Xenopus tropicalis : joining the armada in the fight against blood cancer

Aquatic vertebrate organisms such as zebrafish have been used for over a decade to model different types of human cancer, including hematologic malignancies. However, the introduction of gene editing techniques such as CRISPR/Cas9 and TALEN, have now opened the road for other organisms featuring large externally developing embryos that are easily accessible. Thanks to its unique diploid genome that shows a high degree of synteny to the human, combined with its relatively short live cycle, Xenopus tropicalis has now emerged as an additional powerful aquatic model for studying human disease genes. Genome editing techniques are very simple and extremely efficient, permitting the fast and cheap generation of genetic models for human disease. Mosaic disruption of tumor suppressor genes allows the generation of highly penetrant and low latency cancer models. While models for solid human tumors have been recently generated, genetic models for hematologic malignancies are currently lacking for Xenopus. Here we describe our experimental pipeline, based on mosaic genome editing by CRISPR/Cas9, to generate innovative and high-performing leukemia models in X. tropicalis. These add to the existing models in zebrafish and will extend the experimental platform available in aquatic vertebrate organisms to contribute to the field of hematologic malignancies. This will extend our knowledge in the etiology of this cancer and assist the identification of molecular targets for therapeutic intervention

Xenopus tropicalis: Joining the Armada in the Fight Against Blood Cancer

Aquatic vertebrate organisms such as zebrafish have been used for over a decade to model different types of human cancer, including hematologic malignancies. However, the introduction of gene editing techniques such as CRISPR/Cas9 and TALEN, have now opened the road for other organisms featuring large externally developing embryos that are easily accessible. Thanks to its unique diploid genome that shows a high degree of synteny to the human, combined with its relatively short live cycle, Xenopus tropicalis has now emerged as an additional powerful aquatic model for studying human disease genes. Genome editing techniques are very simple and extremely efficient, permitting the fast and cheap generation of genetic models for human disease. Mosaic disruption of tumor suppressor genes allows the generation of highly penetrant and low latency cancer models. While models for solid human tumors have been recently generated, genetic models for hematologic malignancies are currently lacking for Xenopus. Here we describe our experimental pipeline, based on mosaic genome editing by CRISPR/Cas9, to generate innovative and high-performing leukemia models in X. tropicalis. These add to the existing models in zebrafish and will extend the experimental platform available in aquatic vertebrate organisms to contribute to the field of hematologic malignancies. This will extend our knowledge in the etiology of this cancer and assist the identification of molecular targets for therapeutic intervention

CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis

Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development. Here, we show by CRISPR/Cas9 techniques that similar to the mouse, neither rb1 nor rbl1 single mosaic mutant Xenopus tropicalis develop tumors, whereas rb1/rbl1 double mosaic mutant tadpoles rapidly develop retinoblastoma. Moreover, occasionally presence of pinealoblastoma (trilateral retinoblastoma) was detected. We thus present the first CRISPR/Cas9 mediated cancer model in Xenopus tropicalis and the first genuine genetic non-mammalian retinoblastoma model. The rapid kinetics of our model paves the way for use as a pre-clinical model. Additionally, this retinoblastoma model provides unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (rb1 + rbl1 + modifier gene) in order to address the clinically unmet need of targeted retinoblastoma therapy

Τεχνητή διατροφή στο βαρέως πάσχοντα ασθενή

Εισαγωγή: Η τεχνητή διατροφή στο βαρέως πάσχοντα ασθενή αποτελεί βασικό στοιχείο της θεραπευτικής του αγωγής, καθώς του παρέχει τα απαραίτητα θρεπτικά συστατικά και επιτυγχάνεται με τη μέθοδο της εντερικής και παρεντερικής σίτισης. Οι δύο μέθοδοι παρουσιάζουν πλεονεκτήματα και μειονεκτήματα και μπορούν να συμβάλουν στη βελτίωση της κατάστασης του βαρέως πάσχοντα ή να παρατείνουν την παραμονή στο νοσοκομείο, ακόμη και να αυξήσουν τη θνητότητα. Σκοπός: Ο σκοπός της παρούσας συστηματικής ανασκόπησης ‘ήταν η διερεύνηση της χρήσης των μεθόδων τεχνητής διατροφής στο βαρέως πάσχοντα ασθενή, πότε και σε ποιες περιπτώσεις μπορούν να χρησιμοποιηθούν καθώς και η συμβολή τους στη θεραπεία. Ερευνητική μεθοδολογία: Πραγματοποιήθηκε βιβλιογραφική ανασκόπηση άρθρων στην ξένη βιβλιογραφία από το 2004 έως το 2019, στην ηλεκτρονική βάση δεδομένων Google Scholar, χρησιμοποιώντας λέξεις κλειδιά στην αγγλική γλώσσα όπως «Critically ill patient», «artificial nutrition», «enteraparenteral nutrition». Στη μελέτη συμπεριελήφθησαν τελικά 12 άρθρα. Αποτελέσματα: Τα αποτελέσματα των 12 μελετών κατέδειξαν ότι, οι βαρέως πάσχοντες ασθενείς που έλαβαν πρώιμα, εντερική σίτιση, είχαν καλύτερη πρόγνωση από εκείνους οι οποίοι έλαβαν καθυστερημένα εντερική σίτιση αλλά και από εκείνους που έλαβαν πρώιμα παρεντερική σίτιση. Συμπεράσματα: Η τεχνητή διατροφή συμβάλει σημαντικά στη βελτίωση της κλινικής εικόνας στο βαρέως πάσχοντα ασθενή. Η εντερική σίτιση πλεονεκτεί σημαντικά έναντι της παρεντερικής ανάλογα με το χρονικό σημείο χορήγησης της κάθε μίας. Μεγάλη σημασία έχει, η προηγούμενη θρεπτική κατάσταση του ασθενή, καθώς φαίνεται να παίζει σημαντικό ρόλο στην φυσιολογική ανταπόκριση του οργανισμού στην χορήγηση τεχνητής διατροφής. Οι νοσηλευτές και οι επαγγελματίες υγείας μέσα από την τήρηση των πρωτοκόλλων και των κατευθυντήριων οδηγιών χορήγησης της τεχνητής διατροφής διαδραματίζουν σημαντικό ρόλο στη θεραπευτική διαδικασία.Introduction: Artificial nutrition in the critically ill patient is a basic element of the treatment process, since it provides the essential nutritional ingredients, through the method of enteral and parenteral feeding. The two methods demonstrate advantages and disadvantages, and it can contribute to the improvement of the condition of the critically ill patient or prolong the hospital stay or even increase mortality. Purpose: The purpose of this systematic review is to investigate the use of the methods of artificial nutrition in the critically ill patient, when and in which circumstances can those be used and their contribution to the treatment. Research Methodology: A bibliographic review of articles in the foreign literature was carried out the period from 2004-2019, electronic database Google Scholar, using keywords in the English language: “critically ill patient”,” artificial nutrition”,” enteral – parenteral nutrition”. Twelve (12) articles were finally included in the study. Results: Results of the 12 studies had shown a better prognosis to critically ill patients that they had early enteral nutrition, than those receiving it at delay and from those that received early parenteral nutrition. Conclusions: Artificial nutrition contributes significantly to the clinical outcome of the critically ill patient. Enteral nutrition has advantages vs parenteral, depending up on the starting point of both. It is of great importance the past nutritional condition of the patient, as it seems to play a great role to the physiological response of the body to artificial nutrition feeding. Nurses and healthcare professionals have a significant part at the healing process, through follow of the protocols and the guidelines in artificial nutrition

Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation

Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation

Developing Tadpole <i>Xenopus laevis</i> as a Comparative Animal Model to Study <i>Mycobacterium abscessus</i> Pathogenicity

Mycobacterium abscessus (Mab) is an emerging, nontuberculosis mycobacterium (NTM) that infects humans. Mab has two morphotypes, smooth (S) and rough (R), related to the production of glycopeptidolipid (GPL), that differ in pathogenesis. To further understand the pathogenicity of these morphotypes in vivo, the amphibian Xenopus laevis was used as an alternative animal model. Mab infections have been previously modeled in zebrafish embryos and mice, but Mab are cleared early from immunocompetent mice, preventing the study of chronic infection, and the zebrafish model cannot be used to model a pulmonary infection and T cell involvement. Here, we show that X. laevis tadpoles, which have lungs and T cells, can be used as a complementary model for persistent Mab infection and pathogenesis. Intraperitoneal (IP) inoculation of S and R Mab morphotypes disseminated to tadpole tissues including liver and lungs, persisting for up to 40 days without significant mortality. Furthermore, the R morphotype was more persistent, maintaining a higher bacterial load at 40 days postinoculation. In contrast, the intracardiac (IC) inoculation with S Mab induced significantly greater mortality than inoculation with the R Mab form. These data suggest that X. laevis tadpoles can serve as a useful comparative experimental organism to investigate pathogenesis and host resistance to M. abscessus

Engraftment of Allotransplanted Tumor Cells in Adult rag2 Mutant Xenopus tropicalis

Modeling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated via injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting single or multiple tumor suppressor genes. What has been lacking so far is the possibility to propagate and characterize the induced cancers via transplantation. Here, we describe the generation of a rag2 knockout line in Xenopus tropicalis that is deficient in functional T and B cells. This line was validated by means of allografting experiments with primary tp53−/− and apc+/−/tp53−/− donor tumors. In addition, we optimized available protocols for the sub-lethal irradiation of wild-type X. tropicalis froglets. Irradiated animals also allowed the stable, albeit transient, engraftment of transplanted X. tropicalis tumor cells. The novel rag2−/− line and the irradiated wild-type froglets will further expand the experimental toolbox in the diploid amphibian X. tropicalis and help to establish it as a versatile and relevant model for exploring human cancer.</jats:p