Extracellular phosphoesterases of invertebrate origin: underrated actors in phosphorus recycling?

Extracellular enzymes contribute substantially to the remineralisation of organic matter in aquatic systems. Marine invertebrates release endogenous enzymes through activities such as sloppy feeding or egestion, but the significance of such processes is widely unexplored. We compared functional key properties such as activity, stability, and apparent molecular masses of extracellular phosphatases from faeces of the marine crustaceans Idotea balthica, Palaemon varians, and Homarus gammarus with those of their digestive organs. A fluorescent substrate was used to examine enzyme activity qualitatively on agarose plates and quantitatively in microplate assays. Apparent molecular masses and enzyme stability were examined by native substrate gel electrophoresis (NSGE). Active extracellular phosphatase were present in the faeces of all tested species. NSGE activity band patterns were similar in digestive tissue extracts and faeces extracts. The initial enzyme activity retained for about 2 days, but thereafter rapidly decreased. Activity band patterns of digestive organs and faeces of I. balthica remained consistent for up to 72 h. Antibiotic treatment did not reduce phosphatase activity in the faeces of I. balthica and H. gammarus but in the faeces P. varians. Because of the comparatively high activities in the faeces, the electrophoretic similarity between faeces and digestive organs, and the limited durability of the enzymes in faeces extracts, we conclude that the studied crustaceans release predominantly endogenous, rather than bacterial phosphatases through faeces. These can substantially contribute to the pool of active extracellular phosphatases and the recycling of phosphorus in aquatic systems. The wider physiological and ecological context is discussed

Mouse model of intrahepatic cholangiocarcinoma validates FIG-ROS as a potent fusion oncogene and therapeutic target

Cholangiocarcinoma is the second most common primary liver cancer and responds poorly to existing therapies. Intrahepatic cholangiocarcinoma (ICC) likely originates from the biliary tree and develops within the hepatic parenchyma. We have generated a flexible orthotopic allograft mouse model of ICC that incorporates common genetic alterations identified in human ICC and histologically resembles the human disease. We examined the utility of this model to validate driver alterations in ICC and tested their suitability as therapeutic targets. Specifically, we showed that the fused-in-glioblastoma-c- ros-oncogene1 (FIG-ROS1(S); FIG- ROS) fusion gene dramatically accelerates ICC development and that its inactivation in established tumors has a potent antitumor effect. Our studies establish a versatile model of ICC that will be a useful preclinical tool and validate ROS1 fusions as potent oncoproteins and therapeutic targets in ICC and potentially other tumor types

Genetic Mouse Models as In Vivo Tools for Cholangiocarcinoma Research

Cholangiocarcinoma (CCA) is a genetically and histologically complex disease with a highly dismal prognosis. A deeper understanding of the underlying cellular and molecular mechanisms of human CCA will increase our current knowledge of the disease and expedite the eventual development of novel therapeutic strategies for this fatal cancer. This endeavor is effectively supported by genetic mouse models, which serve as sophisticated tools to systematically investigate CCA pathobiology and treatment response. These in vivo models feature many of the genetic alterations found in humans, recapitulate multiple hallmarks of cholangiocarcinogenesis (encompassing cell transformation, preneoplastic lesions, established tumors and metastatic disease) and provide an ideal experimental setting to study the interplay between tumor cells and the surrounding stroma. This review is intended to serve as a compendium of CCA mouse models, including traditional transgenic models but also genetically flexible approaches based on either the direct introduction of DNA into liver cells or transplantation of pre-malignant cells, and is meant as a resource for CCA researchers to aid in the selection of the most appropriate in vivo model system

Genetic mouse models as in vivo tools for cholangiocarcinoma research

Cholangiocarcinoma (CCA) is a genetically and histologically complex disease with a highly dismal prognosis. A deeper understanding of the underlying cellular and molecular mechanisms of human CCA will increase our current knowledge of the disease and expedite the eventual development of novel therapeutic strategies for this fatal cancer. This endeavor is effectively supported by genetic mouse models, which serve as sophisticated tools to systematically investigate CCA pathobiology and treatment response. These in vivo models feature many of the genetic alterations found in humans, recapitulate multiple hallmarks of cholangiocarcinogenesis (encompassing cell transformation, preneoplastic lesions, established tumors and metastatic disease) and provide an ideal experimental setting to study the interplay between tumor cells and the surrounding stroma. This review is intended to serve as a compendium of CCA mouse models, including traditional transgenic models but also genetically flexible approaches based on either the direct introduction of DNA into liver cells or transplantation of pre-malignant cells, and is meant as a resource for CCA researchers to aid in the selection of the most appropriate in vivo model system

_

This is the published version: Allardyce, Benjamin J., Linton, Stuart M. and Saborowski, Reinhard 2010, The last piece in the cellulase puzzle : the characterisation of β-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis, Journal of experimental biology, vol. 213, no. 17, pp. 2950-2957. Available from Deakin Research Online: http://hdl.handle.net/10536/DRO/DU:30031463 Reproduced with the kind permissions of the copyright owner. Copyright : 2010, Company of Biologists 2950 INTRODUCTION Herbivorous terrestrial crustaceans, such as gecarcinid land crabs, consume a wide range of plant material, which is rich in cellulose and hemicellulose. Land crabs possess multiple endogenous endo--1,4-glucanases, one of the key enzyme classes involved in cellulose hydrolysis The traditional model for cellulose digestion in invertebrates reflects what is known about fungal cellulase systems. This model suggests that cellulose hydrolysis involves the synergistic activity of endo--1,4-glucanases (1,4--D-glucan-4-glucanohydrolases, EC 3.2.1.4), exoglucanases, including both cellobiohydrolases (1,4--D-glucan cellobiohydrolases, EC 3.2.1.91) and sometimes glucohydrolases (1,4--D-glucan glucanohydrolases, EC 3.2.1.74) and -glucosidases (-glucoside glucohydrolases, EC 3.2.1.21) Like fungal systems, endo--1,4-glucanases and -glucosidases appear to be key components of the cellulase systems of many marine and terrestrial invertebrate

Conditional Reverse Tet-Transactivator Mouse Strains for the Efficient Induction of TRE-Regulated Transgenes in Mice

Tetracycline or doxycycline (dox)-regulated control of genetic elements allows inducible, reversible and tissue specific regulation of gene expression in mice. This approach provides a means to investigate protein function in specific cell lineages and at defined periods of development and disease. Efficient and stable regulation of cDNAs or non-coding elements (e.g. shRNAs) downstream of the tetracycline-regulated element (TRE) requires the robust expression of a tet-transactivator protein, commonly the reverse tet-transactivator, rtTA. Most rtTA strains rely on tissue specific promoters that often do not provide sufficient rtTA levels for optimal inducible expression. Here we describe the generation of two mouse strains that enable Cre-dependent, robust expression of rtTA3, providing tissue-restricted and consistent induction of TRE-controlled transgenes. We show that these transgenic strains can be effectively combined with established mouse models of disease, including both Cre/LoxP-based approaches and non Cre-dependent disease models. The integration of these new tools with established mouse models promises the development of more flexible genetic systems to uncover the mechanisms of development and disease pathogenesis

Pituitary and systemic autoimmunity in a case of intrasellar germinoma

Germinomas arising in the sella turcica are difficult to differentiate from autoimmune hypophysitis because of similar clinical and pathological features. This differentiation, nevertheless, is critical for patient care due to different treatments of the two diseases. We report the case of an 11-year-old girl who presented with diabetes insipidus and growth retardation, and was found to have an intra- and supra-sellar mass. Initial examination of the pituitary biopsy showed diffuse lymphocytic infiltration of the adenohypophysis and absent placental alkaline phosphatase expression, leading to a diagnosis of hypophysitis and glucocorticoid treatment. Because of the lack of clinical and radiological response, the pituitary specimen was re-examined, revealing this time the presence of scattered c-kit and Oct4 positive germinoma cells. The revised diagnosis prompted the initiation of radiotherapy, which induced disappearance of the pituitary mass. Immunological studies showed that the patient’s serum recognized antigens expressed by the patient’s own germinoma cells, as well as pituitary antigens like growth hormone and systemic antigens like the Sjögren syndrome antigen B and alpha-enolase. The study first reports the presence of pituitary and systemic antibodies in a patient with intrasellar germinoma, and reminds us that diffuse lymphocytic infiltration of the pituitary gland and pituitary antibodies does not always indicate a diagnosis of autoimmune hypophysitis

Assessing ecotoxicity of an innovative bio-based mulch film: a multi-environmental and multi-bioassay approach

Among the highly diverse range of biobased polymers, polylactic acid (PLA) received vast attention in recent years due to its versatility for different applications and being the first commercially used polymer produced from renewable sources. Production and application of bio-based, biodegradable plastics will have one of the most crucial roles in tackling worldwide plastic pollution. Methods: This study is based on integrative ecotoxicological assessment of an innovative PLA-based agricultural mulch film (BPE-AMF-PLA), developed under the H2020 EU project “BIO-PLASTICS EUROPE”, towards organisms from different environmental compartments (soil, fresh water and marine) and from different trophic levels. Such comprehensive evaluation has an overarching goal to promote environmentally safe and sustainable use of these PLA-based plastics for agricultural and other potential applications. Results: Low-to-no phytotoxicity was obtained in both single-species standardized bioassays, and in a multi-species microcosms experiment. Earthworm reproduction was negatively affected at the lowest test concentration of 0.1% w/w of PLA-based plastic particles. For freshwater Daphnia, reproduction was found a sensitive endpoint, upon exposure to the leachates of the PLA-based plastic. However, the reported toxicity seemed to be caused by the presence of 2-methylnaphthalene, which can be avoided in the production process. As for the marine organisms, algae growth was inhibited with a LOEC = 25 g L−1, whereas test with brine shrimp only revealed stimulation of lipase upon digestion of micro-sized PLA-based plastics. Marine lugworm ingested pristine and UV pre-treated micro-sized plastics, yet without impact either on biological activity, or on the health of the test individuals. Discussion: The approach used in the present work will contribute to product development, environmental safety and sustainable applications of the PLA-based mulch film BPE-AMF-PLA, in the scope of project BIO-PLASTICS EUROPE. Furthermore, the tools and results obtained in this work are a relevant contribution in the framework development for additional support in the certification of the bio-based polymers, being aligned with European zero waste and non-toxicity strategies, certification, and regulations