Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

BACKGROUND: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. RESULTS: We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays. CONCLUSION: These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b

Computational Models for Prediction of Yeast Strain Potential for Winemaking from Phenotypic Profiles

Saccharomyces cerevisiae strains from diverse natural habitats harbour a vast amount of phenotypic diversity, driven by interactions between yeast and the respective environment. In grape juice fermentations, strains are exposed to a wide array of biotic and abiotic stressors, which may lead to strain selection and generate naturally arising strain diversity. Certain phenotypes are of particular interest for the winemaking industry and could be identified by screening of large number of different strains. The objective of the present work was to use data mining approaches to identify those phenotypic tests that are most useful to predict a strain's potential for winemaking. We have constituted a S. cerevisiae collection comprising 172 strains of worldwide geographical origins or technological applications. Their phenotype was screened by considering 30 physiological traits that are important from an oenological point of view. Growth in the presence of potassium bisulphite, growth at 40 degrees C, and resistance to ethanol were mostly contributing to strain variability, as shown by the principal component analysis. In the hierarchical clustering of phenotypic profiles the strains isolated from the same wines and vineyards were scattered throughout all clusters, whereas commercial winemaking strains tended to co-cluster. Mann-Whitney test revealed significant associations between phenotypic results and strain's technological application or origin. Naive Bayesian classifier identified 3 of the 30 phenotypic tests of growth in iprodion (0.05 mg/mL), cycloheximide (0.1 mu g/mL) and potassium bisulphite (150 mg/mL) that provided most information for the assignment of a strain to the group of commercial strains. The probability of a strain to be assigned to this group was 27% using the entire phenotypic profile and increased to 95%, when only results from the three tests were considered. Results show the usefulness of computational approaches to simplify strain selection procedures.Ines Mendes and Ricardo Franco-Duarte are recipients of a fellowship from the Portuguese Science Foundation, FCT (SFRH/BD/74798/2010, SFRH/BD/48591/2008, respectively) and Joao Drumonde-Neves is recipient of a fellowship from the Azores government (M3.1.2/F/006/2008 (DRCT)). Financial support was obtained from FEDER funds through the program COMPETE and by national funds through FCT by the projects FCOMP-01-0124-008775 (PTDC/AGR-ALI/103392/2008) and PTDC/AGR-ALI/121062/2010. Lan Umek and Blaz Zupan acknowledge financial support from Slovene Research Agency (P2-0209). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence for Origin-Dependent Metabolic Traits

The species Saccharomyces cerevisiae includes natural strains, clinical isolates, and a large number of strains used in human activities. The aim of this work was to investigate how the adaptation to a broad range of ecological niches may have selectively shaped the yeast metabolic network to generate specific phenotypes. Using 72 S. cerevisiae strains collected from various sources, we provide, for the first time, a population-scale picture of the fermentative metabolic traits found in the S. cerevisiae species under wine making conditions. Considerable phenotypic variation was found suggesting that this yeast employs diverse metabolic strategies to face environmental constraints. Several groups of strains can be distinguished from the entire population on the basis of specific traits. Strains accustomed to growing in the presence of high sugar concentrations, such as wine yeasts and strains obtained from fruits, were able to achieve fermentation, whereas natural yeasts isolated from “poor-sugar” environments, such as oak trees or plants, were not. Commercial wine yeasts clearly appeared as a subset of vineyard isolates, and were mainly differentiated by their fermentative performances as well as their low acetate production. Overall, the emergence of the origin-dependent properties of the strains provides evidence for a phenotypic evolution driven by environmental constraints and/or human selection within S. cerevisiae

La conception de la santé chez des enfants de 5 à 12 ans. Quelques clés pour la promotion de la santé

Sylvie Jutras et Jocelyn Bisson: The conception of health of children aged 5 to 12 years old: considerations for health promotion A study of the perceptual field of health was conducted to describe the conception of health of over 500 children aged 5 to 12 years old. Even though health conception seems to develop and to become more subtle with age, ail children, including the 5 and 6 years old, demonstrated a clear conception of health. Functionality, well-being and the practice of a healthy life style are the most significant elements of this conception. More than a quarter of the children, and in the same proportions in the three age groups, have a concept of mental health, suggesting the presence of parental influence in this regard. Healthy eating habits are most important amongst essential conducts to be healthy. Consequences of being healthy are mainly associated with better functioning and well-being. Overall, an unhealthy life style constitutes the principal threat to health. For the youngest, threats to health corne from poor eating habits, for those aged 9 to 12 years old, from drug addiction and smoking. Only a few gender differences are significant. Children with recurrent health problems are not different from healthy children. This study also reflects preventive messages transmitted by social agents, in which seem to dominate the importance of good eating habits, the dangers of drug addiction, the lack of awareness of the potential threat of injuries, and a vision of mental health as a consequence and not as a process on which one has control.Résumé. Une étude sur le champ perceptuel de la santé a permis de décrire la conception de la santé de plus de cinq cents enfants de cinq à douze ans. Même si elle semble se développer et se raffiner avec l'âge, tous les enfants ont une conception claire de la santé. La fonctionnalité, le bien-être et la pratique de saines habitudes de vie en sont les éléments saillants. La notion de santé mentale est présente chez plus du quart des enfants, et au même degré dans les trois groupes d'âge, suggérant la présence d'une influence familiale à cet égard. L'alimentation domine les conduites essentielles. Les conséquences d'une bonne santé sont principalement associées à un meilleur fonctionnement et au bien-être. Les mauvaises habitudes de vie constituent la principale menace. Chez les petits, elles sont liées à la pratique de mauvaises habitudes alimentaires ; chez ceux de neuf à douze ans, à la drogue, suivie du tabagisme. Très peu de différences inter-genres sont apparues, tandis que les enfants éprouvant de façon régulière un problème de santé ne se sont pas distingués des autres. L'étude a permis de refléter les messages préventifs transmis par les agents de socialisation, où semblent dominer l'importance de l'alimentation et de la toxicomanie, la négligence en matière de traumatisme et la vision de la santé mentale comme un résultat et non un processus sur lequel on peut avoir prise.Sylvie Jutras et Jocelyn Bisson : La concepción de la salud en los niños de 5 a 12 anos. Algunas claves para la promoción de la salud. Un estudio desarrolado en el campo de la percepción de la salud ha permitido describir la concepción de la salud en mas de quinientos niños entre cinco y doce años. Aun cuando parezca desarrolarse y afinarse con la edad, todos los niños tienen una concepción clara de la salud. La funcionalidad, el bienestar y la práctica de hábitos sanos de vida constituyen sus elementos primordiales. La noción de salud mental présente en mas de un cuarto de los niños estudiados, y en un mismo grado en los très grupos de edad, sugiere la presencia de una influencia familiar en este aspecto. La alimentación domina las conductas esenciales. La consecuencias de una buena salud están asociadas con un mejor funcionamiento y al bienestar. Los malos hábitos de vida constituyen la amenaza principal. En los niños mas pequeños, esos están ligados con los malos hábitos alimenticios, mientras que en los niños entre nueve y doce anos, lo son a la droga, seguida del tabaquismo. Muy pocas diferencias entre sexos aparecen, mientras que los ninos que se encuentran enfermos regularmente no se distinguen de los demás. El estudio ha puesto de relieve los mensajes de prevención transmitidos por los agentes de socialización, donde domina la importancia de la alimentación y de la toxicomania, la negligencia en materia de traumatismos y la percepción de la salud mental como un resultado y no como un proceso que se pueda controlar.Jutras Sylvie, Bisson Jocelyn. La conception de la santé chez des enfants de 5 à 12 ans. Quelques clés pour la promotion de la santé. In: Sciences sociales et santé. Volume 12, n°2, 1994. pp. 5-37

Prevalence and Associated Factors of Self-Reported Gingival Bleeding: A Multicenter Study in France

International audienceGingival bleeding (GB) is a common sign of gingival inflammation which indicates the presence of periodontal diseases. This cross-sectional multicenter survey aimed to assess the prevalence of self-reported gingival bleeding (SRGB) in French adults and identify the main associated factors. A questionnaire-based interview was randomly proposed to 794 individuals in four French cities (Nancy, Montpellier, Paris, and Rennes). Subjects were recruited in preventive medicine centers (50%), railway stations, and malls (50%). The questionnaire comprised 25 items: SRGB characteristics, socioeconomic variables, oral hygiene habits, use of drugs, and anxiety level. The overall prevalence of SRGB was 63.2% [59.8%; 66.6%], with 58.7% bleeding after toothbrushing and 4.5% spontaneous bleeding. Males reported significantly lower SRGB prevalence than females (p = 0.04). The distribution of SRGB frequency was inversely proportional to age (p < 0.0001). No association between drug use and SRGB was found. The people interviewed in the preventive medicine centers reported the highest frequency of SRGB (p < 0.0001). In the multivariate logistic model, SRGB was significantly related to occupation, smoking status, brushing frequency, and anxiety level. In conclusion, SRGB was prevalent in more than half of the sample and was mainly associated with age, toothbrushing frequency, and anxiety level. Thus, providing information to patients about the importance of this oral manifestation may play an important role in preventing periodontal diseases

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Abstract Background Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. Results We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays. Conclusion These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.</p

Perception of Gingival Bleeding by People and Healthcare Professionals: A Multicentre Study in an Adult French Population

International audienceGingival bleeding (GB) is a common sign of gingival inflammation, which indicates the presence of periodontal diseases. This study aimed to describe the perception of French adults about their self-reported GB and answers of healthcare professionals regarding the GB reported by these interviewees. A questionnaire administered by one investigator in each of three public settings of four cities in France from September 2016 to November 2017. Among 794 adults interviewed, 502 (63.2%) reported a GB. Among them, 414 (82.5%) believed that GB is benign, and 309 (61.6%) declared one or more responses. The three main responses were to use mouthwash (29.3%), to change to a soft-bristle toothbrush (20.1%) and to modify the brushing technique (19.3%). Almost half (49.0%) questioned at least one healthcare professional concerning their GB: a dentist (43.0%), a physician (14.1%), and a pharmacist (8.0%). The main response of each healthcare professional was: for dentists: a "prescription of mouthwash", for physicians to say "gingival bleeding is not serious"; and for pharmacists: "to sell a mouthwash". Most of the participants considered their GB as benign and had inappropriate responses, which indicates their lack of knowledge regarding periodontal health. The same conclusions can be drawn for healthcare professionals, as reported by interviewees

Comprendre les défis associés à la formation en allaitement et les barrières aux changements curriculaires : une perspective systémique pour transformer la formation des professionnels de la santé

Objectives: A majority of women and families wish that their babies be breastfed. However, too many still receive insufficient or inappropriate initial care from health professionals (HPs) who have limited breastfeeding (BF) competencies. We investigated barriers and potential solutions to improve the undergraduate training programs for various HPs. Methods: Focus groups were carried out in three universities in Quebec and one in Ontario (Canada), with 30 faculty and program directors from medicine, midwifery, nursing, nutrition, and pharmacy. Discussions were subjected to thematic content analysis, before being validated in a strategic planning workshop with 48 participants from the same disciplines, plus dentistry and chiropractic. Findings: Substantive improvements of undergraduate training programs for BF could be obtained by addressing challenges related to the insufficient, or lack of, (i) interactions among various HPs, (ii) opportunities for practical learning, (iii) specific standards to guide course content, (iv) real-life experience with counselling, and (v) understanding of the influence of attitudes on professional practice. Several potential solutions were proposed and validated. The re-interpretation of the results in light of various literature led to an emerging framework that takes a systems perspective for enhancing the education of HPs on BF. Conclusions: To improve the education of HPs so as to enable them to provide relevant support for future mothers, mothers and their families, solutions need to be carried out to address challenges in the health system, the education system as well as regarding the curricular change process.Objectifs : La majorité des femmes et des familles souhaitent que leur bébé soit allaité. Toutefois, plusieurs ne reçoivent pas un soutien adéquat de la part de professionnels de la santé (PS) qui ont des compétences limitées en allaitement. Nous avons étudié les barrières et les solutions potentielles en vue de rehausser la formation initiale de divers PS. Méthodes : Des groupes de discussion ont été organisés dans trois universités du Québec et une en Ontario (Canada) avec 30 directeurs de programmes et membres du corps professoral en médecine, pratique sage-femme, sciences infirmières, nutrition et pharmacie. Les discussions ont fait l’objet d’une analyse de contenu thématique laquelle fut ensuite validée dans un atelier de planification stratégique avec 48 participants des mêmes disciplines auxquelles se sont ajoutées dentisterie et chiropratique. Résultats : Des améliorations substantielles des compétences en allaitement dans les programmes de formation initiale pourraient être obtenues en travaillant sur les défis associés à l’insuffisance, ou à l’absence de, (i) interactions entre les divers PS, (ii) opportunités d’apprentissages pratiques, (iii) normes spécifiques pour guider les contenus de cours, (iv) expériences réelles avec le counseling, et (v) compréhension de l’influence des attitudes sur la pratique professionnelle. La ré-interprétation des résultats à la lumière de la littérature a fait émerger un cadre conceptuel avec une perspective systémique pour guider le rehaussement de la formation en allaitement des divers PS. Conclusions : Afin d’améliorer la formation des PS pour qu’ils/elles puissent fournir un soutien pertinent aux futures mères, aux mères et à leurs familles, des solutions visant à la fois les défis dans le système de santé, dans le système d’éducation et dans le processus de changement curriculaire doivent être mises en œuvre

Understanding the challenges related to breastfeeding education and barriers to curricular change: a systems perspective for transforming health professions education

Objectives: A majority of women and families wish that their babies be breastfed. However, too many still receive insufficient or inappropriate initial care from health professionals (HPs) who have limited breastfeeding (BF) competencies. We investigated barriers and potential solutions to improve the undergraduate training programs for various HPs.Methods: Focus groups were carried out in three universities in Quebec and one in Ontario (Canada), with 30 faculty and program directors from medicine, midwifery, nursing, nutrition, and pharmacy. Discussions were subjected to thematic content analysis, before being validated in a strategic planning workshop with 48 participants from the same disciplines, plus dentistry and chiropractic.Findings: Substantive improvements of undergraduate training programs for BF could be obtained by addressing challenges related to the insufficient, or lack of, (i) interactions among various HPs, (ii) opportunities for practical learning, (iii) specific standards to guide course content, (iv) real-life experience with counselling, and (v) understanding of the influence of attitudes on professional practice. Several potential solutions were proposed and validated. The re-interpretation of the results in light of various literature led to an emerging framework that takes a systems perspective for enhancing the education of HPs on BF.Conclusions: To improve the education of HPs so as to enable them to provide relevant support for future mothers, mothers and their families, solutions need to be carried out to address challenges in the health system, the education system as well as regarding the curricular change process.Objectifs : La majorité des femmes et des familles souhaitent que leur bébé soit allaité. Toutefois, plusieurs ne reçoivent pas un soutien adéquat de la part de professionnels de la santé (PS) qui ont des compétences limitées en allaitement. Nous avons étudié les barrières et les solutions potentielles en vue de rehausser la formation initiale de divers PS.Méthodes : Des groupes de discussion ont été organisés dans trois universités du Québec et une en Ontario (Canada) avec 30 directeurs de programmes et membres du corps professoral en médecine, pratique sage-femme, sciences infirmières, nutrition et pharmacie. Les discussions ont fait l’objet d’une analyse de contenu thématique laquelle fut ensuite validée dans un atelier de planification stratégique avec 48 participants des mêmes disciplines auxquelles se sont ajoutées dentisterie et chiropratique.Résultats : Des améliorations substantielles des compétences en allaitement dans les programmes de formation initiale pourraient être obtenues en travaillant sur les défis associés à l’insuffisance, ou à l’absence de, (i) interactions entre les divers PS, (ii) opportunités d’apprentissages pratiques, (iii) normes spécifiques pour guider les contenus de cours, (iv) expériences réelles avec le counseling, et (v) compréhension de l’influence des attitudes sur la pratique professionnelle. La ré-interprétation des résultats à la lumière de la littérature a fait émerger un cadre conceptuel avec une perspective systémique pour guider le rehaussement de la formation en allaitement des divers PS.Conclusions : Afin d’améliorer la formation des PS pour qu’ils/elles puissent fournir un soutien pertinent aux futures mères, aux mères et à leurs familles, des solutions visant à la fois les défis dans le système de santé, dans le système d’éducation et dans le processus de changement curriculaire doivent être mises en œuvre