bluuebunny/crossref_metadata_2025 huggingface dataset:
Response to Epigenetic Disruption in Tumor Suppressor of Hepatocellular Carcinoma (HCC)
Authors: Luna Aesis , Wu Qunfeng , Hlady Ryan , Sathyanarayan Aishwarya , Rustgi Vinod , Guarrera James , Robertson Keith , Liu Chen
Published: April 2020

Hepatocellular Carcinoma (HCC) is a highly fatal liver cancer with no effective treatments. HCC tumors, formed after long‐term viral or alcohol related inflammation and cirrhosis, have a surprisingly low number of genetic mutations. Prior research has found that epigenetic patterns, such as DNA methylation at gene promoter sites, are common in HCC patients. Recent improvements in genetic sequencing and online genetic databases, have allowed us to identify a set of gene targets, including FMO3, for HCC detection or treatment. The hypothesis is that FMO3 is reduced by epigenetic dysregulation, leading to tumor formation.

To find the connection between FMO3, epigenetic events and HCC tumor pathways, our lab created novel HCV‐stable and non‐infected HCC cell lines. We also used Huh7 and Huh7.5 cells to show the epigenetic changes in stressed cells and methylation inhibited cells. To mimic inflammation and stress leading to epigenetic changes, Huh7 cells were exposed to heat shock at 40°C for 40 min. Huh7.5 cells were treated with epigenetic inhibitor 5‐Aza‐2 deoxycytidine. RT‐qPCR and Western Blot show that FMO3 is significantly reduced in stressed cells and upregulated when treated with the methylation inhibitor. These results indicate that FMO3 may be used for early cancer risk detection and/or to improve treatment options by identifying the relationship between epigenetic regulation and tumorigenesis.

Support or Funding Information

NIH NIDDK 1R01DK110024‐01A1
Antibody mediated targeting of autoimmune cells in multiple sclerosis
Authors: Rustad Karin , Ripplinger Alexandra , Kisheev Anastasye , Buttigieg Josef
Published: April 2020

Multiple Sclerosis (MS) is a debilitating autoimmune disorder of the central nervous system (CNS) affecting over 77,000 Canadians, with a prevalence rate of almost one out of 300 Canadians, with the highest rates in Saskatchewan. Myelination of neuronal axons is essential for the normal and rapid electrical conduction along an axon. Currently there are few feasible options to treat MS. Here, we propose a novel therapy that specifically targets aberrant immune cells.

We created a trimolecular peptide (TPC) that combines and antigen sequence (myelin oligodendrocyte glycoprotein; MOG 35‐55), a modified type IV secretatory system (TIVSS) peptide, and an apoptotic protein. As the TPC displays MOG, the TPC is hypothesized to only bind to immune cells that are actively seeking MOG (e.g. in MS). Since the TPC contains TIVSS, the cell that bound the antigen (e.g. MOG 35‐55) is forced to take up the TPC. Lastly, the apoptotic protein, which is now inside the cell, causes the cell to undergo apoptosis. Thus eliminating the aberrant cell.

In order to test this TPC compound, we induced experimental autoimmune encephalopathy (EAE) using MOG 35‐55. Test compound was administered 15 days post EAE induction. Administration of the TPC resulted in the suppression of autoimmune cells, as determined via flocytometry. We also found significant improvements in motor function as well as a reduction in MS plaque formation in the cerebellum. By targeting the pathogenic immune response whilst leaving the patient’s normal immune response intact, we believe this strategy will lead to improved clinical outcomes without the side effects seen in current MS therapies.

Support or Funding Information

Saskatchewan Health Research Foundation
Old Drugs, New Synergy: Identifying Novel Synergistic Antibiotic Combinations to Combat MDR Bacterial Pathogens
Authors: Dillon Nicholas Allen , Nizet Victor
Published: April 2020

Dwindling therapeutic options have necessitated a push to identify more efficacious antibiotic regimens for treating drug resistant bacterial pathogens. Acinetobacter baumannii, a member of the ESKAPE pathogens, is of special concern due to its high mortality rate and propensity for broad spectrum antibiotic resistance. We recently described hitherto unexplored azithromycin activity against MDR A. baumannii. To expand on those findings, we sought to compare the activity of azithromycin to minocycline, the current standard of care for MDR A. baumannii infections. We identified disparate media dependent susceptibility profiles for each drug that suggested the potential for an efficacious combination therapy. In combination, the two drugs were found to interact synergistically in standardized laboratory media, human whole blood, and in a murine MDR A. baumannii lung pneumoniae model. A synergistic interaction was a surprising finding as both azithromycin and minocycline are translation inhibitors and would be expected to only have an additive interaction. Synergy was found to be mediated through enhancing the kinetics of translation inhibition, yielding a combination that was faster, more potent, and longer lasting then either drug in monotherapy. The combination was found to have broad applicability as it was also synergistic against MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, and methicillin‐resistant Staphylococcus aureus. Although initially found with azithromycin and minocycline, synergy was also uncovered with other macrolides and tetracyclines and may therefore be a class wide phenomenon. Interestingly, this synergistic interaction does not appear to necessitate bacterial sensitivity to the drugs in monotherapy. Considering both azithromycin and minocycline are already FDA approved, their inclusion in a combination therapy has the potential to have an immediate impact in the clinic for treating high‐priority MDR bacterial pathogen infections in humans.

Support or Funding Information

NIAID U01AI124316
Optogenetic Multimerization of Dopamine Transporter Alters Its Uptake and Trafficking Kinetics
Authors: Ingram Shalonda Marie , Rana Tanu , Manson Ashley M. , Yayah Faisal , Goodwin Shawn
Published: April 2020

The dopamine transporter (DAT) is essential for the reuptake of released neurotransmitter dopamine (DA) in the brain. DAT is one of the main targets for psychostimulants, leading to a disruption of DA homeostasis. Methamphetamine (METH), a psychostimulant, is a substrate for DAT and induces the formation of DAT (multimeric) complexes. The function of the DAT is not dependent on the multimer state, and the contribution of the multimerized state of DAT is unclear in the literature. We have developed a substrate‐independent, optogenetic model to test the functional significance of DAT multimers. Cry2‐mCh‐DAT (Cry2‐DAT) forms multimers upon blue light exposure. This construct has normal uptake activity under control conditions, and increased uptake function when multimerized with blue light pulses. Additionally, we found that blue light‐induced multimers of Cry2‐DAT restore the dopamine uptake to control levels after pretreatment with METH, and significantly increase the uptake after nomifensine pretreatment. We evaluated the mechanism of enhanced DAT uptake function by examining alterations in trafficking and conformational changes in DAT. We used endocytic trafficking inhibitors to determine which steps may affect the enhanced uptake after multimerization. We found that enhanced uptake of Cry2‐DAT was sensitive to Calphostin C treatment. We confirmed these findings with immunofluorescence staining and colocalization of Cry2‐DAT with Rab compartments. We could not detect any significant contribution of DAT conformational change to account for the increased uptake after DAT multimerization. These data will lead to a better understanding of the role of DAT multimers in the regulation of DA homeostasis in the presence and absence of conditions that disrupt DA homeostasis.

Support or Funding Information

DA036420, MHD007593, CA163069, MHD007586, GM059994, D34HP16299‐10‐00
Proximal Tubule Transport Function Screening Assay Used to Optimize Function of Kidney Organoids
Authors: Baty Catherine J. , Hukriede Neil A. , Espiritu Eugenel , Przepiorski Aneta , Weisz Ora A. , Kleyman Thomas
Published: April 2020

Background

The mechanisms that mediate solute filtration, reabsorption, secretion and excretion in the kidney are well established. However, there are a paucity of data regarding the functional properties of tubular epithelia cells within organoids. The goal of our current work is to define specific function properties of tubular epithelia cells within kidney organoids.

Study objectives

To develop quantitative assays adapted to human kidney organoids to measure proximal tubule function and test whether they can be used to optimize function in developing kidney organoids.

Methods

Organoids were generated from induced pluripotent stem cells (hiPS) derived from human fibroblasts. 6‐carboxyfluorescein (6CF) was used as a fluorescent prototype of organic anion transport (OAT), and was loaded into live organoids (1 mM in OptimemTM, with orbital rotation for 45 min), to measure basolateral uptake using confocal microscopy with tile scanning to test organoid populations and allow quantitation of transport function. To mimic continuous fluid shear stress which occurs in the PT, one population of organoids were placed on an orbital shaker (at 146 or 74 rpm for at least 30 hrs) prior to loading with 6CF. Organoids from the same culture batch at d 14 of differentiation were divided into two treatment groups, static and shaken, and loaded with 6CF, and imaged by a blinded investigator. Quantitation of average maximum intensity of basolateral uptake was done on organoid stacks using LASX software.

Results

Organoids derived from the LRP2:mtagBFP2 reporter line (available through the Rebuilding a Kidney consortium) confirmed the PT segments within organoids and showed colocalization with 6CF. Concurrent treatment with
probenecid (an OAT inhibitor) and 6CF inhibited uptake of 6CF into PT segments. Blinded quantitation of several organoid populations showed increased intensity of 6CF in PT segments subjected to chronic orbital shear. Assays demonstrated basolateral uptake and/or luminal secretion.

Conclusions

Kidney organoids subject to continuous fluid shear stress have increased basolateral anion transport capacity relative to static cultured controls. A screening assay for PT basolateral uptake, assumed to be OAT mediated, can be used to test and optimize PT function within developing human kidney organoids.

Support or Funding Information

Support from NIDDK: U01 DK107350RBK, 2 P30 DK079307‐07, R01 DK18726‐01.
Ozanimod Protects Against Hypoxia plus Glucose Deprivation‐Induced Morphological Changes and Improves Cell Health in Human Brain Vascular Smooth Muscle
Authors: Albrecht Anthony G. , Li Yu Jing , Wendt Trevor S. , Gonzales Rayna J.
Published: April 2020

Vascular smooth muscle cells regulate arterial contractile tone, blood pressure, and flow in relation to specific metabolic demands during normal and pathophysiological (i.e. hypoxic or ischemic) conditions. The plasticity of vascular smooth muscle cells allows for phenotypic transitions from contractile to synthetic‐like dedifferentiated states during pathophysiological conditions which then can lead to the development and progression of vascular inflammation and blood vessel wall remodeling. Our previous studies demonstrated that hypoxia plus glucose deprivation (HGD), an in vitro model of ischemia, alters human vascular cell morphology and increases cell death. We further demonstrated that the selective sphingosine‐1‐phosphate receptor 1 (S1PR1) ligand, ozanimod, attenuated these HGD‐mediated responses. In this study, we characterized HGD mediated phenotypic transitions and assessed expression of proteins involved in contraction (myosin light chain kinase, MLCK) and structure formation (alpha‐smooth muscle actin, vimentin) following HGD exposure. We further investigated the effect of ozanimod HGD‐mediated phenotypic transitions by assessing protein levels and localization LC3B as an indicator of autophagocytosis. Using immunocytochemistry and standard immunoblotting, we determined the localization and expression levels of α‐smooth muscle actin, MLCK, vimentin, and LC3B protein during normoxia (21% O2, control) or HGD (1%O2) for 9 h in male human brain vascular smooth muscle cells (HBVSMC) at passage 6–7. We observed that LC3B protein, a vacuole marker associated with autophagy, is ubiquitously expressed in the cytoplasm of HBVSMC during normoxia. During HGD exposure, LC3B protein levels were increased and immunohistochemical staining demonstrated LC3B localization in what appears to be autophagic vacuole formation. Both responses were attenuated by ozanimod. Although alpha‐smooth muscle actin in HBVSMC was effective in visualization of morphological changes from spindle (contractile) to rhomboid (synthetic) shape following HGD, basal protein expression levels were not altered. Similarly, MLCK, an enzyme responsible for contractile protein phosphorylation, was not altered following HGD. Levels of the synthetic phenotype marker, vimentin, was basally expressed in HBVSMC, however, levels were not increased following HGD. In conclusion, although vacuole formation is prominent, indicating poor health, HBVSMCs retained a contractile phenotype during ischemic‐like conditions. Attenuation of HGD‐mediated LC3B via ozanimod suggests a novel mechanism associated with the beneficial effect of this selective S1PR1 ligand during ischemic‐like exposure.

Support or Funding Information

American Heart Association 19AIREA34480018 (RJG).
Biochemical Characterization of Receptor Activity‐Modifying Protein and Peptide Agonist Effects on G protein Coupling to the Calcitonin‐Like Receptor
Authors: Roehrkasse Amanda , Warner Margaret , Booe Jason , Pioszak Augen
Published: April 2020

The vasoactive peptides calcitonin gene‐related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD) have demonstrated protective effects in disease processes that include ischemia/reperfusion injury and inflammatory bowel disease. The actions of these peptides are mediated by the calcitonin‐like receptor (CLR), which is a class B G protein‐coupled receptor (GPCR) that forms heterodimers with any one of three receptor activity‐modifying proteins (RAMPs 1/2/3). The RAMPs modulate its peptide binding preferences and may also alter signaling output, but the latter remains poorly understood. We hypothesized that different RAMP and peptide combinations stabilize distinct CLR conformations that alter transducer interactions. We adapted a native PAGE method as an inexpensive and efficient way to visualize detergent‐solubilized, fluorescently‐tagged RAMP:CLR complexes expressed in mammalian cells and to measure their interactions with transducer proteins. Addition of agonist peptides and the purified engineered minimal Gs protein (MiniGs) prior to solubilization resulted in agonist‐dependent coupling of MiniGs to each receptor visible as a quaternary complex. Using the MiniG chimeras MiniGs/i and MiniGs/q along with MiniGs we characterized the coupling preferences of each RAMP:CLR complex in the presence of each agonist peptide. For each of the three agonist peptides MiniGs coupled best to each RAMP:CLR complex, followed by MiniGs/q and finally MiniGs/i. Overall, all three MiniG proteins exhibited better coupling to the RAMP1:CLR complex consistent with RAMP‐dependent modulation of MiniG coupling. At each RAMP:CLR complex, we also observed peptide‐dependent effects with CGRP exhibiting distinct behavior from AM and AM2/IMD. This work demonstrates that the native gel assay is a powerful tool for screening and characterizing membrane protein stabilities and ligand interactions and supports our hypothesis that the RAMPs and agonist peptides alter CLR transducer interactions. Our findings may aid development of therapeutics that exploit the beneficial effects of these peptide hormones.

Support or Funding Information

This work was supported by grants NIH R01GM104251 (AAP) and NIH predoctoral MD/PhD fellowship 1F30 HL142232 (AMR).
The relation between aortic stiffness and retinal microvascular flow pulsatility is attenuated by higher vascular resistance in healthy adults
Authors: Holwerda Seth W. , Kardon Randy , Hashimoto Ryuya , Full Jan , Nellis Julie , DuBose Lyndsey , Fiedorowicz Jess , Pierce Gary
Published: April 2020

Aortic stiffness is associated with increased risk of cerebrovascular disease and cognitive impairments with aging, although the mechanism that contribute to this link are not well‐defined. Increases in carotid artery flow pulsatility coincide with elevated aortic stiffness and may contribute to microvascular brain damage, but studies confirming augmented flow pulsatility in the downstream microvasculature have been limited. Given the ability to assess the retinal microvasculature non‐invasively in humans and evidence that disease‐ and age‐related alterations in the retinal microvasculature mirror changes in the cerebral microvasculature, we examined the hypothesis that age‐related increases in aortic stiffness would be correlated with higher retinal arteriole flow pulsatility (systolic flow – diastolic flow / mean flow). Among 33 healthy adults (age range: 25–60 years, 47% women), aortic stiffness was assessed by carotid‐femoral pulse wave velocity (CFPWV). Retinal arteriole flow pulsatility was quantified using laser speckle flowgraphy before and during a reduction in retinal vascular resistance via 30 s of diffuse luminance flicker. Multiple regression analysis including CFPWV, mean arterial pressure (MAP), and heart rate (HR) as independent variables demonstrated that lower MAP and HR, but not higher CFPWV, were significant determinants of greater flow pulsatility at baseline (CFPWV: β=0.10, P=0.548; MAP: β= −0.40, P=0.021; HR: β= −0.50, P=0.002; model R2=0.43, P=0.001). However, after reducing retinal vascular resistance (P<0.001) via 30s of diffuse luminance flicker, multiple regression analysis demonstrated that higher CFPWV was a significant determinant of greater retinal arteriole flow pulsatility (β=0.35, P=0.026) in addition to lower MAP (β= −0.54, P=0.001) and HR (β= −0.43, P=0.005) (model R2=0.51, P<0.001). In summary, no relation was observed between higher CFPWV and retinal arteriole flow pulsatility at rest, but a positive relation was observed during arteriole vasodilation. These findings suggest that greater vascular resistance may be an important compensatory mechanism to reduce transmission of flow pulsatility.

Support or Funding Information

AHA 17POST33440101, T32 HL07121 NHLBI (P01HL014388), U54 TR001356 and AHA (13DG143400012).
TMEM163 (ZNT11) protein interacts with distinct ZNT efflux transporters
Authors: Escobar Adrian , Styrpejko Daniel , Ali Saima , Cuajungco Math P.
Published: April 2020

Our recent work showed that TMEM163 is a zinc efflux transporter that belongs to the cation diffusion facilitator (CDF) family of proteins. We propose that the TMEM163 be now called ZNT11 as a new member of the Group 1 CDF family ZNT efflux proteins that include 10 mammalian zinc transporters, ZNT1‐ZNT10. Accordingly, we hypothesize that TMEM163/ZNT11 interacts with at least one of the ZNT proteins, namely ZNT1 and ZNT2, based on its structural and functional characteristics. To show that TMEM163/ZNT11 interacts with either ZNT1 or ZNT2, we cloned their cDNAs into mammalian expression constructs containing either Myc‐DDK or HA peptide tag. Using HEK‐293 cells, we individually transfected TMEM163/ZNT11, ZNT1, and ZNT2 constructs as negative controls, while we co‐transfected TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2. As a positive control, we co‐transfected HA‐tagged TMEM163/ZNT11 with a Myc‐DDK‐tagged TMEM163/ZNT11 construct in HEK‐293 cells, based on a previous report that its rodent counterpart exists as a homodimer. We then used a co‐immunoprecipitation assay using either anti‐HA‐ or anti‐DDK‐bound agarose beads to pull down one of the target proteins. Western blot analysis showed that TMEM163/ZNT11 physically bound ZNT1 or ZNT2 protein. To determine the functional relevance of the interaction, we performed zinc flux assays using two zinc‐specific fluorescence dyes, Fluozin‐3 (high affinity, membrane impermeable) and Newport Green (low affinity, membrane permeable) following single‐ and co‐transfection of TMEM163/ZNT11, ZNT1, and ZNT2 constructs in HeLa cells. Our results confirmed that homodimers of TMEM163/ZNT11, ZNT1, and ZNT2 proteins transport zinc out of the cells, but that the efflux activity of homodimer TMEM163/ZNT11 proteins varied slightly in magnitude when compared with heterodimers of TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2 proteins. These results suggest that the interaction between TMEM163/ZNT11 and distinct ZNT proteins is physiologically relevant and may serve to modify the transport activity of ZNT protein interactor. Overall, our investigations showed for the first time that TMEM163/ZNT11 forms functional heterodimers with ZNT1 and ZNT2 proteins. Thus, TMEM163/ZNT11 by itself, or in combination with one of these specific ZNT proteins, may play a crucial role in maintaining intracellular zinc homeostasis in specific cell types.

Support or Funding Information

This work is funded by NIH R15 NS101594.
Small molecules breaking down islet amyloid polypeptide self‐assembly
Authors: Fortin Jessica S. , Isa Nurhanis B.M. , Rashid Anisa M. , Thompson Thomas L. , O’Dell Malikah
Published: April 2020

Amyloid deposition is a recognised feature of the islets in the majority of type 2 diabetic patients. These deposits originate from islet amyloid polypeptide (IAPP). Normally, IAPP is secreted along with insulin from pancreatic β‐cells and has hormonal functions related to satiety. However, IAPP is one of the most potent amyloidogenic proteins. Deposition of IAPP is associated with β‐cell death and diabetes progression. Drugs to inhibit pancreatic amyloidosis are not available. We have prepared a small molecule screening library to inhibit pancreatic amyloidosis. Our central hypothesis is that IAPP fibril formation (fibrillization) and deposition can be modulated by IAPP‐interactive compounds. We aim to establish structure‐activity relationships by which our small molecules impede IAPP fibrillization. Our primary approach, thioflavin T (ThT) assay, represented a major tool for the determination of IAPP fibrillization and inhibition by small molecules. For the most potent molecules, the abrogation of cytotoxic intermediate species (i.e. oligomerization leading to fibril formation) was determined with photo‐induced cross‐linking of unmodified protein. To confirm the inhibitory effect morphologically, electron microscopy analysis was performed to detect IAPP fibrils directly. Disaggregation of preformed fibrils by different compounds was studied with ThT assays and electron microscopy. The 1st‐tier screening method (ThT assay) identified several lead compounds to target IAPP. The oligomerization of IAPP was abrogated by JF‐19‐029. JF‐19‐029 and NBMI‐19‐013 deteriorated preformed fibrils. Few molecules have been reported to break down mature fibrils. We hope to open a new therapeutic approach to reduce type 2 diabetes progression.
Physiological effect of trazodone hydrochloride use for anxiety and sedation in rabbits (Oryctolagus cuniculus)
Authors: Rickerl Kaitlin , Reed Jessica , Brundage Cord
Published: April 2020

Oral sedation options for rabbits (Oryctolagus cuniculus) have remained limited despite the need for medications with anxiolytic effects during transportation and high stress procedures. Trazodone HCl (TZN), a serotonin modulator, is a commonly prescribed medication in humans to treat depression and anxiety. The efficacy of TZN use in rabbits was initially investigated in 2016 and found TZN to be successful in producing mild sedation, however the safety of the medication still needed to be determined. To identify the systemic effects of repeated TZN administration in rabbits, New Zealand white laboratory rabbits (n = 12) were given orally 40 mg/kg TZN every 12 hours (± 1 hour) for 28 days. A control group (n = 4) was used to monitor standard hematological fluctuations. Heart and respiratory rate were collected daily, and a physical examination was performed by a veterinarian every 7 days. Blood was collected for cell counts and serum chemistry analysis prior to and every 14 days during the experiment. No significant changes in heart or respiratory rate were noted throughout treatment (p = 0.201 and p = 0.984 respectively). Some fluctuations were noticed in blood serum chemistry and hematology results in both the experimental and control group, particularly in the liver and kidney values following 28 days of treatment. Serum alanine aminotransferase (ALT) between the blood draws of both groups was significantly elevated (p = 0.002), however the elevations were noticed in both the control and TZN treated animals (p = 0.597). A simmilar trend was noticed with alkaline phosphatase (ALP; p = 0.001 between blood draws, p = 0.428 between groups), creatinine (p < 0.0001 between blood draws, p = 0.340 between groups), and blood urea nitrogen (BUN) levels (p = 0.025 between blood draws, p = 0.343 between groups) indicating the changes overtime were not due to TZN treatment. All physiologic parameters tested remained within normal rabbit physiologic reference ranges. These results indicate TZN could be a viable option to provide mild sedation as needed and act as an alternative option to conventional treatments of behavioral problems. Future studies could be performed on dose range and safety among rabbits of varying age, sex, and breed.
Activation of Astrocytes in the Dorsomedial Striatum Facilitates Transition from Habitual to Goal‐Directed Reward‐Seeking Behavior
Authors: Kang Seungwoo , Hong Sa-Ik , Lee Jeyeon , Peyton Lee , Choi Sun , Kim Hyunjung , Chang Su-Youne , Choi Doo-Sup
Published: April 2020

Habitual reward‐seeking behavior is a hallmark of addictive behaviors. Although habitual behavior could be reversed to goal‐directed behavior, this shifting is not well studied. Here, we identify a role of astrocyte activation in the dorsomedial striatum (DMS) on transition from habitual to goal‐directed reward‐seeking behavior. Utilizing the Inscopix endomicroscopy/fiber photometry in vivo calcium imaging and the cell‐type specific activation of designer receptors exclusively activated by designer drugs (DREADDs), we confirmed that GFAP‐driven activation of hM3Dq DREADDs increases intensity and frequency of calcium signaling in the DMS astrocytes of ALDH1L1‐GCaMP6s expressing mice. We found that this chemogenetic activation of the DMS astrocytes increase and decrease neuronal excitability of direct pathway Medium Spiny Neurons (dMSNs) and indirect pathway Medium Spiny Neurons (iMSNs), respectively. Specifically, it reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in the dMSNs, whereas it increased the amplitude of the sEPSCs and decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the iMSNs. The changes in synaptic events induced by astrocytic activation were significantly inhibited by either of the pretreatments of DPCPX (Adenosine A1R antagonist, 1 μM), PSB12379 (Inhibitor of ectonucleotidase, CD73, 10 μM), NBTI [Inhibitor of the astrocytic adenosine transporter, equilibrative nucleoside transporter 1 (ENT1, slc29a1), 10 μM], and genetic deletion of ENT1. These data suggest that astrocytic activation‐induced synaptic activities are, in part, adenosine‐dependent. In the evaluation tests of goal‐directed and habitual reward‐seeking, the chemogenetic activation of the DMS astrocytes shifted the habitual behavior to goal‐directed action in sucrose reward‐seeking for WT, but not ENT1 KO mice. The astrocytic activation‐induced shifting was rescued when ENT1 expression was normalized in the DMS astrocytes of the ENT1 KO mice. On the other hand, when mice were trained to have goal‐directed behavior, the astrocytic activation in the DMS did not change the reward‐seeking behavioral pattern. Together, our results indicate that astrocyte‐regulated adenosine signaling in the DMS determines goal‐directed and habitual behaviors by selectively regulating synapses in the MSNs. The interaction between astrocytes and neurons via adenosine signaling could be a potential therapeutic target for maladaptive reward‐seeking behaviors.

Support or Funding Information

This work was supported by the Samuel C. Johnson for Genomics of Addiction Program at Mayo Clinic, the Ulm Foundation, and National Institute on Alcohol Abuse and Alcoholism (K01 AA027773 to SK, R01 AA018779 to DSC).
Coupling arginine with DNA replication and chemoresistance
Authors: Wang Yi-Chang , Kelso Andrew A. , Stark Jeremy M. , Ann David K.
Published: April 2020

How cells adapt DNA replication to meet amino acid demands is an active area of interest across biology. Among a broad range of functions, the extracellular arginine is needed for the availability of newly synthesized histones to promote DNA replication fork progression. We show herein that transient deprivation of extracellular arginine, but not glutamine or leucine, causes a reversible S‐phase arrest. This unexpected dynamic S‐phase arrest feature is independent of mTORC1 signaling inhibition, insufficient metabolites, endogenous de novo arginine biosynthesis, and general amino acid control pathway. Moreover, knockdown of arginyl‐tRNA‐transferase recapitulated the loss of histone H4 lysine 5 and 12 acetylation marks and the reduction of replication fork speed without inhibition of origin firing. Importantly, single round of arginine depletion and addition did not induce ATR/Chk1 activation and DNA double‐stand breaks associated γH2AX. The halt of arginine‐dependent histone H4 synthesis is therefore emerging as a “brake” for DNA replication bypassing checkpoint control. However, repeated arginine fluctuation promote the emergence of nuclear p53‐binding protein 1 bodies in the subsequent G1‐phase, creating vulnerabilities of DNA replications and can be exploited by cancer cells that promote DNA repair and resistance to genotoxic agents. Thus, intratumoral variation in the arginine microenvironment synchronizes DNA replication with therapeutic responses.
Saving the Walking Dead: The delayed effects of radiation and mitigation by blocking the TGFβ‐1 pathway
Authors: Strandberg Stacey , Fleischmann Scott , Braun Makayla , Connelly Riley , DebBaruah Caroline , Fricker Ellen , Glaszcz Carly , Griffith-Topps Annabelle , Jenkins Ava , Jessick Madeline , Kuehn Claire , Kulju Tierney , Moews Alex , Rivas Jaylin , Schraufnagel Lauren , Strother Clarissa , Vanderhoef Kayla , Medhora Meetha , Narayanan Jayashree
Published: April 2020

Fibrosis of the lungs in patients who have undergone radiation therapy is a silent killer, inducing dense scar tissue in injured areas. Transforming growth factor beta 1 (TGFβ‐1) plays a role in the development of fibrosis. Radiation fibrosis takes months to develop. Consequently, people who are exposed to radiation to their lungs can die suddenly from pulmonary fibrosis. Latent TGFβ‐1 is stored in the extracellular matrix and is activated by the binding of an AV integrin to the arginine, glycine, and aspartate (RGD) amino acid sequence in the prodomain. Inside of the TGFβ‐1 is a ring‐like structure containing a solvent. Two prodomain arms in the structure connect to form a crossed V‐shape surrounding the growth factor monomers. These arms connect to form a bowtie with disulphide links close to the RGD sequences. The arm domain is composed of two anti‐parallel, four‐stranded beta sheets which contain hydrophobic faces. Active TGFβ‐1 binds to receptors on the surface of cells which signal to other proteins called SMADs, by adding phosphates to the SMADs. This step is inhibited by the medicine currently being tested, IPW‐5371. To understand the long‐term effects of radiation and the structurefunction relationship role of TGFβ‐1, the Divine Savior Holy Angels High School SMART (Students Modeling A Research Topic) Team designed a model of TGFβ‐1 using 3D printing technology. Currently, research is being conducted into fibrosis from activated TGFβ‐1 due to radiation exposure and IPW’s ability to reduce delayed injury to many organs that are exposed to radiation.

Support or Funding Information

NIAID award HHSN272201800012C to Innovation Pathways, Palo Alto and NIAID U01AI133594 to the Medical College of Wisconsin.
HGF/Met Axis Protects Human Retinal Pigment Epithelial Cells from Cell Death
Authors: Zarnegar Arman , Ma Jihong , Tan XinPing , Kwon YongKook , Zarnegar Reza
Published: April 2020

Demise of the retinal pigment epithelium (RPE) caused by etiologies such as age‐related macular degeneration is a major underlying factor leading to vision loss. Cell death can occur through two major pathways: apoptosis and necrosis/necroptosis. In the former, Fas/FasL death receptor signaling induces apoptosis, but its role in human RPE cell death is not thoroughly elucidated. In the latter, reactive oxygen species (ROS) can induce necrosis/necroptosis. Additionally, Hepatocyte Growth Factor (HGF) is a potent growth and survival factor for epithelial cells, but its functional role in RPE is not well characterized, especially its ability to protect RPE cells from death and to induce their growth. Accordingly, we hypothesized that HGF/Met protects human RPE cells from Fas‐promoted apoptosis and H2O2‐induced necrosis/necroptosis.

We analyzed whether the HGF receptor (Met) is expressed in human and non‐human primate (NHP) eye tissue via western blot analysis and immunohistochemistry. To determine whether HGF/Met protects RPE cells against death induced by Fas/FasL and H2O2, we utilized gain‐ and loss‐of‐function models, cell survival assays and gene expression analysis (RNAseq). Human ARPE‐19 cell line was treated with various combinations of HGF, Met kinase inhibitor SU11274 (SU), FasL or H2O2. Cell growth and survival was determined via MTT assay. Met, Akt, and Erk activation were analyzed by western blot.

Western blot analysis and immunohistochemistry confirmed Met activity and signaling in human and NHP eye tissue, including in the RPE cell layer. We found that HGF elicited Met stimulation and signaling culminating in activation of its downstream effectors Akt and Erk, proteins involved in two major pathways controlling cell survival and proliferation. We also uncovered that HGF inhibited FasL‐ and ROS‐induced killing of RPE. Conversely, inhibiting Met signaling rendered RPE susceptible to cell death induced by FasL and H2O2. Global gene expression analysis using an RNAseq approach revealed that HGF controls expression of numerous genes governing RPE cell growth, metabolism, and survival to name a few. In particular, HGF significantly upregulated 400 and repressed expression of 420 genes six hours post‐treatment; at 24 hours post‐treatment, HGF upregulated 291 and downregulated expression of 260 genes. Expression of 11,000 genes was unchanged.

Our studies establish that HGF and Met play a major role in promoting the survival and homeostasis of RPE cells by inhibiting two major pathways of RPE cell death. Our findings suggest HGF/Met is a potential druggable target for therapeutic management of RPE‐related human ocular diseases.
The Role of Cancer Associated Fibroblasts in Maintaining Cancer Stem Cells
Authors: Lewis Shantae , Jones Dennis
Published: April 2020

Breast cancer is one of the leading causes of cancer related deaths in females. Current treatment options for breast cancer ultimately fail, resulting in cancer relapse. One explanation for recurrence after treatment is the presence of cancer stem cells (CSCs). CSCs have high proliferative potential and are able to initiate tumor growth. In order to target CSCs, it is important to understand how CSCs are maintained. Recent studies have shown that cancer‐associated fibroblasts (CAFs) aid in the maintenance of CSCs. CAFs are an abundant cell type in the tumor microenvironment and have been found to promote tumor growth and confer resistance to anti‐cancer drugs by supporting cancer stem cells. We hypothesized that CAFs promote and maintain the growth of cancer stem cells in a novel tumor model (MCaP). Mammary carcinoma cells (MCaP) were propagated in culture from a tumor that developed spontaneously in an aged mouse. Cancer cells, along with CAFs were expanded from the isolated tumor. Cancer cells were grown as a mixed culture with CAFs or alone after enrichment. A colony growth assay was used to assess the formation of CSCs, which grow as colonies. Cultures were fixed and stained with cytokeratin and colonies were counted manually. Colony size was quantified using ImageJ. Flow cytometry was performed to detect and quantify surface expression of CSC markers CD44 and CD24 in mixed cultures and isolated cancer cells. To investigate whether CAFs enable cancer cell resistance to chemotherapy, we measured the dose response to cyclophosphamide or paclitaxel. Immunofluorescence was performed on tissue sections of metastatic lymph node and primary tumor. Tissues were stained with cytokeratin, aldehyde dehydrogenase (ALDH), and alpha‐sma (SMA). From the colony growth assay we noticed that colony formation and the size of colonies were both increased when MCaP cells were cultured with CAFs. Flow cytometry showed that MCaP cancer cells grown with CAFs resulted in the appearance of a CD44 high/CD24low population. This CSC population was not seen in cancer cells grown alone. After treating cells with paclitaxel or cyclophosphamide, we saw no notable difference in the viability of cancer cells in the presence or absence of CAFs. Immunofluorescence staining showed ALDH was expressed in SMA‐positive CAFs, but not in cytokeratin‐positive cancer cells. Although we did not find that CAFs protected cancer cells from chemotherapy, CAFs appear to have a positive effect on the size and quantity of CSC colonies. In addition, we detected a CD44 high, CD24 low CSC population in mixed cultures. CAFs were found to express the CSC marker ALDH, with expression increasing as the size of the tumor increased. This finding suggests that CAFs may provide a niche for CSCs. In conclusion, these results suggest that CAFs play an important role in maintaining CSCs in the MCaP mouse model.

Support or Funding Information

The University of the Virgin Islands MARC grant (GM008422)and The Jones lab ACS Pilot grant (K22CA230315)
Reproductive and Pup Parameters of Female Wistar Rats (Rattusnorvegicus) Mated by Males Treated with Date Fruit Extract and Cypermethrin
Authors: Ubah Simon Azubuike , Agbonu Adikpe Oluwa , Columbus Kwinjoh Philemon , Abah Kenneth Owoicho , Chibuogwu Ijeoma Chika , Abalaka Samson Eneojo , Abayomi Samuel Bankole , Enem Simon Ikechukwu , Itopa Ajayi IEtudaye , Ejiofor Charles Ejike
Published: April 2020

The work was designed to study the effect on fertility and pup parameters of female Wistar rats mated to male Wistar rats that were treated with Date fruit extract and Cypermethrin. A total of 48 mature Wistar rats (Rattusnorvegicus) comprising of 24 males and 24 females were randomly distributed into four groups (I, II, III, and IV) of 12 animals per group of which six were males and six were females. The six males in groups I, II, III and IV received distilled water; Cypermethrin at 60mg/kg body weight; Date fruit extract (DFE) at 250 mg/kg body weight; and a mixture of Cypermethrin (60 mg/kg body weight) and DFE (250mg/kg body weight) respectively. Treatment was given once weekly per os for eight weeks after which treated males were mated with the females at a ratio of 1: 1. Conception and pregnancy rates; gestation length; litter size; litter weights at birth, day‐5, day‐10, day‐15 and day‐21 post‐partum; anogenital distance at day‐10; sex ratio and age of pup at hair growth were measured. Results showed that there were no significant differences (p>0.05) in all parameters measured except sex ratio. More female than male offsprings were born to the Date fruit treated male groups. It was concluded that Cypermethrin at 60 mg/kg body weight did not alter the fertility of male Wistar rats while Date fruit extract at 250 mg/kg body weight favored the birth of more female off springs. Further investigation is required to unravel the potential of Date fruit extract in sex selection.
Ameliorative Effect of Ocimum gratissimum Leaf extract Against Phenylhydrazine‐Induced Toxicity in Albino Rats
Authors: Ugbogu Eziuche Amadike , Akara Emmanuel Ugochukwu
Published: April 2020

This study investigated the effect of aqueous leaf extract of Ocimum gratissimum on haematological and biochemical parameters of phenylhydrazine‐induced toxicity in rats. The experimental rats were divided into five (5) groups of six (12) rats each (6 males and 6 females placed in different cages). The test groups (Groups B to E) were induced intraperitoneally with 50 mg/kg phenylhydrazine each for two consecutive days. Group A served as normal control and received orally 0.25 ml/kg of distilled water as placebo, while group B (negative control) was left untreated. Groups C, D and E received orally 5 mg/kg ferrous sulphate, 200 mg/kg and 400 mg/kg aqueous extract of O. gratissimum respectively for 14 days. In the acute toxicity, the LD50 was above 5000 mg/kg body weight. There were significant decrease (p<0.05) in total cholesterol, triglycerides, low density lipoprotein (LDL) concentrations in the extract treated groups while aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, urea, creatinine, potassium ion (K+) significantly increased (p<0.05) in the negative control. High density lipoprotein (HDL) concentrations statistically increased (p<0.05) in the extract groups (C, D and E) when compared to the negative control. Red blood cell indices, packed cell volume (PCV) and haemoglobin concentrations (P< 0.05) significantly decreased in the negative control when compared to the extract treated groups in both male and female rats. The results show that aqueous leaf extract of Ocimum gratissimum could have the potentials of ameliorating the metabolic distortions in haematology, hepatorenal and lipid profile inflicted by phenylhydrazine. Thus, the extract could be of a good therapeutic use and safe for consumption at the tested doses.

Support or Funding Information

I received funding from Tertiary Education Trust Fund (TETFUND) Nigeria to attend this conference
Parkin‐Mediated Ubiquitination of Mitofilin Mediates Dopaminergic Neuron Death in Response to PD Stressors
Authors: Aliagan Abdulhafiz Imam , Feng Yansheng , Tombo Nathalie , Bopassa Jean C.
Published: April 2020

Background

Parkinson’s disease (PD) is a neurodegenerative disease characterized by a gradual and preferential loss of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction plays a critical role in the pathophysiology of dopaminergic neuron death induced by parkinsonian neurotoxins (PD stressors) that cause Parkinson’s disease (PD). However, the exact mitochondrial‐dependent mechanisms resulting in PD stressor‐induced dopaminergic neuron death remains elusive. The inner mitochondrial membrane (IMM) protein, Mitofilin, has been shown to play a key role of controlling and maintaining mitochondrial cristae morphology and its dysregulation has deleterious effects. Here, we investigated the role of Parkin‐Mitofilin interaction in PD stressor‐induced neuron death.

Methods

N27‐A dopaminergic neural cell line was used to determine the mitochondrial‐dependent mechanisms by which dopamine (Dopa) and Rotenone (Rot) induce cell death. Mitochondrial dysfunction was assessed by measuring mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production as well as mitochondrial integrity. Cell death was measured by flow cytometry using Annexin V/7AAD dyes and cell viability assessed by MTT assay. Protein quantification was measured using biochemical techniques including Western blot analysis, immunoprecipitation, and confocal microscopy.

Results

We found that PD stressors treatment increased Parkin translocation in to mitochondria where it interacts with Mitofilin in the IMM. This interaction promotes Mitofilin degradation via ubiquitination, which was associated with reduced MMP and increased ROS production, effects that were concomitant with abnormal mitochondrial structure and increased neuron death including by apoptosis. Further, we found that Dopa treated cells did not activate mitophagy process, while Rot treated cells exhibited excessive mitophagy. In addition, overexpressing the mitochondrial deubiquitinase, USP30, attenuated cell death induced by Rot, but not Dopa, suggesting that Parkin mediates two distinctive mechanisms leading to neuron death. Conversely, we found that Dopa treatment triggered an increase in mitochondrial calpain activity, cleavage and release of AIF into the cytosol, as well as apoptosis via AIF‐PARP dependent mechanism.

Conclusions

Together, our study reveals a novel role for Parkin‐Mitofilin interaction in PD pathogenesis, which leads to the ubiquitination and degradation of Mitofilin. The degradation of Mitofilin results in mitochondrial structural damage and dysfunction that causes apoptosis via AIF‐PARP pathway.

Support or Funding Information

National Institutes of Health [R01 grant HL138093 (JCB)]; Perry and Ruby Stevens Parkinson’s Disease Center of Excellence pilot grant (JCB).
Post‐treatment With Qing‐Ying‐Tang, a Compound Chinese Medicine Relives Lipopolysaccharide‐Induced Cerebral Microcirculation Disturbance in Mice
Authors: Han Jingyan
Published: April 2020

Objective

Lipopolysaccharide (LPS) causes microvascular dysfunction, which is a key episode in the pathogenesis of endotoxemia. This work aimed to investigate the effect of Qing‐Ying‐Tang (QYT), a compound Chinese medicine in cerebral microcirculation disturbance and brain damage induced by LPS.

Methods

Male C57/BL6 mice were continuously transfused with LPS (7.5 mg/kg/h) through the left femoral vein for 2 h. QYT (14.3 g/kg) was given orally 2 h after LPS administration. The dynamics of cerebral microcirculation were evaluated by intravital microscopy. Brain tissue edema was assessed by brain water content and Evans Blue leakage. Cytokines in plasma and brain were evaluated by flow cytometry. Confocal microscopy and Western blot were applied to detect the expression of junction and adhesion proteins, and signaling proteins concerned in mouse brain tissue.

Results

Post‐treatment with QYT significantly ameliorated LPS‐induced leukocyte adhesion to microvascular wall and albumin leakage from cerebral venules and brain tissue edema, attenuated the increase of MCP‐1, MIP‐1α, IL‐1α, IL‐6, and VCAM‐1 in brain tissue and the activation of NF‐κB and expression of MMP‐9 in brain.

QYT ameliorated the downregulation of claudin‐5, occludin, JAM‐1, ZO‐1, collagen IV as well as the expression and phosphorylation of VE‐cadherin in mouse brain.

Conclusions

This study demonstrated that QYT protected cerebral microvascular barrier from disruption after LPS by acting on the transcellular pathway mediated by caveolae and paracellular pathway mediated by junction proteins. This result suggests QYT as a potential strategy to deal with endotoxemia.