bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II (AngII) regulates activity of kinases and phosphatases in neurons
Authors: Basu Urmi , Alikunju Saleena , Zimmerman Matthew C.
Published: June 2021

AngII signaling in the brain plays critical role in regulating cardiovascular function. AngII increases neuronal firing rate and modulates ion channel activity, which may be controlled by cellular kinases and phosphatases. Here, we tested the hypothesis that AngII stimulation of central neurons influences protein levels and activity of calcium/calmodulin kinaseII (CaMKII), protein kinase C (PKC), and protein phosphatase 2A (PP2A). Mouse CATH. a neurons were stimulated with AngII (100nm; 10 min‐24 hr) and PP2A and PKCδ protein levels were measured by Western blot analysis. CaMKII activity was assessed by detecting its phosphorylated levels and PP2A activity by a ser/thr Malachite Green phosphatase assay. AngII significantly increased phosphorylated CaMKII levels after 2 and 24 hr of stimulation (3.17 and 3.19‐fold increase vs. vehicle, P<0.05) and PKC δ protein levels after 24 hr (2.63‐fold increase vs. vehicle, P<0.05). AngII modestly decreased PP2A protein levels after 0.5–24 hrs of stimulation. PP2A activity decreased within 10 min and up to 1 hr of AngII stimulation (818 μg phosphate released at 10 mins and 794 μg at 1 hr vs. 1666 μg in vehicle). Together, these data indicate that in neurons AngII inhibits PP2A activity while increasing PKCδ, and activating CaMKII. Future studies will determine the role of these kinases and PP2A in modulating ion channel activity in AngII‐stimulated neurons.

AHA0930204N
Versican expression and turnover in hepatic stellate cell activation
Authors: Maurice Sean B , Crick Cody L , Kim Wan-Cheol , Law Christine , Norrie Minetta , Kim Sang-Eun , Winwood Paul
Published: June 2021

The pathophysiologic processes of extracellular matrix deposition and turnover in liver injury are central to our understanding of liver fibrosis and the development of antifibrotic therapies. Here we report expression of versican and the proteoglycan degrading enzymes ADAMTS1 and 4 by hepatic stellate cells (HSCs) activated to a myofibroblastic phenotype in tissue culture.

HSCs were isolated from C57B/6 mice by pronase/collagenase digestion and density gradient centrifugation and activated by culture on plastic. Versican expression was detected in HSCs at all stages of activation in culture. Expression increased gradually from days 1 through to 7, then more dramatically, up to 5 fold, by days 10 and 14. Immunofluorescence microscopy confirmed versican production by activated HSCs. Western blotting of activated HSC media demonstrated secretion of both intact versican and versican proteolytic products. Expression of the known versican processing enzymes, ADAMTS1 and 4, was detected at all time points but was suppressed during early stages of HSC activation (days 2 through 7) relative to day 1 levels.

Versican synthesis and turnover appear to be important components of HSC activation. Its regulated turnover may play a role in modulating normal and fibrotic remodeling in the liver. The reduction in ADAMTS expression could contribute to the deposition of a versican rich matrix in which tissue remodeling occurs.

Grant Funding Source: Northern Medical Program
Investigating the developmental processes of nasal contour shape
Authors: Wheat Amber D.
Published: June 2021

Nasal contour shape (NCS) is considered to be a useful indicator of ancestry in humans, which would imply that it is minimally affected by environmental factors over short periods of time. However, it is unclear whether differences in NCS are largely determined by genetics, exhibit responses to environmental factors (e.g., plasticity resulting from climatic factors), or are a combination of the two. One method for examining this looks at the integration of cranial shape. High correlations between NCS and other regions of the cranium, such as the vault and cranial base, may indicate ontogenetic constraint. This study tests three hypotheses: basicranial dimensions are correlated with nasal contour shape; cranial vault shape correlates with nasal contour shape; human groups significantly vary in the shape of the nasal contours. Seventy‐six crania of adult humans from three populations were utilized: Arikara, American “blacks,” and American “whites.” Preliminary results unexpectedly indicate no significant differences in NCS exist between population groups. Phenotypic correlations were calculated and results indicate that nasal contour shape is not significantly correlated with cranial vault shape or basicranial dimensions.

Grant Funding Source : AAA
CT‐SCAN of the head and neck of a roebuck (Capreolus capreolus): seriated cross‐sections and reconstruction
Authors: Le Floch-Prigent Patrice Pierre , Verdeille Stéphane
Published: June 2021

The entire head and neck of a roebuck was CT‐scanned, then reconstructed (Centre d'Imagerie de l'Ouest Parisien = CIMOP). The sample had been given by Mr Nicolas BRESSON (chasse de la Bélardière, Ardennes). It was a one year and a half old animal which measured 23cm length, 12,5cm high and 11cm width. It weighed 2,7kg. The acquisition was realized in the frontal plane (61 cross‐sections). The views were printed both with normal and osseous density. From this plane, the 21 sagittal and the 21 horizontal cross‐sections were reconstructed, and then the skeleton and the skin from several views. The images were tremendous. The anatomical topographical relationships were rendered with a fine precision. The skeleton could be visualized without any specific preparation. The stylo‐hyoïd apparatus and the laryngeal set were well visible. the seriated CT‐scan and the reconstruction are now irreplaceable in comparative anatomical studies as the samples are quite rare.
Generation of Floxed‐ShhTransgenic Mice for Cre‐directed Misexpression of Sonic Hedgehog
Authors: Limpach Aimee L
Published: June 2021

Sonic hedgehog (Shh) is a member of the Hedgehog family of signaling molecules that was identified by homology to the Drosophila segment polarity gene, hedgehog (Hh). Shh acts as a morphogen during embryonic development and has been shown to be involved in the patterning of many tissues, including the limb, the brain and spinal cord and the craniofacial region. The Shh signaling pathway has also been linked to the formation of a variety of kinds of cancerous tumors. Our laboratory has generated a floxed‐Shh transgenic mouse line that when crossed to various Cre lines, results in tissue‐specific misexpression of Shh. Using the Cre‐Lox transgenic system, we have generated mutant mice that misexpress Shh in tissues such as the inner ear, midbrain, cerebellum and limbs.
FGF‐INDUCED ACTIVATION OF THE LIMB SPECIFIC SHH REGULATORY REGION (LSSRR)
Authors: Bailey Traci W , Pira Charmaine U , Oberg Kerby C
Published: June 2021

Regeneration of amputated embryonic chick wings can be induced by Fgf, and upregulation of Shh is an essential step in this process. During development, a 1.8 kb conserved non‐coding region up‐stream of Shh functions as a limb‐specific Shh regulatory region (LSSRR). We hypothesize that LSSRR is involved in Fgf‐induced limb regeneration. To test our hypothesis, we electroporated (EP) an LSSRR‐green fluorescent protein (GFP)‐reporter construct into stage 23 chick limb bud mesoderm and then implanted an Fgf‐soaked bead to induce Shh expression. Green fluorescence was detected around the Fgf bead after 24 hr, supporting a role for LSSRR in the up‐regulation of Shh by Fgf. To determine the sequences within the LSSRR that were required for Shh up‐regulation, we isolated three separate candidate sequences within the LSSRR that contained Hox or RAR/NFκB binding site. The candidate sequences were ligated into a ptk‐GFP reporter plasmid and then EP into the presumptive wing mesoderm of stage 14 chick embryos. All three candidate sequences showed fluorescence at 48 hr, indicating enhancer activity with these minimal fragments. We conclude that during chick wing regeneration, Fgf utilizes the LSSRR in upregulating Shh and that multiple enhancer elements within the LSSRR may contribute to Shh regulation.

Grant Funding Source : Departmental Funding
Negative Regulation of Enhancer‐Associated RNA in Macrophages
Authors: Lam Michael T , Cho Han , Heinz Sven , Benner Christopher , Kaikkonen Minna U , Tanaka-Oishi Yumiko , Kosaka Mika , Lee Cindy , Evans Ronald M , Glass Christopher K
Published: June 2021

Recent discoveries of genome‐wide transcription at non‐coding regions suggest an unrecognized role of non‐coding RNAs. One class of these non‐coding RNAs is transcribed from cell‐type specific enhancer elements (eRNA). Here we show how transcription of eRNAs is negatively regulated by Rev‐Erb α and β, a subclass of the nuclear receptor family characterized as dedicated transcriptional repressors. Genome‐wide localization of Rev‐Erb α and β in macrophages by chromatin‐immunoprecipitation couple with sequencing showed that 70% of their binding sites are located in enhancer elements distal to protein coding genes. Approximately 55% of these distal regions are enriched for RNA Polymerase II. To test whether these enhancer elements express RNAs, we performed global run‐on sequencing with confirmation using RT‐PCR and detected nascent RNA transcripts at Rev‐Erb bound enhancers. Transcription of eRNA is de‐repressed in Rev‐ Erb deficient macrophages, whereas constitutive expression of Rev‐Erb represses eRNA transcription. Moreover, changes in eRNA expression are positively correlated to changes in expression of nearby protein coding genes. These findings suggest a mechanism whereby Rev‐Erb regulates gene expression by repressing eRNA from distal enhancer elements.

Grant Funding Source : NIH
Mesenchymal stromal cells (MSC) and platelet rich plasma (PRP) on a collagen matrix improve the tensile strength of a repaired incision in a rat model
Authors: Fagan Diana , Heffner Jeremy , Holmes Jon , Ferrari Jon , Krontiris-Litowitz Johanna , Perko John , Dorion Heath , Marie Hazel
Published: June 2021

The goal of this project is to prevent hernia formation by improving wound healing in surgically repaired tissue. Midline abdominal incisions were performed on Lewis rats. In Group 1 (n=14) the incision was repaired with no further additions. Group 2 had PRP on a collagen matrix (CollaTape™, Zimmer Dental) applied to the repaired incision. Group 3 received the same treatment as Group 2 with the addition of 106 MSC. The biomechanical properties of the facial defects, including the control samples, were determined using a standard force‐extension tensiometric analysis after 4 weeks (n=7) or 8 weeks (n=7). Tissues from Group 1 had an average tensile strength of 204 kPa at four weeks and 421.83 kPa at eight weeks; Group 2 at four weeks was 409.58 kPa and 582.18 kPa at eight weeks; Group 3 at four weeks was 817.15 kPa and at eight weeks was 917.17 kPa. Marked improvement in healing was seen with PRP, which was further improved by the addition of MSC. Supported by St. Elizabeth Health Center, Youngstown State University and LifeCell Hernia Resident/Fellow Research Grant.
Mathematical modeling of wound healing using Compucell3D multicell modeling environment
Authors: Gens John Scott , Jeyaraman Srividhya , Glazier James A.
Published: June 2021

Recent experimental studies suggest that contact inhibition (CI) of cell growth is a key mechanism during wound healing. Here we implement a 3D modeling method to explicitly model complex interactions of CI with local EGF concentrations during epithelial wound healing in cultured mammary epithelial cells. Model predictions are compared to patterns of cell growth observed in culture. Models presented are easy to modify and extensible to other epithelial cell types, to mesenchymal cells such as fibroblasts, and to epithelial cells grown on extracellular matrix or mesenchymal tissue. This publication was made possible by US EPA grant number R834289.
Developing Henna‐Haematoxylin stain as alternative to Nissl stain in delineating the cytoarchitecture of the neocortex and archicortex of the cerebrum
Authors: Alawa Judith Nkiruka , Gideon Olugbenga , Adetiba Bamidele , Alawa Clement B.
Published: June 2021

Demonstration of neuronal distribution patterns such as cerebral cortical layers or zones of neuronal cell loss usually requires specific stains such as Nissl stains which are not easily obtainable in developing countries. This study aims at testing henna as a counterstain to haematoxylin in delineating the cortical layers in the cerebrum. Paraffin‐embedded serial sections (5μm) of cerebral cortex of Wistar rat fixed in Bouin's fluid and stained with haematoxylin and henna (0.5% w/v aqueous crude extract of Lawsonia inermis leaves dissolved in 5% aqueous alcohol and mordanted with potassium alum) was observed to be effective in mapping out the six cortical layers of the neocortex and the three layers of the archicortex distinctly. It also stained the cytoplasm of the perikarya and glial cells brown and in addition demonstrated the thalamic nucleus in the central core of the cerebrum as a dense aggregation of neuronal cell bodies criss‐crossed by bundles of fibres. Thus, henna used as a counterstain to haematoxylin demonstrated reliability as a neurological stain to delineate the cellular layers in the archicortex (hippocampus) and the neocortex and therefore could serve as a readily available and cheaper alternative to Nissl stains for developing or third world countries.

Grant Funding Source: Ahmadu Bello University Research Grant
Distribution of urea transporter B (UT‐B) in the mice brain
Authors: ran jianhua , Sun Shanquan , He Guiqiong
Published: June 2021

The study was performed to describe morphologic features of UT‐B and speculated its functional roles in brain. Immunohistochemistry and immunofluorescence double staining were performed to detect the cellular localization and cell type of this transporter. UT‐B is widespread expressed in astrocytes in cerebral cortex, hippcampus, cerebellum, olfactory bulb as well as in some special glia, such as Bergmann glia, Müller cells, ependymal cells and epithelial cells of the choroid plexus. Additionally, UT‐B expressed in the neuron of cerebral cortex, the hippocampus, the mitral cells in the olfactory bulb, indicated it new role in the brain. Furthermore, UT‐B positive signaling was detected in NG2 cells. Wide distribution of UT‐B in the brain indicated that UT‐B may make an important role in controlling urea transport between neurons and astrocytes sequence throughout the astrocytic network and guiding urea to blood vessels and the cerebral spinal fluid (CSF) to keep homeostasis in the brain.

Supported by the National Natural Science Foundation of China (30500171), and the Natural Science Foundation of Chongqing (CSTC2010BB5098).
The death of neurons derived from familial Alzheimer's disease pluripotent stem cells is no different from the death of neurons derived from normal controls
Authors: Shaner Mason A , Yuan Shauna H , Goldstein Lawrence S
Published: June 2021

Neuronal death, a pathological hallmark of Alzheimer's Disease (AD) is seen in patients whose presenilin‐1 (PS1) mutation causes their autosomal dominant AD. We assayed markers of the apoptotic death of neurons induced from pluripotent stem cells obtained from human PS1 and non‐demented controls. Our investigation included immunofluorescent imaging, analyzing TUNEL, MAP2, Caspase 3, and GABA staining for multiple samples from different PS1 individuals at different length of culture conditions. We found no significant differences between the PS1 and control groups. These baseline observations of an in‐vitro model suggest that expression of the AD phenotype requires factors beyond standard culturing conditions. This model can be used to explore stresses on the control and PS1 cell populations that will differentiate them, providing a disease‐in‐a‐dish model for those biochemical alterations likely to express the premature apoptotic phenotype of autosomal dominant AD. Research support was provided by CIRM (California Institute of Regenerative Medicine) and HHMI (Howard Hughes Medical Institute).

Grant Funding Source : CIRM (California Institute of Regenerative Medicine), HHMI (Howard Hughes Medical Institute)
AChR Clustering is Increased by Neuregulin
Authors: Boothe Sarah M. , Grow Wade A.
Published: June 2021

In skeletal muscle development, agrin is released by the approaching motor neuron prior to innervation and the formation of the neuromuscular synapse. Agrin induces the clustering of existing acetylcholine receptors (AChRs) at the site of the future neuromuscular synapse through an agrin signaling pathway. Neuregulin is also released by the approaching motor neuron and induces the production of additional AChRs, especially at the nuclei near the future neuromuscular synapse. Our lab investigates myogenesis and neuromuscular synapse formation using the C2C12 myotube cell culture model. C2C12 myoblasts proliferate in growth medium, and then fuse into myotubes when switched to low‐serum differentiation medium (DM) for 72 hours. Myotubes are stimulated to cluster AChRs by the addition of agrin for the last 16 hours in DM. Our hypothesis was that neuregulin would increase AChR clustering in C2C12 myotubes as previously demonstrated in another cell line. Concentrations of agrin or neuregulin as low as 10 ng/ml was sufficient to increase AChR clustering. We conclude that AChR clustering in C2C12 myotubes is increased either by utilizing existing AChRs or the production of additional AChRs.

Grant Funding Source: None
MyoD and Myogenin are Required for Agrin‐induced AChR Clustering
Authors: Anderson Erica E. , Grow Wade A.
Published: June 2021

During skeletal muscle development, specific transcription factors called myogenic regulatory factors are expressed at specific times to guide myogenesis. Gene expression of MyoD correlates with myoblast proliferation, while gene expression of Myogenin correlates with myoblast fusion into multinucleated myotubes and subsequent muscle fiber differentiation. Our lab investigates myogenesis and neuromuscular synapse formation using the C2C12 myotube cell culture model. C2C12 myoblasts proliferate in growth medium, and then fuse into myotubes when switched to low‐serum differentiation medium (DM) for 72 hours. Myotubes are stimulated to cluster acetylcholine receptors (AChRs) by the addition of agrin for the last 16 hours in DM. Our hypothesis was that Myogenin, but not MyoD, is required for agrin‐induced AChR clustering. Daily exposure to 1 ug/ml of MyoD antibody (Ab) or Myogenin Ab was sufficient to decrease agrin‐induced AChR clustering. In addition, exposure to 1 ug/ml of MyoD Ab or Myogenin Ab limited to the first or second 24 hours in DM also decreased agrin‐induced AChR clustering. However, agrin‐induced AChR clustering was unaffected by exposure to MyoD Ab or Myogenin Ab exposure during the last 24 hours in DM. We conclude that both MyoD and Myogenin are required for agrin‐induced AChR clustering.

Grant Funding Source : None
Optogenetic regulation of peripheral sensory or motor axons during chick embryogenesis
Authors: Sharp Andrew A , Fromherz Sylvia
Published: June 2021

Our goal is to understand the effects of altered embryonic movement and sensation on proper neural development. Previously, we have shown in chick embryos that persistent expression of channelrhodopsin in peripheral axons of the leg can be achieved via electroporation of a modified transposon‐based exrpression system and that illumination of the leg with a blue LED can activate leg movements and modulate spontaneous leg motility. We now extend this work to include the ability to achieve persistent expression in targeted neuronal populations including peripheral sensory or motor axons by controlling the age of the embryo electroporated and the directionality of the applied current field. Also, we have developed an inexpensive system for focusing light from brighter LEDs to confine illumination to selected nerves and to activate them at later stages of development as superficial tissues thicken. This allows us to assay the effects of selective activation of sensory or motor axons in specific nerves in the leg. Lastly, we are testing transposon‐based expression of Arch and Halorhodopsin in chick embryos for the ability to suppress excitation in peripheral nerves.
Exploring the Plasmodium falciparum metabolome
Authors: Llinas Manuel
Published: June 2021

The genome of Plasmodium falciparum indicates that the metabolic pathways utilized by this organism are highly unique. Recent efforts to comprehensively examine the biology of P. falciparum have focused on transcriptome and proteome analysis to gain insight into Plasmodium‐specific pathways. The third crucial component that remains to be established is the metabolome: the complement of small‐molecule metabolites and their relative levels. Our lab has begun to characterize various aspects of parasite metabolism using high accuracy mass‐spectrometry to simultaneously measure metabolites from complex cellular extracts from parasite‐infected cells. The approaches we are using allow us to assay various aspects of the P. falciparum metabolome. One approach has been to examine the interaction of Plasmodium with the host red blood cell using targeted measurements of specific metabolites shared with the host erythrocyte. We are also using 13C and 15N isotopic labeling experiments to directly trace carbon flux through known biochemical pathways. Finally, we are using metabolite measurements to map genetic control of metabolism by assaying global metabolite patterns in the parents and progeny of a Plasmodium falciparum genetic cross. Results from these studies are beginning to unravel the divergence of metabolism in P. falciparum and promise to provide unique avenues for future drug intervention strategies.
Localization of gamma‐secretase subunit Aph‐1 in the central nervous system of Alzheimer's disease mouse model
Authors: He Guiqiong , Sun Shanquan
Published: June 2021

The gamma‐secretase complex cleaves the final step of amyloid protein precursor (APP) to generate amyloid beta peptide (Abeta), a pathogenic component of senile plaques (SP) in the brain of Alzheimer's disease (AD). Recent studies show that Aph‐1 along with presenilins (PS), nicastrin, Pen‐2 are essential components of the gamma‐secretase. The structure and function of Aph‐1 in vitro are well defined, but less is known about its localization in the central nervous system (CNS) of AD model mice. Using Western blot and immunohistochemical methods, we found that APH‐1 is widely expressed at select neuronal populations of major areas (including cortex, hippocampus, olfactory bulb, hypothalamus, striatum, cerebellum, brainstem and spinal cord) in both APP/PS1 double transgenic AD model mice and its wildtype controls. The Aph‐1 expression level is relatively high in the brain at the early stages of postnatal development, but declined gradually to reach adult profiles in both AD and control mice. Aph‐1 is distributed much more extensively than circumscribed SP in AD mice. Aph‐1 expression level is higher than that in control mice. The present study provides an anatomical basis for indicating that APH‐1 is tightly related to AD pathogenesis.

This work was supported by National Natural Science Foundation of China (30700885), Key project of Chinese Ministry of Education(209102
Disregulated Sphk1, Sphk2 and their receptors in the brain of MPTP‐induced mouse model of Parkinson's disease
Authors: sivasubramanian meenalochani , Dheen S Thameem , Peng Zhao Feng , Srinivasan Dinesh Kumar , Tay Samuel SW
Published: June 2021

Sphingosine kinase (sphk) is a key enzyme responsible for phosphorylating sphingosine into sphingosine‐1‐phosphate(S1P). There are two forms of sphingokinases, Sphk1 and Sphk2. Sphingokinases in the nervous system control several signaling pathways involved in neuronal survival. In many neurodegenerative diseases, sphingolipid metabolism is highly disregulated. The present study demonstrates that the mRNA expression of Sphk1(a key enzyme of the sphingolipid metabolic pathway), its isoform Sphk2 and their five GPCRS (five G protein coupled receptors which are involved in the sphingolipid pathway) in dopaminergic neurons of the substantia nigra of the brain showed a marked decrease in the MPTP‐ induced mouse model of Parkinson's disease. Their downregulation was confirmed in MPP iodide‐treated MN9D cell line which contains dopaminergic neurons. Remarkably, the downregulation of these two sphingokinases was accompanied by a marked increase in the expression of TNF alpha. These results suggest that sphingolipid metabolism is altered in the substantia nigra of MPTP‐induced mouse model of Parkinson's disease. Relevance of these changes in the pathogenesis of Parkinson's disease will be discussed.

Supported by grant no : MOE 2009‐T2‐1‐061 from the Ministry of Education, Singapore.
Potent Suppressive Effect of Resveratrol and Apigenin on Pro‐Inflammatory Responses in Lipopolysaccharide and IFN‐γ‐activated Microglia and Macrophages: Implications for Alzheimer's disease therapies
Authors: Ooi Lezanne
Published: June 2021

Purpose

Activation of microglia and macrophages contribute to neuroinflammation and neurotoxicity in Alzheimer's disease. However, microglia and macrophages differ in their ability to induce immune responses. Understanding the differences in the release of pro‐inflammatory factors by microglia and macrophage cells is important for identifying potential drugs to treat disease.

Methods

RAW macrophages, C8B4 microglia and primary murine microglia cells were activated with lipopolysaccharide (LPS) with interferon‐γ (IFN‐γ) and levels of 23 cytokines were measured using a cytokine array. Further to this, the cells were treated with resveratrol and apigenin, anti‐inflammatories suggested as potential anti‐neurodegenerative drugs, and nitrite levels measured as a marker of oxidative stress.

Results

LPS and IFN‐γ increased nitrite levels 13 fold in RAW and 3 fold in microglia. RAW activation resulted in greater release of cytokines, such as IL‐10, whereas microglia showed greater release of chemokines, such as eotaxin. Furthermore treatment with 25 μg/ml resveratrol or 35 μg/ml apigenin lead to a 50% decrease in nitrite levels.

Conclusion

Microglia and macrophages respond to activation by releasing distinct cytokine profiles. A potent suppressive effect of resveratrol and apigenin on pro‐inflammatory responses suggests a therapeutic potential for these compounds in neurodegenerative diseases.

Grant Funding Source : University of Western Sydney
Effects of daidzein on post‐stroke injury and recovery of function in the rat
Authors: Stout Jessica M , Wallace Douglas G , Cheatwood Joseph L
Published: June 2021

Treatments to reduce negative outcomes associated with stroke and/or improve functional recovery are needed. A recent study from our laboratory demonstrated the ability of a soy protein‐based diet to decrease the severity of post‐stroke behavioral deficits when provided before the onset of stroke. One potential contributor to the observed effects is daidzein, a bioactive isoflavone found in soy foods. In the current study, we aimed to determine the effectiveness of daidzein as a post‐stroke treatment and its ability to produce protective effects in the brain which may lead to enhanced behavioral recovery after stroke. To test this hypothesis, young adult male rats underwent a middle cerebral artery occlusion (MCAO) to produce a stroke lesion. Immediately after the MCAO procedure, rats were treated with either daidzein or vehicle via subcutaneous osmotic minipumps. Forelimb function was assessed using the skilled ladder rung walking task to determine post‐stroke behavioral effects and gauge functional recovery in all groups. In addition, gross locomotor performance was analyzed using the accelerating rotarod walking task. No significant difference was detected in rotarod performance between groups, although a possible trend was noted. Analyses of stroke lesion volume, skilled ladder rung walking data, and neuronal survival are ongoing. Supported by: SIUC REACH program, SIUC School of Medicine

Grant Funding Source : None
Heart Regeneration in Notophthalmus viridescens: Where do the Stem Cells Come From?
Authors: Wilder Amanda Somers , Sessions Stanley K. , Piefer Andrew J.
Published: June 2021

The purpose of this study was to investigate the origin of cardiomyocyte stem cells involved in the regeneration of heart tissue in Eastern newts (Notophthalmus viridescens). We tested the idea that cardiomyocyte stem cells originate from the blood clot that forms at the site of injury. We used RT‐PCR on RNA extracted from regenerating and non‐regenerating newt heart tissue to analyze expression patterns of three cardiomyocte stem cell specific genes known to be important in vertebrate heart development: crip2, fgf8, and nanog. We discuss the importance of our results in understanding the repair and regeneration in the heart tissue of vertebrates.
Evaluation of a new single tube RNA amplification method with low 3′/5′ ratios
Authors: Coleman Jack , Jin Richard , Patton Wayne
Published: June 2021

Expression microarrays have become a common tool in basic and clinical biomedical sciences. However, current technology is limited by the need for relatively large amounts of RNA for labeling and hybridization, or multiple rounds of amplification are required that may reduce the quality of resulting RNA. Methods in use today for amplification of low input RNA often under represent the 5′ end of mRNAs. In this study, we have developed a unique one tube T7 based RNA amplification kit that can reproducibly amplify nanogram amounts of total RNA and generate gene expression profiles that are independent of amount of input RNA, and are comparable with larger amounts of RNA amplified by other accepted methods. The actin 3′/5′ ratio remains low whether amplifying and labeling 20 ng or 500 ng total RNA. The resulting labeled RNA gives high %P calls on Affymetrix arrays, and provides low 3′/5′ ratios even with low input RNA.
Deregulation of E‐Cadherin and MTA2 in Tamoxifen Resistant Breast Cancer Cells
Authors: Hathaway Millie Mae , Louie Maggie C
Published: June 2021

Tamoxifen is an antiestrogen often used to treat hormone responsive breast cancer. It is a selective estrogen receptor modulator that inhibits breast cancer cell growth by competing with estrogen for receptor binding. Although tamoxifen has been successful in the treatment of breast cancer, prolonged usage of the drug often leads to the development of acquired resistance in which tumors become non‐responsive. Previously, our lab demonstrated that tamoxifen resistant breast cancer cells exhibit an increased ability to proliferate even in the presence of the antagonist. In addition to changes in growth characteristics, recent analysis suggest that tamoxifen resistant cells also develop more aggressive phenotypes, including the ability to migrate and invade through the extracellular matrix. Our data also suggest that these resistant cells express lower levels of E‐cadherin and higher levels of metastasis associated protein 2 (MTA2). Furthermore, our preliminary results indicate that DNA methylation may be responsible for the down regulation of E‐cadherin, however its role in MTA2 expression requires further investigation. The development of acquired resistance to tamoxifen and other anti‐estrogens has been a major obstacle in breast cancer therapy, and understanding how the deregulation of E‐cadherin and MTA2 contribute to the development of resistance will offer some insights to this problem.
Genetic association of SIX1 gene variants and primary open‐angle glaucoma in Caucasians
Authors: Shaw Robert , Wang Ye , Chen Yuhong , Du Hongjn , Zhang Kang
Published: June 2021

Primary open‐angle glaucoma (POAG) is a condition of disorders that have optic nerve damage and slow progressive degeneration of retina ganglion cells, causing irreversible defects in the visual field loss usually associated with elevated intraocular pressure (IOP). The exact mechanism for the pathogenesis of POAG is still largely unknown. However, studies have shown that POAG has a multifactorial etiology, including genetics, high IOP and central corneal thickness (CCT). Recently, genome‐wide association study (GWAS) identified a common variant (rs10483727) on chromosome 14q22.3‐q23 within 40kbp of the SIX1 gene significantly associated with vertical cup‐disc ratio (VCDR), a parameter in the clinical assessment of the glaucoma management. To investigate the association of SIX1 gene variants with POAG, we genotyped a Caucasian cohort with 768 cases and 815 age and ethnicity matched controls. For the four SNPs we tested near SIX1 gene, we found that rs10483727 showed significant association with POAG in the Caucasian cohort (p = 9.82 × 10–6 for an additive model, OR =1.37 (1.19 – 1.59); T allele: 45.4% in cases versus 37.7% in controls). To confirm our association study result, we induced a mechanical injury to the axon by optic nerve crush in mice and found the expression of SIX1 was increased in nerve injured mice.

This study was supported by grants from NIH/NEI to KZ.
RELATIONSHIP OF GENE EXPRESSION AND SEX HORMONE RECEPTORS IN PRIMARY BREAST CARCINOMA AND METASTASES FROM THE SAME PATIENT
Authors: Andres Sarah Alane , Wittliff James L.
Published: June 2021

Using microarray results and published reports, 32 genes were selected whose expression appears related to clinical outcome of breast cancer. Candidate gene expression and estrogen (ER) and progestin receptor (PR), determined by ligand binding or EIA, were evaluated to ascertain their relationship during lymph node invasion. RNA was isolated from tissue sections of de‐identified frozen carcinoma biopsies. cDNA was prepared with Superscript RT III reagents (Invitrogen) and qPCR reactions were performed using Power Sybr Green PCR Master Mix (Applied Biosystems). Although levels of ER and PR protein varied between the primary and the metastasis, no primary lesion that was ER− or PR−exhibited a positive ER/PR result in the metastasis. Only 7 patients with either ER+ or PR+ primaries exhibited loss of receptor expression in the metastasis. ESR1 and PGR gene expression correlated highly with protein levels of ER and PR. Expression of 6 of 32 genes (TBC1D9, TRIM29, SCUBE2, IL6ST, SLC39A6, LRBA) was discordant in at least half of the primary lesion‐lymph node pairs, while expression of 6 genes (TRIM29, SCUBE2, SLC39A6, TCEAL1, PFKP, XBP1) was discordant in at least half of the primary‐distant metastasis pairs. These results address the molecular basis for progression of a primary lesion to a metastatic phenotype. Supported in part by grants from Phi Beta Psi Charity Trust and a CTSP Grant from the Commonwealth of Kentucky.
Cosmc Is Silenced in Human Tn4 B Cells through Hypermethylation of the Gene Promoter
Authors: Ju Tongzhong , Mi Rongjuan , Song Lina , Wang Yingchun , Ding Xiaokun , Crew Vanja K. , Lehoux Sylvain D. , van Die Irma , Chapman Arlene B. , Cummings Richard D.
Published: June 2021

Background

Mucin‐type O‐glycosylation is regulated through core 1 β3‐galatosyltransferase (T‐synthase) and its specific molecular chaperone Cosmc. Human Cosmc is encoded by its gene on Xq24. Genetic alterations of Cosmc, including point mutations and gene deletions, have been identified in several human diseases. Tn4 B cells from patient with Tn‐syndrome‐like features have no transcript of Cosmc and express the Tn antigen (GalNAcα1‐Ser/Thr).

Study Objective

To elucidate the molecular mechanism as to how human Tn4 B cells lack Cosmc transcripts.

Methods

Treat Tn4 B cells with 5‐Aza‐2′‐deoxycytidine (5‐Aza‐CdR) to test whether it can induce expression of Cosmc; and perform bisulfite sequencing to examine the methylation status of the Cosmc promoter.

Results

Treatment of Tn4 B cells with 5‐Aza‐CdR results in expression of Cosmc transcripts, restores the activity of T‐synthase, and correct the O‐glycosylation. Bisulfite sequencing showed that CG dinucleotides in the Cosmc promoter in Tn4 B cells are exclusively methylated. Interestingly, other X‐linked genes, OGT and PIG‐A are not silenced in the Tn4 B cells.

Conclusion

Hypermethylation of the Cosmc promoter can completely silence the gene, which is another mechanism that can lead to expression of Tn and STn antigens, which may shed the light on our better understanding of the epigenetic alteration leading to the tumor antigen expression in human tumors.
Epigenetic dysregulation via regulator of calcineurin 1 (RCAN1) in Alzheimer's disease
Authors: Wong Helen , Levenga Josien , Rothermel Beverly , Klann Eric , Hoeffer Charles A
Published: June 2021

Alzheimer's disease (AD) is a neurodegenerative disease that is associated with epigenetic dysregulation. However, the mechanisms mediating epigenetic dysfunction in AD are unknown. Our objective was to determine if Regulator of Calcineurin 1 (RCAN1) has a role in AD‐related epigenetic changes. RCAN1 is located on human chromosome 21, trisomy of which results in Down syndrome (DS), a disorder characterized with early onset AD. Moreover, RCAN1 is overexpressed in both DS and AD. RCAN1 modulates the activity of calcineurin (CaN), a calcium‐sensitive phosphatase that can in turn regulate the transcription factor cyclic AMP response element binding protein (CREB). CREB affects epigenetic modification by recruiting histone acetylase activity that promotes transcription at specific DNA sites. To test the idea that RCAN1 overexpression may mediate aberrant CaN/CREB signaling leading to epigenetic dysregulation in AD, we generated transgenic mice that selectively overexpress RCAN1 in the brain using the Cre/lox system. Using immunoblotting, enzymatic assays and chromatin immunoprecipitation of hippocampal tissue from these mice, we found altered CaN activity and trafficking to the nucleus, CREB activation and histone acetylation. Overall, this work provides critical new understanding of a role for RCAN1 in regulating epigenetic mechanisms and gene expression in AD. Supported by NS034007, NS047384 (E.K.)
Transcription factor profile of homocysteine‐induced genes in human monocytes
Authors: Meng Shu , Ciment Stephen , Jan Michael , Yang Xiao-Feng , Wang Hong
Published: June 2021

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. It is reported that homocysteine (Hcy) induces pro‐inflammatory genes in monocytes (MC). However, the underlying mechanism is unclear. We identified 11 Hcy‐induced genes, 17 anti‐inflammatory cytokine interleukin 10‐induced, 8 pro‐inflammatory cytokine interferon γ (IFNγ)‐induced and 8 proinflammatory cytokine tumor necrosis factor α (TNFα)‐induced genes through literature search. Binding frequency of 36 transcription factors (TFs) implicated in inflammation and MC differentiation were analyzed within core promoter regions of identified genes, and classified into 3 classes based on the significant binding frequency to the promoter of Hcy‐induced genes. Class 1 TFs exert high significant binding frequency in Hcy‐induced genes. Class 2 and 3 TFs have low and no significant binding frequency, respectively. Class 1 TF binding occurrence in Hcy‐induced genes is similar to that in IFNγ‐induced genes, but not that in TNFα‐induced. We propose that class 1 TFs, including heat shock factor, MC enhancer factor‐2, nuclear factor of activated T‐cells, nuclear factor kappa light chain enhancer of activated B cells and Krueppel‐like factor 4, are putative Hcyresponsive TFs. We conclude that Hcy functions as a pro‐inflammatory cytokine and induces inflammation and MC differentiation via transcriptional signal pathways mediated by class 1 TF.
Hiding in Plain Sight: Elucidation of mechanisms underlying metastatic melanoma immune escape via suppression of Major Histocompatability Complex (MHC) II through dysregulation of the JAK/STAT pathway
Authors: Osborn joDi Lynn , Greer Susanna F
Published: June 2021

Transcriptional activation of Major Histocompatability Complex (MHC) I and II molecules by the cytokine interferon gamma (IFN‐ γ) is a key step in cell‐mediated immunity against pathogens and tumors. Following IFN‐γ induction, JAK/STAT signaling triggers higher‐order chromatin remodeling and decondensation of the MHC locus, resulting in activation of MHC genes. Recent evidence suggests suppression of MHC I and II expression on multiple tumor types plays important roles in tumor immunoevasion. One such tumor is malignant melanoma, the leading cause of skin cancer related deaths. Despite awareness of MHC expression defects, the molecular mechanisms by which melanoma cells suppress MHC and escape from immune‐mediated destruction remain unknown. Here we analyze dysregulation of the JAK/STAT pathway and its role in suppression of MHC II in melanoma cell lines at the Radial Growth Phase (RGP), the Vertical Growth Phase (VGP) and the Metastatic Phase (MET). RGP and VGP cells express both MHC II and the MHC master regulator, the Class II Transactivator (CIITA). MET cells lack not only MHC II and CIITA, but also both protein and mRNA for STAT 1 and for the STAT 1 coactivator, the Interferon Response Factor (IRF) 1. The objective of this study is to elucidate the mechanisms underlying silencing of STAT1 in metastatic melanoma. Research supported by grants from the American Cancer Society (to S.F. Greer).
PKCδ Regulates MCP‐1 Expression at the Transcription Level in Vascular Smooth Muscle Cells
Authors: Ren Jun , Morgan Stephanie , Si Yi , Liu Bo
Published: June 2021

Monocyte chemoattractant proerin 1 (MCP‐1) is upregulated in injured arteries and has been implicated in vascular inflammation and intimal hyperplasia. We have previously shown that protein kinase C δ (PKCδ) is upregulated in smooth muscle cells (SMCs) of injured arteries. The purpose of this study is to determine whether and how PKCδ regulates MCP‐1 expression. Using SMCs isolated from mouse aortic arteries, we showed that wildtype cells responded to TNFα (10 ng/ml) with rapid increase in MCP‐1 mRNA (29.00±2.56 fold, n=4, p<0.05), an induction that was significantly diminished by PKCδ gene deficiency (11.58±1.65 fold, n=4, p<0.05). Conversely, overexpressing PKCδ by adenovirus in SMCs significantly induced MCP‐1 mRNA expression (3.57±0.27 fold, n=3, p<0.05) and protein secretion (5.79±0.05 fold, n=3, p<0.05) as compared to AdNull (empty viral vector) infected SMCs. To test whether PKCδ modulates MCP‐1 mRNA stability, we blocked gene transcription with actinomycin D followed by the TNFα treatment. The level of MCP‐1 mRNA declined in control SMCs and SMCs treated with the PKCδ inhibitor rottlerin at a similar rate, with a half‐life around 60 min (n=3). In sum, our data suggest that PKCδ regulates MCP‐1 expression in arterial SMCs, likely at the transcription level rather than by increasing its mRNA stability.

This work was supported by NIH grant R01HL088447‐01A2 (to B. Liu).
The Retinoblastoma protein regulates Wnt signaling activity in the MC3T3 osteoblasts
Authors: Vazquez-Rivera Viviana , Cruz Alexander , Noel Richard , Santiago-Cardona Pedro
Published: June 2021

The retinoblastoma protein (pRb) is a transcriptional regulator of osteoblast differentiation. Wnt signaling also regulates this process. While Wnt signaling hinders degradation of β‐catenin leading to its nuclear accumulation and transcription of Wnt target genes, lack of Wnt activity promotes β‐catenin degradation. Since inactivation of both the pRb and Wnt pathways is commonly observed in osteosarcomas, we intend to determine if these pathways are functionally linked. Using MC3T3 pRb+/+ and pRb−/− cells, we performed immunoblots of Dishevelled (Dvl), which promotes Wnt activity when phosphorylated, and qRT‐PCR analyses of c‐myc, a known Wnt target gene, and GSK3‐β, an inducer of β‐catenin degradation. We found a 2‐fold increase in the phosphorylated/total Dvl ratio in the pRb−/− relative to pRb+/+ cells. Our results also showed 50% and 80% decreases in GSK3‐β and c‐myc mRNA levels, respectively, in the pRb−/− relative to pRb+/+ cells. Our results suggest that pRb may have a dual effect on Wnt: it inhibits Wnt by inhibiting Dvl phosphorylation and increasing GSK3‐β mRNA levels, while it promotes Wnt by promoting c‐myc mRNA levels. Research supported by RCMI Grant #5G12RR003050.
Physical and functional characterization of distinct single‐stranded DNA‐binding subdomains in purine‐rich element binding protein B (Purβ)
Authors: Rumora Amy , Wang Shu-Xia , Ferris Lauren , Everse Stephen , Kelm Robert
Published: June 2021

Purβ is a ~34 kDa single‐stranded DNA (ssDNA)‐binding protein that represses transcription of the smooth muscle α‐actin gene (ACTA2) in vascular and stromal cell types. Mechanistically, Purβ has been reported to interact with multiple sites in the purine‐rich strand of a 5′ ACTA2 enhancer sequence to form a high affinity 2:1 protein:ssDNA complex. In this study, the structural basis for site‐specific ssDNA‐binding and ACTA2 repression was explored by evaluating the physical and functional properties of specific subdomains formed by putative intra‐ and intermolecular associations between homologous repeats I, II, and III of Purβ. Cell‐based assays indicated that elaboration of full ACTA2 repressor activity required all three repeats (Purβ I‐II‐III). Calibrated size exclusion chromatography validated that the intramolecular domain (Purβ I‐II) exists as a monomer in solution, while Purβ I‐II‐III and the intermolecular domain (Purβ III) self‐associate to form dimers. Comparative ligand binding assays revealed that the inter‐ and intramolecular subdomains have quantitatively distinct ssDNA and transcription factor interaction properties. Collectively, our findings suggest that the tripartite modular organization adopted by the Purβ dimer is uniquely suited for cooperative ssDNA interaction and consequent repression of ACTA2 expression. Supported by grants AHA 09GRNT2170060 and NIH T32 HL007594.
Replication‐dependent expression of Herpes simplex virus late genes is controlled by P‐TEFb/CDK9 and DSIF/SPT5
Authors: Elias Per , Tang Ka-Wei
Published: June 2021

Herpes simplex virus expresses three classes of genes: immediate early, early and late genes. The immediate early genes are induced by VP16, whereas early and late genes depend on the sequence‐independent transcription factor ICP4. In addition expression of late genes is dependent on on‐going DNA synthesis. Here we demonstrate that late gene expression, e.g. glycoprotein C and the capsid protein UL38, is specifically inhibited by the P‐TEFb/CDK9 inhibitor DRB. In contrast, expression of early genes, e.g. ICP8 and UL39, is not affected by DRB. Furthermore, DNA replication proceeds normally in DRB‐treated virus‐infected cells. Knock‐down of DSIF, a target of P‐TEFb/CDK9, severely reduces expression of late genes but do not affect synthesis of DNA. ChIP experiments demonstrate that transcription complexes are efficiently assembled on the UL38 late gene already at 3 hrs, well before transcription starts. At 6 hrs p.i., when synthesis of DNA has started, a major redistribution of transcription complexes occurs. However, in DRB‐treated cells no such redistribution is seen. Our observations are compatible with a model in which DNA replication promotes the recruitment of P‐TEFb/CDK9 to virus DNA and acts continuously on late genes during the later phase of virus replication.