bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the mammalian cell cycle by the unfolded protein response
Authors: Solley Sabrina , Zappa Francesca , Acosta-Alvear Diego
Published: April 2020

Symmetric cell division requires partitioning of organelles to daughter cells after mitosis. Upon mitosis, organelles found in multiple copies in the cell are equally distributed to the daughter cells. Single organelles, such as the endoplasmic reticulum (ER) and Golgi apparatus, are fragmented prior to their re‐distribution to daughter cells. We hypothesize that mammalian cells monitor the health of their ER prior to commitment to cell division. Our goal is to define the roles of the unfolded protein response (UPR), a main mechanism maintaining ER health, during cell cycle progression. Our data suggest that the most conserved UPR sensor, the kinase/RNase IRE1 arrests cells at the G1/S boundary until ER stress is mitigated. Even though previous work has defined a role for the kinase PERK, another key UPR sensor/transducer, in controlling the cell cycle of mammalian cells (Brewer et al., 2000; Adamson et al., 2016), a role for IRE1 in controlling cell cycle progression has not been described in metazoans. A role for IRE1 in cytokinesis has been proposed in Saccharomyces cerevisiae (Bicknell et al., 2007), however IRE1’s roles in controlling cytokinesis or any other aspect of the cell cycle of animal cells has remained obscure. Our data show that manipulating IRE1 activity negatively impacts the cell cycle of mammalian cells. Our results also indicate that the G2/M cell cycle checkpoint kinase PKMYT1 suppresses IRE1 activity, suggesting that PKMYT1 may exert specific regulatory control over IRE1 prior to the commitment to cytokinesis. To study specific G1 or G2 responses, we have generated cell lines harboring a fluorescent reporter of cell cycle progression known as “FUCCI” (fluorescent ubiquitination cell cycle indicator; Koh et al., 2017). FUCCI takes advantage of the natural regulation of the turnover of fluorescent protein‐tagged cell cycle licensing factors (geminin and CDT1) to visualize and track live cells in the G1 or G2 stages of the cell cycle. FUCCI allows us to separate cells in the G1 or G2 stages of the cell cycle using fluorescence‐activated cell sorting (FACS) to measure UPR—and specifically IRE1—activity in G1 or in G2. Our data obtained from G1 and G2 FUCCI cells separated by FACS suggest that the activation threshold for the UPR differs between G1 and G2, and that PKMYT1 exerts negative regulatory control over IRE1. In addition, we observe that IRE1 loss‐of‐function leads to cytostasis, substantiating a role for the UPR in controlling cell cycle progression. Our data support a model in which IRE1 and PKMYT engage in a regulatory feedback loop wherein PKMYT1 controls IRE1 in G2, extending UPR control of the cell cycle beyond the G1/S transition.
Genome‐wide Screening Uncovers Transforming Growth Factor‐β3 as Regulator of Glycosaminoglycan Assembly
Authors: Rother Sandra , Weiss Ryan J. , Esko Jeffrey D.
Published: April 2020

Heparan sulfate (HS) and chondroitin sulfate (CS) are glycosaminoglycans covalently linked to proteoglycans (PG) present on all cell surfaces and in the extracellular matrix. They play a complex role in carcinogenesis as they are important for extracellular matrix remodeling as well as cell‐cell and cell‐matrix interactions. These processes are altered during malignancy, when tumor cells invade and systemically spread in different tissues. We have developed a genome wide screening method using CRISPR‐Cas9 gene targeting to identify novel genes involved in glycosaminoglycan metabolism. The approach utilizes agents that bind specifically to cell surface HS. One of the top hits was transforming growth factor‐β3 (TGF‐β3), a growth factor that controls multiple genes with tumor‐suppressing and tumor‐promoting effects. Cell surface levels of PG were evaluated in A375 melanoma cells after treatment with soluble TGF‐β1 or TGF‐β3 in the presence or absence of a TGF‐β type I receptor inhibitor (SB‐431542) using specific agents that bind to glycosaminoglycans, including mAbs 10E4 or CS56, guanidinylated neomycin and fibroblast growth factor‐2 (FGF2). Both TGF‐β isoforms stimulated cell surface HSPG and CSPG expression, that was blocked by SB‐431542. Inactivation of TGF‐β3 expression by CRISPR/Cas9 had the opposite effect. These findings show that TGF‐β3 is a newly identified regulatory factor of PG assembly in melanoma cells and that tonic expression of TGF‐β3 regulates PG production in a cell autonomous manner. Further exploration how TGF‐β affects disorders associated with altered PG formation in cancer might lead to innovative solutions to alter tumor cell growth and metastasis.

Support or Funding Information

We acknowledge financial support by the DFG (research fellowship, project number 420160411) and the NIH (fellowship award, K12HL141956).
Lipid droplet degradation through a novel and direct engulfment by endo‐lysosomes
Authors: Schott Micah B. , Mehall Bridget , Weller Shaun , Schulze Ryan J. , McNiven Mark
Published: April 2020

Lipid droplets (LDs) are fat‐storage organelles that play critical roles in energy homeostasis, but can excessively accumulate in metabolic diseases such as obesity and fatty liver where hepatocytes become grossly engorged with excess lipid. The catabolism of LDs aids in disease resolution and occurs in part by lipophagy, a form of selective autophagy, but the molecular and vesicle trafficking events that facilitate this process are not fully understood. During lipophagy, autophagosomes are thought to target LDs prior to fusion with acidic lysosomes, where neutral lipids are terminally degraded by lysosomal acid lipase. Ultrastructural viewing of hepatocytes showing LDs contained within electron‐dense acidic vesicles is consistent with this concept. In addition to canonical autophagy, we find that most LDs undergoing degradation are encased within single‐membrane endo‐lysosomes, and few LDs are observed within traditional double‐membrane autophagosomes. Further, studies of LD catabolism in yeast have shown that LDs can be directly internalized by the large acidic vacuole through a process known as microlipophagy. Thus, the GOAL of this study was to test if similar microlipophagy mechanisms occur in mammalian cells such as hepatocytes, and whether this process involves autophagy.

METHODS

To test this, we used microscopic fatty acid tracing studies and time‐lapse confocal microscopy to determine the role of autophagosomes versus endo‐lysosomes in LD catabolism. To trace the fate of LDs into terminal degradative vesicles, cells were first loaded with the fluorescent lipid marker Bodipy C12, then treated for 24h with LAListat, an inhibitor of lysosomal acid lipase.

RESULTS

We observed a ~5‐fold higher incidence of LDs within endo‐lysosomes (marked with the tetraspanin CD63), compared with autophagosomes (marked by the autophagy protein LC3). Furthermore, siRNA knockdown of macroautophagy proteins (Atg5, Atg7, and Fip200) did not inhibit LD internalization by endo‐lysosomes, suggesting that autophagy is not a prerequisite for this process. In addition, time‐lapse microscopy revealed a direct internalization of cytosolic LDs by endo‐lysosomes positive for lysotracker or CD63‐mCherry.

CONCLUSION

These findings suggest that hepatocytes utilize a distinct form of microautophagy that entails the direct envelopment of cytoplasmic LDs by endocytic vesicles such as multivesicular bodies and lysosomes/late endosomes. In addition to lipolysis and canonical macroautophagy, this process plays an important role in the catabolic utilization of LDs by the hepatocyte.

Support or Funding Information

This study was supported by NIH R01DK044650 (to M.A.M.), RO1AA020735 (to M.A.M.), and the K99AA020735 (to M.B.S.).
Coevolution of secreted proteins and members of the secretory pathway reveals interactions and motifs used for proper secretion
Authors: Shamie Isaac , Lewis Nathan E.
Published: April 2020

The secretory pathway consists of hundreds of macromolecules that function by physically interacting with and modifying secreted proteins to bring them to their final form and location. These include include chaperones, translocases, and enzymes for post‐translational modifications. Some of these are generic proteins for maintaining homeostasis in the secretory pathway, while others are specific for processing groups or individual secreted proteins. Additionally, many of these machinery come from common families that are conserved across eukaryotes. Understanding how these proteins are able to process thousands of secreted proteins across organisms and cell types is important and still lack fine‐grained details. In this study, we take an evolutionary approach to systematically characterize interactions between secreted proteins and the machinery proteins. Using mutual information and directed coupling analysis, we discover amino acids between pairs of proteins that have coevolved. We use this to predict interactions between proteins and compare them to the literature as well as computationally predicted database PrePPI. Additionally, we discover functional amino acid motifs within this pathway seen across organisms. By examining regions not found in the most conserved regions, this technique can capture previously unknown functional residues. This approach adds to work done in prokaryotes, here showing that interactions can be inferred between proteins in the secretory pathway by studying their amino acid content.

Support or Funding Information

Novo Nordisk Foundation
MCF‐7 derived cell spheres and clusters generated during the long‐term progression of 3D artificial tumors yield distinctive colonies and monolayer populations potentially reflective of relative malignant stage.
Authors: Lyden Timothy , Albu Greta , Jewett Sarah , Lozano Ashley , Vue Long
Published: April 2020

In a continuing effort to establish bio‐physiologically relevant in‐vitro models of tumor biology, our laboratory has shown that MCF‐7 derived 3D artificial tumors undergo a process that closely resembles tumor progression. A significant feature of this process is the release of both single cells and cell clusters as well as spheroids of various sizes. These generally range for clusters of a few cells and perhaps 50um in diameter to large structures which contain hundreds of cells and exceed 300–400um in overall dimension. This progressive release of cell structures continues for extended periods of time once start and easily generates dozens of new clusters per day. Once released these clusters remain suspended in the media of the culture for up to several days, eventually settling and attaching to the plate in most cases. At that point, these spheres/clusters explant onto the plate and form new colonies of tumor cells. Eventually these will cover large portions of the plate, however the individual colonies retain their own distinct structure even when multiple such colonies meet to form large coverage monolayers. In this report we examine the behaviors of these shed cell spheres and clusters using 3D artificial tumors as sources as well as applying two additional methods to generate similar structures. Those additional methods are hanging drop plates and rocking cultures. In all cases, the cultures which result present with distinctly colonial behaviors and in many cases these colonies also form new 3D structures following a period of growth in 2D. Previously we have also shown that these features will attach and invade target artificial stromal tissues as well. Taken together, these data suggest that MCF‐7 derive 3D artificial tumors function as a workable model of in‐vitro metastasis and provides a new tool‐set to examine and better understand this critical phase of cancer biology.
CHANGES IN THE METABOLIC PROFILE OF MDA‐MB231 BREAST CANCER DUE TO LDHA AND LDHB DOUBLE KNOCKOUT
Authors: Mack Nzinga , Soliman Karam , Mazzio Elizabeth
Published: April 2020

PURPOSE

Triple Negative Breast Cancer (TNBC) is an aggressive form of cancer that disproportionately affects African Americans and has a high mortality rate with no known cure. All solid tumors display an elevated glycolytic flux accounting for increased levels of lactic acid production, which is believed to be driven by the various isoforms of the enzyme Lactate Dehydrogenase (LDH). LDHA and LDHB are highly expressed in MDA‐MB‐231 cells. Increased lactic acid secretion contributes to significant pro oncogenic malignant and metastatic processes.

METHODS

Edited MDA‐MB 231 TNBC cells with LDHA and LDHB double gene knockouts were provided by Snynthego from their engineered cell product, knockout cell pool. The effects of LDHA and LDHB double knockouts on genetic parameters in MDA‐MB‐231 cells were evaluated by examining whole transcriptomic influence on mRNAs and long intergenic non‐coding RNA transcripts (lincRNA) using Affymetrix human 2.1‐ST microarrays. Genome studies were complemented by metabolic analysis of mitochondrial function, lactic acid production, glucose consumption and ATP production.

RESULTS

The data provide evidence to suggest that there are alternative pathways to the production of lactic acid outside of LDH conversion of pyruvate to lactic acid. The transcriptome analysis shows specific genes that could be involved with an alternative route of lactic acid production in cancer cells.

DISCUSSION/CONCLUSION

There is clearly enough evidence to suggest that acidity drives malignant processes using lactic acid as a means to control pH. Prevention of lactic acid production would be a formidable detriment to survival and metastasis of human malignancies, including TNBC.

Support or Funding Information

NIH 2U54MD00758234A1
Modeling cerebral network hemodynamics: Effect of conducted vasodilation on brain perfusion.
Authors: Bernier Lina , Ashenagar Baarbod , Moshkforoush Arash , Tsoukias Nikolaos
Published: April 2020

Neuronal activity signals local changes in functional hyperemia which match metabolic demands for oxygen (O2) ‐and other nutrients, referred to as Neurovascular Coupling (NVC). NVC is essential for proper functioning of the brain and is impaired in various clinical conditions including Alzheimer’s disease, stroke and aging. The emerging paradigm is that neurons and astrocytes release vasoactive mediators in the perivascular space of penetrating arterioles to increase their diameter and blood supply. Recent evidence supports the concept that NVC mediators initiate electrical vasoactive signaling on arterioles that propagate upstream to cause dilation and increase local blood flow. How electrical conduction of neuronal activity along the vasculature shapes the hyperemic response has not been elucidated. In this study, we model hemodynamic responses by conservation of flow, Fahraeus–Lindqvist effect, and the phase separation. Simulations are performed in macroscale reconstructed brain vascular networks. The model examines changes in tissue perfusion in response to neuronal activity and predicts the requirements for propagating electrical signals to evoke physiologically relevant changes in blood perfusion. The theoretical framework presented allows for testing proposed NVC mechanisms and assisting in the interpretation of macroscale functional imaging responses in health and in disease.
Monolayers of bovine milk‐derived stem cells present with distinctive multinucleated syncytial cells following self‐induced differentiation.
Authors: Lyden Timothy , Williams Katlyn
Published: April 2020

In an ongoing series of studies, our laboratory has been examining the biology of a putative stem cell population harvested from fresh bovine milk samples. Among the observations previously reported have been the long‐term survival of ductal‐like monolayer colonies which clearly undergo differentiation and generate several cell types consistent with the normal mammary gland. These include cells which produce large fat globules as well as supporting cells. In addition, after several month of maintenance, these colonies eventually generate localized 3D tissue features representative of mammary gland ducts and acinar structures. Now we are reporting on the development within these cultures of a subset of multinucleated syncytial cells. These cells appear to form in specific regional distributions and in smaller colonies are often spaced out relative to each other suggestive of a regulatory function in tissue feature development. In these studies, we have examined the relative development of milk ductal syncytial cells and sought evidence of a regulatory role using morphologic analysis and labeling studies. In addition, we are also examining the potential to artificially induce such syncytial cell formation using the cAMP activator forskolin. Preliminary experiments in this regard have focused on the effects of forskolin on the ductal cell line MCF10A. To date, results suggest that forskolin will in fact increase the incidence of syncytial cells in these monolayers. It remains to be examined whether this will also occur in our milk samples and then whether these syncytia are functionally equivalent to those already spontaneously occurring. While formation of syncytial cells is a common observation in breast ductal cancers and precancerous tissues, there is no indication in our bovine milk samples of any neoplastic transformation and the position, developmental time‐frame and relatively ubiquitous observation of these in every sample suggest a potentially functional regulatory role for these cells.
Mitochondrial function after bisphenol S exposure: a safe substitute for BPA?
Authors: Singh Radha Dutt , Easson Sarah , Thompson Jennifer A.
Published: April 2020

Objective

Bisphenol A (BPA) is one of the most common industrial chemicals, detected in the urine of 93 percent of US adults. BPA was declared a toxic substance in 2010 and has been linked to metabolic and cardiovascular disease. Amid growing consumer concern, manufacturers increasingly turned to BPA substitutes, the most common of which is bisphenol S (BPS); however, its safety was not determined before introduction to market. Thus, exposure to BPS is on the rise; but its health effects are unclear. Our aim was to determine the impact of BPS exposure on mitochondrial dynamics in vascular smooth muscle cells.

Methods

Vascular smooth muscle cells (VSMC) were isolated by enzymatic digestion from the thoracic aorta of juvenile (6–7 week old) male C56BL/6 mice. Cells were cultured in DMEM/F12 media supplemented with 10% fetal bovine serum and 1% penicillin‐streptomycin. At 70–80% confluency, cells were treated with vehicle (DMSO) or variable doses of BPS [2.5nM (levels in drinking water); 250nM (permissible limit) and 25μM]. Reactive oxygen species (ROS) levels from cellular and mitochondrial sources were determined by 2′,7′‐dichlorofluorescin diacetate (DCFDA) and MitoSOX staining, respectively. Membrane integrity was assessed by evaluating mitochondrial membrane potential (MMP) by JC1 staining. Mitochondrial fragmentation was measured by MitoTracker Red CMXROS staining and confocal microscopy. Real‐time PCR will be used to measure the expression of genes involved in mito‐fission and fussion and mitochondrial DNA copy number.

Results

After 30 min and 1 hr of BPS exposure, cellular and mitochondrial ROS levels were increased in a dose‐dependent manner, with a more prominent increase at 1 hr (all doses p<0.001). At 1, 12 and 24 hrs after exposure, mitochondrial depolarization was observed at all doses. At 24 hours, the reduction in mitochondrial membrane potential was greatest at the lowest dose (3‐fold vs. vehicle, p<0.0001). MitoTracker Red CMXROS revealed significant mitochondrial fragmentation at 24 hrs after exposure to 25μM BPS.

Conclusion

Mitochondrial function is disrupted in VSMCs exposed to BPS at doses lower than the permissible limit and equivalent to human exposure. Studies are underway to determine the mechanistic basis of BPS‐induced mitochondrial dysfunction and long‐term effects on vascular health.

Support or Funding Information

This study was supported the CIHR, Heart and Stroke Foundation of Canada, and a Kertland Fellowship.
Differential Splicing of the Lectin Domain of an O‐glycosyltransferase Influences Charged Peptide Preferences
Authors: May Carolyn , Syed Zulfeqhar , Zhang Liping , Hagen Kelly Ten
Published: April 2020

Mucin‐type O‐glycosylation is an essential post‐translational modification that is required for secretion, extracellular matrix composition and organ growth. O‐glycosylation is initiated by a large family of enzymes (GalNAcTs in mammals and PGANTs in Drosophila) that catalyze the addition of the sugar N‐acetylgalactosamine (GalNAc), onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes have distinct domains, including the catalytic domain and the C‐terminal ricin‐like lectin domain. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing the transfer of GalNAc, while the lectin domain is thought to recognize extant GalNAc on previously glycosylated substrates to position the catalytic domain for further glycosylation of unmodified serines and threonines. Previous work from our group has demonstrated that one member of this family in Drosophila undergoes differential splicing within the lectin domain, leading to variants that contain either a positively or negatively charged subdomain. Interestingly, while this splicing event affects only a subdomain of the lectin region, we find that it influences peptide specificity. We demonstrate that each charged enzyme variant has a preference for oppositely charged peptide substrates, and that this preference can be predictably altered by altering peptide charge. We further show that the expression of each splice variant is associated with the expression of appropriately charged mucins in secretory tissues of Drosophila, suggesting that differential splicing may be employed in vivo to ensure O‐glycosylation of highly charged substrates. Our results elucidate a mechanism for modulating substrate preferences of O‐glycosyltransferases based on the needs of the cell through alternative splicing within a specific subregion of the non‐catalytic domain.
Asperuloside reduces food intake and body weight via downregulation of orexigenic hypothalamic signalling in a mouse model of metabolic syndrome.
Authors: Caruso Vanni , Ishaq Muhammad , Tran Duyen , Yang Cheng , Vicenzi Silvia , Guven Nuri , Deans Bianca , Randall Cameron , Smith Jason , Jacobson Glenn , Ronci Maurizio , Eri Raj , Canales Juan , Iglesias Miguel
Published: April 2020

Controlling the urge to eat requires adequate integration of hypothalamic neuropeptides regulating energy balance processes with dopaminergic circuits in the mesolimbic pathway. Disruption of this signalling alters the hedonic control of reward‐driven food intake contributing to obesity. Our study aimed to understand the mechanism of action of the novel anti‐obesity compound, asperuloside (ASP), and whether ASP treatment could be used as safe weight loss treatment.

Mice were fed with a 45% high fat diet (HFD) and concurrently treated with ASP for 12 weeks. ASP reduced significantly (10.50%, p<0.05) final body weight of HFD mice compared to their control group. The effects of ASP reached significance after 5 weeks of treatment and were maintained throughout the experiment. Over the experimental period, the compound induced a significant daily energy intake reduction (12.8%, p<0.05) in mice consuming HFD compared to their control group. When standardised by body weight, visceral adipose mass was increased by the effect of HFD and significantly reduced by ASP treatment (35%, p<0.05). Orexigenic hypothalamic genes including NPY and ARGP, as well as important receptors involved in the control of food intake such as ghrelin, leptin and cannabinoid receptors, were significantly (p<0.05) downregulated by ASP treatment. These results suggest that ASP might induce weight loss plausibly via downregulation of hypothalamic signalling and support ASP candidacy as a potential and safe drug for the treatment of obesity.
Stepwise autophosphorylation regulates biased agonism of the insulin receptor
Authors: Yunn Na-Oh , Park Mangeun , Ryu Sung Ho
Published: April 2020

Insulin is a key regulator of energy metabolism in peripheral tissues but also functions as a growth factor. Insulin binding to the insulin receptor (IR) leads to autophosphorylation of intracellular tyrosine residues, which simultaneously initiates a multitude of signals and functions. In contrast, some artificial (non‐insulin) ligands for the IR result in biased agonism, selectively activating the PI3K/AKT pathway and metabolic effects without activating mitogen‐activated protein kinase (MAPK) pathway and mitogenic effects. However, the precise mechanism of biased agonism at the receptor level remains unclear. The biased agonist IR‐A48 aptamer selectively induces mono‐phosphorylation of Tyr1150 residue (m‐pY1150) in the kinase domain of IR. Hence, we hypothesized that IR autophosphorylation is a stepwise process in which formation of m‐pY1150 represents an intermediate step. To explore this idea, we used hybrid receptors in which insulin can bind only one of the two binding sites of the dimeric receptor. Asymmetric insulin binding selectively induced symmetric m‐pY1150 in both kinase domains. Moreover, the juxtamembrane domain, which interacts with the kinase domain, restricted the full activation of IR, and symmetric m‐pY1150 played a crucial role in the rearrangement of intracellular domains to release this restriction. Our findings demonstrate that the symmetry of insulin binding to a dimeric receptor determines the stepwise autophosphorylation of IR. Furthermore, considering that the degree of ligand symmetry on a receptor depends mainly on ligand concentration, our results suggest that IR may play metabolic‐biased roles in peripheral tissues dependent on local insulin concentrations.

Support or Funding Information

This research was supported by the Global Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF‐2016K1A1A2912722).
Cytochrome B5 Reductase 3 Can Be Approached As A Contemporary Therapeutic Target to Restrain Hypertension
Authors: Kumar Gaurav , Dey Sanjay Kumar , Kundu Suman
Published: April 2020

Hypertension or high blood pressure is attributed as a critical health problem. Currently available therapeutics are indeed effective in combating hypertension, but are not always bereft of complications. Hence, to pursue alternate anti‐hypertensive therapeutics, the present study investigated human Cytochrome B5 Reducatse3 (hCYB5R3) enzyme as a “contemporary” therapeutic target, since this enzyme regulates the bioavailability of nitric oxide, a vasodilator involved in governing vascular tone; and hence hypertension. In the present investigation, small molecule databases were virtually screened against the crystal structure of the enzyme to identify potential inhibitors as well as to obtain binding energies and inhibitory constants. The enzyme was purified to homogeneity from recombinant sources and the inhibitors so obtained were tested in vitro using potassium ferricyanide assay in order to calculate IC50 values as a measure of inhibitor efficacy of these compounds against hCYB5R3. Further, the effect of these inhibitors was tested using various biophysical techniques including fluorescence and CD spectroscopy so as to obtain binding affinity, number of binding sites and effect on secondary structure, respectively. Fluorescence analysis has revealed promising binding constants (in micro molar ranges) and stoichiometric ratios (≈1:1) for some of the inhibitors. CD analysis revealed that these inhibitors do not alter the secondary structure of the FAD containing enzyme. Further, selected inhibitors were able to curb hypertension in vivo in spontaneously hypertensive rats. These encouraging findings have poised us to investigate the inhibitors further since overall, the identified and screened leads showed promising potential to be developed as novel anti‐hypertensive drugs against hCYB5R3.

Support or Funding Information

The funding support to conduct this study was provided by Department of Biotechnology (DBT) and Council of Scientific and Industrial Research (CSIR), Government of India. For experiments in rats, ethical clearance was obtained from Rajiv Gandhi Centre for Biotechnology (RGCB), India (IAEC/2015/23/CCKartha/RGCB/20.05.2015 (Protocol No. IAEC/570/CCK/2016)
Identification of Microglial Cell Molecular Markers During Chick Embryo Brain Development and Human Xenograft Tumor Formation
Authors: Lin Amy
Published: April 2020

Glioblastoma (GBM) is the most aggressive and invasive human brain tumor, which despite advancement in surgery and immunology, only 10% of patients survive 18 months after diagnosis. The Galileo lab studies GBM, focusing on the role of the protein L1CAM on increasing the motility, proliferation, and invasiveness of GBM cells using in vitro cell tracking and a novel xenograft chick embryo brain tumor model. The work here focused on microglial cells, which are resident brain immune system cells that are not well understood. Studies in rodents and GBM patients showed that microglia play a critical role in promoting GBM growth via an activation of complex network of cytokines, chemokines and other metabolic factors that occur in the extracellular matrix. In the context of brain tumors, microglia promote neuroinflammation, which induces the BBB breakdown and inhibits antitumor immune responses. The first aim for my project was to find biomarkers that specifically identify microglial cells in normal chick embryo brain during development. The second aim was to use those markers in xenograft tumors in the chick embryo brain to look for any proinflammatory changes in microglial cells. Chick embryo brains were fixed, frozen, and cut into serial cryostat sections or fixed and vibratome sectioned, followed by immunofluorescent staining using antibodies or lectins that specifically identified microglial cells and endothelial cells in other model organisms. The optimal staining protocol required overnight primary antibody/lectin with 0.1% Triton X‐100 detergent and 5% normal goat serum in phosphate buffered saline. The antibodies and lectins investigated included Isolectin B4, Tomato (Lycopersicon esculentum) lectin, anti‐ferritin, anti‐laminin and CD45. Immunofluorescence analysis revealed CD45 to stain microglial cells with the most specificity. CD45 staining reveal microglial cell morphology to progressively change from a compact ameboid shape (E5) into ramified branched shape (E12 and E15). In addition to the increased quantity of ramified microglial cells in chick brain with xenograft human tumors, the microglia also appeared to be localized near endothelia. Further investigations are needed to verify the increased presence of reactive microglial cells and their interaction with tumor cells and angiogenesis.

Support or Funding Information

This work was supported by the Delaware INBRE program, with a grant from the National Institute of General Medical Sciences‐NIGMS (8 P20 GM103446‐16) from the National Institutes of Health, a grant to DSG from the National Cancer Institute, and a generous gift from Pamela S. Simpson ‘97 Translational Cancer Research Fund. I would like to thank Tyler Hellmig and Emily Kollenbroich for their guidance.
Anatomy of the round window: visibility prediction from preoperative CT scanners in cochlear implantation: literature criteria study and three‐dimensional reconstruction evaluation
Authors: Labrousse Marc , Veleine Yohanan , Dubernard Xavier , Brenet Esteban , Braun Marc , Renard Yohann
Published: April 2020

Introduction

During cochlear implantation, round window membrane visibility through posterior tympanotomy is associated with type of cochleostomy used. Limiting bone drilling helps preserve residual hearing. Studies of preoperative scanner sections were proposed to anticipate its visibility with variable results. Based on preoperative scanner three‐dimensional reconstruction the purpose of this study is to evaluate the predictive capacity of the round window membrane.

Materials and methods

Prospective monocentric observational study conducted at Reims University Hospital Center on all patients who were candidates for cochlear implantation from December 2018 to May 2019. The exposure of the round window membrane is classified as “good exposure” and “bad exposure” according to the St Thomas’ Hospital classification, on three‐dimensional (3D) reconstruction and on the basis of intraoperative vision, blind to each other. The criteria in the literature measured on the CT scan sections were also studied. Their correlation is then evaluated, as well as the diagnostic performance of the 3D analysis to predict the intraoperative exposure of the round window membrane.

Results

25 procedures were carried out in 10 children and 15 adults. Among literature criteria only the distance from the facial nerve to the membrane of the oblique sagittal round window is statistically significantly associated with intraoperative exposure of the membrane of the round window (good exposure: 5.5 ± 0.6[4.7–6.7] mm versus poor exposure: 4.8 ± 0.6[3.9–5.3] mm ; p=0.05). Exposure of the round window membrane in 3D reconstruction is associated with intraoperative exposure of the round window membrane in a statistically significant manner (p=0.0005).

Discussion

In our study, among literature criteria only distance from facial nerve to round window membrane in oblique sagittal plane is statistically significantly associated with intraoperative exposure of the round window membrane. For a safe cochlear implantation, we propose a new criterion using petrous 3D CT scanner reconstructions which is strongly associated with intraoperative exposure of the round window membrane.
Retrotransposition Bursts in the Adult Brain: Stress, Running, and Instrumental Learning Increase the Number of L1 Retrotransposon DNA Copies in the Mouse Nervous System
Authors: Anokhin Konstantin , Ivashkina Olga
Published: April 2020

Retrotransposons, the DNA elements which proliferate by reverse transcription of their RNA intermediates, comprise a major fraction of mammalian genomes. In the human genome a particularly abundant retrotransposon ‐ long interspersed nuclear element‐1 (LINE‐1 or L1) has 5x105 copies 80–100 of which are active and competent for retrotransposition. Recent findings indicate that LINE‐1 is active in neuronal precursor cells during differentiation, and its de novo insertions can change expression of neuron‐specific genes and cause somatic mosaicism in the adult brain. However, there has been limited understanding whether such retrotranspositions happen in the adult brain, when and how they occur, and what is their importance for the CNS function. In our study we tested whether prolonged stress, wheel running, or operant learning can lead to changes in L1 DNA copy number in the mouse brain. For this purpose, we subjected mice to immobilization stress (2 hours a day, 10 days), wheel running (unlimited access, 10 days) or nose‐poke operant training (1 session a day, 10 days). We collected the samples of hippocampus, frontal cortex, cerebellum, muscle and testicle tissues from experimental and home cage control mice. We performed qPCR for analysis of 3′‐UTR and ORF‐1 copy number. We observed significant increase in the 3′‐UTR and ORF‐1 copy number in all brain structures of the stress group compared to the home cage group. Nose‐poke training was associated with specific increase in 3′‐UTR copy number in the hippocampus. We also observed a small increase in 3′‐UTR and ORF‐1 copy number in all investigated brain structures in wheel running group. Altogether our results show that retrotranspositions is a common event in the adult brain under different conditions of novel experience.

Support or Funding Information

RFBR # 19‐015‐00550
Metabolic Activation of Pyrrolizidine Alkaloids – Generation of Multiple Reactive Metabolites Leading to DNA Adduct Formation and Potentially Liver Tumor Initiation
Authors: Xia Qingsu , He Xiaobo , Lin Ge , Fu Peter P.
Published: April 2020

Pyrrolizidine alkaloids (PAs) are toxic and carcinogenic phytochemicals widespread in the world. Metabolic formation of dehydro‐PAs and 6,7‐dihydro‐7‐hydroxy‐1‐hydroxymethyl‐5H‐pyrrolizine (DHP) has long been considered the principle metabolic activation pathway. We determined that a set of DHP‐DNA adducts are generally formed from the metabolism of a series of carcinogenic PAs in vitro, in cells and in vivo. The level of DHP‐DNA adducts well correlated with liver tumor potency for a set of PAs. We have proposed that this set of DHP‐DNA adducts is a potential biomarker of PA exposure and PA‐induced liver tumor initiation. Our present studies have found that, in addition to dehydro‐PAs and DHP, there are another seven reactive pyrrolic metabolites that are capable of binding to cellular DNA to produce the same set of DHP‐DNA adducts. These DNA reactive metabolites are: 7‐glutathione‐DHP, 7‐cysteine‐DHP, 7‐N‐acetylcysteine‐ DHP, 1‐CHO‐DHP, cysteinyl‐[2′‐S‐7]‐1‐CHO‐DHP, cysteinyl‐[3′‐N‐7]‐1‐CHO‐DHP, and 1‐cysteinylimino‐DHP. These pyrrolic metabolites are enzymatically and/or chemically formed from the reaction of the corresponding amino acids (and glutathione) with either dehydro‐PAs or DHP. Also, these pyrrolic metabolites are interconvertible, apparently ascribed to their unique reactive bifunctional alkylating character. These overall results represent the first time that concerning the metabolic activation of PAs: (i) there involve the formation of multiple DNA reactive pyrrolic metabolites; and (ii) metabolism of carcinogenic and potential carcinogenic PAs all generate the identical DHP‐DNA adducts. [This abstract is not an official U.S. Food and Drug Administration (FDA) guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred. The authors declare no competing financial interest.]
Thymoquinone Protects Against Cigarette Smoke Extract‐induced Vascular Dysfunction Through Inhibition of the RhoA/Rho‐Kinase Signaling Pathway
Authors: Moustafa Gamal A. , El-Mahdy Mohamed A. , Ismail Raed S. , Mansour Ahmed M.
Published: April 2020

Background

There is an ever‐growing focus on the role of natural products in modulating reactive oxygen species (ROS) and RhoA/Rho kinase‐mediated vascular disease. Thymoquinone (TQ), a constituent of the volatile oil derived from Nigella sativa seeds, possesses promising antioxidant and vasodilating properties via its effect on multiple signaling pathways; however, the effect of TQ on the RhoA/Rho‐Kinase pathway remains to be investigated. The aims of the present study were to examine whether TQ protects against CSE‐induced vascular dysfunction and to identify the underlying mechanisms of TQ on CSE‐induced ED in isolated rat aorta.

Methods

Cigarette smoke extract (CSE)‐exposed rat aortic rings were mounted in a wire myograph and subjected to contraction and relaxation. Quantitative assessment of RhoA activation was determined using G‐LISA RhoA activation assay kit where phosphorylation of myosin light chain‐20, myosin phosphatase‐targeting subunit 1 and protein kinase CPI‐17were determined by western blot analysis in the hole tissue protein extracts.The GSH content of aortic tissues was calculated using HPLC.

Results

TQ protected against CSE‐induced impairment of acetylcholine‐induced endothelium‐dependent relaxation, and decreased CSE‐induced ROS generation and glutathione depletion. Preincubation of aortic rings for 20 min with TQ attenuated the CSE‐enhanced phenylephrine‐induced vascular tension in endothelium‐denuded rings. TQ‐pretreated rings showed a decrease in CSE‐induced RhoA activation and phosphorylation of myosin light chain‐20, myosin phosphatase‐targeting subunit 1 and protein kinase CPI‐17.

Conclusions

These data indicate that TQ inhibited ROS generation‐induced RhoA/Rho kinase pathway activation, protecting against CSE‐induced vascular dysfunction. This study provides mechanistic insight for understanding the molecular basis and efficacy of TQ on vascular disease management.
Investigating the role of RME‐8 as a late onset Parkinson’s Disease marker in SH‐SY5Y neuroblastoma cells
Authors: Strawn Miguel Thomas-Grier , Xhabija Besa
Published: April 2020

Approximately, 10 million people worldwide live with the progressive, neurological Parkinson’s Disease (PD) experiencing a wide range of symptoms from resting tremors, bradykinesia, rigidity, to postural instability. Recent research has characterized a new mutation in Receptor‐Mediated Endocytosis 8 (RME‐8)/DNAJC13 Asn855Ser conferring a toxic gain‐of‐function resulting in impairment of endosomal transport. Furthermore, in the late‐onset of idiopathic PD, subtle deficits in endosomal receptor‐sorting/recycling are evident through the discover of pathogenic mutations of VPS35, LRRK2, and now DNAJC13/RME‐8.

RME‐8 is composed of 2000 amino acids, containing four IWN repeats of unknown functions and a DNAJ binding between the second and third repeat. Through the discovery of genetic screens of Caenorhabditis elegans, RME‐8 mutants exhibited defects in receptor mediated yolk endocytosis in the oocyte and fluid phase endocytosis in the coelomocyte. The continued studies of RME‐8 mutants shows that it is highly conserved. Our laboratory is interested in studying RME‐8 in the context of Parkinson’s and further investigate the effect of RME‐8 N855S in the disease relevant SH‐SY5Y neuroblastoma cell line.
LCZ696 mitigates diabetic‐induced nephropathy through inhibiting oxidative stress, NF‐κB mediated inflammation and glomerulosclerosis in rats
Authors: Belali Osamah , Al-Rejaie Salim
Published: April 2020

Background

One of the most common microvascular complications of diabetes is DN that considered the primary cause of end‐stage renal disease. Recently, combined therapy of angiotensin II receptor antagonist and neprilysin inhibitor are using to attenuate the DN. Thus the present study was designed to investigate the beneficial effects of LCZ696 (LCZ696) therapy on DN‐induced oxidative stress, inflammation and glomerulosclerosis in rats.

Methods

A single dose (60 mg/kg/day) of STZ is used to induced type 1 diabetes. The diabetic rats were treated orally with valsartan (31 mg/kg) or LCZ696 (68 mg/kg) for 6 weeks. At end of the treatment period, serum and kidney samples were collected and analyzed. In serum, glucose, insulin, urea, creatinine, TNF‐α, IL‐1β, IL‐6 and IL‐10 levels were estimated. In renal tissues, oxidative stress biomarkers including TBARs, GSH, SOD, CAT, GPx and GST were measured. The levels of TNF‐α, IL‐1β, IL‐6 and NF‐kB levels also estimated in renal tissues. Histological changes were observed in kidney.

Results

Diabetic animals exhibited hyperglycemia and significant elevation in: renal functions, serum and kidney pro‐inflammatory cytokines, NF‐kB, lipid peroxidation, infiltration of leukocytes, mean glomerular volume and glomerulosclerosis with a marked decrease in IL‐10 and antioxidant enzymes. LCZ696 therapy significantly improved renal function and kidney structure and decreased oxidative stress, inflammation and glomerulosclerosis symptoms. Furthermore, valsartan alone had comparable effects against renal oxidative stress, inflammation and glomerulosclerosis.

Conclusion

Taken together, present data suggest that LCZ696 had therapeutic potential to restrict DN progression through inhibiting oxidative stress, NF‐kB mediated inflammation and glomerulosclerosis.
Pre‐treatment with miR‐182 Antagomir Mitigates Ischemic Brain Damage by Reducing Astrocytes Injury and Inflammation
Authors: Alhadidi Qasim , Xu Lijun , Sun Xiaoyun , Althobaiti Yusuf , Almalki Atiah , Alsaab Hashem , Stary Creed
Published: April 2020

Ischemic stroke remains a devastating cerebrovascular disease that account for a high proportion of mortality and disability worldwide. MicroRNAs (miRNAs) are a class of small non‐coding RNAs that are responsible for regulation of posttranscriptional gene expression, and growing evidence supports a role for miRNAs in stroke injury and recovery. The current study examined the role of miR‐182 in experimental stroke using both in vitro and in vivo models of ischemic injury. A significant increase in miR‐182 expression was detected in the infarct area of transient middle cerebral artery occlusion (tMCAO) treated mice as well as in primary astrocyte cultures subjected to combined oxygen‐glucose deprivation/reperfusion (OGD/R) injury. In vivo, both stroke volume and neurobehavioral parameters were significantly improved by pretreatment with miR‐182 antagomir. Astrocytes stressed with OGD/R resulted in mitochondrial fragmentation, downregulation of cortactin, an actin binding protein, and reduced levels of connexin 43 expression, a cortactin‐associated protein. Conversely miR‐182 inhibition in astrocyte cultures preserved cortactin expression, and resulted in significantly improved cell viability after OGD/R. In parallel, lipopolysaccharide‐induced nitric‐oxide release in astrocytes was significantly reduced by pretreatment with miR‐182 inhibition. In conclusion, inhibition of ischemia‐induced miR‐182 reduced brain injury in experimental stroke and preserved astrocyte viability after in vitro ischemia, associated with reciprocal alterations in cortactin expression. These findings identify miR‐182 and/or cortactin as potential targets for ischemic stroke therapy.

Support or Funding Information

NIH grant NS107445 to Creed M. Stary.
Effect of CO2 on the Expression of Heme Oxygenase‐1 in Vascular Smooth Muscle Cells
Authors: Nakamura Taichi , Sugimoto Naotoshi , Nishimura Naoki , Wada Taizo
Published: April 2020

Objectives

Carbon dioxide is one of the factors that affect the constriction and dilatation of smooth muscle cells. However, the effects of CO2 on smooth vascular cell homeostasis are unclear. The heme oxygenase‐1(HO‐1) enzyme is involved in heme metabolism, and this protein helps protect cells against oxidative stress. This study aimed to clarify the role of CO2 in regulating HO‐1 expression and elucidate its cytoprotective mechanisms in vascular smooth muscle cells.

Methods

A7r5 rat smooth muscle cells were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, at 37°C in a 5% CO2 incubator. The cells were exposed to 20% CO2 for 24 h and 48 h. The expression of HO‐1 was subsequently evaluated by real time PCR, and cell viability was analyzed 24 h after treatment with hydrogen peroxide (H2O2; 100μM).

Results

The expression levels of HO‐1 were significantly reduced in the vascular smooth muscle cells following exposure to 20% CO2. Exposure to 20% CO2 for 24 h and 48 h facilitated H2O2‐induced cell death in the vascular smooth muscle cells.

Conclusions

These results suggested that exposure to CO2 may attenuate cytoprotection against oxidative stress via the downregulation of HO‐1 expression in vascular smooth muscle cells.

Support or Funding Information

Kakenhi 16K10064, 17H01963