Zeiss LSM Research Articles

133 Sensory Denervation Decreases PanIN Progression in a Mouse Model of Pancreatic Cancer

BACKGROUND: Reciprocal molecular signaling between pancreatic ductal adenocarcinoma (PDAC) and nerves may promote perineural invasion (PNI) and tumor growth. The identity and function of sensory neuropeptides in the tumor microenvironment are unknown. We hypothesized that sensory neurons play an important role in tumor progression and that substance P (SP) is a candidate neuropeptide for mediating this effect. AIMS:1) to characterize SP receptor (NK1R) expression in PanIN epithelium 2) to determine NK1R expression in human PDAC cell lines 3) to study effects of sensory denervation on pancreatic intraepithelial neoplasia (PanIN) initiation and progression in the KPC ( Pdx1-Cre; LSL-Kras; LSLTrp53) mouse model. METHODS: KPC mice were injected with the sensory neurotoxin resiniferatoxin (RTX; Sigma) or control solution on postnatal day 7. Pancreata from 8and 12-week-old mice were fixed and subject to HE Santa Cruz). Images were taken with a Nikon Camera or Zeiss LSM 510 Meta microscope. PanINs were graded as early (grade 1) or advanced (grades 2 and 3). PanIN burden was calculated by the percentage of total surface area occupied in 5 random views per HE p = 0.02). All analyzed human PDAC cell lines expressed the NK1R. In vivo studies revealed axons in close proximity to PanIN epithelium (Fig.1). RTX-treated mice had a 50% decrease in pancreatic sensory axonal density when compared to control mice at 8 and 12 weeks (p < 0.05). RTX-treated mice had a significant reduction in advanced PanINs compared to control mice at both 8 weeks (0.05% versus 0.9%; p < 0.05) and 12 weeks (0.3% versus 8%; p < 0.05). CONCLUSIONS: The NK1R is expressed in distinct cells within the PanIN epithelium and the percentage of NK1R+ cells increases with PanIN grade. Human PDAC cell lines express the NK1R. Sensory denervation is associated with a significant reduction in progression from early to advanced PanIN in the KPC model. Sensory nerves are likely an important part of the PanIN microenvironment and may affect tumorigenesis via the NK1R. This study provides new insight into the pathogenesis and potential therapeutic targets in PDAC.

MP1-07 ENRICHMENT OF CIRCULATING TUMOR CELLS IN PATIENTS WITH LOCALIZED PROSTATE CANCER USING A MICROFLUIDIC DEVICE

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2015MP1-07 ENRICHMENT OF CIRCULATING TUMOR CELLS IN PATIENTS WITH LOCALIZED PROSTATE CANCER USING A MICROFLUIDIC DEVICE Tilman Todenhöfer, Emily Park, Hamid Abdi, Alex Li, Richard Ross, Xiaoyan Deng, Chao Jin, Simon Duffy, Martin Gleave, Hongshen Ma, and Peter Black Tilman TodenhöferTilman Todenhöfer More articles by this author , Emily ParkEmily Park More articles by this author , Hamid AbdiHamid Abdi More articles by this author , Alex LiAlex Li More articles by this author , Richard RossRichard Ross More articles by this author , Xiaoyan DengXiaoyan Deng More articles by this author , Chao JinChao Jin More articles by this author , Simon DuffySimon Duffy More articles by this author , Martin GleaveMartin Gleave More articles by this author , Hongshen MaHongshen Ma More articles by this author , and Peter BlackPeter Black More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.170AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Circulating tumor cells have proven prognostic value in patients with advanced prostate cancer (PC) but have been identified only infrequently in patients with localized PC using current standard techniques which are dependent on cell surface expression of epithelial cell adhesion molecule (EpCAM). However, evidence suggests that a proportion of CTCs likely do not express EpCAM. We developed a microfluidic system enabling antigen-independent enrichment of circulating tumor cells based on cell size and deformability. The aim of the present study was to evaluate presence of CTCs detected by this platform in patients with clinically localized PC. METHODS We included 58 consecutive patients undergoing radical prostatectomy and pelvic lymphadenectomy for localized PC. As control, we included 7 healthy men. Peripheral blood was collected preoperatively and processed by a microfluidic device for CTC enrichment. This device uses the deformation of single cells through layers of funnel shaped constrictions with openings smaller than the cell diameter in order to selectively transport cells based on size and deformability. Effluent cells were stained for pancytokeratin, CD45 and the androgen receptor (AR), and were identified by spectral analysis on the Zeiss LSM 780 confocal microscope. Cells positive for CK and negative for CD45 were considered to be CTCs. CTC counts were correlated to clinicopathologic parameters. RESULTS One or more CTCs were detected in 0/7 healthy controls and 35/58 (60.3%) patients with PC. The proportions of patients with ≥5 and ≥20 CTCs per ml were 41.4% and 24.1%, and the maximum CTC count was 527 CTCs/ml. We observed no correlation of CTC counts with tumor stage, nodal stage, Gleason score and serum PSA. CTCs were positive for AR-expression. CONCLUSIONS Circulating tumor cells can be detected in a considerable proportion of patients with localized prostate cancer using an EpCAM-independent system. CTC counts in our study were clearly higher than in other cohorts using EpCAM-dependent detection. The expression of AR in cytokeratin positive CTCs confirms their prostatic origin. The prognostic value of CTCs in peripheral blood before and after prostatectomy remains to be defined. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e3 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tilman Todenhöfer More articles by this author Emily Park More articles by this author Hamid Abdi More articles by this author Alex Li More articles by this author Richard Ross More articles by this author Xiaoyan Deng More articles by this author Chao Jin More articles by this author Simon Duffy More articles by this author Martin Gleave More articles by this author Hongshen Ma More articles by this author Peter Black More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Dyssynchronous Heart Failure is Associated with Spatially Heterogeneous Spark Density in Left Ventricular Cardiomyocytes

Dyssynchronous heart failure (DHF) is associated with structural and functional remodeling in cardiomyocytes from subcellular to whole organ level. Several studies have demonstrated remodeling of Ca2+ signaling and excitation-contraction coupling. This study aimed at quantification of the subcellular spatial distribution of diastolic local Ca2+ release events (sparks) in cardiomyocytes and their remodeling in DHF.Adult canines were used as control and DHF models. DHF was induced by right ventricular tachypacing at 200 beats per minute. Left ventricular cardiomyocytes were isolated via enzymatic digestion, loaded with Fluo-4 and Di-8-ANEPPS, and imaged with rapid scanning confocal microscopy (Zeiss LSM 5 Duo). Acquisition of 2D image sequences was initiated 460 ms after stimulation of the myocytes. Images were acquired at a xy resolution of 0.1 µm with a field of view of 102.4 µm (x) x 25.6 µm (y). Image sequences comprised 100 frames at 9.3 ms per frame. Image analysis involved cell segmentation and spark detection. We compared spark density in regions 0-10 µm and 10-40 µm from the longitudinal cell end.Our analyses yielded a similar diastolic spark density in control and DHF cells (0.0119 ± 0.0023 vs 0.0155 ± 0.0027 sparks/µm2/s, respectively). In control cells spark density was homogeneously distributed. In contrast, DHF cells exhibited a decreased spark density within 0-10 µm versus 10-40 µm from the longitudinal cell end (0.0101 ± 0.0039 vs 0.0227 ± 0.0047 sparks/µm2/s, respectively).DHF is associated with a spatially heterogeneous remodeling of spark density. This finding provides a foundation for understanding basic mechanisms of arrhythmogenesis in heart failure cells. The heterogeneity of spark density may result from heterogeneous structural remodeling, for instance, remodeling of ryanodine receptor clusters and the transverse tubular system.

77 EFFECT OF HEAT SHOCK DURING IN VITRO MATURATION ON HETEROCHROMATIN COMPACTION IN BOVINE EMBRYOS AT 4- AND 8-CELL STAGES: PRELIMINARY STUDY

High temperatures cause several reproductive losses in cattle. Under in vitro conditions, heat shock decreases oocyte developmental competence and influences embryonic gene expression (Gendelman and Roth 2012 Anim. Reprod. Sci. 134, 125–134). This preliminary study aimed to evaluate whether heat shock during oocyte in vitro maturation (IVM) could have any further effect on chromatin remodelling of fertilized embryos at 4- and 8-cell stages, once such modifications are required for the gene activation in bovine embryos. We evaluated the distribution of heterochromatin 1 (HP1β) and of histone H3 trimethylated at lysine 9 (H3K9me3), both reportedly correlated with heterochromatin formation, in 4- and 8-cell stage embryos derived from control (C) and heat-shocked (HS) bovine oocytes. Immature cumulus-oocyte complexes (COC) collected from crossbred cows in Brazil were exposed for 12 h to 38.8°C (C group) or 41.0°C (HS group) followed by 12 h at 38.8°C, totalizing 24 h of IVM at 5% CO2 in air. Oocytes were in vitro fertilized (IVF) with non-sexed sperm and denuded zygotes were in vitro cultured in CR2aa medium at 38.8°C and 5% CO2, 5% O2 and 90% N2. Four- and 8-cell embryos at 44 h post-IVF were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β and anti-rabbit H3K9me3 first antibodies. Immunofluorescence was evaluated by confocal microscopy (Zeiss LSM 700, MIMA platform, INRA) and 3D images processed by ZEN Lite software (Zeiss, Jena, Germany). Three different distribution patterns of fluorescence were identified based on morphological criteria: diffuse, little clusters, and big clusters. Proportions of embryos in every distribution pattern were compared between C and HS groups by Chi-squared test. No difference (P &gt; 0.05) on cleavage rate was found between C and HS groups until 44 h post-fertilization. Embryos at the 4-cell stage from HS group displayed an increased (P &lt; 0.01) proportion of nuclei with H3K9m3 big clusters (44%, n = 7/16 embryos), whereas embryos from C group displayed only few nuclei with this pattern (5%, n = 1/18). At the 8-cell stage, distribution of H3K9m3 was similar (P &gt; 0.05) between C and HS groups. For HP1β, embryos at the 4-cell stage from HS group displayed an increased (P &lt; 0.05) proportion of nuclei with little clusters (81%, n = 13/16 embryos), whereas embryos from C group had low proportion of nuclei with this same pattern (40%, n = 7/18). Mostly 4-cell stage embryos from C group presented the diffuse pattern (61%, n = 11/18 v.18%, n = 3/16 in the HS group; P &lt; 0.05). At the 8-cell stage, some embryos from the C group (31%, n = 5/16) still showed nuclei with diffuse distribution of HP1β, whereas no nucleus with this pattern was found for the HS group. These preliminary data suggest that bovine embryos derived from heat-shocked oocytes can display precocious heterochromatin compaction, represented by the accumulation of H3K9me3 and HP1β at the 4-cell stage, compared with embryos derived from non-heat-shocked oocytes, which may affect embryonic genome activation with consequences for further gene expression.Research was supported by CNPq, FAPEMIG, FAPES and Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73).

228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P &gt; 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P &gt; 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P &lt; 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P &lt; 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

Identification of Surface Topography Scanned by Laser Scanning Confocal Microscope

Topography and surface roughness is defined as a set of surface textures formed due to used machining or other production technology. It is a key factor mainly for dynamically stressed components. Greater surface roughness negatively affects the fatigue strength of component or its wear resistance.The paper deals with the visualization and processing of data obtained from a laser confocal microscope Zeiss LSM 700. Data acquired by LSM software ZEN2009 are exported in the text form of 3D coordinates of the image data points and are arranged as a set of 2D profiles with resolution 1.10-7 m. The aim is to describe an alternative method of evaluating raw data extracted from z-stack of acquired TIFF images in order to achieve higher precision and identify the topography of the scanned sample surface. Different methods of data processing have been tested to eliminate deficiencies located mainly in the parts of the scanned surface with low intensity.

Orbital single particle tracking on a commercial confocal microscope using piezoelectric stage feedback

Single Particle Tracking (SPT) is a technique used to locate fluorescent particles with nanometer precision. In the orbital tracking method the position of a particle is obtained analyzing the distribution of intensity along a circular orbit scanned around the particle. In combination with an active feedback this method allows tracking of particles in 2D and 3D with millisecond temporal resolution. Here we describe a SPT setup based on a feedback approach implemented with minimal modification of a commercially available confocal laser scanning microscope, the Zeiss LSM 510, in combination with an external piezoelectric stage scanner. The commercial microscope offers the advantage of a user-friendly software interface and pre-calibrated hardware components. The use of an external piezo-scanner allows the addition of feedback into the system but also represents a limitation in terms of its mechanical response. We describe in detail this implementation of the orbital tracking method and discuss advantages and limitations. As an example of application to live cell experiments we perform the 3D tracking of acidic vesicles in live polarized epithelial cells.

Galectin-3 Regulates Desmoglein-2 and Intestinal Epithelial Intercellular Adhesion

The desmosomal cadherins, desmogleins, and desmocollins mediate strong intercellular adhesion. Human intestinal epithelial cells express the desmoglein-2 isoform. A proteomic screen for Dsg2-associated proteins in intestinal epithelial cells identified a lectin referred to as galectin-3 (Gal3). Gal3 bound to N-linked β-galactosides in Dsg2 extracellular domain and co-sedimented with caveolin-1 in lipid rafts. Down-regulation of Gal3 protein or incubation with lactose, a galactose-containing disaccharide that competitively inhibits galectin binding to Dsg2, decreased intercellular adhesion in intestinal epithelial cells. In the absence of functional Gal3, Dsg2 protein was internalized from the plasma membrane and degraded in the proteasome. These results report a novel role of Gal3 in stabilizing a desmosomal cadherin and intercellular adhesion in intestinal epithelial cells.

Evaluation of Barrett Esophagus by Multiphoton Microscopy

Multiphoton microscopy (MPM) based on 2-photon excitation fluorescence and second-harmonic generation allows simultaneous visualization of cellular details and extracellular matrix components of fresh, unfixed, and unstained tissue. Portable multiphoton microscopes, which could be placed in endoscopy suites, and multiphoton endomicroscopes are in development, but their clinical utility is unknown. To examine fresh, unfixed endoscopic biopsies obtained from the distal esophagus and gastroesophageal junction to (1) define the MPM characteristics of normal esophageal squamous mucosa and gastric columnar mucosa, and (2) evaluate whether diagnosis of intestinal metaplasia/Barrett esophagus (BE) could be made reliably with MPM. The study examined 35 untreated, fresh biopsy specimens from 25 patients who underwent routine upper endoscopy. A Zeiss LSM 710 Duo microscope (Carl Zeiss, Thornwood, New York) coupled to a Spectra-Physics (Mountain View, California) Tsunami Ti:sapphire laser was used to obtain a MPM image within 4 hours of fresh specimen collection. After obtaining MPM images, the biopsy specimens were placed in 10% buffered formalin and submitted for routine histopathologic examination. Then, the MPM images were compared with the findings in the hematoxylin-eosin-stained, formalin-fixed, paraffin-embedded sections. The MPM characteristics of the squamous, gastric-type columnar and intestinal-type columnar epithelium were analyzed. In biopsies with discrepancy between MPM imaging and hematoxylin-eosin-stained sections, the entire tissue block was serially sectioned and reevaluated. A diagnosis of BE was made when endoscopic and histologic criteria were satisfied. Based on effective 2-photon excitation fluorescence of cellular reduced pyridine nucleotides and flavin adenine dinucleotide and lack of 2-photon excitation fluorescence of mucin and cellular nuclei, MPM could readily identify and distinguish among squamous epithelial cells, goblet cells, gastric foveolar-type mucous cells, and parietal cells in the area of gastroesophageal junction. Based on the cell types identified, the mucosa was defined as squamous, columnar gastric type (cardia/fundic-type), and metaplastic columnar intestinal-type/BE. Various types of mucosa seen in the study of 35 biopsies included normal squamous mucosa only (n = 14; 40%), gastric cardia-type mucosa only (n = 2; 6%), gastric fundic mucosa (n = 6; 17%), and both squamous and gastric mucosa (n = 13; 37%). Intestinal metaplasia was identified by the presence of goblet cells in 10 of 25 cases (40%) leading to a diagnosis of BE on MPM imaging and only in 7 cases (28%) by histopathology. In 3 of 35 biopsies (9%), clear-cut goblet cells were seen by MPM imaging but not by histopathology, even after the entire tissue block was sectioned. Based on effective 2-photon excitation fluorescence of elastin and second-harmonic generation of collagen, connective tissue in the lamina propria and the basement membrane was also visualized with MPM. Multiphoton microscopy has the ability to accurately distinguish squamous epithelium and different cellular elements of the columnar mucosa obtained from biopsies around the gastroesophageal junction, including goblet cells that are important for the diagnosis of BE. Thus, use of MPM in the endoscopy suite might provide immediate microscopic images during endoscopy, improving screening and surveillance of patients with BE.

Orbital Tracking of Single Fluorescent Particles on a Commercial Confocal Microscope

Single Particle Tracking (SPT) is a super-resolution technique used to determine the position of fluorescent particles with nanometer precision. The localization is generally obtained by analyzing the spatial distribution of fluorescence intensity emitted by the particle. In fact, the center of the distribution can be determined with an uncertainty which is much lower than the size of the distribution itself. In the orbital tracking method the position of a particle is obtained analyzing the distribution of intensity along a circular orbit scanned around the particle. In combination with an active feedback this method allows tracking of particles in 2D and 3D with millisecond temporal resolution[1]. More recently, the use of orbital tracking to perform imaging has also been proposed[2].The orbital tracking and the other 3D SPT feedback methods are generally implemented on homebuilt microscopes which are not yet commercially available. On the other hand, commercial setups offer the advantage of a user-friendly software interface and pre-calibrated hardware components. It would be of interest to implement a SPT setup based on a feedback approach with minimal modification of a commercially available microscope. Here we explore this idea using a widely used confocal laser scanning microscope, the Zeiss LSM 510, in combination with an external piezoelectric stage scanner. We discuss advantages and limitations of this implementation of the orbital tracking method and the potential application to live cell experiments.

181 CYTOSKELETAL AND CHROMOSOMAL ORGANIZATION IN DEVELOPMENTALLY ARRESTED EQUINE ZYGOTES AFTER INTRACYTOPLASMIC SPERM INJECTION

Intracytoplasmic sperm injection (ICSI) has been developed as a clinical procedure in the equine industry. Not all sperm-injected oocytes develop into embryos; however, the causes of developmental failure after equine ICSI have not been studied. Our objective was to use confocal microscopy to evaluate sperm-injected equine oocytes from young and old mares that did not cleave in a clinical ICSI program, to study the hypothesis that cleavage failure is associated with cytoskeletal and chromosomal alterations and affected by mare age. Oocytes were collected from the dominant follicles of young (Y; 5–15 years old) and old (O; 20–24 years old) oestrous mares approximately 24 h after administration of deslorelin and hCG. At approximately 44 h after deslorelin and hCG administration, oocytes were injected using frozen-thawed sperm from various stallions. Injected oocytes (n = 15) that failed to cleave by 24 to 48 h were fixed in microtubule stabilization buffer extraction fixative (MTSB-XF) and stained for DNA, α/β tubulin, acetylated α-tubulin, and f-actin. Confocal z-stacks were obtained using a Zeiss LSM 5 microscope (Carl Zeiss Microscopy LLC, Thornwood, NY), and images were assessed for cytoskeletal structures and chromosome organisation using the Zeiss LSM image browser. Potential zygotes (Y: n = 6 and O: n = 9) were categorized for (1) 2 sets of chromosomes, (2) misaligned chromosomes, (3) oversized spindles (approximately twice the size of an MII spindle), and (4) actin bubbling. The number of zygotes in each category for Y and O mares were compared by Fisher's exact test. Some samples were included in more than 1 morphological category. Two separate sets of chromosomes were observed in 1 of 6 and 4 of 9 injected oocytes from Y and O mares, respectively (P = 0.58), with development stopping before fusion of male and female pronuclei. Misaligned chromosomes were present in 2 of 6 and 7 of 9 oocytes from Y and O mares, respectively (P = 0.14). Oversized spindles were observed in more Y than O potential zygotes (5 of 6 and 2 of 9, respectively, P = 0.04), representing fusion of male and female pronuclei before developmental arrest. Multiasters were associated with the oversized spindles in potential zygotes from Y (2 of 6) and O (2 of 9) mares (P = 1), with microtubule activity potentially associated with the first mitotic division. Structures associated with actin bubbling tended (P = 0.12) to be higher in uncleaved zygotes from Y than O mares (5 of 6 and 3 of 9, respectively). Of the 15 injected oocytes, 10 arrested after fusion of male and female pronuclei. We have previously observed a high incidence of chromosomal misalignment in metaphase II oocytes from old mares. In the small number of potential zygotes that were studied, chromosomal misalignment tended (P = 0.14) to be higher for old mares. Other observations, such as oversized spindles and actin bubbling, appeared to be more prevalent in the potential zygotes of Y than O mares; these observations could be associated with normal development or post-ovulatory aging of the oocyte. Additional work will focus on the roles of actin and microtubules in remodelling the cytoplasm and organizing the chromosomes after fertilization in the horse and on the role of maternal aging in fertilization failure.

Bone Marrow Mesenchymal Stem Cell (BM-MSC) Release Microvesicles/Exosomes That Incorporate Into Hematopoietic Cells From MDS Patients and May Modify Their Behaviour

A new mechanism of intercellular communication has been proposed consisting in the secretion of exosomes/ microvesicles (MVs). Such mechanism has been shown to modify the functional properties of recipient cells by the transfer of proteins, mRNA, or micro-RNAs. The hypothesis of the present work was that MSC from MDS patients could differentially modify the HPC properties throughout the shedding of MVs when compared with those from controls due to their different content. Material and methods: MVs were isolated from MSC from bone marrow (BM) samples 18 patients diagnosed with ‘de novo' and untreated low risk MDS and from MSC from 12 healthy BM. BM-MSC at third passage were cultured in DMEM deprived of FCS, and supernatants were collected after 6 or 24 hours. MVs purification was performed in the majority of the experiments (16 MDS/ 9 Controls) using the ExoQuick-TC exosome precipitation solution (ExoQuick; System Biosiences). To confirm the isolation of MVs by exosome precipitation solution, in some cases (2 MDS/3 Controls) the MVs were obtained by ultracentrifugation; MVs identification was done by transmission electron microscopy (TEM) as well as by flow cytometry (FC). To evaluate if the micro-RNA content into MSC-MVs from patients and controls was different, expression analysis of miRNAs was done using Megaplex™ RT Primers pool (Applied Biosystems) and 384-well microfluidic cards (TaqMan® MicroRNA Array A) were loaded with retro-transcription product and PCR runs were performed on a 7900HT Fast Real-time PCR system (eight MVs from MDS and 4 from HD).To demonstrate the incorporation of MVs from MSC into human hematopoietic progenitors (HPC: CD34+ cells obtained by immunomagnetic selection) HPC were co-cultured with MVs from MSC. Incorporation of Vybrant Dil-labelled MVs into HPC was evaluated at 1, 3, 6, and 24 h. by FC. To detect the incorporation of MVs by confocal microscopy (CM) an intracellular primary Ab for CD90 (Santa Cruz, Biotechnology) was used as MVs marker and anti-CD45 to detect HPC. A Zeiss LSM 510 CM connected to a digital camera (Leica DC 100) were used to obtain confocal images. Apoptotic rate of CD34+ cells that had the MVs-MSC from MDS and controls were evaluated by FC by using APC H7 Annexin V DY634 (Immunostep) and 7AAD (BD Biosciences). Results: More than 95% of MVs isolated by ExoKit system from supernatants of cultured MSC from 6 HD and 6 MDS patients showed scatter intensities lower than of 6µm beads. We observed, in all cases, the same FC pattern. Also, MVs/exosomes isolated by ultracentrifugation (3 MDS/ 5 HD) showed the same FC pattern. MVs from MDS and controls isolated by ultracentrifugation were identified by TEM (fig1). When co-cultures of CD34+ HPC and MVs were studied in both HD and MDS, MVs were incorporated into HPC in all cases (fig2). When the content of miRNAS in the MVs from MDS and HD were compared significant differences were observed between both groups. Twenty-one out of 384 evaluated miRNAs were over-expressed in the MVs from patients compared with the controls. To confirm these results, the expression of miR10a and miR-132 was analyzed by RT-PCR. In both cases their expression was significantly increased in MVs from patients. Recently, it has been suggested that the cargo of these structures are bioactive molecules, therefore we explored the possibility that MVs could modify the behavior of the target cell. For this purpose we searched in which pathways the overexpressed miRNAs could be involved and apoptosis was among them. Since it is considered a very important process in MDS pathophysiology we compared apoptosis by FC, after co-culturing CD34+ cells with MVs from MSC of MDS and HD. Interestingly, preliminary results show that the MVs from MDS protected better from apoptosis CD34+ cells than MVs obtained from controls. In summary, in the present study we show that BM-MSC produce MVs/exosomes with different microRNAs content according to their origin, MDS or HD. These structures can be incorporated into HPC and can modify their properties.Funding: Instituto de Salud Carlos III. PI12/01775. Junta de Castilla y León.GRS 873/A/13. Portuguese FST Grant. SFRH/BD/86451/2012 Disclosures:Diez-Campelo:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. San Miguel:Jansen, Celgene, Onyx, Novartis, Millenium: Membership on an entity's Board of Directors or advisory committees. del Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. [Display omitted] [Display omitted]

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Smooth muscle features of mouse extraocular muscle

We have been intrigued to find smooth muscle markers within mouse extraocular muscle (EOM) while studying contractile features of the aqueous humour drainage tissues by immunohistochemistry. Smooth muscle is known to be present in connective tissue fascial pulleys ensheathing EOM1 but not in the EOM per se. The mouse has many available engineered strains, shares many biological similarities with primates, and is widely used to model human biology, including that of EOM.2 Herein, we show that classic smooth muscle proteins of alpha-smooth muscle actin (α-SMA), myosin heavy chain (MHC), caldesmon, and tropomyosin are intimately associated with EOM fibre bundles, a finding that may be relevant to better understanding EOM control. We studied C57BL/6 mice (2-3 months, Charles River, Wilmington, MA) that were housed in a temperature-controlled room with 12h light and dark cycles and fed ad libitum. Animal care and use complied with the Institutional Animal Care and Use Committee as well as the Association for Research in Vision and Ophthalmology guidelines. Anterior (insertion) and posterior (retro-equator) portions of four rectus EOM of C57BL/6 mice (n=5) were carefully isolated, quickly embedded in OCT compound and snap-frozen in liquid nitrogen. Eight μm-thick cryosections were fixed with 4% paraformaldehyde, permeabilised and blocked (0.3% Triton X-100+5% BSA), incubated with primary antibodies (Abcam) to α-SMA, MHC non-muscle, caldesmon, and tropomyosin overnight at 4°C, then secondary antibodies and Alexa 568-phalloidin. Vascular smooth muscle labeling of the same tissues represented positive controls, while normal IgG isotype labeling represented negative controls. Sections were analysed by Leica SP5 or Zeiss LSM 710 confocal microscopy. The same tissue slides were further processed for hematoxylin and eosin staining to confirm structure. Figure 1 shows representative longitudinal and cross sectional immunohistochemistry images of the mouse anterior EOM at their globe insertions. F-actin labeling localized to contractile regions that were mostly consistent with striated muscle fibre bundles. Positive labeling for α-SMA, MHC, and caldesmon labeling was present in the periphery of phalloidin-positive muscle fibre bundles. Positive tropomyosin labeling was seen centrally and peripherally in muscle bundles, corresponding to regions of striated and presumed smooth muscle elements. Smooth muscle marker labeling partially overlapped with phalloidin labeling in fibre bundles. Figure 1 Smooth muscle profile in the anterior part of 4 different rectus extraocular muscles (EOMs). Co-localisation of α-SMA, MHC, caldesmon, or tropomyosin with phalloidin in the anterior portion of EOM. All 4 different rectus EOMs demonstrated a similar ... Figure 2 shows the distribution of smooth muscle markers in representative longitudinal and cross sections of rectus muscles posteriorly. All recti showed this pattern. As in the anterior EOM, α-SMA, MHC, and caldesmon labeling localized to the periphery of phalloidin-labeled bundles, while tropomyosin was present peripherally and centrally. Positive immunolabeling using the same prior antibody panel was seen in vascular smooth muscle from the same tissues (not shown). Figure 2 Smooth muscle profile of the posterior part of 4 different rectus EOMs. Co-localisation of α-SMA, MHC, caldesmon, or tropomyosin with phalloidin in the posterior portion of EOM was shown. All 4 different rectus EOMs demonstrated a similar in situ ... Orbital smooth muscle is present in superior and inferior palpebral muscles, inferior orbital fissure and EOM fascial pulleys1 as part of a periorbital smooth muscle network.5 To our best knowledge smooth muscle has not been described in EOM, which is considered striated muscle. Here we describe classic smooth muscle markers within mouse EOM, mostly in the periphery of striated muscle fiber bundles. Smooth muscle was present in all rectus muscles at their insertions and retro-equatorially, indicating this organization is widely present in mouse EOM. The EOM and their supporting structures represent a complex that helps orchestrate eye movement. Striated EOM are organized as global and orbital layers, each with different structural, vascular, neural, mechanical and metabolic features.3-4 Fascial sheaths and pulleys influence muscle actions on the globe1 under possible smooth muscle modulation. As with autonomically-innervated palpebral smooth muscle that works with striated muscle to regulate eyelid position,5 we postulate that smooth muscle associated with striated EOM plays a role in determining eye position in mice, and possibly in humans.

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.

Multi-omic Data Integration Links Deleted in Breast Cancer 1 (DBC1) Degradation to Chromatin Remodeling in Inflammatory Response

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

Oligodendrocyte Progenitor Cell Susceptibility to Injury in Multiple Sclerosis

Remyelination in multiple sclerosis (MS) is often incomplete. In experimental models, oligodendrocyte progenitor cells (OPCs) rather than previously myelinating oligodendrocytes (OLs) are responsible for remyelination. This study compares the relative susceptibility of adult human OPCs and mature OLs to injury in actively demyelinating MS lesions and under invitro stress conditions. In all lesions (n = 20), the number of OLs (Olig2 weak/NogoA positive) was reduced compared to control white matter (mean 38± 4% of control value). In 11 cases, OPC numbers (Olig2 strong; NogoA negative) were also decreased; in eight of these, the reduction was greater for OPCs than for OLs. In the other nine samples, OPC numbers were greater than control white matter, indicating ongoing OPC migration and/or proliferation. Analysis of co-cultures with rat dorsal root ganglia neurons confirmed that OPCs were more capable of contacting and ensheathing axons than OLs. In isolated culture under stress conditions (withdrawal of serum/glucose and/or antioxidants), OPCs showed increased cell death and reduced process extension compared to OLs. Under all culture conditions, OPCs up-regulated expression of genes in the extrinsic proapoptotic pathway, and had increased susceptibility to tumor necrosis factor-induced cell death as compared to OLs. Our data suggest that susceptibility of OPCs to injury within the MS lesion environment contributes to the limited remyelination in MS.

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks

Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1. The ends of ZO-1 are embedded in different functional subcompartments of the tight junction. Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.