Sequential extracellular matrix-focused and baited-global cluster analysis of serial transcriptomic profiles identifies candidate modulators of renal tubulointerstitial fibrosis in murine adriamycin-induced nephropathy

Transcriptome analysis using microarray technology represents a powerful unbiased approach for delineating pathogenic mechanisms in disease. Here molecular mechanisms of renal tubulointerstitial fibrosis (TIF) were probed by monitoring changes in the renal transcriptome in a glomerular disease-dependent model of TIF ( adriamycin nephropathy) using Affymetrix (mu74av2) microarray coupled with sequential primary biological function-focused and secondary

Secretory products of the haptoral reservoirs and peduncular glands in two species of Bravohollisia (Monogenea : Ancyrocephalidae)

Light and electron microscopy were used to characterize the structure of secretory cells and their products involved in attachment of two monogenean parasites of fish, in order to understand their role in the attachment process. In Bravohollisia rosetta and Bravohollisia gussevi, peduncular gland cells with two nuclei, granular endoplasmic reticulum, and Golgi bodies produce dual electron-dense (DED) secretory bodies with a homogenous electron-dense rind and a less electron-dense fibrillar core (oval and concave in B. rosetta and oval in B. gussevi). The DED secretory bodies are altered as they migrate from the gland cell to the haptoral reservoir, the superficial anchor grooves, and into the gill tissues. The contents of the DED secretory bodies are exocytosed into the reservoirs, fibrillar cores persisting in the matrix, some of which condense, forming highly electron-dense spherical bodies. Small, oval, electron-dense bodies occur in the grooves, while no inclusions are visible in the homogenous exudate within the gill tissues. The single tubular extension of the reservoir enters a bifurcate channel within the anchor via a concealed, crevice-like opening on one side of the anchor. The channel directs secretions into the left and the right grooves via concealed apertures. The secretions, introduced into the tissues by the anchors, probably assist in attachment. The secretions are manifested externally as net-like structures and observed in some cases to be still attached to the point of exudation, on anchors detached from the gill tissues. This suggests that despite having the anchors detached, the worms can still remain anchored to the gill tissues via these net-like structures. Based on this, it is postulated that the net-like secretions probably function as a safety line to anchor the worm during the onset of locomotion and in doing so reduce the risk of tearing host tissues.

A potent novel antimicrobial peptide related to caerin 1.12 from the skin secretion of the Australian frog, Litoria aurea

Skin secretions from Australian frogs of the genus Litoria have been extensively studied for many years and are known to contain a large array of antimicrobial peptides that often bear their specific names — caerins (L. caerulea), aureins (L. aurea), citropins (L. citropa) and maculatins (L. genimaculata) — and each group displays distinct primary structural attributes. During a systematic transcriptome cloning study using a cDNA library derived from skin secretion of L. aurea, a series of identical clones were identified that encoded a novel 25-mer antimicrobial peptide that displayed 92% structural identity with caerin 1.12 from L. caerulea, differing in amino acid sequence at only two positions — Arg for Gly at position 7 and Leu amide for Ser amide at the C-terminus. The novel peptide had conserved Pro residues at positions 15 and 19 that flank a flexible hinge region which previous studies have suggested are important for effective orientation of the two alpha-helices within the bacterial membrane resulting in lysis of cells. As the two substitutions in the novel peptide serve to increase both positive charge and hydrophobicity, we synthesised a replicate and determined its minimal inhibitory concentration (MIC) against Gram positive Staphylococcus aureus and Gram negative Escherichia coli. The MICs for these organisms were 3 µM and 4 µM, respectively, indicating a high potency and haemolysis was

Fasciola hepatica: Histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and the related benzimidazole derivative, Compound Alpha

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10 mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd alpha), a benzimidazole derivative of TCBZ (15 mg/kg). The lambs were euthanised 48,72 and 96 h after TCBZ treatment, or 24, 48 and 72 h after Cpd a treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanised 24 h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis' secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis' gland, histological evidence indicating failure of ovigenesis was evident from 24 h post-treatment onwards. The development of these lesions may be related to the known antitubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd a is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings. (C) 2009 Elsevier B.V. All rights reserved.

Desmoplastic Small Round Cell Tumor of Major Salivary Glands: Report of 1 Case and a Review of the Literature

Desmoplastic small round cell tumor is a rare malignant neoplasm mostly occurring in the vicinity of or within the peritoneal cavity, and is uncommon in the head and neck region. Tumor location within a major salivary gland is exceptional. We report a case of a 41-year-old Chinese man with a history of diabetes mellitus and end-stage renal failure on peritoneal dialysis with a desmoplastic small round cell tumor occurring in the left submandibular gland. Fine-needle aspiration cytology showed variably cohesive clusters of small cells with hyperchromatic nuclei and fine granular chromatin. On histology the neoplasm displayed classic features of a desmoplastic small round cell tumor with angulated nests of small round blue cells in a fibromyxoid/desmoplastic stroma. Neoplastic cells were immunoreactive for cytokeratins (AE1/3), desmin (paranuclear dot-like), WT-1 (nuclear), epithelial membrane antigen, and CD56. EWS gene translocation and EWS-WT1 gene fusion were detected by fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction, respectively. The case presented is the sixth case of and the oldest reported patient with a desmoplastic small round cell tumor occurring in a major salivary gland to date. Desmoplastic small round cell tumor should be considered in the differential diagnosis of a salivary gland neoplasm with a basaloid or small cell pattern on fine-needle aspiration cytology.

Functional and Structural Diversification of the Anguimorpha Lizard Venom System

Venom has only been recently discovered to be a basal trait of the Anguimorpha lizards. Consequently, very little is known about the timings of toxin recruitment events, venom protein molecular evolution, or even the relative physical diversifications of the venom system itself. A multidisciplinary approach was used to examine the evolution across the full taxonomical range of this similar to 130 million-year-old clade. Analysis of cDNA libraries revealed complex venom transcriptomes. Most notably, three new cardioactive peptide toxin types were discovered (celestoxin, cholecystokinin, and YY peptides). The latter two represent additional examples of convergent use of genes in toxic arsenals, both having previously been documented as components of frog skin defensive chemical secretions. Two other novel venom gland-overexpressed modified versions of other protein frameworks were also recovered from the libraries (epididymal secretory protein and ribonuclease). Lectin, hyaluronidase, and veficolin toxin types were sequenced for the first time from lizard venoms and shown to be homologous to the snake venom forms. In contrast, phylogenetic analyses demonstrated that the lizard natriuretic peptide toxins were recruited independently of the form in snake venoms. The de novo evolution of helokinestatin peptide toxin encoding do-mains within the lizard venom natriuretic gene was revealed to be exclusive to the helodermatid/anguid subclade. New isoforms were sequenced for cysteine-rich secretory protein, kallikrein, and phospholipase A 2 toxins. Venom gland morphological analysis revealed extensive evolutionary tinkering. Anguid glands are characterized by thin capsules and mixed glands, serous at the bottom of the lobule and mucous toward the apex. Twice, independently this arrangement was segregated into specialized serous protein-secreting glands with thick capsules with the mucous lobules now distinct (Heloderma and the Lanthanotus/Varanus clade). The results obtained highlight the importance of utilizing evolution-based search strategies for biodiscovery and emphasize the largely untapped drug design and development potential of lizard venoms. Molecular & Cellular Proteomics 9:2369-2390, 2010.

Research strategies to improve honeybee health in Europe

Understanding the fundaments of colony losses and improving the status of colony health will require cross-cutting research initiatives including honeybee pathology, chemistry, genetics and apicultural extension. The 7th framework of the European Union requested research to empirically and experimentally fill knowledge gaps on honeybee pests and diseases, including 'Colony Collapse Disorder' and the impact of parasites, pathogens and pesticides on honeybee mortality. The interactions among these drivers of colony loss will be studied in different European regions, using experimental model systems including selected parasites (e. g. Nosema and Varroa mites), viruses (Deformed Wing Virus, Black Queen Cell Virus, Israeli Acute Paralysis Virus) and model pesticides (thiacloprid, tau-fluvalinate). Transcriptome analyses will be used to explore host-pathogen-pesticide interactions and identify novel genes for disease resistance. Special attention will be given to sublethal and chronic exposure to pesticides and will screen how apicultural practices affect colony health. Novel diagnostic screening methods and sustainable concepts for disease prevention will be developed resulting in new treatments and selection tools for resistant stock. Research initiatives will be linked to various national and international ongoing European, North-and South-American colony health monitoring and research programs, to ensure a global transfer of results to apicultural practice in the world community of beekeepers.

Disruption of egg formation by Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host

Eight indoor-reared cross-bred sheep with no prior exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Twelve weeks after infection, sheep were treated with 10 mg/kg triclabendazole. Two sheep were euthanised per time period; at 48 h, 72 h and 96 h post-treatment (pt). Two untreated control sheep were euthanised at 96 h pt. Flukes were recovered from the liver and, if present, from the gall bladder of the sheep. They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system; specifically, the uterus, vitelline follicles. Mehlis' gland and ovary.

Early onset of changes to the reproductive system of Fasciola hepatica following in vivo treatment with triclabendazole

Lambs infected with the Cullompton triclabendazole (ICBZ)-susceptible isolate of Fasciola hepatica were treated with TCBZ at a dosage of 10 mg/kg at 16 weeks post-infection. Adult flukes were recovered from the liver at 3 h, 24 h, 48 h and 60 h post-treatment (pt). They were processed for histological analysis of the uterus, Mehlis' gland, vitellaria, ovary and testis. At 3 h pt, the flukes were essentially similar to the controls and were producing normal eggs. Egg production had ceased by 24 h pt. At this time period, the cells of the Mehlis' gland showed some evidence of vacuolation, but otherwise were relatively normal. A shift in the population of vitelline cells towards mature cells was observed at 24 h pt, and this trend continued at later time-periods. It was accompanied by a breakdown of the cells and the presence of apoptotic bodies. Marked changes to the ovary were first noted at 48 h Pt, as evidenced by vacuolation and the presence of apoptotic bodies. Some disruption to the testis was seen at 24 h pt, with a reduction in the population of spermatogenic cells, the appearance of apoptotic bodies and some peripheral vacuolation of the tubules. These abnormalities increased in severity with longer time periods pt. The results bring forward the time-line of cessation of egg production by 24 h, demonstrating that this process is affected very rapidly pt. (C) 2011 Elsevier B.V. All rights reserved.

Ultrastructural observations on rhabdite formation in the planarian, Artioposthia triangulata

The epidermis of the predatory terrestrial flatworm. Artioposthia triangulata has been examined by transmission electron microscopy for the presence of rhabdiform secretions. Two types of secretion are present: epidermal rhabdoids, produced by a special type of epidermal cell and true adenal rhabdites produced by gland cells beneath the epidermis. The epidermal rhabdoids are formed from Golgi-derived vesicles, which Fuse together to form the developing rhabdoid. Within the latter is a filamentous network on which granular material is deposited and coalesces to form a rod-shaped inclusion. The rhabdoids accumulate in the apical region of the cell and release their contents from the apical surface. The adenal rhabdites are formed by Golgi-derived vesicles. which become more elongated and their contents more electron-dense as they mature. The vesicles Fuse together to form the primordial rhabdite, which continues to lengthen with the addition of further vesicles. The neck of the rhabdite-forming cell passes between the muscle layers and through the basement membrane to open into the base of the epidermal cell. The rhabdites move from the cell body through the neck into the cytoplasm of the epidermal cell and make their way to the apical surface where they are released to the exterior.

Immunocytochemical localization of glutamate-like immunoreactivity within the nervous system of the cestode Mesocestoides corti and the trematode Fasciola hepatica

The localization and distribution of glutamate-like immunoreactivity (IR) in the nervous system of both the cestode Mesocestoides corti and the trematode Fasciola hepatica has been determined by an indirect immunofluorescent technique, in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the central (CNS) and peripheral (PNS) nervous systems of both species examined. In the CNS, IR was evident in nerve cells and fibres in the cerebral ganglia, the cerebral commissure and the dorsal, ventral and longitudinal nerve cords. In the peripheral nervous system (PNS) of M. corti, IR was apparent in nerve plexuses associated with the subtegmental musculature and the musculature associated with the anteriorly positioned suckers. In F. hepatica, IR was evident in the innervation of both the oral and the ventral suckers, In the reproductive system of F. hepatica, glutamate-IR was observed around the ootype/Mehlis' gland complex.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF A SALMFAMIDE-LIKE NEUROPEPTIDE IN THE NERVOUS-SYSTEM OF ADULT AND LARVAL STAGES OF THE HUMAN BLOOD FLUKE, SCHISTOSOMA-MANSONI

The localization and distribution of SALMFamide immunoreactivity (IR), SI(GFNSALMFamide), in the nervous system of both the adult and larval stages of the trematode Schistosoma mansoni has been determined by an indirect immunofluorescent technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the nervous system of adult male and female S. mansoni. In the central nervous system (CNS), IR was evident in nerve cells and fibres in the anterior ganglia, cerebral commissure and dorsal and ventral nerve cords. In the peripheral nervous system (PNS), IR was apparent in nerve plexuses associated with the subtegmental musculature, oral and ventral suckers, the lining of the gynaecophoric canal, and in fine nerve fibres innervating the dorsal tubercles of the male worm. In the reproductive system of male and female worms, S1-IR was only observed around the ootype/Mehlis' gland complex in the female. Immunostaining was also evident in the nervous system of both miracidium and cercarial larval stages. A post-embedding, IgG-conjugated colloidal gold immunostaining technique was employed to examine the subcellular distribution of SALMFamide-IR in the CNS of S. mansoni. Gold labelling of peptide was localized over dense-cored vesicles within nerve cell bodies and fibres constituting the neuropile of the anterior ganglia, cerebral commissure and nerve cords of the CNS. Antigen pre-absorption studies indicated that the results obtained do suggest S1-like immunostaining and not cross-reactivity with other peptides, in particular FMRFamide.

Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARγ-targeted therapy

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-? (PPAR?), cultures were treated with the PPAR? ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPAR? antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPAR? ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.

PRIMARY STRUCTURES AND BIOLOGICAL-ACTIVITIES OF SUBSTANCE-P-RELATED PEPTIDES FROM THE BRAIN OF THE DOGFISH, SCYLIORHINUS-CANICULA

Two peptides with substance-P-like immunoreactivity were isolated in pure form from an extract of the brain of the elasmobranch fish, Scyliorhinus canicula (european common dogfish). One peptide was identical to scyliorhinin I, previously identified in dogfish intestine, and the second was the undecapeptide Lys-Pro-Arg-Pro-Gly-Gln-Phe-Phe-Gly-Leu-Met-CONH2 which is structurally similar to mammalian substance P Scyliorhinin II or a peptide analogous to mammalian neurokinin A were not detected in the extract. Synthetic dogfish substance P ([Lys1, Arg3, Gly5]substance P) was approximately threefold more potent than mammalian substance P (K(d) = 0.21 +/- 0.11 nM versus K(d)= 0.74 +/- 0.17 nM; mean +/- SD; n = 6) in inhibiting the binding of I-125-labelled substance P to neurokinin (NK1) receptors in rat submandibular gland membranes. The vasodilator action of tachykinins in mammals is mediated primarily through interaction with NK1 receptors. Bolus intravenous injections of [Lys1, Arg3, Gly5]substance P (100 pmol) and scyliorhinin I (100 pmol) produced appreciable (>4 kPa) decreases in arterial blood pressure in the rat whereas intravenous injections of up to 5 nmol of the peptides into conscious, unrestrained dogfish produced no change in arterial blood pressure, pulse amplitude or heart rate. Injections of greater amounts of the peptides (10-50 nmol) produced a slight increase (400-667 Pa) in blood pressure. The data indicate that mammalian-type NK1 tachykinin receptors are not involved in cardiovascular regulation in elasmobranch fish.

An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.