Metabolites of milk intake: a metabolomic approach in UK twins with findings replicated in two European cohorts

Purpose: Milk provides a significant source of calcium, protein, vitamins and other minerals to Western populations throughout life. Due to its widespread use, the metabolic and health impact of milk consumption warrants further investigation and biomarkers would aid epidemiological studies.  Methods: Milk intake assessed by a validated food frequency questionnaire was analyzed against fasting blood metabolomic profiles from two metabolomic platforms in females from the TwinsUK cohort (n = 3559). The top metabolites were then replicated in two independent populations (EGCUT, n = 1109 and KORA, n = 1593), and the results from all cohorts were meta-analyzed.  Results: Four metabolites were significantly associated with milk intake in the TwinsUK cohort after adjustment for multiple testing (P < 8.08 × 10−5) and covariates (BMI, age, batch effects, family relatedness and dietary covariates) and replicated in the independent cohorts. Among the metabolites identified, the carnitine metabolite trimethyl-N-aminovalerate (β = 0.012, SE = 0.002, P = 2.98 × 10−12) and the nucleotide uridine (β = 0.004, SE = 0.001, P = 9.86 × 10−6) were the strongest novel predictive biomarkers from the non-targeted platform. Notably, the association between trimethyl-N-aminovalerate and milk intake was significant in a group of MZ twins discordant for milk intake (β = 0.050, SE = 0.015, P = 7.53 × 10−4) and validated in the urine of 236 UK twins (β = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (β = 0.034, SE = 0.005, P = 9.75 × 10−14) and diacylphosphatidylcholine C28:1 (β = 0.034, SE = 0.004, P = 4.53 × 10−16), were also replicated.  Conclusions: We identified and replicated in independent populations four novel biomarkers of milk intake: trimethyl-N-aminovalerate, uridine, hydroxysphingomyelin C14:1 and diacylphosphatidylcholine C28:1. Together, these metabolites have potential to objectively examine and refine milk-disease associations

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Etude de l’impact des contraintes de croissance sur des micro-tumeurs

National audienceIn a solid tumor, cancer cells form a mass where the volume is spatially limited by the surrounding tissue. The tumor undergoes compressive stresses that originate from its microenvironment and are induced in particular by its growth. In the case of pancreatic cancer, these stresses are significant and can reach the kilopascal range. They have a considerable influence on cell division, growth and cell motility, potentially leading to the inefficiency of certain drugs.Despite the importance of these studies, few researches have been carried out into understanding the mechanisms inherent in the response under compression. We have developed microfluidic devices to spatially confining tumor subunit models, known as spheroids. Each of the culture chambers contains a sensor (membrane) sensitive to the pressure induced by cell growth. We have also initiated experiments on a promising treatment which could circumvent the apparent inefficacy of conventional therapies under mechanical stress: oncolytic viruses.Using the microfluidic device, we were able to culture spheroids for 5 days and measure growth-induced pressures ranging from 0 to 3kPa. Using a cell line labelled with the FUCCI vector for measuring the phases of the cell cycle, we observed an accumulation of cells in the G1 phase of the cell cycle under the effect of growth-induced pressure. We were also able to study the impact of this pressure on macromolecular crowding present in the cytoplasm of the cells by carrying out rheological measurements using nanoparticles genetically expressed by the cells (GEMs). We observed a decrease in nanoparticle diffusion as the growth-induced pressure increased in the culture chambers. These results suggest that the concentration of macromolecules increases in parallel with the growth-induced pressure.In order to characterize the growth of cells within a spheroid, we carried out simulations using a simple finite element model. It revealed the presence of a pressure gradient in a spheroid that likely facilitate cell division at its periphery. In addition, by immunofluorescence labelling of the Ki-67 protein, we observed a majority of dividing cells at the edges of spheroids. We also carried out rheological measurements on cells located at different positions along the radius of the spheroids. Results show a diffusion gradient ranging from 0.55µm2/s for cells at the edge of the spheroid to 0.3µm2/s for cells located at the heart of the spheroid.Finally, we were able to compare the infection dynamics of oncolytic viruses on cells grown in 2D on substrates of different rigidity. On plastic, the infection dynamics obtained was slightly faster than one on a soft substrate (E=0.5kPa). Furthermore, when the cells were cultured at 2D under the effect of mechanical compression, the results showed a significant delay (40h) on the beginning of infection.In conclusion, we were able to measure spheroid growth-induced pressure using a microfluidic chip and study its impact on cell growth and macromolecular crowding. Results on oncolytic viruses show that infection depends to a greater or lesser extent on the type of mechanical stress applied to the cells. This work provides a new methodological perspective on the spatial confinement of spheroids and raises further questions about our understanding of the mechanisms characterizing the cellular response under compression.Dans une tumeur solide, les cellules cancéreuses forment une masse dont le volume est limité spatialement par le tissu qui l’entoure. La tumeur subit des contraintes compressives qui proviennent de son microenvironnement et qui sont notamment induites par sa croissance. Dans le cas du cancer du pancréas, ces contraintes sont importantes et peuvent atteindre l’ordre du kilopascal. Elles ont une influence considérable sur la division cellulaire, la croissance ainsi que la motilité des cellules, entrainant potentiellement l’inefficacité de certains médicaments.Malgré l’importance de ces études, peu de recherches ont été menées sur la compréhension des mécanismes inhérents à la réponse sous compression. Nous avons mis au point des dispositifs microfluidiques permettant de confiner spatialement des modèles de sous-unités tumorales, appelés sphéroïdes. Chacune des chambres de culture comporte un capteur (membrane) sensible à la pression induite par la croissance des cellules. Par ailleurs, nous avons initié des essais sur un traitement prometteur qui pourrait permettre de contourner l’inefficacité sous contrainte mécanique de thérapies usuelles: les virus oncolytiques.Grâce au dispositif microfluidique, nous avons pu cultiver des sphéroïdes pendant 5 jours et mesurer des pressions de croissance allant de 0 à 3kPa. En utilisant une lignée cellulaire marquée avec le vecteur FUCCI permettant de mesurer les phases du cycle cellulaire, nous avons observé sous l’effet de la pression de croissance, une accumulation de cellules dans la phase G1 du cycle cellulaire. Nous avons également pu étudier l’impact de cette pression sur l’encombrement macromoléculaire présent dans le cytoplasme des cellules en réalisant des mesures rhéologiques, faites au moyen de nanoparticules génétiquement exprimées par les cellules (GEMs). Nous avons observé une diminution de la diffusion des nanoparticules à mesure que la pression de croissance augmentait dans les chambres de culture. Ces résultats suggèrent que la concentration en macromolécules augmente en parallèle de la pression de croissance.Afin de caractériser la croissance des cellules au sein d’un sphéroïde, nous avons réalisé des simulations à partir d’un modèle par éléments finis simple. Ce dernier met en évidence la présence d’un gradient de pression dans un sphéroïde qui faciliterait vraisemblablement la division des cellules en sa périphérie. De plus, en réalisant des marquages en immunofluorescence de la protéine Ki-67, nous avons observé une majorité de cellules en division en bordure des sphéroïdes. Nous avons également réalisé des mesures rhéologiques sur des cellules localisées à différentes positions le long du rayon des sphéroïdes. Les résultats montrent un gradient de diffusion, allant de 0,55µm2/s pour des cellules présentes au bords du sphéroïde à 0,3µm2/s pour des cellules situées au cœur du sphéroïde.Enfin, nous avons pu comparer la dynamique d’infection des virus oncolytiques sur des cellules cultivées à 2D sur des substrats de différentes rigidités. Sur du plastique, la dynamique d’infection obtenue est légèrement plus rapide que sur un substrat mou (E=0,5kPa). Par ailleurs, lorsque les cellules sont cultivées à 2D sous l’effet d’une compression mécanique, les résultats montrent un important retard (40h) sur le début de l’infection.Pour conclure, nous avons pu mesurer la pression de croissance des sphéroïdes au moyen d’une puce microfluidique et étudier son impact sur la croissance des cellules et l’encombrement macromoléculaire. Des résultats préliminaires sur les virus oncolytiques montrent une dépendance de l’infection plus ou moins forte en fonction du type de contraintes mécaniques appliquées sur les cellules. Ces travaux apportent une nouvelle perspective méthodologique sur le confinement spatial de sphéroïdes et soulèvent des questions supplémentaires quant à la compréhension des mécanismes caractérisant la réponse cellulaire sous compression

Etude de l’impact des contraintes de croissance sur des micro-tumeurs

National audienceIn a solid tumor, cancer cells form a mass where the volume is spatially limited by the surrounding tissue. The tumor undergoes compressive stresses that originate from its microenvironment and are induced in particular by its growth. In the case of pancreatic cancer, these stresses are significant and can reach the kilopascal range. They have a considerable influence on cell division, growth and cell motility, potentially leading to the inefficiency of certain drugs.Despite the importance of these studies, few researches have been carried out into understanding the mechanisms inherent in the response under compression. We have developed microfluidic devices to spatially confining tumor subunit models, known as spheroids. Each of the culture chambers contains a sensor (membrane) sensitive to the pressure induced by cell growth. We have also initiated experiments on a promising treatment which could circumvent the apparent inefficacy of conventional therapies under mechanical stress: oncolytic viruses.Using the microfluidic device, we were able to culture spheroids for 5 days and measure growth-induced pressures ranging from 0 to 3kPa. Using a cell line labelled with the FUCCI vector for measuring the phases of the cell cycle, we observed an accumulation of cells in the G1 phase of the cell cycle under the effect of growth-induced pressure. We were also able to study the impact of this pressure on macromolecular crowding present in the cytoplasm of the cells by carrying out rheological measurements using nanoparticles genetically expressed by the cells (GEMs). We observed a decrease in nanoparticle diffusion as the growth-induced pressure increased in the culture chambers. These results suggest that the concentration of macromolecules increases in parallel with the growth-induced pressure.In order to characterize the growth of cells within a spheroid, we carried out simulations using a simple finite element model. It revealed the presence of a pressure gradient in a spheroid that likely facilitate cell division at its periphery. In addition, by immunofluorescence labelling of the Ki-67 protein, we observed a majority of dividing cells at the edges of spheroids. We also carried out rheological measurements on cells located at different positions along the radius of the spheroids. Results show a diffusion gradient ranging from 0.55µm2/s for cells at the edge of the spheroid to 0.3µm2/s for cells located at the heart of the spheroid.Finally, we were able to compare the infection dynamics of oncolytic viruses on cells grown in 2D on substrates of different rigidity. On plastic, the infection dynamics obtained was slightly faster than one on a soft substrate (E=0.5kPa). Furthermore, when the cells were cultured at 2D under the effect of mechanical compression, the results showed a significant delay (40h) on the beginning of infection.In conclusion, we were able to measure spheroid growth-induced pressure using a microfluidic chip and study its impact on cell growth and macromolecular crowding. Results on oncolytic viruses show that infection depends to a greater or lesser extent on the type of mechanical stress applied to the cells. This work provides a new methodological perspective on the spatial confinement of spheroids and raises further questions about our understanding of the mechanisms characterizing the cellular response under compression.Dans une tumeur solide, les cellules cancéreuses forment une masse dont le volume est limité spatialement par le tissu qui l’entoure. La tumeur subit des contraintes compressives qui proviennent de son microenvironnement et qui sont notamment induites par sa croissance. Dans le cas du cancer du pancréas, ces contraintes sont importantes et peuvent atteindre l’ordre du kilopascal. Elles ont une influence considérable sur la division cellulaire, la croissance ainsi que la motilité des cellules, entrainant potentiellement l’inefficacité de certains médicaments.Malgré l’importance de ces études, peu de recherches ont été menées sur la compréhension des mécanismes inhérents à la réponse sous compression. Nous avons mis au point des dispositifs microfluidiques permettant de confiner spatialement des modèles de sous-unités tumorales, appelés sphéroïdes. Chacune des chambres de culture comporte un capteur (membrane) sensible à la pression induite par la croissance des cellules. Par ailleurs, nous avons initié des essais sur un traitement prometteur qui pourrait permettre de contourner l’inefficacité sous contrainte mécanique de thérapies usuelles: les virus oncolytiques.Grâce au dispositif microfluidique, nous avons pu cultiver des sphéroïdes pendant 5 jours et mesurer des pressions de croissance allant de 0 à 3kPa. En utilisant une lignée cellulaire marquée avec le vecteur FUCCI permettant de mesurer les phases du cycle cellulaire, nous avons observé sous l’effet de la pression de croissance, une accumulation de cellules dans la phase G1 du cycle cellulaire. Nous avons également pu étudier l’impact de cette pression sur l’encombrement macromoléculaire présent dans le cytoplasme des cellules en réalisant des mesures rhéologiques, faites au moyen de nanoparticules génétiquement exprimées par les cellules (GEMs). Nous avons observé une diminution de la diffusion des nanoparticules à mesure que la pression de croissance augmentait dans les chambres de culture. Ces résultats suggèrent que la concentration en macromolécules augmente en parallèle de la pression de croissance.Afin de caractériser la croissance des cellules au sein d’un sphéroïde, nous avons réalisé des simulations à partir d’un modèle par éléments finis simple. Ce dernier met en évidence la présence d’un gradient de pression dans un sphéroïde qui faciliterait vraisemblablement la division des cellules en sa périphérie. De plus, en réalisant des marquages en immunofluorescence de la protéine Ki-67, nous avons observé une majorité de cellules en division en bordure des sphéroïdes. Nous avons également réalisé des mesures rhéologiques sur des cellules localisées à différentes positions le long du rayon des sphéroïdes. Les résultats montrent un gradient de diffusion, allant de 0,55µm2/s pour des cellules présentes au bords du sphéroïde à 0,3µm2/s pour des cellules situées au cœur du sphéroïde.Enfin, nous avons pu comparer la dynamique d’infection des virus oncolytiques sur des cellules cultivées à 2D sur des substrats de différentes rigidités. Sur du plastique, la dynamique d’infection obtenue est légèrement plus rapide que sur un substrat mou (E=0,5kPa). Par ailleurs, lorsque les cellules sont cultivées à 2D sous l’effet d’une compression mécanique, les résultats montrent un important retard (40h) sur le début de l’infection.Pour conclure, nous avons pu mesurer la pression de croissance des sphéroïdes au moyen d’une puce microfluidique et étudier son impact sur la croissance des cellules et l’encombrement macromoléculaire. Des résultats préliminaires sur les virus oncolytiques montrent une dépendance de l’infection plus ou moins forte en fonction du type de contraintes mécaniques appliquées sur les cellules. Ces travaux apportent une nouvelle perspective méthodologique sur le confinement spatial de sphéroïdes et soulèvent des questions supplémentaires quant à la compréhension des mécanismes caractérisant la réponse cellulaire sous compression

Lithographie laser bi-photons pour l'impression de moules PDMS dédiés à des applications micro-fluidique

National audienceLes moules utilisés pour la fabrication de puces micro-fluidiques sont en général fabriqués à partir de la mise en forme de films secs ou résine SU-8 en photolithographie UV. Pour certaines géométries de moule cette technique n’est pas la mieux appropriée. On observe des limites au respect des côtes et des difficultés d’alignement des géométries. Pour améliorer la qualité des moules nous avons développé un nouveau procédé de fabrication par photo-polymérisation bi-photons de la résine IP-S sur silicium. Après avoir optimisé les stratégies de détection d’interface et de raccordement de champs, nous avons fabriqué des moules de longueur supérieure à 8 mm et comportant des flancs de hauteurs comprises entre 2 et 530 µm

Lithographie laser bi-photons pour l'impression de moules PDMS dédiés à des applications micro-fluidique

National audienceLes moules utilisés pour la fabrication de puces micro-fluidiques sont en général fabriqués à partir de la mise en forme de films secs ou résine SU-8 en photolithographie UV. Pour certaines géométries de moule cette technique n’est pas la mieux appropriée. On observe des limites au respect des côtes et des difficultés d’alignement des géométries. Pour améliorer la qualité des moules nous avons développé un nouveau procédé de fabrication par photo-polymérisation bi-photons de la résine IP-S sur silicium. Après avoir optimisé les stratégies de détection d’interface et de raccordement de champs, nous avons fabriqué des moules de longueur supérieure à 8 mm et comportant des flancs de hauteurs comprises entre 2 et 530 µm

Time-resolved X-ray absorption spectroelectrochemistry of redox active species in solution

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The microfluidic laboratory at Synchrotron SOLEIL Chaussavoine Igor

International audienceA microfluidic laboratory recently opened at Synchrotron SOLEIL, dedicated to in-house research and external users. Its purpose is to provide the equipment and expertise that allow the development of microfluidic systems adapted to the beamlines of SOLEIL as well as other light sources. Such systems can be used to continuously deliver a liquid sample under a photon beam, keep a solid sample in a liquid environment or provide a means to track a chemical reaction in a time-resolved manner. The laboratory provides all the amenities required for the design and preparation of soft-lithography microfluidic chips compatible with synchrotron-based experiments. Three examples of microfluidic systems that were used on SOLEIL beamlines are presented, which allow the use of X-ray techniques to study physical, chemical or biological phenomena. © 2020 International Union of Crystallography

A microfluidic mechano-chemostat for tissues and organisms reveals that confined growth is accompanied with increased macromolecular crowding

International audienceConventional culture conditions are oftentimes insufficient to study tissues, organisms, or 3D multicellularassemblies. They lack both dynamic chemical and mechanical control over the microenvironment. While specific microfluidic devices have been developed to address chemical control, they often do not allow the control of compressive forces emerging when cells proliferate in a confined environment. Here, we present a generic microfluidic device to control both chemical and mechanical compressive forces. This device relies on the use of sliding elements consisting of microfabricated rods that can be inserted inside a microfluidic device. Sliding elements enable the creation of reconfigurable closed culture chambers for the study of whole organisms or model micro-tissues. By confining the micro-tissues, we studied the biophysical impact of growth-induced pressure and showed that this mechanical stress is associated with an increase in macromolecular crowding, shedding light on this understudied type of mechanical stress. Our mechano-chemostat allows the long-term culture of biological samples and can be used to study both the impact of specific conditions as well as the consequences of mechanical compression

EPAC1 Inhibition Protects the Heart from Doxorubicin-Induced Toxicity

Summary Rationale The widely used chemotherapeutic agent Doxorubicin (Dox) induces cardiotoxicity leading to dilated cardiomyopathy and heart failure. This cardiotoxicity has been related to ROS generation, DNA intercalation, bioenergetic distress and cell death. However, alternative mechanisms are emerging, focusing on signaling pathways. Objective We investigated the role of Exchange Protein directly Activated by cAMP (EPAC), key factor in cAMP signaling, in Dox-induced cardiotoxicity. Methods and Results Dox was administrated in vivo (10 ± 2 mg/kg, i . v .; with analysis at 2, 6 and 15 weeks post injection) in WT and EPAC1 KO C57BL6 mice. Cardiac function was analyzed by echocardiography and intracellular Ca 2+ homeostasis by confocal microscopy in isolated ventricular cardiomyocytes. 15 weeks post-injections, Dox-treated WT mice, developed a dilated cardiomyopathy with decreased ejection fraction, increased telediastolic volume and impaired Ca 2+ homeostasis, which were totally prevented in the EPAC1 KO mice. The underlying mechanisms were investigated in neonatal and adult rat cardiac myocytes under Dox treatment (1-10 μM). Flow cytometry, Western blot, BRET sensor assay, and RT-qPCR analysis showed that Dox induced DNA damage and cardiomyocyte cell death with apoptotic features rather than necrosis, including Ca 2+ -CaMKKβ-dependent opening of the Mitochondrial Permeability Transition Pore, dissipation of the Mitochondrial membrane potential (Δψm), caspase activation, cell size reduction, and DNA fragmentation. Dox also led to an increase in both cAMP concentration and EPAC1 protein level and activity. The pharmacological inhibition of EPAC1 (CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations including DNA damage, Δψm, apoptosis, mitochondrial biogenesis, dynamic, and fission/fusion balance, and respiratory chain activity, suggesting a crucial role of EPAC1 in these processes. Importantly, while preserving cardiomyocyte integrity, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Conclusion Thus, EPAC1 inhibition could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity