Metabolic engineering for the production of plant isoquinoline alkaloids.

Several plant isoquinoline alkaloids (PIAs) possess powerful pharmaceutical and biotechnological properties. Thus, PIA metabolism and its fascinating molecules, including morphine, colchicine and galanthamine, have attracted the attention of both the industry and researchers involved in plant science, biochemistry, chemical bioengineering and medicine. Currently, access and availability of high-value PIAs [commercialized (e.g. galanthamine) or not (e.g. narciclasine)] is limited by low concentration in nature, lack of cultivation or geographic access, seasonal production and risk of overharvesting wild plant species. Nevertheless, most commercial PIAs are still extracted from plant sources. Efforts to improve the production of PIA have largely been impaired by the lack of knowledge on PIA metabolism. With the development and integration of next-generation sequencing technologies, high-throughput proteomics and metabolomics analyses and bioinformatics, systems biology was used to unravel metabolic pathways allowing the use of metabolic engineering and synthetic biology approaches to increase production of valuable PIAs. Metabolic engineering provides opportunity to overcome issues related to restricted availability, diversification and productivity of plant alkaloids. Engineered plant, plant cells and microbial cell cultures can act as biofactories by offering their metabolic machinery for the purpose of optimizing the conditions and increasing the productivity of a specific alkaloid. In this article, is presented an update on the production of PIA in engineered plant, plant cell cultures and heterologous micro-organisms.

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum.

With the completion of the genome sequence, and development of an efficient conjugation-based transformation system allowing the introduction of stable episomes, Phaeodactylum tricornutum has become an ideal platform for the study of diatom biology and synthetic biology applications. The development of plasmid-based genetic tools is the next step to improve manipulation of this species. Here, we report the identification of endogenous P. tricornutum promoters and terminators allowing selective expression of antibiotic resistance markers from stably replicating plasmids in P. tricornutum. Significantly, we developed a protocol for sequential conjugation of plasmids from Escherichia coli to P. tricornutum and demonstrated simultaneous replication of two plasmids in P. tricornutum. We developed a simple and robust conjugative system for Cas9 editing that yielded up to 60% editing efficiency of the urease gene. Finally, we constructed a plasmid encoding eight genes involved in vanillin biosynthesis that was propagated in P. tricornutum over four months with no evidence of rearrangements, with whole-plasmid sequencing indicating that the majority of mutations occurred after plasmid assembly and initial conjugation rather than during long-term propagation. The plasmid-based tools described here will facilitate investigation of the basic biology of P. tricornutum and enable synthetic biology applications.

Validity of an Interactive Functional Reach Test.

INTRODUCTION: Videogaming platforms such as the Microsoft (Redmond, WA) Kinect(®) are increasingly being used in rehabilitation to improve balance performance and mobility. These gaming platforms do not have built-in clinical measures that offer clinically meaningful data. We have now developed software that will enable the Kinect sensor to assess a patient's balance using an interactive functional reach test (I-FRT). The aim of the study was to test the concurrent validity of the I-FRT and to establish the feasibility of implementing the I-FRT in a clinical setting. SUBJECTS AND METHODS: The concurrent validity of the I-FRT was tested among 20 healthy adults (mean age, 25.8±3.4 years; 14 women). The Functional Reach Test (FRT) was measured simultaneously by both the Kinect sensor using the I-FRT software and the Optotrak Certus(®) 3D motion-capture system (Northern Digital Inc., Waterloo, ON, Canada). The feasibility of implementing the I-FRT in a clinical setting was assessed by performing the I-FRT in 10 participants with mild balance impairments recruited from the outpatient physical therapy clinic (mean age, 55.8±13.5 years; four women) and obtaining their feedback using a NASA Task Load Index (NASA-TLX) questionnaire. RESULTS: There was moderate to good agreement between FRT measures made by the two measurement systems. The greatest agreement between the two measurement system was found with the Kinect sensor placed at a distance of 2.5 m [intraclass correlation coefficient (2,k)=0.786; P<0.001] from the participant. Participants with mild balance impairments whose balance was assessed using the I-FRT software scored their experience favorably by assigning lower scores for the Frustration, Mental Demand, and Temporal Demand subscales on the NASA/TLX questionnaire. CONCLUSIONS: FRT measures made using the Kinect sensor I-FRT software provides a valid clinical measure that can be used with the gaming platforms.

Interferon-gamma is not a universal requirement for islet allograft survival.

BACKGROUND: Although many transplantation studies have implicated a graft-destructive role for T helper (Th)1 cytokines and a graft-protective role for Th2 cytokines, more recent studies have challenged this paradigm by showing that long-term allograft survival can actually require the presence of Th1 cytokines, such as interleukin 2 and interferon (IFN)-gamma. The purpose of this study was to examine the requirement for IFN-gamma in the induction of islet allograft acceptance after monoclonal antibody therapy targeting conceptually distinct molecular pathways: the costimulatory molecule CD154, the CD4 coreceptor, or the beta2 integrin lymphocyte function-associated antigen (LFA)-1 (CD11a). METHODS: Diabetic C57Bl/6 (B6; H2b) mice were grafted with fully MHC mismatched BALB/c (H2d) islets, or reciprocally, diabetic BALB/c mice underwent transplantation with B6 islets and were treated with anti-CD154, anti-CD4, or anti-LFA-1. RESULTS: When IFN-gamma gene knockout mice were used as graft recipients, the requirement for IFN-gamma in allograft survival was found to be highly conditional, depending on both the host strain and the induction therapy used. In both strain combinations studied, anti-CD154 was effective in the presence or absence of IFN-gamma, whereas anti-CD4 lost therapeutic potential in the absence of this cytokine. Alternatively, the requirement for IFN-gamma for allograft prolongation by anti-LFA-1 therapy was noted only in B6 transplant recipients. CONCLUSIONS: IFN-gamma is not always requisite in islet allograft survival but rather varies according to the molecular target of induction therapy and the genetic background of the transplant recipient.