bluuebunny/crossref_metadata_2025 huggingface dataset:
Ephrin Reverse Signaling Induces Mouse Palatal Fusion and Epithelial To Mesenchymal Transition In Cultured Medial Edge Epithelia
Authors: Serrano Maria J , Nawshad Ali , Ibrahim Isra , Dyke Jeffrey , Svoboda Kathy K , Benson M Douglas
Published: June 2021

Understanding the molecular events in palate development is a prerequisite to more effective treatment of cleft palate. The secondary palate in humans and mice forms from shelves of mesenchyme covered with epithelium. These shelves adhere to form the midline epithelial seam (MES). MES cells then proceed through epithelial to mesenchymal transition (EMT) and apoptosis to yield a fused palate. This fusion requires Transforming Growth Factor β3 (Tgfβ3). Eph receptor tyrosine kinases and their ephrin ligands are responsible for many contact‐mediated developmental processes. Binding of ephrins causes receptor activation in Eph‐bearing cells (forward signaling), and intracellular signaling inside ephrin‐bearing cells (reverse signaling). Previously, we established that ephrin‐B reverse signaling is required for palate fusion in mice and is sufficient to cause fusion in chicken palates without Tgfβ3. In this study, we asked if activation of ephrin reverse signaling is sufficient to fuse mouse palatal shelves in vitro in the absence of Tgfβ3. We cultured embryonic day 13.5 mouse palatal shelves with a blocking antibody against Tgfβ3 that prevents fusion and clustered recombinant EphB2/Fc protein. EphB2/Fc treatment restored fusion. We also found that treatment with EphB2/Fc of cultured cells isolated from MES induced phenotypic and gene expression changes indicative of EMT. These results demonstrate that ephrin reverse signaling is sufficient to cause fusion of the mammalian palate.

Grant Funding Source: RO3DE020119 (NIDCR)
A novel role for Tbx3 in regulating digit number in the developing limb by Shh dependent and independent mechanisms
Authors: Emechebe Uchenna Johnbosco , Kumar Pavan , Thomas Kirk , Moon Anne
Published: June 2021

The crucial roles of TBX3 in development are evident in the fact that heterozygous mutations of TBX3 cause human Ulnar–mammary syndrome (UMS) including limb malformations, apocrine and mammary gland hypoplasia, dental and genital defects. Although germline deletion of Tbx3 in mice generates UMS‐like phenotypes, embryonic lethality has previously prevented investigating its precise role during limb development. To overcome this, we conditionally ablated Tbx3 in critical signaling compartments in the developing forelimb and discovered distinct functions in anterior and posterior mesenchyme. We found that Tbx3 promotes posterior digit development by positively regulating the Hand2/Shh signaling pathway. In anterior mesenchyme, Tbx3 constrains digit number by increasing the level of Gli3R and preventing expression of Hand1. The molecular basis for the effect of Tbx3 on Gli3R is at least in part via post‐transcriptional regulation of Suppressor of fused (Sufu). Loss of Tbx3 results in increased Sufu protein and altered Gli3R processing in primary cilia. Furthermore, mutants with complete loss of Tbx3 phenocopy the severe limb defects of Shh and Hand2 null mutants. In total, Tbx3 controls digit number by regulating Shh signaling in both ligand dependent (posterior mesenchyme) and ligand independent (anterior mesenchyme) mechanisms.

This work was supported by a March of Dimes Basil O'Connor Award (AMM), and NIH R01HD046767 (AMM).

Grant Funding Source: N/A
Upregulation of sonic hedgehog by fibroblast growth factor: Is TFAP2C a downstream intermediate?
Authors: Coggins Ashley , Pira Charmaine U , Feenstra Jennifer M , Oberg Kerby C
Published: June 2021

Fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER) and sonic hedgehog (SHH) from the zone of polarizing activity (ZPA) in the distal posterior limb bud mesoderm are critical for limb outgrowth and patterning. Maintenance of SHH expression is dependent on FGF, but the mechanism by which FGF regulates SHH has not been determined. Previous gene arrays have identified 300+ genes up‐regulated in response to FGF application. Among the genes up‐regulated by FGF was Transcription Factor Activating Protein‐2 gamma (TFAP2C). TFAP2C was up‐regulated within 3 hours of FGF application, which led us to hypothesize that TFAP2C was a downstream intermediate in the FGF to SHH regulation pathway.

We analyzed the effects of ectopic TFAP2C expression on SHH induction in a region of the posterior limb bud known to be inducible for SHH by FGF. A TFAP2C expression plasmid was injected into Hamburger/Hamilton stage 22–23 chick embryos and harvested at 24 hrs. In situ hybridizations for TFAP2C and SHH were performed.

We confirmed ectopic TFAP2C expression following injection of a beta‐actin promoter‐driven construct. However, no SHH expression was seen in 18 out of 18 embryos.

We conclude that TFAP2C expression alone in the SHH inducible domain is not sufficient to up‐regulate SHH expression.

Grant Funding Source: Departmental
The effects of cyclopamine on the ontogeny of fibroblast growth factor 8 mRNA expression during embryonic development of the chick limb
Authors: Maze Jennifer , Smith Jessica , Roche Blake
Published: June 2021

The formation of the vertebrate limb is a complex and tightly regulated developmental process. Outgrowth and patterning of the limb depends on two signaling centers in the limb bud, the Apical Ectodermal Ridge (AER) and the Zone of Polarizing Activity (ZPA). Outgrowth of the limb is controlled by the AER, which expresses the signals for Fibroblast Growth Factors, particularly Fibroblast Growth Factor 8 (FGF‐8). Anterior/posterior patterning of the limb is controlled by the ZPA, which expresses Sonic Hedge Hog (Shh). Previous studies have shown that both FGF‐8 and Shh signaling are crucial for limb development and outgrowth, as well as the expression of each other. In zebrafish, Shh mutants demonstrated a reduction in FGF signaling as well as a truncated limb. The aim of this study was to determine the mRNA expression pattern of FGF‐8 during limb development of the chicken (E 3–15) and the effect of cyclopamine, an inhibitor of Shh, on FGF‐8 mRNA expression using RT‐PCR. Although FGF‐8 expression was maintained in both the treated and untreated embryos throughout limb development, chicks exposed to cyclopamine demonstrated truncated limbs, consistent with previous data. Our data suggests that FGF‐8 expression is not affected by cyclopamine treatment. Future studies will investigate expression patterns of other genes, like BTRC, DLX‐5 and DLX‐6 that are involved in chick limb development after cyclopamine treatment. Sources for this research were provided by Lander University.
Evidence for dynamic rearrangements but lack of fate or position
Authors: McKinney Mary Cathleen , Fukatsu Kazumi , Morrison Jason , McLennan Rebecca , Bronner Marianne E , Kulesa Paul M
Published: June 2021

Neural crest cells emerge from the dorsal trunk neural tube and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the neural tube have similar or differential ability to contribute to neural crest cells and their derivatives. To this end, we precisely labeled neural tube precursors at specific dorsoventral levels of the chick neural tube using fluorescent dyes and a photoconvertible fluorescent protein. Neural tube and neural crest cell dynamics were then examined in vivo and in slice culture using 2‐photon and confocal time‐lapse imaging. The results show that neural crest precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the neural tube, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the neural tube to contribute to neural crest derivatives, with the exception of sympathetic ganglia, which appeared to be “filled” by the first population to emigrate. Rather than restricted developmental potential, however, this is likely due to a matter of timing.

Grant Funding Source: NIH grant 1RO1HD057922
Insights from transcriptional profiling the mouse hindbrain: Novel feedback between retinoids and Hox genes
Authors: Yurieva Marina , De Kumar Bony , Krumlauf Robb
Published: June 2021

Adult vertebrate hindbrain regulates critical body functions such as sleep, respiration and heart rate. During embryogenesis hindbrain forms seven transient swellings called rhombomeres, each with unique morphology and gene expression patterns. Rhombomere specification and regulation is controlled by transcription factors including Hox family and Krox20. Disruption of these genes changes the number and properties of rhombomeres and leads to neuronal disorganization causing animal lethality.

We are using transcriptional profiling and functional validation to reveal rhombomeric‐specific patterns of gene expression regulated by these key transcription factors. We isolated individual rhombomeres from 9.5 dpc mouse embryos using laser capture microscopy and performed transcriptional profiling on either single rhombomeres from the wild type hindbrains or whole hindbrains of wild type and Hoxa1, Hoxa2, Hoxb1 and Krox20 mutants. Analyses and validation of these results have uncovered several novel targets and pathways associated with hindbrain specification. For example, Hox genes which are induced by retinoids in turn modulate retinoid metabolism. These feedback loops are important for hindbrain patterning.

Grant Funding Source: Stowers Institute for Medical Research
LMX1B‐Regulated Nephrogenesis: A Role for Proteoglycans
Authors: Espinoza Aldo Jhonathan , Feenstra Jennifer , Pira Charmaine , Oberg Kerby
Published: June 2021

Lmx1b is a transcription factor necessary for kidney development. In humans, haploinsufficiency causes Nail Patella Syndrome frequently with chronic renal failure. In Lmx1b knockout mice, glomerular basement membrane formation is impaired, blocking filtration, urine production and long term survival. The mechanisms used by Lmx1b to direct basement membrane formation remain unclear. Recent microarray studies in the limb have implicated the proteoglycans Keratocan, Lumican, and Decorin (KLD) as downstream targets of Lmx1b. We hypothesized that Lmx1b might also regulate the KLD proteoglycans during kidney development.

To determine the role of Lmx1b in regulating the KLD proteoglycans, we compared the expression patterns of Lmx1b, Podocin, Keratocan, Lumican, and Decorin in the developing chicken kidney using whole mount in situ hybridization.

We found that Keratocan expression localized to nephric tubules and collecting ducts, Lumican expression was associated with nephric tubules, and Decorin expression surrounded Bowman's capsules and nephric tubules. Correspondingly, Lmx1b is expressed in Bowman's capsule and in nephric tubules. Thus, the expression of Decorin complements the expression of Lmx1b.

Our data suggests Decorin is a target of Lmx1b regulation during nephrogenesis. Further research is needed to determine the functional role of KLD proteoglycans in kidney development.

Grant Funding Source: Departmental
Quantitative co‐localization of hyaluronic acid and damaged myelin in the aging rhesus monkey brain
Authors: Estrada Larissa Isabel , Boule‐Getsinger Peter , Witkowski Ellen , Mortazavi Farzad , Rosene Douglas L
Published: June 2021

Normal aging in the rhesus monkey is characterized by subtle sublethal changes that affect cell function and lead to cognitive impairments even though neurons are not lost. One sublethal change that correlates with cognitive decline is damaged myelin which can lead to action potential failure, decreased trophic factor support, loss of axons and synapses, and ultimately to cognitive decline but the cause of this damage is unknown. Hyaluronic acid (HA) has been shown to interfere with remyelination in various disease models by inhibiting differentiation of oligodendroglia precursor cells. HA has also been shown to increase in the prefrontal cortex of aged rhesus monkeys. We hypothesize that age‐related increases in HA contribute to myelin deterioration by interfering with myelin repair mechanisms. Tissue from behaviorally characterized young and old monkeys is being processed to quantitatively co‐localize HA (HA binding protein histochemistry) with damaged myelin (encephalitogenic peptide antibody) in white matter tracts critical to cognitive functions including learning and memory and executive function. This histological data will be compared with subjects’ cognitive impairment measures to identify the age of onset of significant changes for specific white matter tracts. (Supported by R01AG021133 and P01AG000001)
Activity and frequency of the ITPA P32T variant among colorectal patients
Authors: Brown Carrie
Published: June 2021

Inosine Triphosphate Pyrophosphatase (ITPase) is an NTPase responsible for preserving genomic integrity by maintaining low cellular dITP and dXTP levels. Of the many variants of ITPA, the P32T variant is of interest due to high allelic frequency and pharmacological significance of its phenotype. ITPA variants P32T, F31Q and P32T/F31Q were tested for in vivo activity against HAP mutagenesis in E.coli and for in vitro activity by PiPer assay with dITP and dXTP substrates. All variants were successful in protecting against HAP mutagenesis. Both single mutant variants (P32T and F31Q) showed enzymatic activity against both substrates, results for the P32T/F31Q variant were inconclusive. In addition, the frequency of the P32T allele among colorectal cancer patients was tested by HPY166II digest. Out of 53 samples tested, 8 are heterozygous for the P32T allele as confirmed by DNA sequencing, giving an allelic frequency of 0.075. This frequency is comparable to frequency among Caucasian Americans of 0.06 (Cao and Hegele, 2002) and therefore the P32T allele does not appear to be enriched amongst colorectal cancer patients. Activity of the variants is comparable to the wild type and the low levels of activity in P32T homozygous individuals does not appear to be due to a lack of enzymatic function of the protein. This publication funded by NIH grant 5P20RR016469 and NIGMS grant 8P20GM103427.
Nucleosome dynamics regulate DNA processing
Authors: Adkins Nicholas , Niu Hengyao , Sung Patrick , Peterson Craig
Published: June 2021

The repair of DNA double strand breaks (DSBs) is critical for the maintenance of genome integrity. The first step in DSB repair by homologous recombination (HR) is processing of the ends by one of two resection pathways, exemplified by yeast Exo1 and Sgs1/Dna2. Here we report in vitro and in vivo studies that characterize the impact of chromatin on each resection pathway. We find that the helicase activity of Sgs1 is reduced on nucleosomal substrates, and that efficient resection by the Sgs1/Dna2 ‐dependent machinery requires a nucleosome‐free gap adjacent to the DSB. We also report that resection by Exo1 is blocked by nucleosomes, and that processing activity can be partially restored by removal of the H2A/H2B dimers. Our study also supports a role for the H2A.Z histone variant in Exo1 processing. Our study suggests that these two DNA processing pathways require distinct chromatin remodeling events in order to navigate chromatin structure adjacent to a DSB, indicating complex interactions between chromatin dynamics and DNA repair.
Large‐scale structural variations linked to the NOTCH4 locus of the Human Major Histocompatibility Complex
Authors: Bowman Jacob D. , Zhou Bi , Yu Chack‐yung , Lawrance Simon K
Published: June 2021

The human major histocompability complex (MHC) encodes antigen‐presenting proteins and other proteins critical to the function of the the immune system. It is also the most polymorphic component of the human genome. Variations within the MHC region have been linked to both susceptibility and resistance to multiple autoimmune diseases. The objective of the present study is to determine whether the portion of the MHC linked to the NOTCH4 gene is characterized by large‐scale structural variation and if it is, whether that variation is linked to auto‐immune disease. The NCBI human genome reference sequence indicates that NOTCH4 is contained within a 450 kilobase PmeI restriction fragment. To date, this study has used polymerase chain reaction to isolate NOTCH4 specific probes from cDNA for NOTCH4. The probes will be hybridized with Southern blots of pulsed field electrophoresis gels of PmeI digested genomic DNA isolated from patients with systemic lupus erythematosus and normal controls. This will allow the observation of size variation and imply the possibility of copy number variations, insertions and/or deletion within the region and their possible role in autoimmune disease.
Determination of the extent of hybridization between the threatened Hibiscus dasycalyx, Hibiscus laevis, and Hibiscus moscheutos using ISSR genetic markers
Authors: Baker Laura Marie , Clack Bea
Published: June 2021

East Texas is home to several native Hibiscus species, including Hibiscus dasycalyx, Hibiscus laevis, and Hibiscus moscheutos. These species are herbaceous perennials that live in wetland conditions. Of the three morphologically distinct species, H. dasycalyx has been listed as threatened by the US Fish and Wildlife Service. Before restoration can occur, the gene flow and level of hybridization with the other two species must be determined in its native habitat. Previous investigations in our laboratory of these species have suggested that the Hibiscus dasycalyx is an individual species with little to no hybridization, however, a more comprehensive analysis is needed using a larger sampling set. The objective of this study is to identify species specific Intersimple Sequence Repeat (ISSR) markers for Hibiscus dasycalyx, H. laevis and H. moscheutos so that these markers could be used for tracking the extent of hybridization that occurs between the three species as well as the gene flow of H. dasycalyx. Genomic DNA was extracted and PCR amplified using ISSR primers (Wolfe,1998). PCR products were separated on an ALF sequencer to detect polymorphisms between the three Hibiscus species. Population analysis software was used to determine the gene flow and extent of hybridization between H. dasycalyx, H. laevis and H. moscheutos.
Monitoring the expression and activity of the DinB protein in vivo in Escherichia coli
Authors: Diamond Brett Haden , Godoy‐Carter Veronica
Published: June 2021

The Escherichia coli protein DinB plays an important role in the SOS response to DNA damage. DinB is a member of the Y‐family of polymerases which serve to synthesize DNA from a damaged bulky template in a process known as translesion synthesis (TLS). DinB in particular is the only Y‐family polymerase that is conserved throughout all domains of life. In addition to synthesizing DNA across from lesions, DinB is associated with ‐1 frameshift mutations that are particularly lethal. RecA and UmuD have already been shown to modulate DinB activity preventing the ‐1 frameshift mutation during TLS in vitro, however no such study has been carried out in vivo. By constructing DinB, RecA, and UmuD fluorescent fusion proteins, the localization and prevalence of these proteins will be able to be monitored in vivo thus proving or disproving the in vitro findings that DinB, RecA and UmuD form a multi‐protein complex preventing mutagenesis during TLS.
Mechanisms of cytotoxicity of bifunctional epoxide crosslinking agents
Authors: Spierer Adam , Millard Julie T.
Published: June 2021

Since the discovery of nitrogen mustard as a powerful anti‐tumor agent, structurally similar bifunctional alkylating agents have been widely studied for their clinical potential. These compounds prevent replication and transcription by cross‐linking duplex DNA. We are characterizing the mechanisms by which diepoxybutane and related compounds such as epichlorohydrin (ECH) exert their cytotoxic effects. Our first goal is to determine the relationship between interstrand cross‐linking and cytotoxicity. We are assaying cross‐linking in HL‐60 cells using ethidium bromide to monitor the amount of duplex DNA following denaturation. Only cross‐linked DNA reanneals rapidly and interacts with the dye, which is highly fluorescent when bound to duplex DNA. Preliminary results suggest a correlation between cross‐linking and LD50 values for these compounds, with DEB as a more efficient cross‐linker. Our second goal is to characterize the pathways by which these compounds induce cell death. We are using flow cytometric apoptosis assays to understand the dosing requirements necessary to maximize apoptosis and minimize necrosis. Preliminary results suggest a greater apoptotic potential for DEB than ECH. This project is supported by grants from the National Center for Research Resources (5P20RR016463–12) and the National Institute of General Medical Sciences (8 P20 GM103423–12) from the NIH.
Werner's syndrome protein limits the error‐prone 8‐oxo‐G lesion bypass activity of human DNA polymerase kappa by promoting the error‐free bypass
Authors: Maddukuri Leena , Eddy Sarah D , Eoff Robert L
Published: June 2021

Oxidative stress induced DNA damage plays a key role in cancer and aging. 7,8‐dihydro‐8oxo‐2′‐deoxyguanosine (8‐oxo‐dG), produced during DNA oxidation is highly mutagenic and known to block replication. Previous studies show that the human DNA polymerase kappa (hpol κ) bypasses 8‐oxo‐dG by preferentially inserting dATP opposite the lesion. Recent studies indicated that hpol κ coordinates with Werner's syndrome protein (WRN) during the 8‐oxo‐dG bypass. Our data show that WRN stimulates the 8‐oxo‐dG bypass activity of hpol κ by enhancing the correct base insertion opposite the lesion and diminishing the efficiency of dATP mispair insertion. Kinetic analysis reveals that in the presence of WRN, hpol κ inserts the dCTP opposite 8‐oxo‐dG ~ 1.7‐fold more efficiently than dCTP opposite dG. Currently we are investigating the fidelity and catalytic efficiency of hpol κ bypass of 8‐oxo‐dG in the presence of WRN using pre‐steady state kinetic analysis and mass spectrometric detection of polymerization products. WRN may function in coordination with hpol κ to limit the error‐prone bypass of 8‐oxo‐dG, which could suppress the mutagenicity associated with hpol κ and prevent genomic instability. This work is supported in part by USPHS R00 GM084460 (R.L.E).
Regulation of DNA damage tolerance by the AhR pathway and its role in glioma progression
Authors: Bostian April , Eoff Robert
Published: June 2021

Gliomas, tumors arising in glial cells in the brain, are associated with a particularly poor patient prognosis. These tumors generally exhibit little response to chemotherapy and radiation treatments and have constitutive and marked DNA damage response activation even before the onset of treatment. This study's objective is to examine glioma‐specific activation of the aryl hydrocarbon receptor (AhR) pathway and the subsequent effect upon DNA damage tolerance mechanisms, especially on the regulation of the Y‐family polymerases and the effect of their up‐regulation on mutagenic bypass of DNA adducts. The hypothesis that the AhR signaling pathway transcriptionally regulates levels of two members of this family, human polymerase kappa (hpol κ) and human polymerase iota (hpol κ), was tested using in vitro cell culture assays after exposure to AhR agonists. Initial luciferase reporter assays and immunoblotting results suggest hpol κ may indeed be up‐regulated by this pathway in gliomas. Results from this study will contribute to our understanding of how DNA damage tolerance affects glioma development and may provide insight into the mechanisms leading to the chemoresistance often inherent in this disease. Supported in part by USPHS R00 GM084460 (R.L.E.).
Aurora‐A‐elicited Astrin phosphorylation regulates mitotic progression
Authors: Chiu Shao‐Chih , Chen Jo‐Mei Maureen , Liu Ming‐Chao , Yu Chang‐Tze Ricky
Published: June 2021

Astrin is a spindle protein regulating several aspects of mitosis including the maintenance of bipolar spindle pole, regulation of sisterchromatid cohesion and chromosome alignment. However, it remains unclear how to regulate Astrin protein in mitosis. Our present study has shown that Astrin behaves as a mitotic phosphoprotein and was directly phosphorylated by the mitotic kinase Aurora‐A in an in vitro kinase assay. Furthermore, the mitosis dependent phosphorylation of Astrin was blockaded by immunodepletion or shRNA‐mediated knockdown of Aurora‐A. It indicates that Astrin acts as an in vitro and in vivo substrate of Aurora‐A. The phosphorylation site was subsequently mapped to serine 115 of Astrin. And site‐directed mutagenesis analysis revealed that Astrin S115A mutants, no longer serving as a substrate of Aurora‐A, delayed the mitosis progression and induced chromosome misalignment and DNA lagging along the mitotic progression. By contrast, Astrin S115D, which mimics Aurora‐A elicited phosphorylation, accelerated the mitosis progression and slightly deceased the level of DNA lagging. The binding activity of Astrin S115A mutant to its mitotic interaction proteins LC8 and CLASP‐1alpha was remarkably reduced, leading to the mislocalization of the sequential two proteins and the misalignment of chromosome. In conclusion, we are the first to demonstrate that Aurora‐A, a well‐characterized oncokinase controlling a wide range of mitotic events by phosphorylating numerous mitotic factors, phosphorylates Astrin in vitro and in vivo, which guides the binding of Astrin to its cellular partners, and ensures accurate chromosome alignment and segregation and thereby proper mitosis progression.
Characterization of lysine‐specific demethylase 1 (LSD1) as a potential olfactory receptor regulator
Authors: Savarino Alyssa Leigh , Kilinc Seda , Lane Robert
Published: June 2021

Mouse olfactory sensory neurons (OSNs) express a variety of over 1000 odorant receptor (OR) genes located throughout the genome. Previous studies have shown that each OSN expresses only one OR gene out of the >;1000 possible genes and that the expressed locus lacks an H3K9 trimethylation mark. Lysine specific demethylase 1 (LSD1) is a candidate demethylase that may play a role in the epigenetic pathway of OR gene regulation. Preliminary immunofluorescence experiments have shown that LSD1 displays three phenotypes throughout the cell cycle. LSD1 exhibits a broad, diffuse nuclear staining or a highly localized, punctate nuclear expression pattern. Some punctate nuclei exhibit one distinct compartment of LSD1. Cell synchronization experiments show a significantly higher incidence of punctate LSD1 during G2/M phase and a higher frequency of the distinct LSD1 phenotype during telophase. One possible model suggests that all OR loci are initially silenced by trimethylation and that distinct signals represent active LSD1 complexes which derepress a gene locus for future expression. DNA immuno‐FISH experiments show that active OR gene clusters co‐localize with LSD1 distinct and punctate signals within the nucleus of cycling OP6 cells occasionally, while the control, actin, does not co‐localize with LSD1 in cycling cells. Co‐localization would suggest possible LSD1 and OR gene interaction, in which LSD1 may function in the derepression of the expressed OR locus.
Epigenetic markers in patients with endometriosis
Authors: Ray Kristeena Lauren , Isme Mardochee , Cook Carla , Massie Melissa , Dawley Brenda , Santanam Nalini
Published: June 2021

Endometriosis is a clinical condition affecting young women. Cells from the endometrium appear and flourish outside the uterus, often causing pain. Typically viewed as a hormonal disorder, new studies suggest that endometriosis may be an epigenetic disease. The major objective of this study was to investigate if epigenetic‐mediated changes play a role in endometriosis‐associated pain. Peritoneal fluid (PF) from women with and without endometriosis (n=6) were used to treat endometrial cells (EM‐42 and Ishikawa) for 48 hours. Global Histone modification changes were detected using Western blots. The mRNA expression of nociceptors such as cycloxygenase‐2 (COX‐2) and transient receptor potential channel TRPV1 was detected using quantitative real‐time polymerase chain reaction (qPCR). Our results indicated changes in Histone 3 and Histone 4 modifications depending on the presence or absence of endometriosis‐associated pain. There was >;25‐fold induction in the pain‐associated genes in endometriotic tissue. Human miRNome array showed significant differences in over 30 miRNAs in endometrial tissue from endometriosis patients compared to controls. Our findings provide evidence for epigenetic changes in patients with endometriosis‐associated pain.
Enzymatic analysis of EF‐Tu and EF‐Ts from Pseudomonas aeruginosa
Authors: Tran Alexis , Palmer Stephanie , Bullard James
Published: June 2021

Introduction

Elongation factors (EF) facilitate many steps in the process of the biosynthesis of proteins. EF‐Tu in particular plays a central role in the GTP‐dependent placement of aminoacylated tRNA at the A site of the ribosome. EF‐Ts acts by catalyzing the change of EF‐Tu from a GDP‐bound inactive state to a GTP‐bound active state. We present here analysis of the kinetic parameters of EF‐Tu and EF‐Ts.

Results

Kinetic parameters for the interaction of EF‐Tu with its substrate GDP in the absence of EF‐Ts were observed to be: KM = 33 mM, kcat = 0.003 sec−1 and the specificity constant kcat/KM was then calculated as 0.1 × 10−3 sec−1 uM−1. In the presence of EF‐Ts, these values were shifted to: KM = 2 mM, kcat = 0.005 sec−1 and the specificity constant kcat/KM was then calculated as 2.5 × 10−3 sec−1 uM−1. We determined the equilibrium dissociation constant (KGDP) to be in the range of 100–160 nM in the absence of EF‐Ts at various temperatures. The presence of EFTs stimulated the exchange of GDP by EF‐Tu by up to 10‐fold. P. aeruginosa EF‐Tu was also shown to be active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in polyU dependent placement of Phe‐tRNAPhe at the A site of the P. aeruginosa ribosome.

Conclusion

EF‐Tu and EF‐Ts identified in P. aeruginosa were cloned, expressed and purified and shown to be functional in assays suggesting that each is functional in protein synthesis. Research was supported by NIH grant 1SC3GM098173–01A1.
Formation and maturation of the autophagosome
Authors: Mizushima Noboru
Published: June 2021

Autophagy is one of the major degradation pathways in the cell. In autophagy, intracellular components are sequestered by autophagosomes and then degraded upon fusion with lysosomes. Formation of the autophagosome is governed by organized functions of autophagy‐related (Atg) proteins, which were originally identified in yeast. We showed that the Atg hierarchy is well conserved in other eukaryotes including mammals. We also investigated the recognition mechanism of selective autophagy substrates and found that p62 and depolarized mitochondria, and ferritin can localize to the ER‐associated autophagosome formation site, independently of Atg proteins. These observations suggest that there is a common mechanism that recruits autophagy substrates to the autophagosome formation site, which is independent of binding to the autophagosomal membrane. Another important question that remains to be solved is how the autophagosome can specifically fuse with the lysosome. If the lysosome fuses with the elongating isolation membrane (unclosed autophagosome), it cannot accomplish degradation of cytoplasmic materials. We recently identified an autophagosomal SNARE, which is absent in the isolation membrane. Thus, the late recruitment of the SNARE to the autophagosome can prevent premature fusion with the lysosome.
Reconstitution of the Class III Phosphatidylinositol‐3‐kinase in autophagy
Authors: Zhong Qing
Published: June 2021

The class III phosphatidylinositol‐3‐kinase (PI3KC3) plays a central role in autophagy and endocytosis. How its function is differentially regulated in these processes remain elusive. We are the first group to purify PI3KC3 holocomplex in human cells, in which we found seven subunits including Vps34, p150, Beclin 1, Barkor/Atg14(L), NRBF2, Rubicon and UVRAG. These proteins form two mutually exclusive subcomplexes sharing Vps34, p150 and Beclin 1. Barkor and NRBF2 regulate PI3KC3 activity on autophagosome membrane, while UVRAG and Rubicon control PI3KC3 activity on endosome membrane. We have reconstituted the biochemical activity of autophagic PI3KC3 activity in vitro, and explored their functions in membrane curvature sensing and membrane tethering/hemifusion.
A stress‐activated, p38 MAPK‐ATF/CREB pathway regulates the post‐transcriptional decay of target mRNAs
Authors: Gao Jun , Wagnon Jacy L , Protacio Reine M , Glazko Galina V , Beggs Marjorie , Raj Vinay , Davidson Mari K , Wahls Wayne P
Published: June 2021

Broadly conserved, mitogen‐activated/stress‐activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos and Jun) to regulate the transcription of target genes. We report that, upon stress, the fission yeast Spc1 kinase‐regulated Atf1‐Pcr1 heterodimer controls the post‐transcriptional decay of a set of target RNAs. Whole transcriptome RNA‐SEQ data revealed that decay is associated nonrandomly with transcripts that contain an M26 (CRE‐like) motif. Moreover, the ablation of an M26 site in a target mRNA is sufficient to block its stress‐induced decay. Conversely, engineered M26 sites can render an otherwise stable mRNA into an mRNA that is targeted for decay in their vicinity, demonstrating that the position of M26 site dictates where the transcript is cleaved. This post‐transcriptional, Stress‐Activated Gene Silencing (SAGS) provides a mechanism to reduce the expression of target genes without shutting off transcription itself. Thus, a single p38‐ATF/CREB signal transduction pathway can coordinately induce (promote transcription) and repress (SAGS) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli.
A Redox Insensitive Triple Cysteine Mutant of SirT1 Rescues Oxidative Stress induced Apoptosis
Authors: Shao Di , Fry Jessica , Zee Rebecca , Kumar Vikas , Pimentel David , Cohen Richard , Bachschmid Markus
Published: June 2021

Sirtuin 1 (SirT1) is a NAD+‐dependent class III histone deacetylase that orchestrates cell cycle progression, energy metabolism and aging. SirT1 activity is modulated by [NAD+]/[NADH] ratio and post translational modifications. Oxidative stress is a hallmark of metabolic disease and inflammation that contributes to the progression of pathology in part through oxidative post‐translational modification (OPTM) of proteins. HepG2 (liver carcinoma) and HEK293 (transformed embryonic kidney) cells were treated with increasing levels of oxidants (H2O2, Cys‐NO) or high palmitate high glucose (HPHG) to mimic conditions of metabolic disease. We found that SirT1 activity was inhibited without change in protein expression in both an activity assay and by p53 acetylation measured by western and by PUMA luciferase activity. The oxidant‐induced increase in apoptosis was ameliorated by overexpression of a mutant SirT1 that cannot be oxidized on three critical cysteine thiols that we found are reversibly modified by HPHG, Cys‐NO and H2O2, suggesting that thiol oxidation on SirT1 in metabolic disease contributes to pathology.

NIH PO1 HL 068758, R37 HL104017; NHLBI HHSN268201000031C
Oxidation of the Hsp70 BiP protects cells during ER stress
Authors: Sevier Carolyn , Pareja Kristeen , Wang Jie
Published: June 2021

Protein folding in the endoplasmic reticulum (ER) has emerged recently as a source of reactive oxygen species (ROS). Despite a current appreciation of the potential for ER‐derived ROS to contribute to cell dysfunction, scientists lack a fundamental understanding of how the ER manages ROS production. Taking advantage of our ability to selectively create a hyper‐oxidized ER, we have identified a novel redox pathway that plays a central role in sensing and responding to redox imbalances within the ER. Key to this pathway is the lumenal chaperone BiP, a member of the Hsp70 ATPase family. Intriguingly, this BiP‐centric cellular response to ER oxidative stress is mechanistically distinct from previously characterized ER stress responses and involves redox signaling via a conserved BiP cysteine. The pronounced sensitivity of a cyteine‐less BiP mutant strain to oxidative stress suggests that alteration of the BiP cysteine (e.g. its oxidation) is part of a fundamental mechanism for cell protection. We suggest that post‐translational modification of the BiP cysteine alters BiP's catalytic cycle. We propose that alteration of BiP activity during oxidative stress allows the cell to cope with an existing misoxidized substrate load.
Effects of Aging on the Heat Shock Response and Autophagy in Human Peripheral Blood Mononuclear Cells
Authors: Lanphere Kathryn Rosie , Schneider Suzanne , Mermier Christine , Zuhl Micah , Dokladny Karol , Moseley Pope
Published: June 2021

INTRODUCTION

Aging is associated with an accumulation of damaged aberrant macromolecules. Autophagy is the process that contributes to the maintenance of cellular homeostasis through degradation and subsequent recycling of damaged proteins and organelles and may be coordinated by the heat shock response. In this pilot study, we tested the hypothesis that aging reduces the heat shock and autophagy responses to heat shock in human peripheral blood mononuclear cells (PBMC).

METHODS

Four young control (21.25 ± 1.5 years, 3 male, 1 female) and four older subjects (63 ± 2.9 years, 2 male, 2 female) participated in this study. PBMCs were isolated from whole blood and cells were analyzed for baseline levels of LC3‐II and HSP70 proteins. In subsequent studies, PBMCs collected from young and older subjects were exposed to 1 h heat incubation at 42°C, and HSP70 and LC3‐II expression were measured by Western blot analysis and normalized to β‐actin. Densitometric values (mean ± standard deviation) for baseline and heat shock studies are reported.

RESULTS

Surprisingly, the PBMCs from older subjects had a higher (p < .01) baseline level, 0.64 ± 0.14, of LC3‐II than the younger subjects, 0.07 ± 0.14. An increase (p < .05) in heat shock protein (HSP70) from baseline levels of 0.03 ± 0.07 to 1.77 ± 1.32 following heat shock was found in young subjects, but not older subjects. No within or between group differences were found in LC3‐II following heat shock.

CONCLUSIONS

Contrary to findings in animals, our results suggest that autophagy is increased, not decreased with aging in humans. This increased baseline level may partially compensate for the compromised ability to upregulate HSP70 and stimulate autophagy after a heat stress exposure.
B19, a novel monocarbonyl analogue of curcumin induced human ovarian cancer cells apoptosis via activation endoplasmic reticulum stress and autophagy signaling pathway
Authors: Xiao Jian , Qu Wanglei , Wang Zhouguang , Shi Hongxue , Zhang Hongyu
Published: June 2021

Background

Unfolded protein response, autophagy and Endoplasmic Reticulum stress‐induced apoptosis regulate tumor cells fate and have become novel signaling targets for development of cancer therapeutic drugs. Currently, curcumin has been used to treat several different cancers in clinical trials and researches, including ovarian cancer. But the role of ER stress and autophagy in the therapeutic effects of curcumin or new curcumin analogues remain unclear.

Methods

Cell viability was determined by MTT assays. Cell apoptosis was detected by flowcytometer. The expressions of ER stress and autophagy related proteins were analyzed by Western blotting. The activation of autophagy was detected by Immunofluorescence staining.

Results

B19 induced HO8910 cells apoptosis in a dose‐dependent manner and this effect was associated with activate UPR and ER stress‐mediated apoptosis pathway. B19 treatment induced autophagy in HO8910 cells. Inhibition of autophagy using 3‐methyladenine increased the levels of intracellular misfolded proteins, which enhanced ovarian cancer cell apoptosis.

Conclusion

Our data indicated that ER stress and autophagy plays a key role in B19‐induced apoptosis in epithelial ovarian cancer cell lines and autophagy inhibition can increased B19‐induced apoptosis by inducing severe ER stress.
The activation of factor VII by a variety of potential activators
Authors: KE KE , Morrissey James
Published: June 2021

Factor VII (VII) is an inactive zymogen, which upon proteolysis of a single peptide bond, is activated to the serine‐protease form factor VIIa (VIIa). It is known that in vitro factor Xa (Xa), factor IXa (IXa), thrombin, factor XIIa (XIIa), VIIa, and plasmin can activate VII to VIIa.

In the present study, the activation of VII by potential in vivo activators was evaluated under identical conditions, in the absence and presence of cofactors. In order to avoid the influence of VIIa autoactivation to the analysis of VII activation by other activators, VII mutant (Sc195A) was generated, in which the active site serine residue has been mutated to alanine. Experiments were performed on phospholipid vesicles either in the presence of relipidated membrane‐TF, soluble TF, or none.

The results show that among Xa, IXa, VIIa‐TF, meizothrombin, thrombin and XIIa, Xa and IXa are the most potent activators of VII. TF facilitates VII activation by 3 to 9‐fold, various from different activators. The cofactors for protease Xa and IXa do not influence the activation of VII. Preliminary data also shows that IXaα is one of the potent activators of VII. This study shed light on how VII get activated in physiological conditions.
The structural basis of the secondary function of PMM1 in the presence of IMP
Authors: Ji Tianyang , Dunaway‐Mariano Debra , Allen Karen N.
Published: June 2021

The human phosphomannomutases PMM1 and PMM2 catalyze the interconversion of hexose 6‐phosphates and hexose 1‐phosphates. The two isoforms share 66% sequence identity and similar kinetic properties but appear to have different functional roles. While PMM2 is expressed in all cell types and provides the mannose 1‐phosphate essential for protein glycosylation, PMM1, is expressed mainly in the brain and the presence of inosine monophosphate (IMP), activates a second function—the hydrolysis of glucose 1,6‐bisphosphate (G16BP) to glucose 6‐phosphate. Mechanistically, both enzymes require activation by G16BP to form the phosphoaspartate nucleophile. Our aim is to understand the mechanism of how IMP converts PMM1 from a mutase to a phosphatase. Herein, we present the structure to 1.93 Å resolution of PMM1 in complex with IMP. Together with the kinetics of binding (IMP Kactivation = 1.42 μM), this structure is consistent with the hypothesis that IMP inhibits mutase activity by competing with the substrate binding site at the cap domain necessary for the formation of the active site. Acquisition of phosphatase activity may be a result of IMP induced cap closure thus allowing water to enter the active site and accept the phosphoryl group from the phosphoaspartate nucleophile. Subsequent G16BP binding affords the phosphorylation of the nucleophile allowing PMM1 to effectively act as a G16BP phosphatase. NIH Grant# GM61099
Plasticity of the kinesin‐microtubule interaction is encoded by the motor domain beta‐sheet
Authors: Richard Jessica , Kim Elizabeth , Wojcik Edward , Kim Sunyoung
Published: June 2021

Kinesins have an unanticipated functional breadth via interaction between their motor domain and microtubules (MTs): they move along cytoskeletal tracks in either direction or modify tracks by elongation or depolymerization. Thus, despite conservation of chemistry, structure, and overall sequence, motor domains are specialized in their intra‐ and inter‐molecular interactions. Here, our bioinformatic studies propose an evolutionary history for the functional plasticity of kinesins. Sequence modifications of the central beta‐sheet are ancient events that correlate with the advent of molecular transporters versus MT‐modifying kinesins. By occupying a physical position between the nucleotide‐ and MT‐binding sites, we propose this beta‐sheet allows for cross‐transmission of information. Furthermore, using meta‐analysis of x‐ray structures, we identified two beta‐sheet motifs that tailor motor domain function for either motility or MT‐modification. This functional rewiring of kinesin through beta‐sheet sequence and structure may be a model by which other motors and NTPases can transduce specific intra‐ and interprotein communication. Supported by NIH (GM097350 and GM066328) and Louisiana Board of Regents [NSF‐Pfund‐143].
Pre‐Metazoan Ancestry of CREB as a Kinase‐Inducible Transcription Factor
Authors: Gravis Demetrius Peter , Wolf Katharine
Published: June 2021

The cyclic AMP response element binding protein (CREB) is a bZIP transcription factor activated by phosphorylation within its kinase‐inducible domain (KID), which is mediated by PKA and other kinases. CREB activation promotes the expression of genes regulating cell proliferation and cell survival, and CREB activation in neurons plays an essential role in memory formation. Herein, we have investigated the evolution of CREB in metazoan and unicellular opisthokont lineages. We identified CREB homologs with conserved bZIP domains and KID‐homologous phosphoacceptor domains in basal metazoans including cnidarians, ctenophores, placozoans, and sponges as well as choanoflagellates, the closest unicellular relatives of metazoans. The metazoan and choanoflagellate CREB homologs also possessed conserved residues known to be required for the formation of CREB‐coactivator complexes. This report therefore establishes the pre‐metazoan ancestry of CREB as a kinase‐inducible transcriptional regulator.
Silibinin induces endoplasmic reticulum stress‐mediated and mitochondrial‐mediated apoptosis through disruption of Ca2+ homeostasis in human prostate cancer PC‐3 cells
Authors: Kim Sang‐Hun , Kim Kwang‐Youn , Yu Sun‐Nyoung , Park Seul‐Ki , Kong In‐Soo , Ahn Soon‐Cheol
Published: June 2021

Silibinin is major biologically active compound of milk thistle and has anticancer activity and chemopreventive effects on cancer cell line. Although various evidences showed multiple signaling pathways of silibinin in apoptosis, there is no reported to address the role of ER stress in silibinin‐induced apoptosis in cancer cells. Androgen‐refractive human prostate cancer PC‐3 cells showed dose‐ and time‐dependent reduced viability upon silibinin treatment; non‐malignant prostate RWPE‐1 cells were relatively less sensitive to drug‐induced lethality. The flow cytometric analysis showed that silibinin increased the production of reactive oxygen species (ROS) and Ca2+ and mitochondrial membrane potential in PC‐3 cells. In addition, silibinin promoted the expression of ER stress‐related proteins, such as glucose‐regulated protein 78 (GRP78), inositol‐requiring enzyme‐1a (IRE1¥á) and growth arrest‐ and DNA damage‐inducible gene 153 (GADD153/CHOP). Pretreament of PC‐3 cells with Ca2+ chelator BAPTA/AM prevented silibinin‐induced apoptosis and recovered the expression level of apoptosis‐related proteins and ER stress‐related proteins. These results are the first to link ER‐stress and Ca2+ homeostasis disruption to silibinin‐induced apoptosis of prostate cancer cells.