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The structure of cytoskeleton in the tobacco male meiocytes involved in the migration of nuclei between cells (cytomixis) is studied. The tubulin and actin components of cytoskeleton are examined using specific antibodies and phalloidin. The presence of microtubules and actin filaments inside cytomictic channels directly when the nuclei migrate through these channels is demonstrated for the first time. The actin and tubulin cytoskeleton is shown to retain its reticular structure in early prophase I before the beginning of cytomixis, during nuclear migration, and after completion of this process and emergence of micronuclei in the cytoplasm of recipient cells. It is demonstrated that if migration takes place in late prophase I, the nucleus leaves the perinuclear tubulin cage, encompassing it at this stage. After migration, the cytomictic micronuclei in recipient cell reside beyond the perinuclear tubulin cage in both meiotic prophase I and II. A microtubule-organizing activity of the migrated chromatin in meta-telophase I and II is demonstrated for the first time as well as the ability of this chromatin to form independent minispindles and radial microtubule arrays and contact the spindle of recipient cell. The role of cytomixis in production of aneuploid and unreduced pollen is discussed. [ABSTRACT FROM AUTHOR]

The annelid body can be subdivided into three main regions: the prostomium, the body segments, and the pygidium. The prostomium and pygidium, originating from the episphere and the posterior part of the hyposphere of a trochophore larva, are usually mentioned as non-homologous to body segments. However, recent studies revealed that the pygidium of several annelid species is far more complex than previously mentioned and possesses some segment-like features. To assess the diversity of a pygidial organization, I describe the innervation and muscular system of the pygidium in 19 annelid species belonging to the order Phyllodocida using phalloidin labeling, immunohistochemistry and confocal scanning microscopy. The musculature of the pygidium varies between families and usually consists of the circular and/or horseshoe-shaped hindgut sphincter muscles. In several families it can be accompanied by small additional transversal or dorso-ventral muscles. In contrast to the variable musculature, the pygidial innervation is far more uniform and in general comprises two huge main longitudinal nerves, a terminal commissure between them, and paired circumpigidial nerves. The pygidial epithelium bears numerous receptor cell endings, suggesting that the pygidium may act as an important sensory organ. The obtained results are in accordance with the recent data and indicate that such muscular and nervous organization may be characteristic of the whole order Phyllodocida. [ABSTRACT FROM AUTHOR]

Abstract Pacific oyster (Crassostrea gigas) is a sessile bivalve living in the intertidal zone. It has become an attractive model for developmental studies due to its metamorphic transition from a mobile planktonic larvae to a sessile adults. To determine the effect of metamorphosis on muscle development in oyster larvae, we characterized myogenesis during larval development and metamorphosis by phalloidin staining which labels filamentous actin filaments. Our data revealed a dynamic pattern of myogenesis during embryonic and larval development. It appears that simple "U-shaped" muscle ring first developed at the trochophore stage. This was followed by a more complex musculature including an anterior adductor, velum ventral retractors at the veliger stage, and the addition of posterior adductors and foot retractors at the veliger and pediveliger stages. During metamorphosis, muscle structures in the anterior adductor, velum retractors and ventral retractors were degenerated. At the same time, mantle and gill musculature appeared and became the primary muscle system in juveniles together with the posterior adductor. In addition, indirect immunofluorescence with the monoclonal antibody against C. gigas muscle proteins (myosin heavy chains (MYHC) and α-actinin) were used to monitor changes in the developing musculature at different larval stages. The immunofluorescence staining results of muscle proteins were consistent with phalloidin staining. The expression locations of two muscle proteins were similar and mainly located in larval velum retractor and adductor muscle. The α-actinin expression positions were located in Z -lined of velum striated retractors. Data from these studies provide a comprehensive description of myogenesis in C. gigas embryos and larvae. Moreover, our data showed that metamorphosis has a significant impact on remolding the musculature after transition from a mobile planktonic larvae to a sessile mollusk, associated with certain muscle group degradation. [ABSTRACT FROM AUTHOR]

In this study, we explored the effect of NO-induced changes in [Ca2+]i homeostasis in rat ventricular cardiomyocytes through variation in extracellular Ca2+ and application of SNAP (S-nitroso-N-acetyl-D,L-penicillamine) as an NO-donor. The Fura-2 signal dynamics and phalloidin intensity profile of z-disks in rat cardiomyocytes served as endpoints to describe the mechanisms involved in the control of the intracellular Ca2+ levels, depending on the kinetics of distribution of the SNAP-produced NO. The results demonstrated that SNAP caused small phalloidin intensity profile changes between the z-lines in the presence of [Ca2+]o. However, in the absence of [Ca2+]o, SNAP in a concentration of 300 μmol/L induced a significant decrease in the distance between z-lines. This SNAP-induced decrease was reflected as a decrease in the phalloidin fluorescence intensity in the middle of the sarcomere, due to the preferential imaging of greater fluorescence in the bulk of the thin filaments. Based on the Ca2+ fluorescence intensity profile, we could suggest that intracellular Ca2+ is mainly affected by the mechanisms of Ca2+ outflow, rather than the mechanisms of Ca2+ inflow in the presence of NO. Actually, through variation in the electrochemical gradient of Ca2+, we induced mechanisms of faster/slower cytosolic Ca2+ emptying. The obtained data showed that the delay in the cytosolic Ca2+ emptying in the presence of [Ca2+]o is due to the increased electrochemical gradient of Ca2+. [ABSTRACT FROM AUTHOR]

The study is dedicated to an identification of specific FMRF immunoreactivity in planarian (Girardia tigrina) with special attention to the FMRF-like neuropeptides location near muscle filaments. Using of whole mounts in conjunction with a confocal laser scanning microscopy reveals close spatial relationship of FMRF-immunopositive(-IP) nerves and planarian musculature. Morphological data provide an evidence for FMRF-like peptides participation in the innervations of planarian muscles. The results indicate that the larger part of muscular structures and organs (pharynx, mouth opening, body wall, and tail) of planarian body are deeply supplied by FMRF-IP nerve elements. Such observation of the peptidergic FMRF-IP neurons and its processes in vicinity of the muscle fibers can indicate an important role of the neuropeptides in the regulation of planarian muscle function. [ABSTRACT FROM AUTHOR]

Purpose: Large trials on anti-VEGF/PDGF (vascular endothelial/platelet-derived growth factor) combination therapy have been established to improve management of neovascular activity in age-related macular degeneration. Targeting pericytes, PDGF is thought to induce vessel regression and reduce fibrovascular scarring. The fate of pericytes exposed to anti-VEGF/PDGF combination therapy is not clear. Therefore, this study was designed to study the influence of anti-VEGF/PDGF on pericyte phenotype and cellular behavior.Methods: Human pericytes from placenta (hPC-PL) were treated with axitinib, a tyrosine kinase inhibitor targeting VEGFR1-3 and PDGFR. Toxic effects were excluded using live/dead staining. Phenotypic changes were evaluated using phalloidin staining for actin cytoskeleton and the expression of stress fibers. MRNA and protein expression levels of α-smooth muscle actin (αSMA) as a marker of proto-myofibroblastic transition were evaluated with real-time PCR and Western blotting. Influences of fibrotic cellular mechanisms were evaluated with a scratch wound migration and a collagen gel contraction assay.Results: Treatment with 0.5, 1, and 2.5 μg/ml axitinib strongly induced a proto-myofibroblast-like actin cytoskeleton with a marked increase in stress fibers. Quantitative real-time PCR and Western blotting revealed these changes to be linked to dose-dependent increases in αSMA mRNA and protein expression. However, fibrotic cellular mechanisms were significantly reduced in the presence of axitinib (scratch wound closure: up to − 78.4%, collagen gel contraction: up to − 37.4%).Conclusions: Combined anti-VEGF/PDGF inhibition seems to induce a proto-myofibroblast-like phenotype in human pericytes in vitro, but reduce profibrotic cellular mechanisms due to prolonged anti-PDGF inhibition. [ABSTRACT FROM AUTHOR]

Tissue closure involves the coordinated unidirectional movement of a group of cells without loss of cell–cell contact. However, the molecular mechanisms controlling the tissue closure are not fully understood. Here, we demonstrate that Lamin C, the sole A-type lamin in Drosophila , contributes to the process of thorax closure in pupa. High expression of Lamin C was observed at the leading front of the migrating wing imaginal discs. Live imaging analysis revealed that knockdown of Lamin C in the thorax region affected the coordinated movement of the leading front, resulting in incomplete tissue fusion required for formation of the adult thorax. The closure defect due to knockdown of Lamin C correlated with insufficient accumulation of F-actin at the front. Our study indicates a link between A-type lamin and the cell migration behavior during tissue closure. [ABSTRACT FROM AUTHOR]

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm. [ABSTRACT FROM AUTHOR]

The endocochlear potential (EP) provides part of the electrochemical drive for sound-driven currents through cochlear hair cells. Intense noise exposure (110 dB SPL, 2 h) differentially affects the EP in three inbred mouse strains (C57BL/6 [B6], CBA/J [CBA], BALB/cJ [BALB]) (Ohlemiller and Gagnon, 2007, Hearing Research 224:34-50; Ohlemiller et al., 2011, JARO 12:45-58). At least for mice older than 3 mos, B6 mice are unaffected, CBA mice show temporary EP reduction, and BALB mice may show temporary or permanent EP reduction. EP reduction was well correlated with histological metrics for injury to stria vascularis and spiral ligament, and little evidence was found for holes or tears in the reticular lamina that might ‘short out’ the EP. Thus we suggested that the genes and processes that underlie the strain EP differences primarily impact cochlear lateral wall, not the organ of Corti. Our previous work did not test the range of noise exposure conditions over which strain differences apply. It therefore remained possible that the relation between exposure severity and acute EP reduction simply has a higher exposure threshold in B6 mice compared to CBA and BALB. We also did not test for age dependence. It is well established that young adult animals are especially vulnerable to noise-induced permanent threshold shifts (NIPTS). It is unknown, however, whether heightened vulnerability of the lateral wall contributes to this condition. The present study extends our previous work to multiple noise exposure levels and durations, and explicitly compares young adult (6–7 wks) and older mice (>4 mos). We find that the exposure level-versus-acute EP relation is dramatically strain-dependent, such that B6 mice widely diverge from both CBA and BALB. For all three strains, however, acute EP reduction is greater in young mice. Above 110 dB SPL, all mice exhibited rapid and severe EP reduction that is likely related to tearing of the reticular lamina. By contrast, EP-versus-noise duration examined at 104 dB suggested that different processes contribute to EP reduction in young and older mice. The average EP falls to a constant level after ∼7.5 min in older mice, but progressively decreases with further exposure in young mice. Confocal microscopy of organ of Corti surface preparations stained for phalloidin and zonula occludens-1 (ZO-1) indicated this corresponds to rapid loss of outer hair cells (OHCs) and formation of both holes and tears in the reticular lamina of young mice. In addition, when animals exposed at 119 dB were allowed to recover for 1 mo, only young B6 mice showed collapse of the EP to ≤5 mV. Confocal analysis suggested novel persistent loss of tight junctions in the lateral organ of Corti. This may allow paracellular leakage that permanently reduces the EP. From our other findings, we propose that noise-related lateral wall pathology in young CBA and BALB mice promotes hair cell loss and opening of the reticular lamina. The heightened vulnerability of young adult animals to noise exposure may in part reflect special sensitivity of the organ of Corti to acute lateral wall dysfunction at younger ages. This feature appears genetically modifiable. [ABSTRACT FROM AUTHOR]

Background/Aims: The Trabecular meshwork (TM) is the tissue responsible for outflow resistance and therefore intraocular pressure. TM cells contain a contractile apparatus that is composed of actin stress fibres which run parallel to the axis of the cell and are responsible for facilitating contraction. Cross-Linked Actin Networks (CLANs) are polygonal arrangements of actin that form a geodesic network found predominantly in TM cells both in situ and in vitro. The aim of this work is to determine the functional significance of CLANs in TM cells and to assess the effect of mechanical stretch stimulation on the induction (or not) of CLANs. Methods: We used collagen gel contraction models to demonstrate functional impairment of cells when induced to express CLANs in situ. Cyclic mechanical stretch was used to stimulate cells and measure CLANs Results: CLANs inhibited contraction and cyclic mechanical stretch induced CLANs. Furthermore, we also demonstrated that using shape alone we could predict the appearance of CLANs using a simple light microscopy technique. Conclusion: Taken together we have now shown, for the first time, a functional deficit In TM cells with CLANs Furthermore that shape alone can predict the appearance of CLAN containing cells. CLANs can now be linked to a functional effect and may underlie the appearance of CLANs with the pathology of primary open angle glaucoma (POAG). [ABSTRACT FROM AUTHOR]

The marine microscopic jaw worms, Gnathostomulida, belong to the early branching spiralian lineage Gnathifera and consist of two main clades-Filospermoidea and Bursovaginoidea. Previous studies of gnathostomulid musculature focused on the bursovaginoid taxa, and only LM and TEM techniques were employed in the previous investigations of two filospermoids, Haplognathia simplex and Pterognathia swedmarki. In the present study, we used F-actin staining combined with CLSM to reconstruct the musculature of H. simplex and H. filum and found far-reaching similarities in their jaw musculature. In addition to known pharyngeal muscles we found new apophyseal abductors not uncovered in bursovaginoid gnathostomulids, but which resemble certain muscles of the sister group (Micrognathozoa + Rotifera) and may present an autapomorphy of Gnathifera. The body wall musculature exhibits greater interspecific variation: H. simplex possesses a simple grid of thick longitudinal and thin circular muscles, whereas H. filum shows a very sparse body wall musculature devoid of circular fibers, a pattern similar to another interstitial worm, the enigmatic annelid Lobatocerebrum. The flexibility gained hereby may similarly represent an adaptation and restriction to ciliary gliding among sand grains. [ABSTRACT FROM AUTHOR]

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+] i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists. [ABSTRACT FROM AUTHOR]

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone-specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO)mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistentwith this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p<0.001) in cKOmice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age-matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis. [ABSTRACT FROM AUTHOR]

Irradiation is the standard therapy for glioblastoma multiforme. Glioblastoma are highly resistant to radiotherapy and the underlying mechanisms remain unclear. To better understand the biological effects of irradiation on glioblastoma cells, we tested whether nonlethal irradiation influences the invasiveness, cell stiffness, and actin cytoskeleton properties. Two different glioblastoma cell lines were irradiated with 2 Gy and changes in mechanical and migratory properties and alterations in the actin structure were measured. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Irradiation led to changes in motility and a less invasive phenotype in both investigated cell lines that were associated with an increase in a "generalized stiffness" and changes in the actin structure. In this study we demonstrate that irradiation can induce changes in the actin cytoskeleton and motility, which probably results in reduced invasiveness of glioblastoma cell lines. Furthermore, "generalized stiffness" was shown to be a profound marker of the invasiveness of a tumor cell population in our model. [ABSTRACT FROM AUTHOR]

Nematode-trapping fungi include a variety of species capable of generating specific trapping devices to capture nematodes and the production of devices is also an indicator of a switch from saprophytic to predacious lifestyles. Traps are developed from vegetative mycelia, but they are quite different from hyphae in both morphological and physiological characteristics. Therefore, the molecular mechanisms underlying their formation have attracted much attention. In this investigation, Arthrobotrys oligospora , a nematode-trapping fungus, has three-dimensional networks and genomics and proteomics were recently performed, so as to reveal the relationship between actin cytoskeleton and trap formation. Both actin staining via FITC-phalloidin and treatment of actin polymerization inhibitor Lat-B illustrated that the actin cytoskeleton played an important role in trap development. Furthermore, absence of the conserved actin-associated protein Crn1p caused a structural defect of traps and failure to infect nematodes. It was observed that mutant Δ crn1 represented a reduced number of rings and a lower complexity of three-dimensional networks, likely due to the disturbance of actin branching. Collectively, our study confirmed the involvement of the actin cytoskeleton as well as the conserved actin-associated protein Crn1p in trap formation. It further suggested the manners in which Crn1p influences the development of three-dimensional networks in A. oligospora . [ABSTRACT FROM AUTHOR]

Dynamic cytoskeleton organization is essential for polarized cell behaviours in a wide variety of morphogenetic events. In zebrafish, epiboly involves coordinated cell shape changes and expansion of cell layers to close the blastopore, but many important regulatory aspects are still unclear. Especially, the spatio-temporal regulation and function of actin structures remain to be determined for a better understanding of the mechanisms that coordinate epiboly movement. Here we show that Rac1 signalling, likely functions downstream of phosphatiditylinositol-3 kinase, is required for F-actin organization during epiboly progression in zebtafish. Using a dominant negative mutant of Rac1 and specific inhibitors to block the activation of this pathway, we find that marginal contractile actin ring is sensitive to inhibition of Rac1 signalling. In particular, we identify a novel function for this actin structure in retaining the external yolk syncytial nuclei within the margin of enveloping layer for coordinated movement toward the vegetal pole. Furthermore, we find that F-actin bundles, progressively formed in the vegetal cortex of the yolk cell, act in concert with marginal actin ring and play an active role in pulling external yolk syncytial nuclei toward the vegetal pole direction. This study uncovers novel roles of different actin structures in orchestrating epiboly movement. It helps to provide insight into the mechanisms regulating cellular polarization during early development. [ABSTRACT FROM AUTHOR]

Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotin A carries multiple N-methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin-producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C-terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide. [ABSTRACT FROM AUTHOR]

Background: A defined Microbial Ecosystem Therapeutic (MET-1, or "RePOOPulate") derived from the feces of a healthy volunteer can cure recurrent C. difficile infection (rCDI) in humans. The mechanisms of action whereby healthy microbiota protect against rCDI remain unclear. Since C. difficile toxins are largely responsible for the disease pathology of CDI, we hypothesized that MET-1 exerts its protective effects by inhibiting the effects of these toxins on the host.Methods: A combination of in vivo (antibiotic-associated mouse model of C. difficile colitis, mouse ileal loop model) and in vitro models (FITC-phalloidin staining, F actin Western blots and apoptosis assay in Caco2 cells, transepithelial electrical resistance measurements in T84 cells) were employed.Results: MET-1 decreased both local and systemic inflammation in infection and decreased both the cytotoxicity and the amount of TcdA detected in stool, without an effect on C. difficile viability. MET-1 protected against TcdA-mediated damage in a murine ileal loop model. MET-1 protected the integrity of the cytoskeleton in cells treated with purified TcdA, as indicated by FITC-phalloidin staining, F:G actin assays and preservation of transepithelial electrical resistance. Finally, co-incubation of MET-1 with purified TcdA resulted in decreased detectable TcdA by Western blot analysis.Conclusions: MET-1 intestinal microbiota confers protection against C. difficile and decreases C. difficile-mediated inflammation through its protective effects against C. difficile toxins, including enhancement of host barrier function and degradation of TcdA. The effect of MET-1 on C. difficile viability seems to offer little, if any, contribution to its protective effects on the host. [ABSTRACT FROM AUTHOR]

Myogenesis requires a well-coordinated withdrawal from cell cycle, morphological changes and cell fusion mediated by actin cytoskeleton. Grb2 is an adaptor protein whose central SH2 domain binds to phosphorylated tyrosine residues of activated receptors and activates intracellular signaling pathway, while its N-terminal and C-terminal SH3 domains bind to proline rich proteins such as N-WASP (Neural-Wiskott Aldrich Syndrome Protein). We found that the expression of Grb2 was increased at the beginning of differentiation and remained constant during differentiation in C2C12 myoblasts. Knocking down endogenous Grb2 expression caused a significant increase in the fusion index and expression of MyHC, a terminal differentiation marker when compared with the control. Over expression of Grb2 in C2C12 (C2C12 Grb2-Myc ) reduced myotube formation and expression of MyHC. Similarly over expression of Grb2 P49L -Myc (N-terminal SH3 domain mutant) or Grb2 R86K -Myc (SH2 domain mutant) inhibited myogenic differentiation of C2C12 cells. However, the expression of Grb2 P206L -Myc (C-terminal SH3 domain mutant) did not inhibit myotube formation and expression of MyHC. This suggests that the C-terminal SH3 domain of Grb2 is critical for the inhibition of myogenic differentiation. The C2C12 Grb2-Myc cells have reduced phalloidin staining at late stages of differentiation. Expression of N-WASP in C2C12 Grb2-Myc cells rescued the myogenic defect and increased phalloidin staining (increased F-actin) in these cells. Thus our results suggest that Grb2 is a negative regulator of myogenesis and reduces myogenic differentiation by inhibiting actin polymerization/remodeling through its C-terminal SH3 domain. [ABSTRACT FROM AUTHOR]

Objective Collagen membranes serve as barriers to separate bone grafts from soft tissues. Bone grafts harvested with a bone scraper release growth factors activating transforming growth factor-β ( TGF-β) signaling in mesenchymal cells. The aim of the present pilot study was to determine whether collagen membranes adsorb molecules from bone-conditioned medium ( BCM) with the capacity to provoke the expression of TGF-β target genes in vitro. Materials and Methods Collagen membranes were soaked in aqueous extracts from fresh and demineralized bone chips placed in cell culture medium. Recombinant human TGF-β1 served as control. Gingival fibroblasts were seeded onto the washed collagen membranes and evaluated for the expression of adrenomedullin, pentraxin 3, interleukin 11, and proteoglycan 4. Cell viability and morphology with phalloidin staining were also determined. Results Incubation of collagen membranes with BCM for at least one minute caused fibroblasts to decrease the expression of adrenomedullin and pentraxin 3, and to increase the expression of interleukin 11 and proteoglycan 4. Four different membrane treatments - incubated with recombinant TGF-β1, pre-wetted with saline solution, exposed to UV light, and dry out and stored one week at room temperature - also provoked significant changes in gene expression. Likewise, conditioned medium from demineralized bone chips caused gene expression changes. BCM did not alter the viability or morphology of gingival fibroblasts. Conclusions These findings demonstrate that collagen membranes rapidly adsorb the TGF-β activity released from bone chips, a molecular process that might contribute to guided bone regeneration. [ABSTRACT FROM AUTHOR]

Proximally, the arrector pili muscle (APM) attaches to the follicular stem cell niche in the bulge, but its distal properties are comparatively unclear. In this work, a novel method employing an F-actin probe, phalloidin, was employed to visualize the APM anatomy. Phalloidin staining of the APM was validated by comparison with conventional antibodies/stains and by generating three-dimensional reconstructions. The proximal attachment of the APM to the bulge in 8 patients with androgenic alopecia was studied using Masson's trichrome stain. Phalloidin visualized extensive branching of the APM. The distal end of the human APM exhibits a unique 'C'-shaped structure connecting to the dermal-epidermal junction. The proximal APM attachment was observed to be lost or extremely miniaturized in androgenic alopecia. The unique shape, location, and attachment sites of the APM suggest a significant role for this muscle in maintaining follicular integrity. Proximally, the APM encircles the follicular unit and only attaches to the primary hair follicle in the bulge; this attachment is lost in irreversible hair loss. The APM exhibits an arborized morphology as it ascends toward the epidermis, and anchors to the basement membrane. [ABSTRACT FROM AUTHOR]

Background: The cyclic peptide toxins of Amanita mushrooms, such as α-amanitin and phalloidin, are encoded by the "MSDIN" gene family and ribosomally biosynthesized. Based on partial genome sequence and PCR analysis, some members of the MSDIN family were previously identified in Amanita bisporigera, and several other members are known from other species of Amanita. However, the complete complement in any one species, and hence the genetic capacity for these fungi to make cyclic peptides, remains unknown. Results: Draft genome sequences of two cyclic peptide-producing mushrooms, the "Death Cap" A. phalloides and the "Destroying Angel" A. bisporigera, were obtained. Each species has ~30 MSDIN genes, most of which are predicted to encode unknown cyclic peptides. Some MSDIN genes were duplicated in one or the other species, but only three were common to both species. A gene encoding cycloamanide B, a previously described nontoxic cyclic heptapeptide, was also present in A. phalloides, but genes for antamanide and cycloamanides A, C, and D were not. In A. bisporigera, RNA expression was observed for 20 of the MSDIN family members. Based on their predicted sequences, novel cyclic peptides were searched for by LC/MS/MS in extracts of A. phalloides. The presence of two cyclic peptides, named cycloamanides E and F with structures cyclo(SFFFPVP) and cyclo(IVGILGLP), was thereby demonstrated. Of the MSDIN genes reported earlier from another specimen of A. bisporigera, 9 of 14 were not found in the current genome assembly. Differences between previous and current results for the complement of MSDIN genes and cyclic peptides in the two fungi probably represents natural variation among geographically dispersed isolates of A. phalloides and among the members of the poorly defined A. bisporigera species complex. Both A. phalloides and A. bisporigera contain two prolyl oligopeptidase genes, one of which (POPB) is probably dedicated to cyclic peptide biosynthesis as it is in Galerina marginata. Conclusion: The MSDIN gene family has expanded and diverged rapidly in Amanita section Phalloideae. Together, A. bisporigera and A. phalloides are predicted to have the capacity to make more than 50 cyclic hexa-, hepta-, octa-, nona- and decapeptides. [ABSTRACT FROM AUTHOR]

Morphological studies on digenetic trematodes are quite numerous, but the large majority of researchers deal with the hermaphroditic generation (cercariae, metacercariae, adults). Parthenitae (miracidia, sporocysts, rediae), although they constitute a significant part of a digeneans' life cycle, attract much less attention. The sparse literature concerning the musculature of parthenitae indicates that it differs in many respects from that of digenean hermaphroditic generation and many other flatworms. We have tried to fill the knowledge gap on digenean muscle systems by focusing on daughter parthenitae (three species with rediae and two with daughter sporocysts). The study was performed using TRITC-phalloidin labeling and confocal microscopy and was aimed at both describing general trends and comparing different morphotypes. The basic body-wall muscle layers were confirmed to be composed of circular and longitudinal muscle fibers. Circular fibers form quite a uniform layer, and longitudinal fibers are typically joined into bundles. The density of muscle layers varies widely among studied species, and the possible causes of this variation are discussed. The internal muscle fibers and bundles are present in all studied species, being most prominent in the anterior region. The brood chamber and birth pore channel often have a muscular lining. The rediae pharynx has a typical set of muscle elements, mostly coinciding with one in the pharynx of the hermaphroditic generation. Taken together, our findings suggest that data on the musculature of daughter parthenitae are important from an evolutionary perspective. [ABSTRACT FROM AUTHOR]

The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177–187 in exon 1 of TPRN , which results in a truncated form of taperin protein. Heterozygous TPRN +/− mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates. Homozygous TPRN −/− mice exhibited progressive sensorineural hearing loss as reflected by auditory brainstem response to both click and tone burst stimuli at postnatal days 15 (P15), 30 (P30), and 60 (P60). Alex Fluor-594 phalloidin labeling showed no obvious difference in hair cell numbers in the cochlea between TPRN −/− mice and WT mice under light microscope. However, scanning electronic microscopy revealed progressive degeneration of inner hair cell stereocilia, from apparently normal at postnatal days 3 (P3) to scattered absence at P15 and further to substantial loss at P30. The outer hair cell stereocilia also showed progressive degeneration, though much less severe, Collectively, we conclude that taperin plays an important role in maintenance of hair cell stereocilia. Establishment of TPRN knockout mice enables further investigation into the function of this gene. [ABSTRACT FROM AUTHOR]

An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S-nitroso-N-acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0-28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin-labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki-67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP-induced migration and proliferation were antagonized by the NO-sensitive GC inhibitor 1H-[1,2,4]oxadiazolo[-4,3-a]quinoxalin-1-one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP-induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR-β antagonist (GSK0660) may interfere with NO-mediated wound healing process. J. Cell. Physiol. 231: 2185-2195, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: Platelets (PLTs) are currently stored at room temperature (22°C), which limits their shelf life, primarily due to the risk of bacterial growth. Alternatives to room temperature storage include PLT refrigeration (2-6°C), which inhibits bacterial growth, thus potentially allowing an extension of shelf life. Additionally, refrigerated PLTs appear more hemostatically active than conventional PLTs, which may be beneficial in certain clinical situations. However, the mechanisms responsible for this hemostatic function are not well characterized. The aim of this study was to assess the protein profile of refrigerated PLTs in an effort to understand these functional consequences.Study Design and Methods: Buffy coat PLTs were pooled, split, and stored either at room temperature (20-24°C) or under refrigerated (2-6°C) conditions (n = 8 in each group). PLTs were assessed for changes in external receptor expression and actin filamentation using flow cytometry. Intracellular proteomic changes were assessed using two-dimensional gel electrophoresis and Western blotting.Results: PLT refrigeration significantly reduced the abundance of glycoproteins (GPIb, GPIX, GPIIb, and GPIV) on the external membrane. However, refrigeration resulted in the increased expression of high-affinity integrins (αIIbβ3 and β1) and activation and apoptosis markers (CD62P, CD63, and phosphatidylserine). PLT refrigeration substantially altered the abundance and localization of several cytoskeletal proteins and resulted in an increase in actin filamentation, as measured by phalloidin staining.Conclusion: Refrigerated storage of PLTs induces significant changes in the expression and localization of both surface-expressed and intracellular proteins. Understanding these proteomic changes may help to identify the mechanisms resulting in the refrigeration-associated alterations in PLT function and clearance. [ABSTRACT FROM AUTHOR]

The pennate diatom, Bacillaria paxillifer, forms a colony in which adjacent cells glide smoothly and almost continuously, yet no obvious apparatus driving the movement, such as flagella or cilia, is observed. Thus far, neither the mechanism nor physiological significance of this movement has been well understood. Here, we report quantitative analysis of the gliding motion of B. paxillifer and morphological analysis of this diatom with light and electron microscopes. The gliding of pairs of adjacent B. paxillifer cells in a colony was cyclic with rather constant periods while the average gliding period varied from a few seconds to multiples of 10 s among colonies. The gliding was compromised reversibly by inhibitors for actin and myosin, suggesting involvement of the actomyosin system. Indeed, we observed two closely apposed actin bundles near the raphe by fluorescence- labeled phalloidin staining. Using electron microscopy, we observed filamentous structures that resemble the actin bundles seen with fluorescence microscopy, and we also found novel electron-dense structures located between the plasma membrane and these actin-like filaments. From these and other observations, we suggest that B. paxillifer also uses actin bundles and propose a putative myosin as a molecular motor in the gliding of unicellular diatoms. [ABSTRACT FROM AUTHOR]

Baculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate the ac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma, ac54 ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasin D-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected by ac54 deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly. [ABSTRACT FROM AUTHOR]

Obligate biotrophic fungi that cause powdery mildew on host plants form a specialized infection organ called the haustorium in the host apoplast. It was previously reported that the haustorium is surrounded by host actin microfilaments (AFs). The previous study used fixed cells, in which AFs were stained with fluorescently labeled phalloidine, therefore the structural dynamics of haustorium-surrounding AFs has not been examined. In the present study, we performed a live imaging analysis to examine the dynamics and developmental changes in the organization of haustorium-surrounding host AFs using host Arabidopsis thaliana and A. thaliana-adapted powdery mildew fungus Golovinomyces orontii. Image correlation-based velocimetry analysis suggested that AFs around haustorium are rather static compared to the dynamicity of AFs at the cell surface. Quantification of AF density and bundling showed that the density, but not the level of bundling, of haustorium-surrounding AFs increased as the haustorium matures. The possible role of AFs around haustoria is discussed. [ABSTRACT FROM AUTHOR]

Plants and animals express multiple actin isoforms in a manner that is dependent on tissues, organs and the stage of development. Previous genetic analyses suggested that individual actin isoforms have specific roles in cells, but there is little biochemical evidence to support this hypothesis. In this study, we purified four recombinant Arabidopsis actin isoforms, two major vegetative actin isoforms, ACT2 and ACT7, and two major reproductive isoforms, ACT1 and ACT11, and characterized them biochemically. Phalloidin bound normally to the filaments of the two reproductive actins as well as to the filaments of skeletal muscle actin. However, phalloidin bound only weakly to ACT7 filaments and hardly at all to ACT2 filaments, despite the conserved sequence of the phalloidin-binding site. Polymerization and phosphate release rates among these four actin isoforms were also significantly different. Moreover, interactions with profilin (PRF) were also different among the four Arabidopsis actin isoforms. PRF1 and PRF2 inhibited the polymerization of ACT1, ACT11 and ACT7, while ACT2 was only weakly affected. Plant actin isoforms have different biochemical properties. This result supports the idea that actin isoforms play specific roles to achieve multiple cell functions. [ABSTRACT FROM AUTHOR]

Background: The somatic musculature in trematode hermaphroditic generation (cercariae, metacercariae and adult) is presumed to comprise uniform layers of circular, longitudinal and diagonal muscle fibers of the body wall, and internal dorsoventral muscle fibers. Meanwhile, specific data are few, and there has been no analysis taking the trunk axial differentiation and regionalization into account. Yet presence of the ventral sucker (= acetabulum) morphologically divides the digenean trunk into two regions: preacetabular and postacetabular. The functional differentiation of these two regions is already evident in the nervous system organization, and the goal of our research was to investigate the somatic musculature from the same point of view. Results: Somatic musculature of ten trematode species was studied with use of fluorescent-labelled phalloidin and confocal microscopy. The body wall of examined species included three main muscle layers (of circular, longitudinal and diagonal fibers), and most of the species had them distinctly better developed in the preacetabuler region. In majority of the species several (up to seven) additional groups of muscle fibers were found within the body wall. Among them the anterioradial, posterioradial, anteriolateral muscle fibers, and U-shaped muscle sets were most abundant. These groups were located on the ventral surface, and associated with the ventral sucker. The additional internal musculature was quite diverse as well, and included up to twelve separate groups of muscle fibers or bundles in one species. The most dense additional bundles were found in the preacetabular region and were connected with the suckers. Conclusions: Previously unknown additional somatic musculature probably provides the diverse movements of the preacetabular region, ventral sucker, and oral sucker (or anterior organ). Several additional muscle groups of the body wall (anterioradial, posterioradial, anteriolateral fibers and U-shaped sets) are proposed to be included into the musculature ground pattern of trematode hermaphroditic generation. This pattern is thought to be determined by the primary trunk morphofunctional differentiation into the preacetabular and the postacetabular regions. [ABSTRACT FROM AUTHOR]

Background: The somatic musculature in trematode hermaphroditic generation (cercariae, metacercariae and adult) is presumed to comprise uniform layers of circular, longitudinal and diagonal muscle fibers of the body wall, and internal dorsoventral muscle fibers. Meanwhile, specific data are few, and there has been no analysis taking the trunk axial differentiation and regionalization into account. Yet presence of the ventral sucker (= acetabulum) morphologically divides the digenean trunk into two regions: preacetabular and postacetabular. The functional differentiation of these two regions is already evident in the nervous system organization, and the goal of our research was to investigate the somatic musculature from the same point of view. Results: Somatic musculature of ten trematode species was studied with use of fluorescent-labelled phalloidin and confocal microscopy. The body wall of examined species included three main muscle layers (of circular, longitudinal and diagonal fibers), and most of the species had them distinctly better developed in the preacetabuler region. In majority of the species several (up to seven) additional groups of muscle fibers were found within the body wall. Among them the anterioradial, posterioradial, anteriolateral muscle fibers, and U-shaped muscle sets were most abundant. These groups were located on the ventral surface, and associated with the ventral sucker. The additional internal musculature was quite diverse as well, and included up to twelve separate groups of muscle fibers or bundles in one species. The most dense additional bundles were found in the preacetabular region and were connected with the suckers. Conclusions: Previously unknown additional somatic musculature probably provides the diverse movements of the preacetabular region, ventral sucker, and oral sucker (or anterior organ). Several additional muscle groups of the body wall (anterioradial, posterioradial, anteriolateral fibers and U-shaped sets) are proposed to be included into the musculature ground pattern of trematode hermaphroditic generation. This pattern is thought to be determined by the primary trunk morphofunctional differentiation into the preacetabular and the postacetabular regions. [ABSTRACT FROM AUTHOR]

The role of actin filaments in allodynia induced by chronic compression of the dorsal root ganglion (DRG) (CCD) and the effects of microfilaments dynamics on transient receptor potential vanilloid 4 (TRPV4) were investigated in this study. Anti-microfilaments agents resulted in dose-dependent and partial reduction in CCD-induced allodynia, which could be prevented by the prior stabilizer administration. In association with the reduction of allodynia by microfilaments’ disruption, TRPV4-mediated currents were inhibited by disruptors. In addition, plasma membrane-associated TRPV4 was also depressed by disruptors. The time courses for the changes of TRPV4 activity and distribution in vitro were similar to the time courses for the attenuation of allodynia in vivo. Phalloidin, the stabilizer of microfilaments, did not affect the allodynia in CCD rats. However, phalloidin resulted in reduction and delay of TRPV4 current, which was not consistent with the effect of phalloidin on CCD-induced allodynia. In accordance with the inhibition of TRPV4 activity, the reversal potentials shifted toward more positive voltages and the plasma membrane-associated TRPV4 was depressed by phalloidin. In conclusion, intact actin filaments were necessary for CCD-induced allodynia, and disruptors of microfilaments attenuated CCD-induced allodynia. However, stabilizer of actin filaments did not affect allodynia in CCD rats. Further, TRPV4 contributed to the disruptors-induced attenuation of allodynia in CCD rats. [ABSTRACT FROM AUTHOR]

Terminally differentiated neurons have a single, primary cilium. The primary cilia of hypothalamic neurons play a critical role in sensing metabolic signals. We recently showed that mice with leptin deficiency or resistance have shorter cilia in the hypothalamic neurons, and leptin treatment elongates cilia in hypothalamic neurons. Here, we investigated the molecular mechanisms by which leptin controls ciliary length in hypothalamic neurons. In N1 hypothalamic neuronal cells, leptin treatment increased the expression of intraflagellar transport proteins. These effects occurred via phosphatase and tensin homolog/glycogen synthase kinase-3β-mediated inhibition of the transcriptional factor RFX1. Actin filament dynamics were also involved in leptin-promoted ciliary elongation. Both leptin and cytochalasin-D treatment induced F-actin disruption and cilium elongation in hypothalamic neurons that was completely abrogated by co-treatment with the F-actin polymerizer phalloidin. Our findings suggest that leptin elongates hypothalamic neuronal cilia by stimulating the production of intraflagellar transport proteins and destabilizing actin filaments. [ABSTRACT FROM AUTHOR]

Background Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant 3D in vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. Methods We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Results Spheroids had uniform geometry, with projected areas (42.60 x 10³ µm-475.22 x 10³ µm² for A2780 spheroids and 37.24 x 10³ µm²-281.01 x 10³ µm² for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell-cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70-80% viability) to cisplatin chemotherapy compared to 2D cultures (30-50% viability). Conclusions Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. [ABSTRACT FROM AUTHOR]

BACKGROUND: In coronary artery disease, highly stenosed arteries are frequently treated by stent implantation, which thereafter necessitates a dual-antiplatelet therapy (DAPT) in order to prevent stent-thrombosis. We hypothesized that specific patterns of microstructures on stents can accelerate endothelialisation thereby reducing their thrombogenicity and the DAPT duration. METHODS: Differently designed, 2-5 μm high elevations or hollows were lithographically etched on silicon plates, subsequently coated with silicon carbide. Smooth silicon plates and bare metal substrates were used as controls. To assess attachment and growth of human umbilical vein endothelial cells under static or flow conditions, actin cytoskeleton was visualised with green phalloidin. Endothelial migration was assessed in a modified barrier assay. To investigate surface thrombogenicity, platelets were incubated on the structured surfaces in static and flow conditions, and visualised with fluorescein-conjugated P-selectin antibody. Images were taken with incident-light fluorescent microscope for non-transparent objects. RESULTS: Compared to smooth surface, flat cubic elevations (5 μm edge length) improved endothelial cell attachment and growth under static and dynamic conditions, whereas smaller, spiky structures (2 μm edge length) had a negative influence on endothelialisation. Endothelial cell migration was fastest on flat cubic elevations, hollows, and smooth surfaces, whereas spiky structures and bare metal had a negative effect on endothelial migration. Thrombogenicity assays under static and flow conditions showed that platelet adhesion was reduced on the flat elevations and the smooth surface, as compared to the spiky structures, the hollow design and the bare metal substrates. CONCLUSION: Surface microstructures strongly influence endothelialisation of substrates. Designing stents with surface topography which accelerates endothelialisation and reduces thrombogenicity may be of clinical benefit by improving the safety profile of coronary interventions. [ABSTRACT FROM AUTHOR]

This study utilized pharmacological activation of Nrf2 with oleanolic acid (OA, 22.5 mg/kg, sc for 4 days) and the genetic alteration of Nrf2 (Nrf2-null, wild-type, and Keap1-HKO mice) to examine the role of Nrf2 in protection against phalloidin hepatotoxicity. Mice were given phalloidin (1.5 mg/kg, ip for 8 h) to examine liver injury and the expression of toxicity-related genes. Phalloidin increased serum enzyme activities and caused extensive hepatic hemorrhage and necrosis in Nrf2-null and wild-type mice, but less injury was seen in Keap1-HKO mice and OA-pretreated mice. Phalloidin increased the expression of neutrophil-specific chemokine mKC and MIP-2 in Nrf2-null and WT mice, but such increases were attenuated in Keap1-HKO and OA-pretreated mice. Phalloidin increased, while Nrf2 activation attenuated, the expression of genes involved in acute-phase response (Ho-1) and DNA-damage response genes (Gadd45 and Chop10). Phalloidin is taken up by hepatocytes through Oatp1b2, but there was no difference in basal and phalloidin-induced Oatp1b2 expression among Nrf2-null, wild-type, and Keap1-HKO mice. In contrast, OA decreased phalloidin-induced Oatp1b2. Phalloidin activated MAPK signaling (p-JNK), which was attenuated by activation of Nrf2. In conclusion, this study demonstrates that protection against phalloidin hepatotoxicity by OA involves activation of Nrf2 and suppression of Oatp1b2. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton organization in symbiotic marine dinoflagellates is largely undescribed; most likely, due to their intense pigment autofluorescence and cell walls that block fluorescent probe access. Using a freeze-fracture and fixation procedure, we observed the actin cytoskeleton of Symbiodinium kawagutii cultured in vitro with fluorescently labeled phalloidin and by indirect immunofluorescence with monoclonal antibodies specific for actin. The cytoskeleton appeared as an organized network with interconnected cortical and cytoplasmic thick filaments, along with some intertwined fine filaments. It showed a grid-type, reticular pattern organized in a lattice-like structure within the cell and throughout the cytoplasm. This organization was similar when the observations were done with either fluorescein isothiocyanate (FITC)-phalloidin or anti-actin, although the latter showed a more evenly distributed fluorescence characteristic of nonpolymerized actin. The network organization collapsed upon treatment with latrunculin, resulting in bright foci and diffuse fluorescence. A similar effect was obtained upon butanedione monoxime treatment, except that no bright foci were observed. We have been able to successfully visualize the actin cytoskeleton of S. kawagutii cells using fluorescence-based procedures. This is the first report on the visualization of the organization of the actin cytoskeleton under various conditions in these walled, highly autofluorescent cells. [ABSTRACT FROM AUTHOR]

Introduction Mechanical loads can regulate cell proliferation and differentiation at various stages of development and homeostasis. However, the extension of this regulatory effect of mechanical loads on cancer cells is largely unknown. Increased physical compliance is one of the key features of cancer cells, which may hamper the transmission of mechanical loads to these cells within tumor microenvironment. Here we tested whether brief daily application of an external low magnitude mechanical stimulus (LMMS), would impede the growth of MDA-MB- 231 aggressive type breast cancer cells in vitro for 3 wks of growth. Methods The signal was applied in oscillatory form at 90 Hz and 0.15g, a regimen that would induce mechanical loads on MDA-MB-231 cells via inertial properties of cells rather than matrix deformations. Experimental cells were exposed to LMMS 15min/day, 5days/week in ambient conditions while control cells were sham loaded. Cell proliferation, viability, cycle, apoptosis, morphology and migration were tested via Trypan Blue dye exclusion, MTT, PI, Annex in V, Calcein-AM and phalloidin stains and scratch wound assays. Results Compared to sham controls, daily application of LMMS reduced the number and viability of cancerous MDA-MB-231 cells significantly after first week in the culture, while non-cancerous MCF10A cells were found to be unaffected. Flow cytomety analyses suggested that the observed decrease for the cancer cells in the LMMS group was due to a cell cycle arrest rather than apoptosis. LMMS further reduced cancer cell circularity and increased cytoskeletal actin in MDA-MB-231 cells. Conclusion Combined, results suggest that direct application of mechanical loads negatively regulate the proliferation of aggressive type cancer cells. If confirmed, this non-invasive approach may be integrated to the efforts for the prevention and/or treatment of cancer. [ABSTRACT FROM AUTHOR]

Urea and trimethylamine N -oxide (TMAO) are known to denature and stabilize proteins, respectively. We examined two actin-binding processes, namely, end-to-end annealing of actin filaments (F-form) and the polymerization of actin monomers (G-form) into filaments, in the presence of urea, TMAO, and both solutes. Fluorescence microscopy for direct observation of actin filaments bound by fluorescent phalloidin revealed that the annealing rate constant decreased as the concentrations of urea or TMAO increased. Fluorescence spectroscopy with pyrene-labeled actin monomers showed that urea decreased the polymerization rate, whereas TMAO enhanced the rate. The decrease in the polymerization rate constant and thermal stability induced by 0.6 M urea was almost completely ameliorated by the addition of 0.3 M TMAO. These results suggest that TMAO-dependent stabilization of actin structure facilitates the binding of G-form actin to the ends of F-form actin filaments. Conversely, the binding between ends of mature filaments was impaired by TMAO. [ABSTRACT FROM AUTHOR]

Mycotoxin intoxications can result from the consumption of amatoxins like α- and β-amanitin or of phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatoxins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography–high-resolution-mass spectrometry, LC–HR-MS), Q Exactive™ (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 × 2.1 mm, 2.6 µm) using a gradient of 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for α-amanitin, β-amanitin, phalloidin and IS of 1.9, 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC–HR-MS system was performed by extracting the exact mass value of each protonated species using a 5-p.p.m. mass window, which was 919.3614, 920.3455, 789.3257 and 388.1586 for α-amanitin, β-amanitin, phalloidin and IS, respectively. Calibration curves were obtained by spiking drug-free urine at 1–100 ng/mL. Mean correlation coefficients, r2, were above 0.99 for each amatoxins and phalloidin. According to currently accepted validation procedures, the method was tested for selectivity, calibration, accuracy, matrix effect, precision and recovery. Authentic urine samples from 43 patients suffering from a suspected intoxication with mushrooms were analyzed by LC–HR-MS, and the results were compared with ELISA competitive immunoassay. The LC–HR-MS presented large benefits over immunoassay of being specific, faster and more sensitive, making it suitable for daily emergency toxicological analysis. [ABSTRACT FROM PUBLISHER]

The presence and state of actin in the nucleus has long been controversial. This is poised to change. Over the past two years, the regulation of nuclear actin and its polymerization have begun to be characterized. The transport of actin into and out of the nucleus has been defined and the importance of nuclear actin polymerization in the retention of the serum response factor coactivator MRTF-A is now quite clear. Moreover, serum-starved fibroblasts that are stimulated with serum rapidly form long actin bundles that can be visualized using the F-actin binding drug phalloidin. This provides the first compelling direct evidence that actin polymerizes in the nucleus and provides the foundation for a serious investigation of the function(s) of nuclear polymeric actin. [ABSTRACT FROM AUTHOR]

Highlights: [•] Staining protocols were optimized to study myofibrillogenesis in Xenopus embryos. [•] A dissection approach was developed for ideal staining of Xenopus somitic regions. [•] Optimized phalloidin labeling and immunostaining for Xenopus embryo cryosections. [•] Demonstrated methods to preserve phalloidin labeling after Murray’s clear. [Copyright &y& Elsevier]

Highlights: [•] Rhodnius prolixus female reproductive organs are muscular (phalloidin staining). [•] Spontaneous activity present in all female reproductive tissues. [•] RFamide-like immunoreactivity found in processes on female reproductive tissues. [•] Two extended RFamides are stimulatory on the female reproductive tissues. [Copyright &y& Elsevier]

The musculature of cercariae, pre-ovigerous, and ovigerous adults of Diplodiscus subclavatus was studied by means of TRITC-conjugated phalloidin staining of filamentous actin and confocal scanning laser microscopy. The body wall appears to include four muscle layers as follows: circular, outer longitudinal, diagonal, and inner longitudinal. Two layers of longitudinal muscle fibers are arranged in different modes due to the secondary transformed paramphistomid body construction. The organization of the acetabulum turned out to be more complex than ever described, with a radial layer, two layers of circular, two layers of meridional, an additional starry layer of muscle fibers, as well as a few separate muscle layers of the accessory sucker. Within the pharynx, I found a group of alar muscle fibers, never described before for any paramphistomids, and some morphological features which were not considered to be characteristic for D. subclavatus (namely-the middle semicircular layer and the transverse muscle fibers in the pre-sphincteric space). No significant reorganizations of the somatic musculature occur throughout the development from the cercaria to the ovigerous adult worm, so the metamorphosis goes in the manner of completion. The cercarial tail includes a layer of circular muscle fibers and a longitudinal muscle layer beneath. The latter consists of two medial longitudinal bundles of smooth muscle fibers and two lateral longitudinal bands of obliquely striated muscle fibers which are partially divided in halves. [ABSTRACT FROM AUTHOR]

Highlights: [•] Myosin and phalloidin are predicted to modulate cofilin-mediated filament severing. [•] Competitive displacement of bound cofilin by myosin and phalloidin is measured. [•] Competition can promote filament severing, depending on initial cofilin occupancy. [•] Maximal severing occurs near half cofilin occupancy, even with competitors present. [•] We propose a general “severodyne” framework to describe cofilin regulation. [Copyright &y& Elsevier]

Nemertodermatida is a small taxon of marine worm-like animals; its position in the tree of life is highly contested. The musculature of Nemertoderma westbladi and Meara stichopi is studied here in detail using fluorescent phalloidin and confocal microscopy. In both species, the musculature is composed of an outer layer of circular and an inner layer of longitudinal musculature, diagonal muscles form a distinct layer in N. westbladi, but in M. stichopi these fibres connect to both other layers. The supraterminally opening male pore and antrum are formed by invagination of the whole body-wall in both species, and the seminal vesicle is lined by a thin net of musculature only in full male maturity. Modifications of the ventral body-wall adjacent to the mouth are small and transient in N. westbladi including no extra musculature, whereas it consists of additional strong U-shaped musculature in M. stichopi. Myogenesis in N. westbladi is not finished in hatchlings and will be completed dorsally in juvenile specimens and ventrally in male mature ones, after the loss of the mouth. Musculature between the two species differs considerably and might give insights into the internal relationships of Nemertodermatida and might prove to be useful in studies investigating their phylogenetic position. More data of other species and developmental changes are needed. [ABSTRACT FROM AUTHOR]

Little is known about the actin cytoskeleton architecture in female Strongyloides venezuelensis and thus to investigate the distribution and concentration of actin, female worms were labelled with phalloidin-rhodamine and visualized under confocal microscopy. Our results demonstrate that filamentous actin accumulates in the vulva and the concentration of F-actin at this site suggests its important role, especially during oviposition, in the life cycle of S. venezuelensis. [ABSTRACT FROM PUBLISHER]