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Journal of Virology Feb 2019Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV...
Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host. Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.
Topics: Capsid Proteins; Carcinoma, Hepatocellular; Cell Culture Techniques; Cell Line; Cell Polarity; Epithelial Cells; Hep G2 Cells; Hepatitis E; Hepatitis E virus; Hepatocytes; Humans; Liver Neoplasms; Serum; Viral Proteins; Virus Release
PubMed: 30463960
DOI: 10.1128/JVI.01207-18 -
Sub-cellular Biochemistry 2012Caenorhabditis elegans provides a simplified, in vivo model system in which to study adherens junctions (AJs) and their role in morphogenesis. The core AJ... (Review)
Review
Caenorhabditis elegans provides a simplified, in vivo model system in which to study adherens junctions (AJs) and their role in morphogenesis. The core AJ components-HMR-1/E-cadherin, HMP-2/β-catenin and HMP-1/α-catenin-were initially identified through genetic screens for mutants with body axis elongation defects. In early embryos, AJ proteins are found at sites of contact between blastomeres, and in epithelial cells AJ proteins localize to the multifaceted apical junction (CeAJ)-a single structure that combines the adhesive and barrier functions of vertebrate adherens and tight junctions. The apically localized polarity proteins PAR-3 and PAR-6 mediate formation and maturation of junctions, while the basolaterally localized regulator LET-413/Scribble ensures that junctions remain apically positioned. AJs promote robust adhesion between epithelial cells and provide mechanical resistance for the physical strains of morphogenesis. However, in contrast to vertebrates, C. elegans AJ proteins are not essential for general cell adhesion or for epithelial cell polarization. A combination of conserved and novel proteins localizes to the CeAJ and works together with AJ proteins to mediate adhesion.
Topics: Adherens Junctions; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Morphogenesis
PubMed: 22674076
DOI: 10.1007/978-94-007-4186-7_12 -
Annals of Botany Dec 2022Aquatic carnivorous plants have typical rootless linear shoots bearing traps and exhibit steep physiological polarity with rapid apical growth. The aim was to analyse...
BACKGROUND AND AIMS
Aquatic carnivorous plants have typical rootless linear shoots bearing traps and exhibit steep physiological polarity with rapid apical growth. The aim was to analyse auxin and cytokinin metabolites in traps, leaves/shoots and shoot apices in several species of genera Aldrovanda and Utricularia to elucidate how the hormonal profiles reflect the specific organ functions and polarity.
METHODS
The main auxin and cytokinin metabolites were analysed in miniature samples (>2 mg dry weight) of different organs of Aldrovanda vesiculosa and six Utricularia species using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry.
KEY RESULTS
Total contents of biologically active forms (free bases, ribosides) of all four main endogenously occurring cytokinin types were consistently higher in traps than in leaves in four Utricularia species with monomorphic shoots and/or higher than in shoots in two Utricularia species with dimorphic shoots. In Aldrovanda traps, the total content of different cytokinin forms was similar to or lower than that in shoots. In U. australis leaves, feeding on prey increased all cytokinin forms, while no consistent differences occurred in Aldrovanda. In four aquatic Utricularia species with monomorphic shoots, the content of four auxin forms was usually higher in traps than in leaves. Zero IAA content was determined in U. australis leaves from a meso-eutrophic site or when prey-fed.
CONCLUSIONS
Different cytokinin and auxin profiles estimated in traps and leaves/shoots of aquatic carnivorous plants indicate an association with different dominant functions of these organs: nutrient uptake by traps versus photosynthetic function of traps. Interplay of cytokinins and auxins regulates apical dominance in these plants possessing strong polarity.
Topics: Cytokinins; Carnivorous Plant; Indoleacetic Acids; Magnoliopsida; Droseraceae; Lamiales
PubMed: 36215097
DOI: 10.1093/aob/mcac122 -
Journal of Dental Research, Dental... 2023This study compared apical extrusion of debris and instrumentation time following root canal instrumentation with Reciproc, Reciproc Blue, and Gentlefile (GF) rotary...
In vitro apical extrusion of debris and instrumentation time following root canal instrumentation with Reciproc and Reciproc Blue instruments and a novel stainless steel rotary system (Gentlefile) versus manual instrumentation.
BACKGROUND
This study compared apical extrusion of debris and instrumentation time following root canal instrumentation with Reciproc, Reciproc Blue, and Gentlefile (GF) rotary instruments versus the manual step-back technique.
METHODS
This in vitro study was conducted on 80 extracted mandibular premolars with mature apices and a root curvature of<10°. The teeth were randomly assigned to 4 groups (n=20), standardized regarding working length, and placed in pre-weighed vials. The root canals were instrumented with Reciproc, Reciproc Blue, and GF systems and the manual step-back technique in the four groups. The vials containing the collected debris were then dried and weighed. The instrumentation time was also recorded for each group. Data were analyzed with one-way ANOVA and post hoc Games-Howell test (α=0.05).
RESULTS
Minimum apical debris extrusion was noted in Reciproc, followed by Reciproc Blue, GF, and manual technique (<0.05). Pairwise comparisons showed significantly lower apical extrusion of debris in the Reciproc compared with GF (=0.015) and manual instrumentation (=0.011) groups. The Reciproc system also had the shortest instrumentation time, followed by Reciproc Blue, GF, and manual instrumentation (<0.05). Pairwise comparisons showed significant differences between all the systems (<0.05) except between Reciproc and Reciproc Blue (>0.05) in this respect.
CONCLUSION
Although all systems caused apical extrusion of debris, manual instrumentation caused maximum extrusion of debris. In contrast, the Reciproc system was superior to others regarding minimal apical extrusion of debris and the shortest instrumentation time.
PubMed: 38023797
DOI: 10.34172/joddd.2023.39271 -
Frontiers in Cell and Developmental... 2021The inner/apical surface of the embryonic brain wall is important as a major site for cell production by neural progenitor cells (NPCs). We compared the mechanical...
The inner/apical surface of the embryonic brain wall is important as a major site for cell production by neural progenitor cells (NPCs). We compared the mechanical properties of the apical surfaces of two neighboring but morphologically distinct cerebral wall regions in mice from embryonic day (E) E12-E14. Through indentation measurement using atomic force microscopy (AFM), we first found that Young's modulus was higher at a concave-shaped apical surface of the pallium than at a convex-shaped apical surface of the ganglionic eminence (GE). Further AFM analysis suggested that contribution of actomyosin as revealed with apical surface softening by blebbistatin and stiffness of dissociated NPCs were both comparable between pallium and GE, not accounting for the differential apical surface stiffness. We then found that the density of apices of NPCs was greater, with denser F-actin meshwork, in the apically stiffer pallium than in GE. A similar correlation was found between the decreasing density between E12 and E14 of NPC apices and the declining apical surface stiffness in the same period in both the pallium and the GE. Thus, one plausible explanation for the observed difference (pallium > GE) in apical surface stiffness may be differential densification of NPC apices. In laser ablation onto the apical surface, the convex-shaped GE apical surface showed quicker recoils of edges than the pallial apical surface did, with a milder inhibition of recoiling by blebbistatin than in pallium. This greater pre-stress in GE may provide an indication of how the initially apically concave wall then becomes an apically convex "eminence."
PubMed: 34368153
DOI: 10.3389/fcell.2021.702068 -
Molecular Biology of the Cell Nov 2018A major focus for our laboratory is identifying the molecules and mechanisms that regulate basolateral-to-apical transcytosis in polarized hepatocytes. Our most recent...
A major focus for our laboratory is identifying the molecules and mechanisms that regulate basolateral-to-apical transcytosis in polarized hepatocytes. Our most recent studies have focused on characterizing the biochemical and functional properties of the small rab17 GTPase. We determined that rab17 is a monosumoylated protein and that this modification likely mediates selective interactions with the apically located syntaxin 2. Using polarized hepatic WIF-B cells exogenously expressing wild-type, dominant active/guanosine triphosphate (GTP)-bound, dominant negative/guanosine diphosphate (GDP)-bound, or sumoylation-deficient/K68R rab17 proteins, we confirmed that rab17 regulates basolateral-to-apical transcytotic vesicle docking and fusion with the apical surface. We further confirmed that transcytosis is impaired from the subapical compartment to the apical surface and that GTP-bound and sumoylated rab17 are likely required for apical vesicle docking. Because expression of the GTP-bound rab17 led to impaired transcytosis, whereas wild type had no effect, we further propose that rab17 GTP hydrolysis is required for vesicle delivery. We also determined that transcytosis of three classes of newly synthesized apical residents showed similar responses to rab17 mutant expression, indicating that rab17 is a general component of the transcytotic machinery required for apically destined vesicle docking and fusion.
Topics: Animals; Biological Transport; Cell Line; Cell Membrane; Cell Polarity; Endocytosis; Endosomes; Guanosine Triphosphate; Hepatocytes; Liver; Rats; Transcytosis; rab GTP-Binding Proteins
PubMed: 30256711
DOI: 10.1091/mbc.E18-07-0433 -
Current Biology : CB Sep 2022A key aim in biology is to identify which genetic changes contributed to the evolution of form through time. Apical dominance, the inhibitory effect exerted by shoot...
A key aim in biology is to identify which genetic changes contributed to the evolution of form through time. Apical dominance, the inhibitory effect exerted by shoot apices on the initiation or outgrowth of distant lateral buds, is a major regulatory mechanism of plant form. Nearly a century of studies in the sporophyte of flowering plants have established the phytohormone auxin as a front-runner in the search for key factors controlling apical dominance, identifying critical roles for long-range polar auxin transport and local auxin biosynthesis in modulating shoot branching. A capacity for lateral branching evolved by convergence in the gametophytic shoot of mosses and primed its diversification; however, polar auxin transport is relatively unimportant in this developmental process, the contribution of auxin biosynthesis genes has not been assessed, and more generally, the extent of conservation in apical dominance regulation within the land plants remains largely unknown. To fill this knowledge gap, we sought to identify genetic determinants of apical dominance in the moss Physcomitrium patens. Here, we show that leafy shoot apex decapitation releases apical dominance through massive and rapid transcriptional reprogramming of auxin-responsive genes and altering auxin biosynthesis gene activity. We pinpoint a subset of P. patens TRYPTOPHAN AMINO-TRANSFERASE (TAR) and YUCCA FLAVIN MONOOXYGENASE-LIKE (YUC) auxin biosynthesis genes expressed in the main and lateral shoot apices and show that they are essential for coordinating branch initiation and outgrowth. Our results demonstrate that local auxin biosynthesis acts as a pivotal regulator of apical dominance in moss and constitutes a shared mechanism underpinning shoot architecture control in land plants.
Topics: Bryophyta; Bryopsida; Gene Expression Regulation, Plant; Germ Cells, Plant; Indoleacetic Acids; Plant Growth Regulators; Plant Shoots
PubMed: 35841890
DOI: 10.1016/j.cub.2022.06.064 -
Frontiers in Cell and Developmental... 2021Integrins are transmembrane proteins that are most typically thought of as integrating adhesion to the extracellular matrix with intracellular signaling and cell... (Review)
Review
Integrins are transmembrane proteins that are most typically thought of as integrating adhesion to the extracellular matrix with intracellular signaling and cell regulation. Traditionally, integrins are found at basolateral and lateral cell surfaces where they facilitate binding to the ECM and intercellular adhesion through cytosolic binding partners that regulate organization of actin microfilaments. However, evidence is accumulating that integrins also are apically localized, either endogenously or due to an exogenous stimulus. Apically localized integrins have been shown to regulate several processes by interacting with proteins such as connexins, tight junction proteins, and polarity complex proteins. Integrins can also act as receptors to mediate endocytosis. Here we review these newly appreciated roles for integrins localized to the apical cell surface.
PubMed: 34485286
DOI: 10.3389/fcell.2021.699407 -
BMC Oral Health Nov 2023To evaluate the amount of remaining filing material and apical debris extrusion after retreatment using Reciproc Blue, Hyflex EDM and ProTaper Retreatment Files.
OBJECTIVES
To evaluate the amount of remaining filing material and apical debris extrusion after retreatment using Reciproc Blue, Hyflex EDM and ProTaper Retreatment Files.
MATERIALS AND METHODS
Thirty-six extracted permanent mandibular first molars with moderately curved mesial roots were selected. Mesiobuccal canals were prepared using the ProTaper Next system up to size X2 and filled using gutta-percha and Adseal sealer via cold lateral compaction. Teeth were randomly divided into three equal groups (n = 12): Group 1: Reciproc Blue (RB)(VDW, Germany), Group 2: Hyflex EDM (HEDM)(Coltene/Whaledent, Switzerland), Group 3: ProTaper Universal Retreatment file system + ProTaper Next file system (PTUR + PTN)( Dentsply Maillefer, Switzerland). Eppendorf tubes were used to collect the apically extruded debris. Cone-beam computed tomographic scans were taken prior to and after retreatment and the volume of remaining filling material was assessed at the coronal, middle and apical levels. Statistical analysis was performed using the Kruskal-Wallis test, Friedman's test and Wilcoxon Sign Rank test. Significance level was set at p value 0.05.
RESULTS
There were no statistically significant differences among the three groups in the reduction of the volume of the filling material or in the amount of apically extruded debris.
CONCLUSION
All the tested filing systems showed similar efficacy in removing the filling material, however, none of them could achieve its complete removal. Apical extrusion of debris occurred with all the systems used with no significant difference between the three groups.
Topics: Humans; Root Canal Preparation; Root Canal Obturation; Root Canal Filling Materials; Gutta-Percha; Tooth; Dental Pulp Cavity; Retreatment
PubMed: 37990209
DOI: 10.1186/s12903-023-03579-7 -
Nature Cell Biology Sep 2017Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization...
Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.
Topics: Actomyosin; Adaptor Proteins, Signal Transducing; Animals; Animals, Genetically Modified; Caco-2 Cells; Cell Cycle Proteins; Cell Differentiation; Cell Membrane; Cell Polarity; Dogs; Drosophila Proteins; Drosophila melanogaster; Epithelial Cells; Genotype; Guanine Nucleotide Exchange Factors; Humans; Madin Darby Canine Kidney Cells; Membrane Proteins; Morphogenesis; Myosin Type II; Myotonin-Protein Kinase; Phenotype; Photoreceptor Cells, Invertebrate; Protein Kinase C; Protein Serine-Threonine Kinases; RNA Interference; Signal Transduction; Time Factors; Transfection; cdc42 GTP-Binding Protein
PubMed: 28825699
DOI: 10.1038/ncb3592