bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreasing the survival ability of brain metastatic breast cancer cell by targeting glucose transporter 3
Authors: Kuo Min Hsun , Lee Hsueh-Te
Published: April 2020

Breast cancer brain metastases (BBMs) are found in 15–20 % of breast cancer patients. And it results in a high mortality once the brain metastases are diagnosed. Therefore, making it more comprehensive about the mechanisms of BBMs is emergently necessary. Several studies have demonstrated that tumor microenvironment has potent roles in the tumor progression and metastasis. Besides, according to the former studies, cells in different organs using glucose might be various; as well as the dominant glucose transporters used are distinct. Here, we analyzed the brain metastatic breast cancer (BR) cells collected from intracardiac injection of nude mice and found the utilization of glucose transporters in the BR cells were different from the parental breast cancer cells; the elevated expressions of glucose transporter 3 were observed. In addition, the higher anaerobic glycolysis ability was found in BR cells. And after GLUT3 knockdown, the glucose reprogramming in BR cells was significantly decreased as well as the cancer malignant behaviors both in vitro and in vivo. Besides manipulating GLUT3 expressions, we also provide a potential mechanism that CREB could be a potent regulator of GLUT3 in BR cells by chromatin immunoprecipitation (ChIP) and knockdown assay. After decreasing the expressions of CREB in BR cells, the expressions of GLUT3 were also significantly reduced. In sum, our results reveal that GLUT3 plays an important role in provoking breast cancer cells surviving in brain by mediating glucose utilizations. And these results could provide a potential therapeutic way to target BBMs.

Support or Funding Information

1. Ministry of Science and Technology, R.O.C 2. Molecular Medicine Taiwan International Graduate Program, Academia Sinica
Anti‐cancer Effects of Melatonin, Estrogen, and Progesterone Hormone Therapy in MCF‐7 and MDA‐MB‐231 Breast Cancer Cells
Authors: Hasan Mahmud , Browne Erin , Guarinoni Laura , Darveau Travis , Hilton Katherine , Witt-Enderby Paula A.
Published: April 2020

A novel melatonin, estrogen (E2), and progesterone (P4) hormone therapy (MEPT) was developed as a safe bio‐identical alternative hormone therapy for menopausal women based on the Women’s Health Initiative findings that PremPro™ increased breast cancer (BC) risk and mortality of all types of BC in postmenopausal women. MEPT delayed tumor onset and reduced tumor incidence in Neu female mice (Dodda et al., 2019; Front Oncol). For other BCs, its actions are currently unknown. In this study, MEPT’s anti‐cancer actions (cell viability, migration) were tested in MCF‐7 (human ER+/PR+) and MDA‐MB‐231 (human TNBC with constitutively active ras mutation) using equivalent concentrations of melatonin (1nM), E2 (50pM), and P4 (10nM) observed in serum after an oral dose that produced anti‐tumor actions in neu mice (Dodda et al 2019; Front Oncol). Potential mechanisms underlying MEPT’s anti‐cancer actions were assessed by western blot (pERK1/2, pERK5, NF‐kB, RUNX2, β1‐INTEGRIN, RANKL, and ELF‐5) using MEK1/2 (PD98059) and MEK5 (Bix02189) inhibitors and radioligand binding using 2‐[125I]‐iodomelatonin or [3H]estradiol binding for melatonin and estrogen receptor expression, respectively. For MCF‐7 cells, BIX02189 combined with MEPT blocked MEPT‐mediated inhibition of migration and MEPT‐mediated ELF5 induction in MCF‐7 cells (vs. vehicle) and decreased pERK5 (vs. Bix02189) suggesting that MEK5, through pERK1/2 and ELF5, were involved in the anti‐cancer actions of MEPT in MCF‐7 cells. For MDA‐MB‐231 cells, although PD98059 and Bix02189 alone inhibited viability and migration, when added in combination with MEPT, no further blockade or enhancement occurred suggesting that MEK1/2 and MEK5 were not involved in MEPT’s anti‐cancer actions in MDA‐MB‐231 cells. MEPT’s differential actions on MCF‐7 and MDA‐MB‐231 cells were most likely attributed to unique melatonin/melatonin receptor actions within each cell. Both MCF‐7 and MDA‐MB‐231 cells express high‐affinity melatonin receptors (MCF‐7: Kd = 13 pM; Bmax = 20.55 fmol/mg protein vs. MDA‐231: Kd = 1.7 nM; Bmax = 60.09 fmol/mg protein) but only in MCF‐7 cells were [3H]‐estradiol binding sites detected (MCF‐7: Kd = 2.2 nM; Bmax = 8.6 pmol/mg protein) and stimulatory actions of E2 or P4 on MCF‐7 cell proliferation observed. These findings demonstrate novel anti‐cancer actions of MEPT in ER+ BC and TNBC cells through distinct mechanisms. Future studies assessing MEPT’s actions against ER+ BC and TNBC in vivo are warranted.

Support or Funding Information

This study was funded by the Marie‐Clement Rodier, C.S.Sp. Endowed Chair.
Chronic Hypertension Increases Pial Perivascular Microglia in Female Dahl‐S Rats
Authors: Warrington Junie Paula , Shao Qingmei , Clayton Ahsia M. , Maeda Kenji J. , Garrett Michael R. , Sasser Jennifer M.
Published: April 2020

Recent studies have shown that hypertension increases the proliferation and migration of microglia towards the perivascular space before destruction of the vasculature. Other studies have shown that hypertension increases perivascular macrophages, shown to contribute to impaired neurovascular coupling and cognitive impairments in mice. Despite these studies, there has been no description of what happens to perivascular microglia (PVMG) at the pia in the setting of chronic hypertension or following pregnancy.

The goal of this study was to test the hypothesis that chronic hypertension increases the density of PVMGs and that complicated pregnancy history will exacerbate this increase.

Female (8 months old) Sprague Dawley and Dahl‐S virgin or prior pregnant (2 pregnancies) rats were used. Brains were collected 3.5 months after the second pregnancy and processed for immunofluorescence staining for ion calcium binding adapter molecule (Iba‐1, a microglia marker) and DAPI. Z‐stacks were captured (0.5μm steps) throughout the thickness of the slice (20μm) using confocal microscopy. The number of microglia surrounding the pial artery (posterior communicating artery) was counted and normalized to the circumference of the vessel (density). We also measured the distance between each microglia surrounding the vessel.

There was no effect of pregnancy on PVMG density (p=0.675) or mean distance between PVMG (p=0.161) but a significant effect of hypertension (p<0.001) on both measures. PVMG density increased from 0.017±0.005 to 0.031±0.004/μm in the virgins and from 0.020±0.002 to 0.032±0.002/μm in the prior pregnant rats. Mean distance between PVMGs decreased from 64±13 to 28±5μm in the virgins and from 52±7 to 21±2 μm in the prior pregnant rats. Some of the PVMGs were antigen presenting cells (co‐expressed major histocompatibility complex II, MHCII).

Our results indicate that at the pia, PVMGs accumulate around the blood vessels, with some presenting antigens to lymphocytes. The exact function of these cells are not known, but we hypothesize that PVMGs could be contributing to the pathophysiology of hypertension or could present a compensatory mechanism to protect the blood vessels. Future studies will directly test the function of pial PVMGs in the context of acute and chronic hypertension and will assess whether this finding is specific to female brains.

Support or Funding Information

UMMC Department of Neurology
Smell Loss and Onset of Phantosmia are Critical Aspects of Head Injury Cognition
Authors: Henkin Robert Irwin , Abdelmeguid Mona
Published: April 2020

Head injury is a common event occurring in 1–2 million people in the U.S. yearly. Head injuries may cause minor inconvenience or it may induce major cognitive changes such as memory loss, concentration difficulties, headache, hearing loss, and visual difficulties, associated with or without loss of consciousness. Two of the major events occurring after head injuries which are not commonly recognized but which significantly alter behavior are loss of smell acuity and subsequent onset of smell distortions or phantosmia. Most patients are unaware that these two events can occur but they are present in most patients who experience head injury of any severity. Initially, patients recognize that they cannot recognize any odors and subsequently recognize that they cannot obtain any flavor from any food or drink. Over time they recognize that they experience a peculiar, usually unpleasant, odor in their nose in absence of any external odor. These odors may be persistent or transient, usually unpleasant, and may be described as fecal‐like, burned, chemical‐like, or metallic. The phantom, if persistent, can be severe enough to initiate loss of appetite with subsequent weight loss and depression. We have evaluated over 200 patients with smell loss and phantosmia after a head injury at The Taste and Smell Clinic in Washington, DC over the past 40 years attempting to understand their cognitive changes in smell function. We have found that the mechanism for these cognitive changes relate to metabolic changes mainly in biochemical changes in both sensory and brain function in which specific growth factors which maintain olfactory function have been diminished and specific brain neurotransmitters, mainly gamma‐aminobutyric acid, have also been diminished. Correction of these abnormalities commonly corrects the smell loss, inhibits the phantosmia and improves cognition.
Action of Phosphodiesterase Inhibitors on Olfactory Epithelial Stem Cells
Authors: Henkin Robert Irwin , Abdelmeguid Mona
Published: April 2020

Smell loss is caused by many pathologies including viral‐type illness, allergic rhinitis, head injuries, and external radiation. Each of these pathologies act to inhibit smell loss by acting on the olfactory epithelium which undergoes apoptotic degeneration as a result of these pathologies. The major mechanism underlying all of these pathologies is similar and involves inhibition of stem cell activation which normally occurs by the action of adenylyl cyclase III through the action of cAMP and cGMP. These pathologies act to inhibit growth factors secretion from the nasal mucus which normally stimulates stem cell function at the olfactory epithelium. Since stem cell activation is necessary to initiate growth and development of olfactory epithelial receptor cells any inhibition of this action causes the apoptotic changes observed in olfactory epithelium resulting in onset of smell loss. Phosphodiesterase (PDE) inhibitors can activate cAMP and cGMP if delivered directly to the olfactory epithelium by way of a nasal spray containing this substance. At The Taste and Smell Clinic in Washington, DC we have treated many patients with smell loss of many pathologies with several PDE inhibitors which have increased nasal mucus cAMP and cGMP which subsequently corrected the smell loss and in some patients restored olfactory function to or toward normal.
Role of autophagy in Dynamic Remodeling of Cultured Mouse Hepatocytes
Authors: Ma Xiaowen , Manley Sharon , Ni Hongmin , Ding Wen-Xing
Published: April 2020

Primary culture of hepatocytes is a valuable tool to study mechanisms involved in liver metabolic, endocrine and secretory functions. However, freshly isolated hepatocytes dedifferentiate and lose many features of hepatocyte including the transporters and metabolic enzymes in the course of primary culture. Here, we investigated the role and mechanisms of autophagy in the dynamic remolding of long‐term cultured mouse hepatocytes. Mouse hepatocytes were freshly isolated from wild type mice and liver‐specific Atg5 as well as Drp1 knockout mice and cultured for different time points from 2 hours to 7 days. Autophagy and mitophagy flux were determined using the tandem GFP‐mCherry‐LC3 and Cox8‐GFP‐mCherry. We found that hepatocytes gradually lost their typical cuboidal morphology and displayed fibroblast like morphology, which was accompanied by increased stress fibers and highly fused mitochondrial network formation in the course of culture. These mitochondrial dynamic changes were associated with decreased major mitochondrial fusion proteins Mfn1/2 and fission protein Drp1. The levels of hepatic cytochrome P450 enzymes (such as 2E1 and 2B10) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) markedly decreased, suggesting loss of hepatic metabolic features of drug and glucose metabolism in cultured hepatocytes. GFP‐mCherry‐LC3 puncta analysis and Cox8‐GFP‐mCherry analysis revealed that basal autophagic flux and mitophagy increased as early as day 2 and continue to increase in dedifferentiated hepatocytes during the culture. This mitophagy was independent of Parkin, as the levels of Parkin were dramatically decreased after 12 hours culture in hepatocytes. Mitophagy was markedly blunted in cultured Drp1 knockout hepatocytes, suggesting that elongated mitochondrial may be protective against autophagic degradation. Importantly, the levels of P450 enzymes, Parkin and GADPH remained higher in Atg5 knockout hepatocytes compared with wild type hepatocytes during the culture, suggesting the autophagy may be involved in the degradation of these metabolic enzymes. In conclusion, these findings indicate that autophagy is important for the remodeling of cultured hepatocytes and manipulation of autophagy process may be helpful to maintain some of the hepatocyte features during the culture.
Detection and Analysis of Protein‐Protein Interactions between KU70 and BHRF1, a BCL2 viral analog within Human gamma herpesvirus 4
Authors: McManus Sheighlah , Emami Alexander , Zamora Alberto
Published: April 2020

The Epstein‐Barr virus, also known as human gamma herpesvirus 4 (HHV‐4), affects more than 90% of the adult human population worldwide. To prevent premature death of the host cell, HHV‐4 encodes for BHRF1, an anti‐apoptotic protein with sequence and structural similarities to other proteins involved in apoptosis such as BCL2, BAX and BAK, among others. How BHRF1 prevents apoptosis in host cells remains to be elucidated and major emphasis of our lab. Using protein fluorescence complementation assays, we have preliminary data suggesting that BHRF1 interacts with the Ku70, a DNA repair protein that is known to also play roles during apoptosis. Since Ku70’s role during apoptosis is known to be regulated through acetylation of its C‐terminus, we are also investigating whether or not Ku70 acetylation influences its interaction with BHRF1, as well as identifying the surfaces and residues in each protein that may be directly involved in this protein‐protein interaction. Furthermore, we are extending our analysis to include BAK, a pro‐apoptosis protein with great similarities with BAX, a known Ku70 interactor. Examination of these interactions and their associated binding sites can create new knowledge that can be beneficial in targeting HHV‐4 viral mechanisms.
Elucidating the molecular interactions of the L‐nucleotide analogs with the Complex of HIV‐1 Reverse Transcriptase and DNA and the mechanism of drug resistance conferred by the M184V mutation
Authors: Suo Zucai
Published: April 2020

Emtricitabine (FTC) and lamivudine (3TC) are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in antiretroviral therapy for HIV. They contain an oxathiolane ring with unnatural (−)‐stereochemistry as a mimic of the ribose ring found in natural deoxynucleotides (L‐nucleoside). Treatment with FTC or 3TC primarily selects for the HIV‐1 reverse transcriptase (RT) M184V/I resistance mutants that are characterized by a marked decrease of incorporation of these NRTIs. Pre‐steady state kinetic data and crystal structures of both (−) and (+) oxathiolane analog triphosphates were determined in wild‐type and M184V HIV‐1 RT to elucidate the structural features of RT that recognize these substrates and gain insight into how M184V confers resistance. The pre‐steady state kinetic data reveal that (−)‐FTC‐TP and (−)‐3TC‐TP have higher binding affinities (1/Kd) for WT RT than dCTP however they have a slower rate of incorporation (kp). The crystal structures corroborate these results revealing that the oxathiolane sulfur orients toward the DNA primer terminus and two different triphosphate orientations are observed which would reduce the rate of incorporation. The structure of (+)‐FTC‐TP shows a close match to that of the natural substrate dCTP illustrating how the oxathiolane D‐isomers adopt a triphosphate orientation conducive for incorporation. The M184V mutation displays significantly greater (>200‐fold) Kd for the L isomers and moderately higher (>9‐fold) Kd for the D‐isomers compared to dCTP. The structure of M184V RT illustrates how the mutation repositions the oxathiolane ring, inducing a shift of the triphosphates into a conformation that hinders nucleotide incorporation. This combined study provides a comprehensive kinetic and structural basis for inhibiting WT HIV‐1 RT by the active metabolites of 3TC and FTC and the mechanism of M184V resistance.

Support or Funding Information

This work was supported by a grant from the National Science Foundation (grant number MCB‐1716168).
Endothelin‐1 Stimulates Microglia Cells: Role in Multiple Sclerosis
Authors: Arenas Claudia Patricia , Moliere Jean Paul , Capo Diego , Romero Jose , Inostroza-Nieves Yaritza
Published: April 2020

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disease characterized by inflammation, demyelination, and axonal degeneration. There is evidence that activated microglia cells which are resident innate immune cells of the central nervous system are observed in patients with MS. These cells play an important role in inflammatory responses in part by presenting major histocompatibility complex (MHC) molecules. Elevated levels of circulating endothelin‐1 (ET‐1), a potent vasoconstrictor and important mediator of inflammatory responses, have been shown in some patients with MS and reported to induce severe and prolonged cerebral vasoconstriction. However, the relationship between ET‐1 and microglia cell activation is unclear. We first studied a mouse model of MS –the induced experimental autoimmune encephalomyelitis (EAE) mouse. In female EAE C57BL/6 mice that were 8–12 weeks of age, we observed that ET‐1 mRNA expression by qRT‐PCR and protein levels by western blot analyses were significantly upregulated (1.5 folds ± 0.4, p<0.05, n=5) in brain tissue from EAE mice when compared to control mice. We then studied the effects of ET‐1 on HMC3, a human microglial cell line. Treatment with ET‐1 significantly increased the expression of HLA‐A [MHC class I] (5.6 folds ± 0.7, p<0.0001) and HLA‐DRA [MHC class II] (6.5 folds ± 1.4, p<0.0001) when compared to vehicle treatment. These increases were blocked by preincubation with either endothelin receptor A (ETRA) or B (ETRB) antagonists, BQ123 and BQ788, respectively (p<0.0001). In addition, ET‐1 significantly increased IL‐6 secretion from HMC3 cells by over 90% (p<0.05); an effect that was likewise sensitive to ETRA and ETRB antagonists. We also quantified Nitric Oxide (NO) production by using Griess Reagent and reactive oxygen species (ROS) production by the MUSE Oxidative Stress assay. In our cells, NO and ROS production were significantly increased by ET‐1 (p<0.05) and treatment with ETRA and ETRB blocked these effects (p<0.05). These results suggest a novel ET‐1 pathway for activation of microglia cell inflammatory responses that may provide key information critical for our understanding of the pathophysiology of MS.
Putative Sympathetic Oscillator in the Thoracic Spinal Cord of the Bullfrog
Authors: Herrington Brittney , Braun Connor , Wilson Richard
Published: April 2020

BACKGROUND

Cardiorespiratory control is essential for survival of all vertebrates, yet our understanding of the neuronal circuitry involved is rudimentary ‐‐ in part because there is little data beyond mammals to guide us. Neuronal oscillators are discrete neural circuits located within the central nervous system that are capable of generating rhythmic outputs without phasic input. These circuits are found throughout the animal kingdom and are often critical for regulating overt rhythmic behaviours such breathing. Previously, we found three oscillators in the frog brainstem that control ventilation: one for buccal ventilation, two for lung. Three respiratory oscillators have subsequently been found in mammals. Central neuronal oscillators within the sympathetic nervous system of frogs and mammals have also been suspected, but these oscillators have yet to be precisely located.

FINDINGS

Using extracellular recording and transection techniques in the frog superfused, isolated brainstem and spinal cord preparation, we discovered a robust, yet previously unreported buccal‐like rhythm in the third spinal nerve (SNIII). Unlike the ventilatory rhythms we normally study, the SNIII rhythm demonstrates poor bilateral synchrony. Our data also suggest that this rhythm is: (a) not coupled to ventilatory rhythms recorded in cranial nerves; (b) has distinct responses to pharmacological stimulation compared to ventilatory rhythms; (c) does not require the brainstem; and (d) persists in a chunk of spinal cord centered around SNIII. These data suggest an additional neuronal oscillator in the frog spinal cord located at the level of SNIII. However, removing the brainstem alters the frequency, increases variability and degrades left‐right synchrony of the rhythm, suggesting brainstem connections are important for normal function.

SIGNIFICANCE

The literature has long debated the existence of central neuronal oscillators that modulate sympathetic nervous system activity in vertebrates. We propose that the bursting activity recorded from the SNIII in frog is the output of such an oscillator, discovery of which could prove critical in shaping our current understanding of the function and evolution of the sympathetic nervous system.

Support or Funding Information

NSERC and HBI.
Chronic, but not acute, buprenorphine leads to antidepressant‐like effects in both male and female mice
Authors: Ferrante Julia Renee , Sachs Benjamin , Skaf Marie
Published: April 2020

Current antidepressant medications lack efficacy in a large fraction of patients and typically take weeks to reduce symptoms in individuals who do respond to treatment. However, the factors that govern treatment responses to various classes of antidepressants remain largely unknown. The incidence and presentation of mood and anxiety disorders are both characterized by sex differences, and sex differences in response to antidepressants have been reported for both selective serotonin reuptake inhibitors (SSRIs) and tricyclics (TCAs). As new antidepressants are developed, it will be critical to determine their efficacy in both male and female subjects to enable appropriate treatment selection. Previous research has investigated the antidepressant effects of buprenorphine, which has high affinity for mu opioid receptor (MOR), kappa opioid receptor (KOR), and some affinity for delta opioid receptor (DOR). Though previous studies have shown support for buprenorphine as an antidepressant, the effects have only been demonstrated in males. This study examined potential sex differences in the behavioral and molecular responses to acute and chronic buprenorphine administration in mice. The effects of buprenorphine were measured with the forced swim test, open field test, and light dark emergence test. In contrast to published research, acute buprenorphine showed no significant main effect of treatment in any of these behavioral tests in either sex. However, chronic treatment with buprenorphine showed a main effect of treatment in both the open field and forced swim tests, suggesting that both males and females are responsive to this drug. While gene expression analysis did show that female mice expressed higher levels of pro‐opiomelanocortin (POMC) in the striatum compared to males, no sex by drug interactions were observed. These studies show that chronic, but not acute, buprenorphine treatment has antidepressant effects, and that females do not appear to have reduced sensitivity to these effects.
Sub‐Chronic Stress Induces Similar Depression and Anxiety‐Like Behavioral Alterations in Male and Female Mice
Authors: Baugher Brittany J. , Sachs Benjamin D.
Published: April 2020

Women exhibit higher rates of post‐traumatic stress disorder, major depression, and anxiety disorders than men, but the biological basis for this sex difference remains largely unknown. Stress has been implicated in anxiety, depression, and other mental disorders, and it has been hypothesized that sex differences in stress susceptibility could underlie the clinically observed gender differences in the incidence of mental illness. Preclinical studies examining sex differences in vulnerability to stress have reported mixed findings, but several recent studies have found that females, but not males, exhibit depression‐ and anxiety‐like behavior following sub‐chronic variable stress (SCVS). These prior studies further suggested that the differential responses of males and females to SCVS result at least in part from sex differences in the expression of DNA methyltransferase 3a (Dnmt3a) and nuclear factor kappa B (NFKB) in the striatum. To evaluate the generalizability of these published findings, the current study examined sex differences in response to a modified version of the SCVS model. Our results reveal that SCVS significantly increases the expression of Dnmt3a and the p105 subunit of NFKB in the striatum of females but not males. However, in contrast to prior research, both sexes displayed increased depression‐ and anxiety‐like behavior following SCVS, suggesting that males are not entirely resilient to SCVS. The identification of SCVS‐induced behavioral disturbances in males in the absence of SVCS‐induced alterations in Dnmt3a or NFKB suggests that the molecular mechanisms leading to depression‐ and anxiety‐like behavior may differ in males and females. Future research elucidating the sex differences in stress‐induced molecular pathology could prove critical for improving our ability to design rational therapeutic interventions that counteract the effects of stress in males and females.
The effect of acetylation on isocitrate dehydrogenase 1 regulation
Authors: Strom Alexandra , Widjaja Vinnie , Weeks Joi , Alexander Sati , Pucher Dahra , Nguyen Tin Duc , Luna Lucas , Matteo Diego Avellaneda , Hoang An , Sohl Christal D.
Published: April 2020

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme responsible for catalysis of isocitrate to α‐ketoglutarate using NADP+ as a cofactor. Cancerous mutations of IDH1 yield the neomorphic conversion of α‐ketoglutarate to D‐2‐hydroxyglutarate, a proposed oncometabolite. Various cancers such as gliomas, glioblastomas, acute myeloid leukemias, and bone cancers, are driven by IDH1 mutations. Although it has been established that IDH1 is an enzymatic driver of disease, more research is necessary to elucidate the molecular mechanisms of its regulation. Post‐translational modifications (PTMs), such as acetylation, serve as effective methods of protein regulation. Here, we hypothesize that acetylation may have a significant impact on IDH1 efficiency. To investigate this, we treated wild type IDH1 with acetyl group donors and generated wild type IDH1 mutants (K81Q, K224Q, K321Q) to mimic acetylation in a physiologically relevant setting. We then employed steady state enzyme kinetics to measure the rate of the normal reaction yielding α‐ketoglutarate. We report that acetylation has a notable effect on IDH1 catalytic efficiency in both the acetylation mimics and acetyl‐treated IDH1. Our data suggests that acetylation may be an essential post‐translational modification for regulating IDH1 catalytic efficiency, thus helping us understand pathways relevant to IDH1 activity.

Support or Funding Information

This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).
Chirality of novel bitopic agonists determine unique pharmacology at the dopamine 2 and 3 receptors
Authors: Adhikari Pramisha , Yano Hideaki , Bonifazi Alessandro , Battiti Francisco O. , Newman Amy Hauck , Shi Lei
Published: April 2020

The dopamine 2 and 3 receptors (D2R and D3R) are well‐characterized targets for neuropsychiatric disorders. D2R/D3R agonists are used as add‐on therapies for Parkinson’s disease (PD) and have been studied for other motor‐associated disorders. Selectively targeting D3R over D2R is attractive for two reasons: i) restricted tissue distribution of D3R compared to D2R exhibits potential for lesser side‐effects; ii) there is evidence for dominant role of D3R over D2R for neuroprotective and neurorestorative actions of dopaminergic agonists used for the treatment of PD. However, lack of highly selective D3R agonists has made it difficult to separate the D3R vs. D2R physiological effects. Indeed, the development of highly selective D3R agonists has remained a challenge due to their 78% sequence identity in the transmembrane domain. To improve the outlook of D3R agonist‐based medication development, based on a potent D3R‐selective agonist PF592,379, we have designed, synthesized, and pharmacologically characterized bitopic ligands with a secondary pharmacophore tethered to the PF592,379 primary pharmacophore by a linker.

Using BRET based functional assays that measure G‐protein activation and β‐arrestin recruitment, we characterized these bitopic ligands at both D3R and D2R. We found that the addition of secondary pharmacophore to the PF592,379 scaffold improved potency at D3R in G‐protein activation with maximal efficacy similar or higher than that of the parent compound. In contrast, the potencies at D3R in recruitment of β‐arrestin remained the same. Some of these compounds also showed modest D3R over D2R selectivity. Interestingly, certain combinations of secondary pharmacophores and linkers resulted in G‐protein signaling bias at either D3R or D2R but not both, resulting in receptor‐selective bias. We also found that given the same ligand structure, chirality can confer subtype selectivity and promote G‐protein bias. Together, our novel bitopic D3R agonists based on PF592,379 scaffold provide novel tools to further probe unique pharmacology at D3R and evaluate the therapeutic potential of targeting D3R vs. D2R for PD.

Support or Funding Information

National Institute on Drug Abuse – Intramural Research Program, National Institutes of Health
Neuropilin 2 Drives Tumor Lymphangiogenesis in Oral Squamous Cell Carcinoma
Authors: Almazyad Asma , Gao Yao , Shahrabi-Farahani Shokoufeh , Sui Lufei , Gartung Allison , Das Ridhima , Costea Daniela Elena , Watnick Randy , Panigrahy Dipak , Adam Rosalyn M. , Bielenberg Diane R.
Published: April 2020

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. The majority (67%) of patients with this aggressive cancer already exhibit local or distant metastases at the time of initial diagnosis. Understanding the molecular mechanisms that regulate OSCC progression and metastasis is paramount to developing novel therapeutic strategies for this disease. Enhanced tumor‐associated lymphangiogenesis correlates with increased lymph node metastasis and adversely affects prognosis in OSCC. Neuropilin 2 (NRP2) is an endothelial cell membrane receptor that binds the lymphangiogenic factors, VEGF‐C and VEGF‐D, in complex with VEGFR3 to stimulate the sprouting of lymphatic vessels during developmental lymphangiogenesis. However, once vessels are properly guided and patterned, NRP2 expression is downregulated and not upregulated until times of need such as during inflammation‐, ischemia‐, or cancer‐related neovascularization. Our hypothesis is that NRP2 plays a role in OSCC progression and metastasis. Results from immunohistochemical staining demonstrate that normal human and mouse oral mucosa are devoid of NRP2 expression. Conversely, NRP2 is upregulated in the epithelium in late stages of oral dysplasia and in OSCC and its associated endothelium. Several human OSCC cell lines express NRP2 in vitro and in vivo when implanted orthotopically in the tongue of immunodeficient mice. In fact, NRP2 expression levels in the cancer cells correlate with the degree of tumor‐associated lymphatic vessel density within the tumors. OSCC mouse intralingual isografts show reduced tumor angiogenesis and lymphangiogenesis in adult syngeneic Nrp2‐deficient mice compared to Nrp2‐intact littermates. Taken together, our data suggest that NRP2 may be useful as a late‐stage marker of oral epithelial dysplasia and highlight the importance of the NRP2 axis in effective tumor neovascularization in OSCC. Our ongoing studies using the 4NQO carcinogenesis model in mutant mice lacking Nrp2 specifically in epithelial cells will further elucidate the role of NRP2 in OSCC tumorigenesis.
The Brain Atlas Glow Up: Utilizing Autofluorescence to Enhance Contrast Between White and Gray Matter
Authors: Shearer Jake , Stabio Maureen , Spitzer Victor
Published: April 2020

Myelin stained brain atlases are essential tools used to develop an understanding of brain organization and wiring. However, in a medical setting, accurate diagnoses require providers to be able to mentally translate the information gained from atlases to their patient’s MRI data. Myelin stained atlases provide excellent contrast and detail but are routinely constructed with a centimeter or more between images. This poor volumetric resolution poses a challenge when comparing anatomy from myelin stained atlases to MRI datasets as there are often significant gaps of data between subsequent images. Students must learn how to interpolate the missing data between the myelin stained sections. This study was designed to test the hypothesis that an ultrahigh‐resolution, high‐contrast, volumetric atlas of the brain could be re‐constructed from images obtained from blockface milling of whole brains while taking advantage of the autofluorescent properties of myelin to enhance contrast. Using custom‐built 470nm LED panels in conjunction with a 511/20nm bandpass gel filter, we were able to capture images with enhanced contrast between white and gray matter with sufficient detail to identify individual nuclei of the brain. Formalin‐fixed brains were cryopreserved, frozen, and serially imaged at 50μm intervals. At each interval, we imaged the brain using both full color and fluorescent photography, then converted the data into image volumes. The high volumetric resolution of the visual data allows any viewing plane to be generated and directly compared with the equivalent plane in an MRI volume. The volumes were then coregistered and efficiently segmented using the high contrast between white and gray matter to generate full‐color, 3D models of gray matter nuclei and white matter fiber tracts. We also created an accompanying 2D atlas using traditional atlas sections. This atlas will enhance the literacy of MRI anatomy by allowing both students and providers to visualize the information gaps in current myelin stained atlases.

Support or Funding Information

University of Colorado: Modern Human Anatomy Program, Touch of Life Technologies, Colorado State Anatomical Board, Inworks
Continuous or Transient High Level of Glucose Exposure Differentially Increases Coronary Artery Endothelial Cell Proliferation and Human Colon Cancer Cell Proliferation.
Authors: Okada Junichi , Okada Shuichi , Yamada Masanobu
Published: April 2020

We studied effect of high glucose levels on coronary artery endothelial cell proliferation and human colon cancer cell proliferation. To examine the long‐term effect of glucose exposure on cell growth, cells were cultured for 14 days in the absence or presence of 183 mg/dL D‐glucose addition in the culture medium. Short effect of elevated glucose levels was examined by addition of 183 mg/dL D‐glucose addition in the culture medium for just one hour per day followed by changing the culture to standard medium (5.5 mM D‐glucose) during the next 23‐hours period. Cell proliferation was estimated by 2,3‐Bis (2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carbox‐anilide (XTT) assay and phosphor‐Erk western blot analysis. We found that coronary artery endothelial cell proliferation was significantly increased in the culture medium with the acute one‐hour addition of 183 mg/dL D‐glucose compared to the absence or chronic presence of 183 mg/dL D‐glucose addition in the culture medium. In contrast, colon cancer cell proliferation was significantly increased in the continuous presence of 183 mg/dL D‐glucose addition in the culture medium compared to the acute one‐hour addition of glucose. The extent of Erk2 phosphorylation paralleled with the relative changes in cellular proliferation in both cell types. Taken together, these results suggested that continuous or transient high level of glucose exposure differentially effects coronary artery endothelial and human colon cancer cell proliferation.
PPARγ Regulates the Inflammatory Response to Ozone‐Induced Lung Injury in Mice
Authors: Malcolm Callum , Smith Ley Cody , Gow Andrew J. , Laskin Debra
Published: April 2020

Ozone is an urban air pollutant that is highly reactive causing tissue damage and inflammation in the lung. Long term exposure is linked to various health complications and increased mortality due to respiratory diseases. The inflammatory response to ozone is regulated by distinct populations of macrophages, which consist of M1/pro‐inflammatory and M2/anti‐inflammatory subsets; these cells are involved in the acute response to injury and long term resolution of tissue damage, respectively. Previous studies from our laboratory demonstrated that exposure of mice to ozone caused significant downregulation of PPARγ, a transcription factor important in anti‐inflammatory signaling in macrophages, phagocytosis of apoptotic neutrophils (efferocytosis), and the resolution of inflammation. We hypothesized that administration of a pharmacological agonist of PPARγ would reduce ozone‐induced lung injury by altering macrophage phenotype. Female C57BL/6J mice were exposed to air or ozone (0.8 ppm, 3 hr) and treated with vehicle control or rosiglitazone, a PPARγ agonist, daily via intraperitoneal injection beginning 24 hr prior to ozone exposure; mice were euthanized 24, 48, and 72 hr post ozone. Bronchoalveolar lavage fluid (BAL) was collected and analyzed for total protein and phospholipid content. BAL was enriched for alveolar macrophages by gentle lung massage and isolated cells analyzed by flow cytometry and qPCR. Ozone caused increases in total protein and phospholipid content, consistent with lung injury. In rosiglitazone‐treated animals, there was a trend towards reduced BAL protein levels at 48 hr and significant reduction in phospholipid content at 72 hr, suggesting accelerated injury resolution. Flow cytometric analysis showed increases in both pro‐ and anti‐inflammatory macrophages in the lungs of ozone‐exposed animals throughout the time‐course; a trend towards reduced numbers of these cells was observed in rosiglitazone‐treated animals at 72 hr. These results were consistent with reduced mRNA expression of pro‐ and anti‐inflammatory markers Ptgs2 and Arg1, respectively, at 72 hr. mRNA expression of Caveolin 1, a downstream target of PPARγ involved in lipid catabolism, was reduced in response to ozone but maintained in rosiglitazone‐treated animals. Collectively, these results suggest that PPARγ may modulate ozone‐induced lung injury by regulating inflammatory signaling and lipid metabolism in alveolar macrophages.

Support or Funding Information

Supported by NIH Grants ES004738, ES005022, ES029254, ES007148, ES030984.
Study Preferences of Generation Z Students Admitted to Medical School
Authors: Addae Jonas , Ettarh Rajunor , Crowley Jason
Published: April 2020

Persons born between 1995 and 2012 have been described as belonging to Generation Z (iGen), a group that follows on Generation Y, the millennials. Generation Z individuals have been described as being technologically sophisticated, dependent on mobile devices, love the internet, have short attention span, dislike reading printed material, prefer collaborative activities and learning in teams. In an effort to understand how students will adjust to a medical school’s curriculum, we asked the question: To what extent did students exhibit the characteristics of Generation Z just before they started a medical programme?

We examined the study habits of students at the School of Medicine at the University of the West Indies, St. Augustine Campus in Trinidad & Tobago. We administered a paper questionnaire during the orientation week to students admitted in August, 2019. The total number of students admitted was 236 of which 223 students belonged to the category of Generation Z.

The mean age of the Generation Z group was 19.5 years (range 17 – 24) with 63% females and 37% males. The average time a student took to study before his/her mind began to wander was 48.7 minutes (CI: 44.0, 53.4). There was no significant correlation between age and attention span with r = .061 (p=.388). The number of students who preferred studying alone was 193 (86.5%); those who preferred group study totally or partially (i.e. both alone and in groups) was 30 (13.5%). Most students, 156 (70.0%), preferred studying from textbooks and other printed materials. In contrast, 67 (30.0%) preferred using online materials only or in combination with printed materials. Overall, chi‐square analysis demonstrated a significant difference between student preferences regarding materials used and studying alone or in groups (χ2 (1) = 8.945, p=0.003). Compared to students who preferred group study, students who preferred studying alone significantly preferred printed materials over online material, odds ratio 3.18 [1.45, 6.98]

Conclusion

The majority of students admitted into the medical programme fit in the demographic of Generation Z. This study shows that, on admission, the vast majority of the Generation Z students preferred studying alone using printed material, and not in groups or using online material. Thus, the students’ preferred study habits (regarding studying in groups or using online materials) did not fit the expected characteristics of Generation Z. These results are similar to those of previous studies obtained for Millennials who were admitted to medical schools in Kuwait and Canada, and provide guidance to how we may think of adjusting the curriculum to facilitate learning by Generations Z & Y students.
Use of a Water‐soaked Garment, with and without an Electric Fan, to Mitigate Thermal Strain in Older Individuals under Heat Wave Conditions
Authors: Huang Mu , Cramer Matthew N. , Moralez Gilbert , Crandall Craig G.
Published: April 2020

As average global surface temperatures are projected to rise, and heat waves are expected to increase in frequency, duration, and intensity, exposure to high levels of heat stress will occur with greater regularity. Older individuals are particularly vulnerable to heat stress, evidenced by excess hospitalizations and mortality among individuals aged >60 years. With the continue rise of electricity costs coupled with vulnerable regions that do not have easy access to air conditioning, it is imperative to find alternative cooling strategies that are cost‐effective and accessible for older individuals. The present study evaluated whether wearing a water‐soaked t‐shirt, with and without electric fan use, mitigates thermal and cardiovascular strain under heat wave conditions. Nine healthy older individuals (68 ± 4 years; five females) completed three, 120‐min heat exposures (42.4 ± 0.2°C, 34.2 ± 0.9% relative humidity) on separate occasions while wearing either a dry t‐shirt (CON), a t‐shirt soaked with 500 ml of tap water (WET), or a t‐shirt soaked with 500 ml of tap water while facing an electric fan forcing air at 2.4 ± 0.4 m/s (WET+FAN). Measurements included core and mean skin temperatures, evaporative mass loss, heart rate, and blood pressure. The rise in core temperature was attenuated by ~0.3°C in WET compared to DRY and WET+FAN during the first hour of heat stress (P < 0.01). At 120 min, the rise in core temperature remained attenuated by ~0.2 °C in WET compared to DRY (P=0.0016), while other comparisons were not different. After the 120‐min heat exposure, changes in body mass, which largely reflect evaporation of sweat and moisture trapped in clothing, were greatest in WET+FAN, followed by WET, and then DRY (P < 0.01). However, sweat losses were lowest in WET, followed by DRY, and then WET+FAN (P < 0.01). Heart rate was lower during the first hour in WET vs. DRY, and at 30 min only in WET vs. WET+FAN (P < 0.01). No differences in mean arterial pressure were found (P = 0.51). In conclusion, simply wearing a water‐soaked t‐shirt is an effective heat management strategy to mitigate thermal and cardiovascular strain and lower sweat losses in older people exposed to heat wave conditions. Conversely, the cooling benefits of a water‐soaked t‐shirt are eliminated when used in combination with a fan.

Support or Funding Information

Funding: National Institutes of Health (R01GM068865) and Department of Defense – US Army (W81XWH‐15‐1‐0647)
Fatty Acid Amide Hydrolase (FAAH) Inhibition in the Dorsal Periaqueductal Gray Improves Chronic Neuropathic Pain in Rats
Authors: Dean Caron , Hillard Cecilia J. , Hopp Frances A. , Sherman Katherine , Cao Freddy , Hogan Quinn
Published: April 2020

Neuropathic pain is a critical treatment challenge that accompanies many injury and disease states in which peripheral nerves are damaged. The periaqueductal gray (PAG) in the midbrain is an integrative region that receives sensory input and, in turn, mediates descending sensory inhibition producing antinociception. Increased endocannabinoid concentrations in the dorsal PAG (dPAG) are associated with analgesic effects in neuropathy, and recent data from our lab reveal reduced content of the endocannabinoid anandamide and upregulation of its catabolic enzyme fatty acid amide hydrolase (FAAH) in the dPAG of rats with chronic neuropathic pain, suggesting that maladaptive changes in endocannabinoid signaling in the dPAG could contribute to chronic pain. Therefore, the present study was undertaken to determine the therapeutic potential of FAAH inhibitors in the dPAG for the treatment of chronic neuropathic pain.

Male and female rats (230–300 gm) underwent ligation of the tibial and peroneal branches of the sciatic nerve, leaving the sural nerve intact. Guide cannulae were implanted bilaterally targeting the dPAG in a sub‐group of injured rats. Hyperalgesia (sustained lifting/shaking/grooming after plantar pin touch) was tested prior to surgery, and on days 1,3,7,10,18 and 21 post injury. Twothirds of injured rats demonstrated a hyperalgesia response rate >20% to noxious mechanical stimulation of the hindpaw on day 21 and were considered to have developed chronic hyperalgesia. In these rats, hyperalgesia testing was performed prior to, and 3hr after, administration of a FAAH inhibitor (PF‐3845 as either 2mg/kg i.p., or 0.6ng intra‐dPAG) or vehicle on days 21, 22 and 23. Values for hyperalgesia response rate at 21 days were considered as baseline prior to drug administration. Systemic administration of the FAAH inhibitor reduced the hyperalgesia response rate in both male and female rats. In males, a reduction of 50% was maintained after PF‐3845 administered i.p., while female rats demonstrated a similar consistent response with a 40% reduction in hyperalgesia behavior. A progressive decrease in hyperalgesia response rate up to 80% in males and 50% in females was observed after direct administration of PF‐3845 into the dPAG over the 3d test period. However, the analgesia subsided in the 24hr period between drug administrations for both sexes. These data suggest that A) dysregulation of the endocannabinoid system in the dPAG may contribute to chronic neuropathic pain, and B) that inhibition of the catabolic enzyme FAAH in the dPAG could have therapeutic potential by enhancing endocannabinoid signaling.

Support or Funding Information

Supported by Merit Review Award # RX002747 from the U.S. Department of Veterans Affairs Rehabilitation, Research and Development Program.