La lectine de Xerocomellus chrysenteron, un nano-objet théranostique pour l'imagerie et le traitement des cancers épithéliaux : preuve de concept appliquée aux carcinoses péritonéales d'origine ovarienne

Le développement de thérapies ciblées et des techniques d'imagerie est un défi majeur en santé, particulièrement dans les pathologies cancéreuses. Les carcinoses péritonéales sont habituellement causées par une dissémination de cellules tumorales au sein de la cavité abdominale, ce qui est le cas de 85% des patientes atteintes d'un cancer ovarien et plus de 10% des patients atteints d'un cancer colorectal. Dans les deux cas, les traitements consistent en une chirurgie (cytoréduction), aussi complète que possible, accompagnée de chimiothérapies. L'amélioration de la survie globale des patients passe par le développement de technologies parallèles comme de nouveaux outils diagnostiques pour détecter des implantations précoces dans le péritoine, le blocage de la dissémination de cellules cancéreuses pendant et après la chirurgie, ou encore la combinaison de chimiothérapies et de traitements ciblés intrapéritonéaux. Ce projet de thèse consiste à aborder ces différents aspects par l'utilisation d'un nanocontainer protéique multifonctionnel. L'objectif est d'optimiser cette protéine, appartenant à la famille des lectines, pour envisager son développement en tant qu'outil théranostique dans le cadre du diagnostic et des traitements de cancers épithéliaux. La lectine de Xerocomellus chrysenteron, à l'origine extraite d'un champignon supérieur comestible, présente une forte affinité pour un biomarqueur glycosidique des carcinomes, l'antigène de Thomsen-Friedenreich (antigène TF ou CD176). De plus, la présence d'une large cavité hydratée au centre de cet assemblage protéique (homotétramère) permet d'envisager le confinement et l'adressage spécifique de molécules thérapeutiques à des cellules épithéliales cancéreuses. Nous avons commencé par établir la preuve de concept de la délivrance ciblée de molécules thérapeutiques dans plusieurs lignées d'adénocarcinomes ovariens humains (OVCAR-3, SKOV-3, IGROV-1). Le marquage de la lectine dans le proche infrarouge a permis de confirmer le mécanisme de délivrance et prouver que la molécule thérapeutique avait bien été endocytée grâce à son confinement dans le nanocontainer. La protéine marquée a également été utilisée pour valider son utilisation comme nanosonde pour la détection de nodules tumoraux submillimétriques dans le péritoine. Cette détection est faite par imagerie de fluorescence in vivo dans des modèles précliniques de carcinose péritonéale ovarienne préalablement développés à partir de lignées cellulaires. La combinaison des deux propriétés de la protéine (sonde et container) permet d'envisager son utilisation en nanothéranostique intrapéritonéale. Afin de confirmer ce développement prometteur, il sera nécessaire d'établir la preuve de concept sur des modèles murins plus pertinents de la situation clinique développés à partir de tumeurs issues de patientes (Patient Derived Xenografts, PDX).The development of targeted therapy and imaging tools is a major challenge in human health, particularly in cancer pathologies. Peritoneal carcinomatosis is usually caused by scattering of cancer cells within the abdominal cavity, which is the case for 85% of ovarian cancer patients and more than 10% of colorectal cancer patients. In both cases treatments include a cytoreductive surgery, as complete as possible, and chemotherapies. Patients overall survival improvement can be reach with the development of parallel technologies such as new diagnostic tools to detect early implantations in the peritoneal cavity, agents to block the spreading of cancer cells detached during the surgical procedure, or combining chemotherapies and intraperitoneal targeted drug delivery. This project involves reaching all those aspects by using a unique multifunctional nanocontainer protein. The aim is to maximize this protein, which belongs to the lectin family, to consider its development as a theranostic tool as part of epithelial cancers diagnostic and treatment. Xerocomellus chrysenteron lectin, originally extracted from an edible higher mushroom, has a strong affinity for a carcinoma glycan biomarker, the Thomsen-Friedenreich antigen (TF antigen). Furthermore, a large hydrated inner cavity located in the middle of the tetrameric assembly of the protein led us to consider the containment and specific addressing of therapeutic molecules to epithelial cancerous cells expressing TF antigen. We first established the proof of concept for the targeted drug delivery of therapeutic molecules in several human ovarian adenocarcinoma cell lines (OVCAR-3, SKOV-3, IGROV-1). The labelling of the lectin in near infrared allowed us to confirm the mechanism implicated in the delivery and prove that the uptake of the molecule within the cells was due to its containment in the nanocontainer. The labelled protein was also used also to validate it as a nanoprobe for the detection of submillimeter nodules in the peritoneal cavity. This detection was made by in vivo fluorescence imaging in preclinical models of ovarian peritoneal carcinomatosis developed beforehand using established cell lines. The combination of these two properties of the protein (probe and container) permits to consider its use in intraperitoneal nanotheranostic. To confirm this promising development, it will be necessary to establish the proof of concept for theranostic aspects in mice models closer to clinic situations developed from patients' tumors (patient derived xenografts, PDX)

Continuous cultivation of photosynthetic microorganisms: approaches, applications and future trends

The possibility of using photosynthetic microorganisms, such as cyanobacteria and microalgae, for converting light and carbon dioxide into valuable biochemical products has raised the need for new cost-efficient processes ensuring a constant product quality. Food, feed, biofuels, cosmetics and pharmaceutics are among the sectors that can profit from the application of photosynthetic microorganisms. Biomass growth in a photobioreactor is a complex process influenced by multiple parameters, such as photosynthetic light capture and attenuation, nutrient uptake, photobioreactor hydrodynamics and gas-liquid mass transfer. In order to optimize productivity while keeping a standard product quality, a permanent control of the main cultivation parameters is necessary, where the continuous cultivation has shown to be the best option. However it is of utmost importance to recognize the singularity of continuous cultivation of cyanobacteria and microalgae due to their dependence on light availability and intensity. In this sense, this review provides comprehensive information on recent breakthroughs and possible future trends regarding technological and process improvements in continuous cultivation systems of microalgae and cyanobacteria, that will directly affect cost-effectiveness and product quality standardization. An overview of the various applications, techniques and equipment (with special emphasis on photobioreactors) in continuous cultivation of microalgae and cyanobacteria are presented. Additionally, mathematical modelling, feasibility, economics as well as the applicability of continuous cultivation into large-scale operation, are discussed.This research work was supported by the grant SFRH/BPD/98694/2013 (Bruno Fernandes) from Fundacao para a Ciencia e a Tecnologia (Portugal). The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013. The authors also thank the Project "BioInd Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDE

Morbidity and mortality after anaesthesia in early life: results of the European prospective multicentre observational study, neonate and children audit of anaesthesia practice in Europe (NECTARINE)

Background: Neonates and infants requiring anaesthesia are at risk of physiological instability and complications, but triggers for peri-anaesthetic interventions and associations with subsequent outcome are unknown. Methods: This prospective, observational study recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. The primary aim was to identify thresholds of pre-determined physiological variables that triggered a medical intervention. The secondary aims were to evaluate morbidities, mortality at 30 and 90 days, or both, and associations with critical events. Results: Infants (n=5609) born at mean (standard deviation [SD]) 36.2 (4.4) weeks postmenstrual age (35.7% preterm) underwent 6542 procedures within 63 (48) days of birth. Critical event(s) requiring intervention occurred in 35.2% of cases, mainly hypotension (>30% decrease in blood pressure) or reduced oxygenation (SpO2 <85%). Postmenstrual age influenced the incidence and thresholds for intervention. Risk of critical events was increased by prior neonatal medical conditions, congenital anomalies, or both (relative risk [RR]=1.16; 95% confidence interval [CI], 1.04–1.28) and in those requiring preoperative intensive support (RR=1.27; 95% CI, 1.15–1.41). Additional complications occurred in 16.3% of patients by 30 days, and overall 90-day mortality was 3.2% (95% CI, 2.7–3.7%). Co-occurrence of intraoperative hypotension, hypoxaemia, and anaemia was associated with increased risk of morbidity (RR=3.56; 95% CI, 1.64–7.71) and mortality (RR=19.80; 95% CI, 5.87–66.7). Conclusions: Variability in physiological thresholds that triggered an intervention, and the impact of poor tissue oxygenation on patient's outcome, highlight the need for more standardised perioperative management guidelines for neonates and infants. Clinical trial registration: NCT02350348

Morbidity and mortality after anaesthesia in early life: results of the European prospective multicentre observational study, neonate and children audit of anaesthesia practice in Europe (NECTARINE)

BACKGROUND: Neonates and infants requiring anaesthesia are at risk of physiological instability and complications, but triggers for peri-anaesthetic interventions and associations with subsequent outcome are unknown. METHODS: This prospective, observational study recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. The primary aim was to identify thresholds of pre-determined physiological variables that triggered a medical intervention. The secondary aims were to evaluate morbidities, mortality at 30 and 90 days, or both, and associations with critical events. RESULTS: Infants (n=5609) born at mean (standard deviation [sd]) 36.2 (4.4) weeks postmenstrual age (35.7% preterm) underwent 6542 procedures within 63 (48) days of birth. Critical event(s) requiring intervention occurred in 35.2% of cases, mainly hypotension (>30% decrease in blood pressure) or reduced oxygenation (SpO2 <85%). Postmenstrual age influenced the incidence and thresholds for intervention. Risk of critical events was increased by prior neonatal medical conditions, congenital anomalies, or both (relative risk [RR]=1.16; 95% confidence interval [CI], 1.04–1.28) and in those requiring preoperative intensive support (RR=1.27; 95% CI, 1.15–1.41). Additional complications occurred in 16.3% of patients by 30 days, and overall 90-day mortality was 3.2% (95% CI, 2.7–3.7%). Co-occurrence of intraoperative hypotension, hypoxaemia, and anaemia was associated with increased risk of morbidity (RR=3.56; 95% CI, 1.64–7.71) and mortality (RR=19.80; 95% CI, 5.87–66.7). CONCLUSIONS: Variability in physiological thresholds that triggered an intervention, and the impact of poor tissue oxygenation on patient's outcome, highlight the need for more standardised perioperative management guidelines for neonates and infants

Difficult tracheal intubation in neonates and infants. NEonate and Children audiT of Anaesthesia pRactice IN Europe (NECTARINE): a prospective European multicentre observational study

BACKGROUND: Neonates and infants are susceptible to hypoxaemia in the perioperative period. The aim of this study was to analyse interventions related to anaesthesia tracheal intubations in this European cohort and identify their clinical consequences. METHODS: We performed a secondary analysis of tracheal intubations of the European multicentre observational trial (NEonate and Children audiT of Anaesthesia pRactice IN Europe [NECTARINE]) in neonates and small infants with difficult tracheal intubation. The primary endpoint was the incidence of difficult intubation and the related complications. The secondary endpoints were the risk factors for severe hypoxaemia attributed to difficult airway management, and 30 and 90 day outcomes. RESULTS: Tracheal intubation was planned in 4683 procedures. Difficult tracheal intubation, defined as two failed attempts of direct laryngoscopy, occurred in 266 children (271 procedures) with an incidence (95% confidence interval [CI]) of 5.8% (95% CI, 5.1–6.5). Bradycardia occurred in 8% of the cases with difficult intubation, whereas a significant decrease in oxygen saturation (SpO2<90% for 60 s) was reported in 40%. No associated risk factors could be identified among co-morbidities, surgical, or anaesthesia management. Using propensity scoring to adjust for confounders, difficult anaesthesia tracheal intubation did not lead to an increase in 30 and 90 day morbidity or mortality. CONCLUSIONS: The results of the present study demonstrate a high incidence of difficult tracheal intubation in children less than 60 weeks post-conceptual age commonly resulting in severe hypoxaemia. Reassuringly, the morbidity and mortality at 30 and 90 days was not increased by the occurrence of a difficult intubation event

Difficult tracheal intubation in neonates and infants. NEonate and Children audiT of Anaesthesia pRactice IN Europe (NECTARINE): a prospective European multicentre observational study

Background: Neonates and infants are susceptible to hypoxaemia in the perioperative period. The aim of this study was to analyse interventions related to anaesthesia tracheal intubations in this European cohort and identify their clinical consequences. Methods: We performed a secondary analysis of tracheal intubations of the European multicentre observational trial (NEonate and Children audiT of Anaesthesia pRactice IN Europe [NECTARINE]) in neonates and small infants with difficult tracheal intubation. The primary endpoint was the incidence of difficult intubation and the related complications. The secondary endpoints were the risk factors for severe hypoxaemia attributed to difficult airway management, and 30 and 90 day outcomes. Results: Tracheal intubation was planned in 4683 procedures. Difficult tracheal intubation, defined as two failed attempts of direct laryngoscopy, occurred in 266 children (271 procedures) with an incidence (95% confidence interval [CI]) of 5.8% (95% CI, 5.1e6.5). Bradycardia occurred in 8% of the cases with difficult intubation, whereas a significant decrease in oxygen saturation (SpO2&lt;90% for 60 s) was reported in 40%. No associated risk factors could be identified among comorbidities, surgical, or anaesthesia management. Using propensity scoring to adjust for confounders, difficult anaesthesia tracheal intubation did not lead to an increase in 30 and 90 day morbidity or mortality. Conclusions: The results of the present study demonstrate a high incidence of difficult tracheal intubation in children less than 60 weeks post-conceptual age commonly resulting in severe hypoxaemia. Reassuringly, the morbidity and mortality at 30 and 90 days was not increased by the occurrence of a difficult intubation event. Clinical trial registration: NCT02350348

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Xerocomellus chrysenteron lectin, a theranostic nanotool for imaging and treatment of epithelial cancers : proof of concept applied to ovarian peritoneal carcinomatosis

Le développement de thérapies ciblées et des techniques d’imagerie est un défi majeur en santé, particulièrement dans les pathologies cancéreuses. Les carcinoses péritonéales sont habituellement causées par une dissémination de cellules tumorales au sein de la cavité abdominale, ce qui est le cas de 85% des patientes atteintes d'un cancer ovarien et plus de 10% des patients atteints d’un cancer colorectal. Dans les deux cas, les traitements consistent en une chirurgie (cytoréduction), aussi complète que possible, accompagnée de chimiothérapies. L'amélioration de la survie globale des patients passe par le développement de technologies parallèles comme de nouveaux outils diagnostiques pour détecter des implantations précoces dans le péritoine, le blocage de la dissémination de cellules cancéreuses pendant et après la chirurgie, ou encore la combinaison de chimiothérapies et de traitements ciblés intrapéritonéaux.Ce projet de thèse consiste à aborder ces différents aspects par l'utilisation d'un nanocontainer protéique multifonctionnel. L’objectif est d’optimiser cette protéine, appartenant à la famille des lectines, pour envisager son développement en tant qu'outil théranostique dans le cadre du diagnostic et des traitements de cancers épithéliaux. La lectine de Xerocomellus chrysenteron, à l’origine extraite d’un champignon supérieur comestible, présente une forte affinité pour un biomarqueur glycosidique des carcinomes, l’antigène de Thomsen-Friedenreich (antigène TF ou CD176). De plus, la présence d’une large cavité hydratée au centre de cet assemblage protéique (homotétramère) permet d’envisager le confinement et l’adressage spécifique de molécules thérapeutiques à des cellules épithéliales cancéreuses.Nous avons commencé par établir la preuve de concept de la délivrance ciblée de molécules thérapeutiques dans plusieurs lignées d’adénocarcinomes ovariens humains (OVCAR-3, SKOV-3, IGROV-1). Le marquage de la lectine dans le proche infrarouge a permis de confirmer le mécanisme de délivrance et prouver que la molécule thérapeutique avait bien été endocytée grâce à son confinement dans le nanocontainer. La protéine marquée a également été utilisée pour valider son utilisation comme nanosonde pour la détection de nodules tumoraux submillimétriques dans le péritoine. Cette détection est faite par imagerie de fluorescence in vivo dans des modèles précliniques de carcinose péritonéale ovarienne préalablement développés à partir de lignées cellulaires. La combinaison des deux propriétés de la protéine (sonde et container) permet d’envisager son utilisation en nanothéranostique intrapéritonéale. Afin de confirmer ce développement prometteur, il sera nécessaire d’établir la preuve de concept sur des modèles murins plus pertinents de la situation clinique développés à partir de tumeurs issues de patientes (Patient Derived Xenografts, PDX).The development of targeted therapy and imaging tools is a major challenge in human health, particularly in cancer pathologies. Peritoneal carcinomatosis is usually caused by scattering of cancer cells within the abdominal cavity, which is the case for 85% of ovarian cancer patients and more than 10% of colorectal cancer patients. In both cases treatments include a cytoreductive surgery, as complete as possible, and chemotherapies. Patients overall survival improvement can be reach with the development of parallel technologies such as new diagnostic tools to detect early implantations in the peritoneal cavity, agents to block the spreading of cancer cells detached during the surgical procedure, or combining chemotherapies and intraperitoneal targeted drug delivery.This project involves reaching all those aspects by using a unique multifunctional nanocontainer protein. The aim is to maximize this protein, which belongs to the lectin family, to consider its development as a theranostic tool as part of epithelial cancers diagnostic and treatment. Xerocomellus chrysenteron lectin, originally extracted from an edible higher mushroom, has a strong affinity for a carcinoma glycan biomarker, the Thomsen-Friedenreich antigen (TF antigen). Furthermore, a large hydrated inner cavity located in the middle of the tetrameric assembly of the protein led us to consider the containment and specific addressing of therapeutic molecules to epithelial cancerous cells expressing TF antigen. We first established the proof of concept for the targeted drug delivery of therapeutic molecules in several human ovarian adenocarcinoma cell lines (OVCAR-3, SKOV-3, IGROV-1). The labelling of the lectin in near infrared allowed us to confirm the mechanism implicated in the delivery and prove that the uptake of the molecule within the cells was due to its containment in the nanocontainer. The labelled protein was also used also to validate it as a nanoprobe for the detection of submillimeter nodules in the peritoneal cavity. This detection was made by in vivo fluorescence imaging in preclinical models of ovarian peritoneal carcinomatosis developed beforehand using established cell lines. The combination of these two properties of the protein (probe and container) permits to consider its use in intraperitoneal nanotheranostic. To confirm this promising development, it will be necessary to establish the proof of concept for theranostic aspects in mice models closer to clinic situations developed from patients’ tumors (patient derived xenografts, PDX)

Optimization of protein electroextraction from microalgae by a flow process

International audienceClassical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24 h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size