Acceptability of insect ingredients by innovative student chefs : an exploratory study

Background: In Western societies, the acceptability of entomophagy is low despite the sustainable and nutritional benefits of insects. It is recognized that insect meals incorporated in into familiar foods increases willingness to eat insects. Chefs can offer positive culinary insect-based experiences to their customers which can then contribute to increasing the acceptability of entomophagy by consumers. However, little is known about chefs' perceptions of the use of insect-based ingredients. Objective: The aim of this study was to explore the reasons why innovative student chefs are willing (or not) to incorporate mealworms meals into their dishes. Methodology: Semi-structured interviews were conducted with 7 innovative student chefs at the Institut de tourisme et d'hôtellerie du Québec (ITHQ). Thematic analysis based on a priori Rogers' Diffusion of Innovation Theory was conducted using transcript verbatim. Results: Most participants had a past consumption experience with entomophagy and all of them had a positive attitude toward this practice. The main perceived disadvantages of mealworm meal was the texture (granular and uneven), the odor as well as the low acceptability by consumers. Despite that, student chefs were generally willing to use insect-based ingredients, but they thought that transparency and more opportunities for consumers to try good insect-based dishes are keys to enhancing the acceptability of insect consumption. Conclusion: Understanding perceptions of innovative chefs about the use of insect-based ingredients can help to promote their use in gastronomy and ultimately improve their acceptability by consumers

Breast Cancer Risk Estimation and Personal Insurance: A Qualitative Study Presenting Perspectives from Canadian Patients and Decision Makers

Genetic stratification approaches in personalized medicine may considerably improve our ability to predict breast cancer risk for women at higher risk of developing breast cancer. Notwithstanding these advantages, concerns have been raised about the use of the genetic information derived in these processes, outside of the research and medical health care settings, by third parties such as insurers. Indeed, insurance applicants are asked to consent to insurers accessing their medical information (implicitly including genetic) to verify or determine their insurability level, or eligibility to certain insurance products. This use of genetic information may result in the differential treatment of individuals based on their genetic information, which could lead to higher premium, exclusionary clauses or even the denial of coverage. This phenomenon has been commonly referred to as “Genetic Discrimination” (GD). In the Canadian context, where federal Bill S-201, An Act to prohibit and prevent genetic discrimination, has recently been enacted but may be subject to constitutional challenges, information about potential risks to insurability may raise issues in the clinical context. We conducted a survey with women in Quebec who have never been diagnosed with breast cancer to document their perspectives. We complemented the research with data from 14 semi-structured interviews with decision-makers in Quebec to discuss institutional issues raised by the use of genetic information by insurers. Our results provide findings on five main issues: (1) the reluctance to undergo genetic screening test due to insurability concerns, (2) insurers' interest in genetic information, (3) the duty to disclose genetic information to insurers, (4) the disclosure of potential impacts on insurability before genetic testing, and (5) the status of genetic information compared to other health data. Overall, both groups of participants (the women surveyed and the decision-makers interviewed) acknowledged having concerns about GD and reported a need for better communication tools discussing insurability risk. Our conclusions regarding concerns about GD and the need for better communication tools in the clinical setting may be transferable to the broader Canadian context

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and β1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD

Développement de méthodes de production et d'analyse de vecteurs recombinants dérivés de l'adéno-associated virus

De nombreuses études in vivo ont montré que les vecteurs recombinants dérivés de l'adéno-associated virus (AAVr) peuvent transduire efficacement de nombreux organes et conduisent à une expression stable et à long terme du gène. Ces résultats encourageants ont conduit au développement rapide des essais cliniques utilisant les vecteurs AAVr-2. Cependant ces essais chez l'homme nécessitent de grandes quantités de vecteurs AAVr. Les techniques conventionnelles de production et purification montrent alors leurs limites. Nous avons donc développé des méthodes de productions alternatives plus simples et reproductibles, en établissant des lignées stables, d'encapsidation, contenant intégré dans leur génome les gènes rep-cap hybrides pour les sérotypes-1 et -5. Nous avons aussi décrit une procédure de purification par chromatographie liquide basée sur des résines échangeuses d'ions. Cette procédure a l'avantage d'être directement applicable à grande échelle, et pourrait s'adapter à différents sérotypes. Cette méthode permet de purifier le sérotype-5, le plus divergent au niveau de la séquence protéique de la capside comparée à 1'AAVr-2. Les stocks d'AAVr obtenus présentent une pureté supérieure aux méthodes traditionnelles et transduisent aussi efficacement les organes cibles. Afin de mieux caractériser et différentier les capsides AAVr des différents sérotypes de plus en plus nombreux, nous avons montré que la digestion protéolytique des virions AAVr-2, donne un profil de digestion unique. Cette analyse protéolytique des capsides AAVr permet de distinguer les AAVr-2 des AAV-1 et -5.Numerous in vivo studies have demonstrated that rAAV vectors can efficiently transduce many tissues and lead to stable gene expression. These encouraging results have led to a rapid development of clinical trials involving the use of rAAV-2 vectors. However, the obtainment of large-scale rAAV-2 vector stocks for clinical assay is still hampered by the conventional production methods that are not efficient and difficult to scale-up. We develop a simple and reproducible alternative method based on stable packaging hybrid cell lines containing integrated into their genome the rep-cap genes of serotypes -1 and -5. Another critical step for the assessment of these vectors in clinical trials is the method used for purification of rAAV particles. We developed a purification method, which comprises two-step chromatography process based on the use of ion- exchange resins. This process can be easily scaled up and could be applied to different AAV serotypes. We purified the serotype-5, the most divergent in terms of capsid composition compared to AAV-2. The rAAV stocks obtained are pure and transduced efficiently target tissues. To characterize rAAV capsid from different serotype, we have shown that proteolytic digestion of rAAV virions is able to generate a characteristic cleavage pattern that can be use to distinguish AAV-2 from AAV-1 and -5.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Use of Adeno-Associated Virus to Enrich Cardiomyocytes Derived from Human Stem Cells

International audienceCardiomyocytes derived from human induced pluripotent stem cells (iPSCs) show great promise as autologousdonor cells to treat heart disease. A major technical obstacle to this approach is that availableinduction methods often produce heterogeneous cell population with low percentage of cardiomyocytes.Here we describe a cardiac enrichment approach using nonintegrating adeno-associated virus (AAV). Wefirst examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes.Results showed that AAV1 demonstrated the highest in vitro transduction efficiency among sevenwidely used serotypes. Next, differentiated iPSC derivatives were transduced with drug-selectable AAV1expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to57% in 2 weeks. Compared with other enrichment strategies such as integrative genetic selection, mitochondrialabeling, or surface marker cell sorting, this simple AAV method described herein bypassesantibody or dye labeling. These findings provide proof of concept for large-scale cardiomyocyte enrichmentby exploiting AAV’s intrinsic tissue tropis

Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260 nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles

The SSV‐Seq 2.0 PCR‐Free Method Improves the Sequencing of Adeno‐Associated Viral Vector Genomes Containing GC‐Rich Regions and Homopolymers

International audienceAdeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors