noted except anti‐Xa assay. In TEG, significant viscoelastic changes were observed at 5ug/mL. Prolongation of ACT was observed at 25ug/mL. No significant differences were noted in between batches of PPS in TEG and ACT studies. In TGA, concentration dependent but no significant difference in between the batches were noted. Neutralization of protamine sulfate was observed up to 12.5 ug/mL in anticoagulant assays and marginal effect on anti‐protease assays. Lastly, logarithmic comparison of the protamine sulfate neutralization indicated that protamine sulfate is a weaker neutralizing agent of PPS compared to heparin.

Conclusion

These studies suggest that multiple batches of PPS have a similar anticoagulant profile. In comparison to heparin, PPS presents itself as a 10‐fold weaker anticoagulant alternative for use in a variety of clinical applications. Neutralization of PPS at low concentrations by protamine sulfate in anticoagulant assays warrants further investigation on an effective neutralizing concentration for clinical use.
The Genome‐wide Landscape of Oxidative Base Damage in Hypoxia; Potential Role in Metabolic Reprogramming in Chronic Lung Disease
Authors: Daly Grant , Houserova Dominika , Francis Christopher Michael , Andrews Joel , Langley Raymond J. , Pastukh Viktor M. , Bass Hank W. , Borchert Glen M. , Gillespie Mark N.
Published: April 2020

Hypoxia, a common attribute of many lung diseases, causes an adaptive response characterized by glycolytic reprogramming, which is initiated in part by the master transcriptional regulator, Hif‐1, accumulating in response to reactive oxygen species (ROS) generated as second messengers in hypoxic signaling. ROS produced in hypoxia also appear to engage a DNA damage and repair pathway in promoter regulatory elements that impacts core events in transcription (Pastukh, et al., 2015). Because the genome‐wide disposition of base damage in hypoxia could provide clues about mechanisms of persistent metabolic reprogramming in the lung, we executed a proof‐of‐concept study to define the landscape of the common base oxidation product 8‐oxoguanine (8‐oxoG) in the genomes of normoxic and acutely hypoxic human umbilical vein endothelial cells (HUVECs), focusing on localization of base damage within genes comprising the hypoxic transcriptome. Using ChIP‐seq analysis to delineate sequences harboring 8‐oxoG, we found that base damage was enriched in multiple regulatory regions, including those defined by transcription factor binding motifs and epigenetic marks. Of particular interest, base damage in these regions occurred in distinctly different genes in normoxic and hypoxic cells. SIM super‐resolution immunofluorescence microscopy revealed prominent base damage in facultative heterochromatin residing in close proximity to chromatin‐free channels that appeared to be in direct communication with the nuclear membrane. A comparison of sequences demarcated by 8‐oxoG ChIP‐seq signals to the RNA‐seq‐defined hypoxic transcriptome revealed that base damage was prominent in genes occupying regulatory nodes in pathways controlling the metabolic response to hypoxia as well as other key adaptive pathways. Finally, after noting that sequences harboring 8‐oxoG displayed elevated variant densities, we discovered that a high proportion of these variants were likely mutational artifacts since they were corrected by enzymatic excision of damaged bases in template DNA prior to PCR‐based sequencing library preparation. Collectively, these findings suggest that hypoxia fundamentally alters the genomic landscape of 8‐oxoG in HUVECs, causing accumulation or dissipation of the base oxidation product in non‐coding regulatory regions in differentially‐transcribed genes. The presence of base damage‐associated mutational artifacts supports the concept that the DNA damage and repair pathway engaged during hypoxic transcriptional signaling could impose a mutagenic threat leading to formation of permanent and currently uncharacterized somatic variants with the potential to drive persistent metabolic dysregulation in hypoxia‐related lung diseases.

Support or Funding Information

NIH
Nix induced mitochondrial fission and mitophagy inhibits myocyte insulin signaling through mTOR and p70S6 kinase
Authors: Gordon Joseph W. , da Silva Rosa Simone Cristina , Field Jared T. , Martens Matthew D. , West Adrian R. , Aliani Michel , Dolinsky Vernon W.
Published: April 2020

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor Nix is responsive to lipotoxicity and accumulates in response to diacylglycerols induced by high‐fat (HF) feeding. In addition, previous studies have implicated autophagy and mitophagy in muscle insulin sensitivity. To provide a better understanding of these observations, we undertook gene expression array and shot‐gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy‐related genes including Beclin‐1, ATG3, and ‐5. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the previously reported roles of these phospholipids and Nix in mitochondrial dynamics, we investigated aberrant mitochondrial fission and turn‐over as a mechanism of myocyte insulin resistance. In a series of gain‐of‐function and loss‐of‐function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium‐dependent activation of DRP1, and mitophagy. In addition, Nix‐induced mitochondrial fission leads to myotube insulin resistance through activation of mTOR‐p70S6 kinase and inhibition of IRS1, which is contingent on phosphatidic acids and Rheb1. These findings provide insight into the role of Nix‐induced mitophagy and myocyte insulin resistance during an overfed state when overall autophagy‐related gene expression is reduced.

Support or Funding Information

Funded by Research Manitoba, the Children’s Hospital Research Institute of Manitoba, and the Diabetes Research Envisioned and Accomplish in Manitoba (DREAM) theme.
Negative Modulation of α4β2* Nicotinic Acetylcholine Receptors During Postnatal Maturation of the Mouse Prefrontal Cortex
Authors: Hewitson Elizabeth M. , Bailey Craig D. C.
Published: April 2020

Acetylcholine (ACh) signalling within the medial prefrontal cortex (mPFC) is important for the normal development and function of prefrontal cognitive networks. Layer VI of the mPFC is highly populated with pyramidal neurons that express the α4β2* subtype of nicotinic acetylcholine receptor (nAChR), and the activation of these receptors by ACh is central to its function in this region. These receptors can be negatively modulated through several forms of inhibition. For example, nicotine potently desensitizes nAChRs to ACh activation, while the endogenous progesterone‐derived neurosteroid 3α‐hydroxy‐5α‐prenan‐20‐one (allopregnanolone; ALLO) inhibits nAChR activation by ACh through a presumed negative allosteric modulation. Both forms of inhibition mediated by nicotine and ALLO have been demonstrated for α4β2* nAChRs located on mPFC layer VI neurons in young postnatal mice. However, the relative strength for both forms of inhibition in mature mice, compared with the young postnatal age, is not clear. In addition, the mechanism of action for ALLO inhibition of α4β2* nAChRs is not well understood. Using whole‐cell electrophysiology, we measured the ability of both nicotine (300 nM) and ALLO (10 μM) to inhibit 1 mM ACh activation of α4β2* nAChRs located on mPFC layer VI neurons from male and female CD1‐strain mice at postnatal day (P)15‐20 (young postnatal age) and P80‐120 (adulthood). For nicotine, nAChR desensitization was greater at P15‐20 than at P80‐120, and this result was driven by an effect of postnatal age in male mice only. Conversely, ALLO inhibited nAChR function to a similar degree at both P15‐20 and P80‐120. Ongoing ACh binding experiments aim to determine the mechanism of action for ALLO inhibition of these receptors at both ages. Results from this study confirm that the endogenous neurosteroid ALLO inhibits mPFC α4β2* nAChRs in adulthood as it does in young postnatal life. Developmental changes to the magnitude of nicotine desensitization but not ALLO inhibition suggests that different factors regulate each type of negative modulation at α4β2* nAChRs, and that these factors are differentially altered during postnatal maturation of the mPFC.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 2019‐04989 to CDCB
Molecular Dynamic Simulation Approach Provides Insights into the Tumorigenesis of the N Terminal Portion of the Retinoblastoma Protein
Authors: Vagnone Pietro , Shor Audrey , Borysov Sergiy
Published: April 2020

The Retinoblastoma (Rb) protein is a master regulator of fundamental biological processes, such as the cell cycle entry, DNA replication, and apoptosis. Its functional inactivation is strongly implicated in the development of cancer, including retinoblastoma, osteosarcoma, breast, and lung cancer. Here we analyzed structural organization of the N terminal portion of Rb (RbN), known to harbor familial tumorigenic in‐frame deletions of exons 4, 5, 7 and 9, and exert a direct inhibitory effect on CMG helicase and DNA polymerase α via a bipartite mechanism. By utilizing the Molecular Dynamic Simulations, we reconstructed these RbN variants to visualize how accessibility of the CMG helicase and DNA polymerase α inhibitory domains is altered in the Δ4, Δ5, Δ7, and Δ9 RbN mutants. Our RbN models further supported the proposed bipartite mechanism of DNA replication inhibition by RbN and highlighted key structural components of RbN’s inhibitory domains. Our results reinforced previous studies, and provided insights on how mutations within the RbN contribute to tumorigenesis.
The Morphology of Ballistic Trauma
Authors: Baker Stephanie Anne
Published: April 2020

In forensic anthropology, bone fracture morphology provides potential evidence in the mechanism of injury or death. Specifically, the type of weapon used, the trajectory of impact, and the direction of fire can also be interpreted from internal and external bone morphology. Morphological characteristics, such as buttresses and sutures, are also applicable when analyzing ballistic trauma. This study analyzed fracture morphology of sagittal and coronal plane cranio‐maxillofacial gunshot wounds of five specimens from the Applied Anatomical Research Center’s (AARC) skeletal collection. Methodologies include enumerating and describing the etiology of ectocranial fractures, any missing bony segments, and the macroscopic fractures for each specimen. Sliding calipers were used to measure the distance (mm) of the entrance and exit wounds in relation to identifiable bony landmarks. The distribution of fractures and fracture locations on the cranio‐maxillofacial region were analyzed and assessed to determine similarities and differences among the sagittal and coronal plane gunshot wound specimens. Results indicate that radiating fractures run parallel to buttressed areas of the cranial vault in both sagittal and coronal plane gunshot wounds but run perpendicular to buttressed areas of the face in sagittal plane specimens only. The value of this research could provide additional evidence to reconstruct death histories of relatively incomplete skulls with suspected ballistic trauma. The ultimate goal is to provide the medicolegal community with a larger, more refined data set to better understand ballistic trauma by examining fracture morphologies of sagittal and coronal gunshot wounds in skeletonized human remains.
Effect of an individualized food‐based nutritional intervention on serum vitamin A, E, and carotenoids concentrations in recently diagnosed breast cancer patients
Authors: Prado Jessica Avila , Salcido-Sauceda Diana G. , Limon Miro Ana T. , Pacheco Moreno Bertha I. , Garcia Humberto Astiazaran , Teros Veronica Lopez
Published: April 2020

Breast cancer (BC) is the most common type of neoplasm among Mexican women. The antineoplastic treatment can affect the patient’s nutritional status; especially micronutrients, such as retinol, carotenoids and α‐tocopherol. The objective was to evaluate the effect of a food‐based individualized nutrition intervention on the retinol, carotenoid and α‐tocopherol serum concentrations of recently diagnosed BC patients, at baseline and 6mo after the intervention. This is a pretest‐posttest study in women with a recent diagnosis of invasive ductal/lobular carcinoma of the breast (clinical stage I–III). The individualized nutrition intervention was developed upon the nutritional assessment of participating women; adjusting the dietary plan on their total energy expenditure, and following the WCRF/AICR guidelines, adapting 1.5 g/kg/d of protein intake, 5–9 servings of fruits and vegetables and a caloric restriction (500–1000 kcal/d) when applicable (BMI ≥25 kg/m2). Baseline and final measurements include anthropometry, body composition by dual x‐ray absorptiometry, 24‐hour dietary recall, and the assessment of retinol, carotenoids and α‐tocopherol serum concentrations. Nineteen women between 33 and 68 years completed the intervention. At the time of diagnosis, 74% of the women were overweight or obese, and serum retinol was <1.05 μmol/L in 42% (n=8). After the 6‐month nutritional intervention, BC patients decreased an average of 2.67 kg of body weight (p <0.001), and lost ~2 kg of body fat (p<0.01). After the intervention, serum retinol increased +0.83 μmol/L (p<0.001) and α‐tocopherol increased +1.89 μmol/L (p<0.02) increased, which was positively correlated with a dietary increase of these micronutrients (p<0.05). No difference was found for provitamin A carotenoids or xantophils (β‐carotene, α‐carotene, β‐cryptoxanthin, lutein, zeaxanthin and lycopene) (p>0.05). The implementation of an individualized nutritional intervention before and during antineoplastic treatment prevents the nutritional deterioration of BC patients. This trial was registered at clinicaltrials.gov as NCT03625635.

Support or Funding Information

JAP and ATLM, received a fellowship from the National Research Council (CONACyT).
Sex‐selective Effects of MCH Antagonism on Metabolic Hormones
Authors: Gilman Lee , Beaver Jasmin N. , Vasquez Erin , Bowman Melodi A. , Vitela Melissa , Daws Lynette C.
Published: April 2020

Melanin‐concentrating hormone (MCH) is a neuroactive peptide that influences eating and emotional behaviors, as well as insulin sensitivity and adiposity. The vast majority of MCH research has focused only on males. Further, how endogenous levels of MCH parallel metabolic and behavioral measures over time, or are affected by diet, has not previously been investigated. We evaluated how endogenous MCH changes, as a function of sex, in adult rats as they consumed either a control diet alone, or a control diet supplemented with a high fat/high sugar (i.e., “Western”) diet. During the final week of this 8 week dietary manipulation period, rats received daily injections of the MCH receptor 1 antagonist GW803430 (3 mg/kg) or vehicle (1 mL/kg). Weekly blood sampling for the duration of the diet manipulation allowed for measurement of circulating MCH, insulin, leptin, and glucose levels. Surprisingly, we noted that GW803430 significantly reduced endogenous MCH levels only in males consuming control diet. Endogenous insulin levels were also dramatically lowered in males receiving GW803430, independent of diet condition. Though female rats did not exhibit these hormonal changes in response to drug, they did exhibit diet‐ and drug‐specific emotional behavior responses in the forced swim test that mapped on to endogenous MCH levels. Ongoing analyses are examining leptin levels, food consumption, and hepatic steatosis in relation to endogenous MCH. To our knowledge, these are the first investigations into how MCH receptor 1 antagonism affects circulating metabolic hormones, and into how this drug affects emotional behavior in females. To confirm involvement of the MCH receptor 1 in these sex‐selective hormonal reductions and behavioral effects, future experiments will employ a different MCH receptor 1 antagonist.

Support or Funding Information

This work was supported by a Morrison Trust grant to LCD, a NARSAD Young Investigator Award from the Brain & Behavior Research Foundation and Vital Projects Fund, Inc. to TLG, and National Institutes of Health grants 1R01 MH106978 and R21 DA046044 to LCD.
Exposure to Environmental Pollutant Benzo[a]pyrene Exacerbates Skin Inflammation in a Mouse Psoriatic Model
Authors: Klein Joshua Alan , Singh Satyendra K. , Goswami Dinesh G. , Braucher Leah N. , Wright Holly N. , Noland Erica L. , Agarwal Rajesh , Tewari-Singh Neera
Published: April 2020

A rapid increase in the global prevalence of skin inflammatory diseases like psoriasis cannot be attributed to genetics alone. Environmental factors including major ubiquitous organic pollutants like polycyclic aromatic hydrocarbons (PAHs) could contribute to their pathophysiology. Benzo[a]pyrene (BaP) is one of the PAH that is generated mainly from cigarette smoke, wood‐burning, automobile exhaust etc. The molecular mechanisms that lead to inflammatory skin diseases following exposure to BaP are not well elucidated, though there are suggestions that these responses could be mediated by aryl hydrocarbon receptor (AhR), a xenobiotic sensor. To investigate the effect of BaP exposure on skin inflammation in a mouse psoriatic model, the dorsal skin of naïve C57BL/6 mice was shaved (48h prior to exposure) and exposed to 62.5mg of 5% imiquimod (IMQ) cream once daily for five days. For assessing the initiation or exacerbation of psoriasis from exposure to BaP, mice were exposed 64μg BaP in 50μl acetone for five days before IMQ application [BaP+IMQ] or for five days together with the IMQ [(BaP+IMQ)] application. Following the analysis and scoring of clinical lesions, mice were sacrificed, and skin sections were analyzed for inflammation‐related histopathological and molecular changes. BaP exposure together with IMQ exacerbated IMQ‐induced psoriatic inflammatory lesions including the skin bi‐fold thickness, epidermal and dermal thickness, hyperkeratosis, dermal fibrosis, neutrophil infiltration, neutrophil degeneration and mast cell degranulation. Molecular analysis on the skin samples to further elucidate the effect BaP on IMQ‐induced inflammatory markers such as neutrophil infiltration, macrophages, inflammatory cytokines that could exacerbate psoriasis is being carried out. In other studies, defining the role of AhR in BaP‐induced biological, molecular, and metabolic alterations in mouse skin, concomitant treatment with AhR antagonist CH‐223191 was given before BaP+IMQ treatment. The antagonist treatment before the (BaP+IMQ) treatment abrogated the BaP‐caused increase in epidermal and dermal thickness in psoriatic mice. The effect of the AhR inhibitor on other biological and molecular psoriatic parameters that are exacerbated on exposure to BaP are also being analyzed to decipher the role of AhR in BaP‐induced and/or exacerbation in inflammatory skin diseases like psoriasis.

Support or Funding Information

Skaggs Scholars Program Grant, University of Colorado Denver and Start‐up Fund to Neera Tewari‐Singh, Michigan State University
Mechano‐therapy using high loads enhances regrowth of skeletal muscle in young, but induces muscle damage in old rats.
Authors: Hettinger Zachary R. , Confides Amy L. , Hamagata Kyoko , Butterfield Tim A. , Dupont-Versteegden Esther E.
Published: April 2020

Introduction

Older adults have an impaired ability to recover from skeletal muscle atrophy compared to young, predisposing them to loss of independence and poor quality of life. We have previously shown that mechano‐therapy in the form of cyclic, compressive loading (CCL) enhances regrowth of atrophied skeletal muscle in young adult rats, but not in old. Our preliminary data show that passive, transverse stiffness of muscles from old rats is elevated compared to young, potentially blunting the mechanical stimulus and subsequent growth response. In the current study, we hypothesized that an increased CCL load proportional to the higher tissue stiffness in old rats would enhance skeletal muscle regrowth when compared to atrophied muscles of old rats subjected to a lower load of CCL or ambulation alone.

Methods

Young (10 months; N = 6) and old (30 months; N = 6) adult male F344/Brown Norway rats underwent 14 days of hind limb suspension before being randomly assigned to one of the following: reloading for 7 days without CCL (RE); reloading with 4.5 N CCL (RE + CCL, 4.5 N); or reloading with 7.6 N CCL (RE + CCL, 7.6 N). Starting on day 0, four bouts of CCL were administered to the right gastrocnemius using a custom designed device capable of applying a precise load, with 48 hrs between bouts. Gastrocnemius muscle sections were analyzed for muscle fiber cross‐sectional area (CSA), satellite cell abundance (Pax7+/fiber), embryonic myosin heavy chain expression (eMyHC+/fiber) and centro‐nucleation using immunohistochemistry.

Results

Muscle fiber CSA was load dependent in young adult rats, but had no effect at either load in old rats. When compared to muscles from RE rats, 4.5 N CCL had no effect on satellite cell abundance regardless of age; however, satellite cell abundance was higher in both young and old adult muscle after 7.6 N CCL compared to RE with old rats having 2‐fold higher levels compared to RE. Regardless of load, CCL was not associated with differences in eMyHC expression or centrally located nuclei in young adult rat muscle, while muscles receiving 7.6 N CCL in old rats had a higher number of centrally located nuclei and were ~16‐fold higher in eMyHC+ fibers compared to RE.

Conclusions

These results show that CCL induces a load dependent anabolic response in young adult animals during muscle mass recovery following disuse, but has no regrowth effect on muscles from old animals at either load. Furthermore, using a higher CCL load, scaled for elevated transverse stiffness in aged muscle, induces damage in muscle of old but not young rats.

Support or Funding Information

This research was supported by NIH grant RO1AT009268 and R21AG042699.
Effect of a Single Session of Intermittent Hypoxia on Erythropoietin and Oxygen‐Carrying Capacity
Authors: Nagel Mercedes J. , Jarrard Caitlin P. , Lalande Sophie
Published: April 2020

Intermittent hypoxia, defined as alternating bouts of breathing hypoxic and normoxic air, has the potential to improve oxygen‐carrying capacity through an erythropoietin‐mediated increase in hemoglobin mass. The purpose of this study was to determine the effect of a single exposure of intermittent normobaric hypoxia on erythropoietin levels and hemoglobin mass in young healthy individuals. Nineteen healthy individuals (10 women and 9 men, age: 24 ± 4 years, height: 174 ± 11 cm, weight: 72.2 ± 12.2 kg) participated in the study. Participants were randomly assigned to an intermittent hypoxia group (Hyp, n = 10) or a placebo intermittent normoxia group (Norm, n = 9). Intermittent hypoxia consisted of five 4‐min hypoxic cycles at a targeted arterial oxygen saturation of 90% interspersed with 4‐min normoxic cycles. Air was made hypoxic by titrating nitrogen to a breathing circuit connected to a tank of compressed air. Nitrogen was not added to the breathing circuit in the intermittent normoxia condition. Pulmonary gas exchange, arterial oxygen saturation, and hemodynamics, using finger plethysmography, were continuously assessed during the intervention. Erythropoietin levels were measured before and two hours following the completion of the protocol. Hemoglobin mass was assessed using the carbon monoxide rebreathing technique the day before and seven days after exposure to intermittent hypoxia or normoxia. As anticipated, the intermittent hypoxia group had a lower arterial oxygen saturation than the intermittent normoxia group during the intervention (Hyp: 89 ± 1 vs. Norm: 98 ± 1%, p < 0.01), which was equivalent to a lower fraction of inspired oxygen (Hyp: 0.119 ± 0.008, Norm: 0.209 ± 0.001, p < 0.01). Erythropoietin levels did not significantly increase following exposure to intermittent hypoxia (Hyp: 8.2 ± 4.5 to 9.0 ± 4.8, Norm: 8.9 ± 1.7 to 11.1 ± 2.1 mU/ml, p = 0.56). Hemoglobin mass did not change following exposure to intermittent hypoxia (Hyp: 10.6 ± 1.4 to 10.2 ± 1.4, Norm: 9.7 ± 1.6 to 9.4 ± 1.5 g/kg, p = 0.48). Exposure to intermittent hypoxia did not affect mean arterial pressure (Hyp: 91 ± 6 to 90 ± 6, Norm: 93 ± 12 to 93 ± 12 mmHg, p = 0.84) or heart rate (Hyp: 68 ± 8 to 74 ± 9, Norm: 68 ± 8 to 68 ± 8 mmHg, p = 0.27). Respiratory rate, tidal volume, end‐tidal CO2 and total minute ventilation were not affected by intermittent hypoxia. Thus, a 40‐min session of intermittent hypoxia was not sufficient to elicit a rise in erythropoietin levels or oxygen‐carrying capacity in young healthy individuals. A longer exposure to intermittent hypoxia at a lower arterial oxygen saturation may be necessary to trigger an erythropoietin‐mediated increase in hemoglobin mass in young healthy individuals.