Actin Labeling Research Articles

A high expression ratio of RhoA/RhoB is associated with the migratory and invasive properties of basal-like Breast Tumors.

Basal-like breast cancer is among the most aggressive cancers and there is still no effective targeted treatment. In order to identify new therapeutic targets, we performed mRNA-Seq on eight breast cancer cell lines. Among the genes overexpressed in basal-like tumors, we focused on the RhoA and RhoB genes, which encode small GTPases known to play a role in the actin cytoskeleton, allowing cells to migrate.qRT-PCR and Western blotting were used for expression studies. Migratory and invasive properties were analysed by wound healing and Boyden chambers assays. Stress fibers formation was evaluated by fluorescent actin labeling. Rho siRNA, small inhibitor Rhosin treatment and BRCA1 transfection were performed to study the role of Rho and BRCA1 proteins.We showed that strong expression of RhoA and low expression of RhoB was associated with the basal-like subtype of breast cancer. Decreasing RhoA expression reduced the migratory and invasive capacities of basal-like cell lines, while decreasing RhoB expression increased these capacities. Rhosin, an inhibitor of RhoA, could also reduce the migration of basal-like cell lines. Rho proteins are involved in the formation of stress fibers, a conformation of the actin cytoskeleton found in migrating cells: inhibition of RhoA expression decreased the formation of these fibers. BRCA1, a gene frequently inactivated in basal-like tumors, appears to play a role in the differential expression of RhoA and RhoB in these tumors, as the restoration of BRCA1 expression in a BRCA1-mutated basal-like cell line decreased expression of RhoA and increased expression of RhoB, resulting in reduced migratory capacity.These results suggest Rho proteins as potential therapeutic targets for basal-like and BRCA1-mutated breast cancer, as migration and acquisition of mesenchymal properties are key functional pathways in these tumors with high metastatic potential.

Lithium-Doped Biological-Derived Hydroxyapatite Coatings Sustain In Vitro Differentiation of Human Primary Mesenchymal Stem Cells to Osteoblasts

This study is focused on the adhesion and differentiation of the human primary mesenchymal stem cells (hMSC) to osteoblasts lineage on biological-derived hydroxyapatite (BHA) and lithium-doped BHA (BHA:LiP) coatings synthesized by Pulsed Laser Deposition. An optimum adhesion of the cells on the surface of BHA:LiP coatings compared to control (uncoated Ti) was demonstrated using immunofluorescence labelling of actin and vinculin, two proteins involved in the initiation of the cell adhesion process. BHA:LiP coatings were also found to favor the differentiation of the hMSC towards an osteoblastic phenotype in the presence of osteoinductive medium, as revealed by the evaluation of osteoblast-specific markers, osteocalcin and alkaline phosphatase. Numerous nodules of mineralization secreted from osteoblast cells grown on the surface of BHA:LiP coatings and a 3D network-like organization of cells interconnected into the extracellular matrix were evidenced. These findings highlight the good biocompatibility of the BHA coatings and demonstrate that the use of lithium as a doping agent results in an enhanced osteointegration potential of the synthesized biomaterials, which might therefore represent viable candidates for future in vivo applications.

The Unfolded Protein Response Causes Prothrombotic Transformation of Pancreatic Cancer Linking Tumor Progression with Cancer-Associated Thrombosis

Thrombosis is a common complication of advanced stage cancer. Yet the underlying mechanisms that link tumor progression to clot formation are poorly understood. The unfolded protein response (UPR) is associated with malignant transformation in pancreatic cancer, but whether or not activation of the UPR is linked to cancer thrombosis has not previously been evaluated. To determine whether UPR signaling functions in the prothrombotic transformation of pancreatic cancer, we exposed pancreatic adenocarcinoma cells (HPAF-II cells) to three UPR inducers (tunicamycin, triptolide or thapsigargin) that act via independent mechanisms. Induction of UPR resulted in the release of thrombogenic material into the supernatant as evidenced by a ~3-fold increase in thrombin generation in pelleted material. Release of thrombogenic material was inhibited by siRNA-mediated knockdown of UPR components including IRE1a (80±3% decrease) or PERK (60±10% decrease). Chemical inhibition of UPR also inhibited release of thrombogenic material from HPAF-II cells. Exposure to the IRE1a inhibitor, MKC-3946, resulted in a 70±10% decrease in thrombin generation in pelleted material, and incubation with the PERK inhibitor, GSK2606414, caused an 80±5% decrease. Characterization of the thrombogenic activity revealed that it was present on extracellular vesicles (EVs) and was inhibited by anti-TF antibodies. Flow cytometry demonstrated on average a 3-fold increase in fluorescence of TF-bearing EVs following UPR induction. Electron microscopy showed that the HPAF II EVs ranged from 100-500 mm and demonstrated increased clustering following UPR induction. Three-color immunofluorescence microscopy of HPAF II cells with labeling of actin, nuclei, and TF showed that induction of the UPR resulted in actin-poor membrane blebs rich in TF. Apoptosis as detected by caspase-3 cleavage was not observed under these conditions. Brefeldin A, which inhibits vesicular transport between the endoplasmic reticulum and the Golgi, inhibited UPR-induced generation of TF-bearing EVs, indicating that UPR-mediated vesicular trafficking contributes to TF-bearing EV formation. To evaluate the possibility of an association between the UPR and cancer thrombosis in the clinical setting, we analyzed plasmas collected from pancreatic cancer patients who were monitored prospectively for the development of venous thromboembolism (including a lower extremity ultrasound performed at baseline and at 2 months). Proteomic analysis was performed using Somalogic technology to evaluate ~1300 analytes in plasmas from nine pancreatic cancer patients who subsequently developed venous thromboembolism and ten patients with similar pancreatic cancer characteristics who remained free of venous thromboembolism. Evaluation of eight UPR markers present in the SOMAscan panel demonstrated significant upregulation in plasmas of patients who developed clots compared to those who did not (p = 0.0001). Our current findings support a model whereby activation of the UPR results in increased vesicular trafficking leading to the release of TF-bearing EVs. These observations indicate a mechanistic link between tumor progression in pancreatic cancer and cancer associated thrombosis. Disclosures Peters: Platelet BioGenesis: Employment. Zwicker:Portola: Consultancy; Parexel: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Daiichi: Consultancy; Quercegen: Research Funding; Incyte: Research Funding. Flaumenhaft:Relay Therapeutics: Consultancy; PlateletDiagnostics: Consultancy, Other: Founder.

Nodular lesion in the tongue: A rare case of low grade myofibroblastic sarcoma

Low grade myofibroblastic sarcoma is an atypical myofibroblastic malignant neoplasm that rarely affects the oral cavity and presents a challenging histological diagnosis. Here we report the case of a 38-year-old woman who had a nodular and symptomatic lesion on the lateral border of the tongue. After the biopsy, the histopathological analysis revealed a spindle cell lesion with fascicular and storiform pattern. We performed immunohistochemical analysis that showed intense labeling for vimentin, smooth muscle actin and moderate labeling for calponin and S-100. We emphasize the rarity of low grade myofibroblastic sarcoma such as a nodular lesion on the tongue and the need for accurate diagnosis.

Pathological Study on Epithelial-Mesenchymal Transition in Silicotic Lung Lesions in Rat

Silicosis, caused by the inhalation of crystalline silicon dioxide or silica, is one of the most severe occupational diseases. Persistent inflammation and progressive massive pulmonary fibrosis are the most common histological changes caused by silicosis. Association of epithelial-mesenchymal transition (EMT) of hyperplastic type II epithelial cells with the fibrotic events of pulmonary fibrosis has been suggested in in vitro silica-exposed cultured cell models, patients with idiopathic pulmonary fibrosis, and bleomycin-induced experimental models. Histological features of EMT, however, are not fully described in silicotic lungs in in vivo. The purpose of this study was to demonstrate EMT of hyperplastic type II epithelial cells in the developmental process of progressive massive pulmonary fibrosis in the lungs of rats exposed to silica. F344 female rats were intratracheally instilled with 20 mg of crystalline silica (Min-U-Sil-5), followed by sacrifice at 1, 3, 6, and 12 months after instillation. Fibrosis, characterized by the formation of silicotic nodules, progressive massive fibrosis, and diffuse interstitial fibrosis, was observed in the lungs of the treated rats; the effects of fibrosis intensified in a time-dependent manner. Hyperplasia of the type II epithelial cells, observed in the massive fibrotic lesions, dominated in the lungs of rats at 6 and 12 months after the treatment. Immunohistochemistry of the serial sections of the lung tissues demonstrated positive labeling for cytokeratin, vimentin, and α-smooth muscle actin in spindle cells close to the foci of hyperplasia of type II epithelial cells. Spindle cells, which exhibited features of both epithelial cells and fibroblasts, were also demonstrated with bundles of collagen fibers in the fibrotic lesions, using electron microscopy. Increased expression of TGF-β was shown by Western blotting and immunohistochemistry in the lungs of the treated rats. These findings suggested that enhanced TGF-β expression and EMT of hyperplastic type II epithelial cells are involved in the development process of progressive massive pulmonary fibrosis during silicosis.

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy.

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.

Impact of polypropylene prolapse mesh on vaginal smooth muscle in rhesus macaque

The use of polypropylene prolapse mesh to treat pelvic organ prolapse has been limited by mesh-related complications. Gynemesh PS mesh, implanted via sacrocolpopexy in rhesus macaques, had a negative impact on the vagina with thinning of vaginal muscularis and decreased vaginal smooth muscle contractility. The negative effect was attenuated when a bioscaffold derived from urinary bladder extracellular matrix was used as a composite with Gynemesh PS. The objective of the study was to further elucidate the impact of Gynemesh PS polypropylene mesh and MatriStem extracellular matrix bioscaffolds on the vaginal smooth muscle in terms of micromorphology of vaginal smooth muscle (muscle bundles and individual myocytes), innervation, and nerve-mediated contractile function following their implantations in a rhesus macaque model via sacrocolpopexy. Thirty-two middle-aged rhesus macaques were randomized to undergo either a sham surgery (sham, n= 8), or the implantation of Gynemesh PS alone (n= 8) vs composite mesh comprised of Gynemesh PS plus 2-ply MatriStem (n= 8) vs 6-ply MatriStem alone (n= 8) via sacrocolpopexy. The graft-vagina complexes were harvested 3 months later. Histomorphometrics of smooth muscle bundles and myocytes were performed by immunofluorescent labeling of alpha smooth muscle actin, caveolin-3 (membrane protein), and cell nuclei followed by confocal imaging. The cross-sectional diameters of smooth muscle bundles and individual myocytes were quantified using images randomly taken in at least 5 areas of each section of sample. Contractile proteins alpha smooth muscle actin and smoothelin were quantified by Western immunoblotting. Nerve density was measured by immunohistochemical labeling of a pan-neuron marker, PGP9.5. Nerve-mediated smooth muscle contractility was quantified using electrical field stimulation. One-way analysis of variance and appropriate post hoc tests were used for statistical comparisons. Compared with sham, the implantation of Gynemesh PS alone resulted in a disorganized smooth muscle morphology with the number of small muscle bundles (cross-sectional diameter less than 20 μm) increased 67% (P= .004) and the myocyte diameter decreased 22% (P < .001). Levels of contractile proteins were all decreased vs sham with alpha smooth muscle actin decreased by 68% (P= .009), low-molecular-weight smoothelin by 51% (P= .014), and high-molecular-weight smoothelin by 40% (P= .015). Nerve density was decreased by 48% (P= .03 vs sham) paralleled by a 63% decrease of nerve-mediated contractility (P= .02). Following the implantation of composite mesh, the results of measurements were similar to sham (all P > .05), with a 39% increase in the myocyte diameter (P < .001) and a 2-fold increase in the level of alpha smooth muscle actin relative to Gynemesh (P= .045). Following the implantation of MatriStem alone, the number of small muscle bundles were increased 54% vs sham (P= .002), while the other parameters were not significantly different from sham (all P > .05). The implantation of Gynemesh PS had a negative impact on the structural and functional integrity of vaginal smooth muscle evidenced by atrophic macro- and microscopic muscle morphology, decreased innervation, and impaired contractile property, consistent with a maladaptive remodeling response. The extracellular matrix bioscaffold (MatriStem), when used with Gynemesh PS as a composite (2 ply), attenuated the negative impact of Gynemesh PS; when used alone (6 ply), it induced adaptive remodeling as evidenced by an increased fraction of small smooth muscle bundles with normal contractility.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting indisruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential forglaucoma, the leading cause of irreversible blindness in humans.

3,3'-thiodipropanol as a versatile refractive index-matching mounting medium for fluorescence microscopy.

High resolution fluorescence microscopy requires optimization of the protocols for biological sample preparation. The optical and chemical characteristics of mounting media are among the things that could be modified to achieve optimal image formation. In our search for chemical substances that could perform as mounting media, 3,3'-thiodipropanol (TDP) emerged as a sulfide with potentially interesting characteristics. In this work, several tests of its performance as a mounting medium for fluorescence microscopy of biological samples were performed, including the labeling of filamentous actin with fluorescent phalloidins. The refractive index dispersion curve of pH-adjusted TDP was experimentally obtained in the visible range and compared to the dispersion curves of commercial and lab-made mounting media. The effects on the fluorescence of commonly used dyes were tested by using TDP as a solvent and measuring the relative fluorescence quantum yield of the dyes. By being able to mix TDP in any concentration with water and 2,2'-thiodiethanol (TDE), it was possible not only to fine-tune the refractive index of the resulting solution, but also to preserve the compatibility of TDP with the most popular and efficient fluorescent actin staining used in biological microscopy.

Chinese medicine CGA formula ameliorates liver fibrosis induced by carbon tetrachloride involving inhibition of hepatic apoptosis in rats

Ethnopharmacological revelvanceCGA consisting of Cordyceps sinensis mycelia polysaccharide, gypenosides and amygdalin, was demonstrated to be the effective components formula in Fuzheng Huayu (FZHY) capsule, a traditional Chinese medicine approved by China food and drug administration for treatment of liver fibrosis and to inhibit transforming growth factor-β1 (TGF-β1) signaling, previously. Aim of the studyTo evaluate the effects of CGA on hepatic apoptosis in liver fibrosis induced by carbon tetrachloride (CCl4). Materials and methodsThe hepatic injury and histology was detected by serum biomarker assay and hematoxylin-eosin staining. The hepatic collagen was illustrated by Sirius red staining and hydroxyproline (Hyp) concentration. The hepatic stellate cells (HSCs) activation and hepatic apoptosis was visualized by immunohistochemical analysis of α-smooth muscle actin (α-SMA) and terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL) assay respectively. The protein expression of collagen type I (Col-I), α-SMA, TGF-β1, Fas, tumor necrosis factor receptor 1 (TNF-R1), cleaved-caspase-8, cleaved-caspase-10, cleaved-caspase-9, cleaved-caspase-3, mitochondrial Bcl-2, Bcl-2 associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), cytochrome C and cytoplasmic cytochrome C was detected by western-blot. ResultsCGA or FZHY ameliorated liver histological changes, decreasing serum alanine aminotransferase, aspartate aminotransferase, hepatic Hyp, TUNEL positive-stained area, and down-regulated the protein expression of α-SMA, TGF-β1, Col-I, Fas, TNF-R1, cleaved-caspase-8, cleaved-caspase-10, cleaved-caspase-9, and cleaved-caspase-3, mitochondrial Bax, Bak, and cytoplasmic cytochrome C, while restored the expression of mitochondrial Bcl-2 and cytochrome C. ConclusionCGA formula ameliorates liver fibrosis induced by CCl4, which is correlated to its inhibition on hepatic apoptosis.

Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts (RAGE)

The interactions between the cytoplasmic protein diaphanous-1 (Diaph1) and the receptor for advanced glycation endproducts (RAGE) drive the negative consequences of RAGE signaling in several disease processes. Reported in this work is how Diaph1 affects the nanoscale clustering and diffusion of RAGE measured using super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). Altering the Diaph1 binding site has a different impact on RAGE diffusion compared to when Diaph1 expression is reduced in HEK293 cells. In cells with reduced Diaph1 expression (RAGE-Diaph1−/−), the average RAGE diffusion coefficient is increased by 35%. RAGE diffusion is known to be influenced by the dynamics of the actin cytoskeleton. Actin labeling shows that a reduced Diaph1 expression leads to cells with reduced filopodia density and length. In contrast, when two RAGE amino acids that interact with Diaph1 are mutated (RAGERQ/AA), the average RAGE diffusion coefficient is decreased by 16%. Since RAGE diffusion is slowed when the interaction between Diaph1 and RAGE is disrupted, the interaction of the two proteins results in faster RAGE diffusion. In both RAGERQ/AA and RAGE-Diaph1−/− cells the number and size of RAGE clusters are decreased compared to cells expressing RAGE and native concentrations of Diaph1. This work shows that Diaph1 has a role in affecting RAGE clusters and diffusion.

Developmental Morphology of the Palatine Glands in Rats: An Electron Microscope Study.

Although minor salivary glands play a significant functional role in the oral cavity, their developmental morphology and cell differentiation has been scarcely studied. This study aimed to describe the development of rat palatine glands with regard to the ultrastructural morphology of the secretory cells and surrounding myoepithelial cells (MECs). Palatine glands from rats at embryonic ages (E) 18 and 20 days, and postnatal days (PN) 0, 3, 7, 10, 13, 21, 30, 42, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling of alpha-smooth muscle actin (α-SMA). At E18, epithelial cords were observed extending from the palatal epithelium and showed negative reactivity to α-SMA. After luminization at E20, the cells of immature acini accumulated secretory granules of various densities: electron-dense, electron-lucent and some empty-appearing granules. MECs were poorly differentiated at E20 and exhibited only slight α-SMA expression. At birth, mucous and serous cells were typically located around a common lumen. Thereafter, serous cells began to move to the periphery to form demilunes by PN7. The mucous secretory granules of intermediate electron density became predominant around PN13. At PN21, these granules were dramatically reduced in number and most of the acini in adults contained acinar cells with numerous electron-lucent granules, and a few serous demilune cells with electron-dense granules. After birth, MECs progressively accumulated actin microfilaments until prominent α-SMA expressing MECs invested the acini and the proximal part of the intercalated ducts in the adult. Anat Rec, 301:1820-1833, 2018. © 2018 Wiley Periodicals, Inc.

Malignant Cutaneous Peripheral Nerve Sheath Tumour with Rhabdomyosarcomatous Differentiation (Triton Tumour) in a Domestic Cat

Divergent differentiation is encountered frequently within human malignant peripheral nerve sheath tumours (MPNSTs). The new component is often a rhabdomyosarcoma, but in animals this specific form of divergent differentiation within MPNSTs has only been reported once (in a dog). Incisional wedge biopsy of a locally extensive, ventral abdominal wall mass, which extended from the dermis to the subcutis, from a 12-year-old female domestic shorthaired cat, was performed. The tissue was examined with routine haematoxylin and eosin staining and immunohistochemical methods. A malignant neoplasm with spindle and polygonal cell components and progression towards a rhabdomyosarcomatous phenotype was observed. Both neoplastic cell populations exhibited strong expression of vimentin and there was multifocal expression of S100 and desmin. There was strong cytoplasmic labelling for α-sarcomeric actin and muscle actinand weak labelling for myoglobin within the cells positive for desmin. There was multifocal positive nuclear labelling for myogenin. Glial fibrillary acidic protein, α-smooth muscle actin, microphthalmia-associated transcription factor and melanoma antigen recognized by T cells were not expressed. Microscopical features, aided by immunohistochemistry, identified a MPNST with progression towards a rhabdomyosarcomatous phenotype, a so-called 'triton tumour'. A Schwann cell component could account for the divergent patterns of growth, given the plasticity of the neural crest. Nerve sheath tumours have been reported in the skin and subcutis of cats and are a differential diagnosis of feline cutaneous spindle cell neoplasms.

Implant abutment microgrooves affect soft tissue cells response via connexin 43 pathway

Background The down-regulation of Connexin 43 (CX43) hemichannels (HCs) function in soft tissue cells promotes the up-regulation of numerous pro-healing genes, as the down-regulation of pro-fibrotic ones, leading to its identification as a potential target for the modulation of wound healing. On the other hand, titanium is commonly used as a transmucosal material, both for dental implants and abutments, and its surface micro-topography is known to affect cell responses. Aim/Hypothesis To investigate whether threaded titanium surfaces with different groove characteristics modulate CX43 expression and its organization in HCs rather than in gap junction (GJs) in an immortalized mouse myoblast cell line. In addition, to verify if the growth pattern and arrangement of cells on the grooves. Material and Methods Murine myoblasts C2C12 were seeded on discs with grooved titanium surfaces with different patterns- UTM (Ultrathin Threaded Microsurface) and XA microgrooved surface. A machined surface was used as control (MA). Titanium surface specimens were kindly provided by Sweden & Martina (Padova, Italy). Cells were cultured up to 7 days and daily monitored for viability through chemiluminescence (cell proliferation). Gene expression analysis for CX43, alkaline phosphatase (ALP) and collagen 1 (COL1a) was performed+ after 3 days of culture total RNA was purified, retro-transcribed to cDNA and amplified through quantitative RT-PCR. To localize the expression of CX43 and thus to identify its organization as HCs or as GJs, cells were observed under fluorescence after actin, CX43 and nuclei labeling. Differences among the groups were evaluated through Two-Way ANOVA statistical test. Results Very weak signal for CX43 was observed on UTM surface (mRNA 6.12-fold less than XA and 2.58-fold less than MA). CX43 expression was extremely up-regulated on XA surface after 3 days, when compared to UTM and MA surface (P < 0.0001). Conversely, cell proliferation was significantly reduced (P < 0.0001). On the XA surface, CX43 expression was labelled in the peri-nuclear area, indicating its organization in HCs. MA showed a slightly enhanced level of CX43 (2.37-fold less than XA) and, where cells were confluent, CX43 signal was marked around cell nuclei (HCs) and at the edges of cell profile, indicating GJs with other cells. Noteworthy, the disposition of cells on XA and UTM surfaces followed the grid of the grooved surface topography, with cells laying on the grid hollows and elongated to colonize the surface. Furthermore, gene expression analysis revealed that MA control surface induced a commitment of C2C12 towards an osteoblastic phenotype, showing greater levels of ALP and COL1a. Conclusions and Clinical Implications The microgrooved titanium surfaces showed a modulatory role on myoblasts, promoting an evident cell arrangement along the surface grooves on UTM and XA surfaces. This may lead in vivo to the deposition of an oriented extracellular matrix. Additionally, UTM surface caused CX43 down-regulation, leading to the hypothesis of a more favorable in vivo soft tissues wound healing.

Immunohistochemical characteristics of renomedullary interstitial cell tumor: a study of 41 tumors with emphasis on differential diagnosis of mesenchymal neoplasms

Renomedullary interstitial cell tumors (RMICTs) are almost always incidentally identified either at autopsy or upon resection of the kidney for other reasons. However, rare cases that are large, resulting in a clinical mass, have been reported. The immunohistochemical phenotype of usual, incidental RMICT using modern soft tissue tumor markers is largely unknown, however, providing little information to aid in classification of larger or atypical tumors. We retrieved 41 RMICTs from 36 patients and studied pathologic characteristics including morphology, immunohistochemistry (S100, keratin AE1/AE3, smooth muscle actin, desmin, estrogen and progesterone receptors, calponin, CD34, CD35), and histochemical staining. Data collected included age, sex, tumor size, laterality, and indication for kidney examination. RMICTs (n = 41) were identified in 23 men and 13 women, with a mean age of 57 years (range, 24-83 years); tumor sizes ranged from less than 1 to 13 mm (median, 4 mm). Kidneys were resected for 32 tumors, 1 chronic pyelonephritis, 1 trauma, and 2 autopsies. All (41; 100%) had entrapped renal tubules, 5 (12%) of which included cystic or dilated tubules. Most (35; 85%) had collagenous fibers, all of which were negative for Congo red. RMICT demonstrates a largely negative immunohistochemical phenotype with weak-to-moderate labeling for smooth muscle actin and calponin that is substantially less than myofibroblastic lesions. Positive staining for estrogen and progesterone receptors is common (61%), which could overlap with mixed epithelial and stromal tumor and other entities; however, staining is typically weak. CD34 is usually negative, with occasional weak labeling, in contrast to solitary fibrous tumor.

The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments

Cofilin/ADF proteins are actin-remodeling proteins, essential for actin disassembly in various cellular processes, including cell division, intracellular transport, and motility. Cofilins bind actin filaments cooperatively and sever them preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments. The cooperative binding to actin has been proposed to originate from conformational changes that propagate allosterically from clusters of bound cofilin to bare actin segments. Estimates of the lengths over which these cooperative conformational changes propagate vary dramatically, ranging from 2 to >100 subunits. Here, we present a general, structure-based method for detecting from cryo-EM micrographs small variations in filament geometry (i.e. twist) with single-subunit precision. How these variations correlate with regulatory protein occupancy reveals how far allosteric, conformational changes propagate along filaments. We used this method to determine the effects of cofilin on the actin filament twist. Our results indicate that cofilin-induced changes in filament twist propagate only 1-2 subunits from the boundary into the bare actin segment, independently of the boundary polarity (i.e. irrespective of whether or not the bare actin segment flanks the pointed or barbed-end side of the boundary) and the pyrene fluorophore labeling of actin. These observations indicate that the filament twist changes abruptly at boundaries between bare and cofilin-decorated segments, thereby constraining mechanistic models of cooperative actin filament interactions and severing by cofilin. The methods presented here extend the capability of cryo-EM to analyze biologically relevant deviations from helical symmetry in actin as well as other classes of linear polymers.

Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication

Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1β and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca2+ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca2+ responses were assessed through the Ca2+ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca2+ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca2+ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.

Deep tissue analysis of distal aqueous drainage structures and contractile features

Outflow resistance in the aqueous drainage tract distal to trabecular meshwork is potentially an important determinant of intraocular pressure and success of trabecular bypass glaucoma surgeries. It is unclear how distal resistance is modulated. We sought to establish: (a) multimodal 2-photon deep tissue imaging and 3-dimensional analysis of the distal aqueous drainage tract (DT) in transgenic mice in vivo and ex vivo; (b) criteria for distinguishing the DT from blood and lymphatic vessels; and (c) presence of a DT wall organization capable of contractility. DT lumen appeared as scleral collagen second harmonic generation signal voids that could be traced back to Schlemm’s canal. DT endothelium was Prox1-positive, CD31-positive and LYVE-1-negative, bearing a different molecular signature from blood and true lymphatic vessels. DT walls showed prominent filamentous actin (F-actin) labeling reflecting cells in a contracted state. F-actin co-localized with mesenchymal smooth muscle epitopes of alpha-smooth muscle actin, caldesmon and calponin, which localized adjacent and external to the endothelium. Our findings support a DT wall organization resembling that of blood vessels. This reflects a capacity to contract and support dynamic alteration of DT caliber and resistance analogous to the role of blood vessel tone in regulating blood flow.

A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death.

To evaluate the cytotoxicity and the mechanism of cell aggression of peracetic acid (PA) in comparison with sodium hypochlorite (NaOCl). L929 fibroblasts were exposed to 1% PA and 2.5% NaOCl, at several dilutions for 10min. The following parameters were evaluated: cell metabolism by methylthiazol tetrazolium assay, external morphology by scanning electron microscopy, ultrastructure by transmission electron microscopy, the cytoskeleton by means of actin and α-tubulin labelling, and the type of cell death by flow cytometry (apoptosis/necrosis). The data were analysed by two-way anova and the Bonferroni post-test (α=0.05). The PA group had lower cell viability and a higher percentage of necrotic cells than the NaOCl group (P<0.05). Both solutions diminished cell metabolism, led to destructuring of the cytoskeleton, created changes in the external morphology, resulted in the accumulation of proteins in the rough endoplasmic reticulum and induced cell death predominantly by necrosis. However, these changes were observed in lower doses of PA when compared with NaOCl. Although they had the same mechanism of cytotoxicity, 1% PA had greater cytotoxic potential than 2.5% NaOCl.