bluuebunny/crossref_metadata_2025_split 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
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
Enzymatic analysis of EF‐Tu and EF‐Ts from Pseudomonas aeruginosa
Authors: Tran Alexis , Palmer Stephanie , Bullard James 
Published: June 2021

IntroductionElongation 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.ResultsKinetic 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.ConclusionEF‐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.
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
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

INTRODUCTIONAging 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).METHODSFour 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.RESULTSSurprisingly, 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.CONCLUSIONSContrary 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

BackgroundUnfolded 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.MethodsCell 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.ResultsB19 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.ConclusionOur 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.
Kinetic properties and protein conformational dynamics of the signal transducer HemAT as revealed by time‐resolved stepscan FTIR spectroscopy
Authors: Pinakoulaki Eftychia , Pavlou Andrea , Yoshimura Hideaki , Aono Shigetoshi 
Published: June 2021

HemAT is a heme‐based O2 sensor protein that acts as a signal transducer for aerotaxis in Bacillus subtilis. In our work, we have employed time‐resolved step‐scan Fourier transform infrared (TRS2‐FTIR) spectroscopy to investigate the protein structural changes induced by ligand (CO) photodissociation and rebinding that are crucial for understanding the initial events of the intramolecular signal transduction mechanism in HemAT. We have studied the full length and truncated sensor domain HemAT‐CO adducts as well as the Y70F, L92A, T95A and Y133F mutants. Monitoring the rebinding of CO to the heme‐Fe2+ reveals biphasic kinetics for both the full length and truncated sensor domain HemAT. The TRS2‐FTIR experiments demonstrate that Y70 affects the conformational changes that are induced to the protein matrix by CO photodissociation. Moreover, L92 appears to operate as the conformational gate in the migration pathway of photodissociated CO.This work was funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation (Infrastructure Project ANAVATHMISI/PAGIO/0308/14).
Nicotine regulates FasL, activates Mst‐1, promotes Histone H2B phosphorylation and accelerates neutrophil apoptosis
Authors: Rane Madhavi Jagdish , Kumar Jay , Jin Shunying , Merchant Michael , Ritzenthaler Jeffery , Roman Jesse , Klein Jon 
Published: June 2021

An efficient way of resolving inflammation is induction of neutrophil apoptosis. We hypothesized that nicotine accelerates neutrophil apoptosis. Nicotine (3 mM) accelerated human neutrophil apoptosis by enhancing caspase‐3 cleavage in a p38 and ERK dependent manner. LPS‐induced neutrophil survival was significantly inhibited by nicotine. Moreover, □4nAChR antagonist Dihydro‐β‐erythroidine hydrobromide (D□E), inhibited nicotine‐stimulated pERK, pP38 and caspase‐3 cleavage. Caspase‐3 cleavage promoted Mst‐1 cleavage and pSer14‐Histone H2B which correlates with apoptotic chromatin and onset of apoptosis. Mst‐1 cleavage and pSer14‐Histone H2B were dependent upon p38 and ERK. In addition, nicotine induced FasL expression and secretion in neutrophils. Blockade of □4 AChR, p38 and ERK pathways inhibited nicotine‐induced FasL expression. Furthermore, blockade of FasL with anti‐FasL antibody prevented nicotine‐induced caspase‐3 cleavage and neutrophil apoptosis. These results suggest that nicotine stimulates an extrinsic apoptotic cascade in human neutrophils. Thus, FasL/Fas system in neutrophils could serve as a target for further investigation of the cholinergic system in neuroimmunomodulation.Funding Source: R01AI075212–01 (MJR).
MIX‐cAMP signaling in 3T3‐L1 preadipocyte differentiation
Authors: Gabrielli Matias , Martini Claudia , Romero Damián , del Carmen Vila María 
Published: June 2021

Adipogenesis is stimulated in 3T3‐L1 fibroblasts by a combination of insulin, dexamethasone (DEX), and methylisobutylxanthine (MIX). Mitotic clonal expansion (MCE) precedes differentiation of 3T3‐L1 fibroblasts to adipocytes. MIX increases cAMP content, which activates protein kinase A (PKA). However, PKA‐independent cAMP signaling through EPAC (exchange protein activated by cAMP) has also been reported. We found that H89, a PKA inhibitor, blocks MCE but not differentiation of 3T3‐L1 fibroblasts. Differentiation, evaluated by Oil‐Red‐O staining or quantification of triglycerides, did not occur when MIX was not present in the differentiation mixture but it was restored by addition of either dibutyryl‐cAMP (db‐cAMP) or 8‐CPT‐2‐Me‐cAMP. The latter activates EPAC but not PKA signaling. Furthermore, we found that MIX, db‐cAMP or 8‐CPT‐2‐Me‐cAMP increased PPARγ, a transcription factor required for adipocyte differentiation. PI3K‐PKB signaling is known to be required for differentiation. We found that insulin but not MIX, alone or in combination with insulin+DEX, was able to increase PKB phosphorylation. These results suggest that MIX signaling through cAMP‐EPAC, which is independent of PI3K‐PKB signaling, is also required for 3T3‐L1 fibroblasts differentiation to adipocytes.Supported by National Agency for Scientific and Technological Promotion (Argentina) BID‐PICT: 08–1826.
miR‐10a regulates epithelial‐mesenchymal transition and adhesion and angiogenesis in hepatoma
Authors: Tang Hua 
Published: June 2021

MiRNA is involved in the regulation of hepatocellular carcinoma (HCC). Here, we studied the functions of miR‐10a in HCC cells.miR‐10a promoted cell migration and invasion capacity whereas did not affect cell viability and proliferation in HCC cell lines. However, the intrahepatic metastasis nodules were significantly reduced by miR‐10a in vivo. EphA4 was identified a target gene of miR‐10a. Knockdown of EphA4 promoted cell migration and invasion whereas did not affect cell viability and proliferation. Cell adhesion and vasculogenic minicry assay showed that overexpression of miR‐10a suppressed cell‐matrix adhesion and vessel formation of HCC cells. Blocking expression of miR‐10a or enhanced expression of EphA4 up‐regulated the epithelial marker E‐Cadherin whereas down‐regulate the mesenchymal markers, Vimentin and ICAM‐1.Blocking miR‐10a and up‐regulation of EphA4 enhanced the expression of ¦Â1‐ integrin which mediates cell‐matrix adhesion and MMP‐2.In summary, miR‐10a can promote EMT process in vitro to affect cell mobility whereas suppress cell‐matrix adhesion and vessel formation ability by regulating ¦Â1‐integrin and MMP‐2 to inhibit the metastasis, and may influence angiogenesis in vivo. All of these will provide new understanding into miR‐10a in the metastasis of HCC.