CRISPR/Cas9 gene editing in Drosophila via visual selection in a summer classroom.

CRISPR/Cas9 methods are a powerful in vivo approach to edit the genome of Drosophila melanogaster. To convert existing Drosophila GAL4 lines to LexA driver lines in a secondary school classroom setting, we applied the CRISPR-based genetic approach to a collection of Gal4 'driver' lines. The integration of the yellow+ coat color marker into homology-assisted CRISPR knock-in (HACK) enabled visual selection of Gal4-to-LexA conversions using brightfield stereo-microscopy available in a broader set of standard classrooms. Here, we report the successful conversion of eleven Gal4 lines with expression in neuropeptide-expressing cells into corresponding, novel LexA drivers. The conversion was confirmed by LexA- and Gal4-specific GFP reporter gene expression. This curriculum was successfully implemented in a summer course running 16 hours/week for seven weeks. The modularity, flexibility, and compactness of this course should enable development of similar classes in secondary schools and undergraduate curricula, to provide opportunities for experience-based science instruction, and university-secondary school collaborations that simultaneously fulfill research needs in the community of science.

Simplified homology-assisted CRISPR for gene editing in Drosophila.

In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome-linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome-linked donor strain readily converted the second chromosome-linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome-linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the "dual transgene" cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.

Transgenic Drosophila lines for LexA-dependent gene and growth regulation.

Conditional expression of short hairpin RNAs with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of 2 independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-short hairpin RNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based short hairpin RNA expression vector to achieve short hairpin RNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.

Vincristine-induced peripheral neurotoxicity: A prospective cohort.

Vincristine-induced peripheral neuropathy (VIPN) is a serious and pervasive problem, affecting 12-78% of pediatric patients, based on retrospective studies. The study objective was to prospectively collect a cohort of well-phenotyped patients receiving vincristine in order to accurately classify and grade their neurotoxicity. All children in British Columbia with leukemia or lymphoma requiring vincristine between 2013 and 2016 were approached for consent. Those recruited were assessed by occupational and physiotherapists at baseline, mid and endpoint of their treatment. Assessments included the Bruininks-Oseretsky Test of Motor Proficiency - 2nd ed. (BOT-2), strength, "Timed up and go" test and vibration sensibility. Seventy-two patients consented (age: 2.0-18.7 years). The majority were below average for age on one or more BOT-2 domains at midpoint (N = 32/45, 71%), which decreased by the endpoint (N = 19/41, 46%, p = .049). Six patients showed severe VIPN throughout treatment (N = 6/53, 11%), defined as a BOT-2 score well below average. Muscle strength for wrist extension/flexion, anterior tibialis and peronei decreased significantly between baseline (Median = 5) and midpoint (Median = 4), with no significant change noted by endpoint. Most patients had normal vibration sensibility in lower (N = 30/60, 50%) and upper limbs (N = 26/38, 68%). In conclusion, with no differences between time points. VIPN is highly prevalent among patients with pediatric cancer, causing significant morbidity and functional deficits. Identification of risk factors would allow for resource appropriation to patients at higher risk, as well as potentially permitting dose escalation in patients with low toxicity to improve survival.

Associating Physical Activity Levels with Motor Performance and Physical Function in Childhood Survivors of Acute Lymphoblastic Leukemia.

Purpose: This cross-sectional, observational study investigated whether physical activity (PA) levels are associated with motor performance and physical function in children after treatment for acute lymphoblastic leukemia (ALL). Method: Participants aged 8-13 years who had completed treatment for ALL (3-36 months post-treatment) were tested at their oncology long-term follow-up appointment at the British Columbia Children's Hospital. PA level was measured using the Physical Activity Questionnaire for Older Children (PAQ-C). Motor performance was measured using the Bruininks-Oseretsky Test of Motor Proficiency, Second Edition, Short Form (BOT-2 SF), and physical function was measured using the 6-minute walk test (6MWT). Results: Thirteen children completed testing. PAQ-C scores were not associated with BOT-2 SF or 6MWT performance. Eleven children (85%) performed below the norm for the 6MWT. Children with elevated body mass index had poorer 6MWT but similar PAQ-C scores. Conclusion: PA was not found to be associated with motor performance and physical function. Participants who were overweight or obese had poorer 6MWT performance, which may indicate the need for closer monitoring of post-treatment weight status and physical function in the oncology follow-up setting.

Objectif : cette étude observationnelle à méthodologie transversale portait sur le lien potentiel entre le niveau d'activité physique (AP) et la capacité motrice et physique des enfants ayant reçu un traitement contre la leucémie lymphoblastique aiguë (LLA). Méthode : des participants âgés de 8 à 13 ans ayant reçu un traitement contre la LLA (de 3 à 36 mois après le traitement) ont été évalués lors de leur rendez-vous de suivi à long terme en oncologie au British Columbia Children's Hospital. Le niveau d'AP a été mesuré à l'aide du Questionnaire de mesure de l'activité physique chez les enfants (PAQ-C). La capacité motrice a été mesurée à l'aide du Bruininks-Oseretsky Test of Motor Proficiency, Deuxième édition, Formulaire court (BOT-2 SF) et la capacité physique a été mesurée à l'aide du test de marche de 6 minutes (6MWT). Résultats : treize enfants ont effectué les tests. Aucun lien n'a été observé entre les scores du PAQ-C et les résultats du BOT-2 SF ni ceux du 6MWT. Onze enfants (85 %) ont obtenu un résultat inférieur à la norme au 6MWT. Les enfants ayant un indice de masse corporelle élevé ont obtenu des résultats plus faibles au 6MWT, mais des résultats similaires au PAQ-C. Conclusion : le niveau d'AP ne semble pas avoir de lien avec la capacité motrice ou physique. Les participants en surpoids et obèses ont obtenu des résultats plus faibles au 6MWT, ce qui peut indiquer un besoin de surveillance plus étroit du poids et de la capacité physique après le traitement lors des rendez-vous de suivi en oncologie.

A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research.

Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals.

Raising the Priority of Lifestyle-Related Noncommunicable Diseases in Physical Therapy Curricula.

Given their enormous socioeconomic burdens, lifestyle-related noncommunicable diseases (heart disease, cancer, chronic lung disease, hypertension, stroke, type 2 diabetes mellitus, and obesity) have become priorities for the World Health Organization and health service delivery systems. Health care systems have been criticized for relative inattention to the gap between knowledge and practice, as it relates to preventing and managing noncommunicable diseases. Physical therapy is a profession that can contribute effectively to patients'/clients' lifestyle behavior changes at the upstream end of prevention and management. Efforts by entry-to-practice physical therapist education programs to align curricula with epidemiological trends toward best health care practices are varied. One explanation may be the lack of a frame of reference for reducing the knowledge translation gap. The purpose of this article is to provide a current perspective on epidemiological indicators and societal priorities to inform physical therapy curriculum content. Such content needs to include health examination/evaluation tools and health behavior change interventions that are consistent with contemporary values, directions, and practices of physical therapy. These considerations provide a frame of reference for curriculum change. Based on 5 years of experience and dialogue among curriculum stakeholders, an example of how epidemiologically informed and evidence-based best health care practices may be systematically integrated into physical therapy curricula to maximize patient/client health and conventional physical therapy outcomes is provided. This novel approach can serve as an example to other entry-to-practice physical therapist education programs of how to align their curricula with societal health priorities, specifically, noncommunicable diseases. The intentions are to stimulate dialogue about effectively integrating health-based competencies into entry-level education and advancing best practice, as opposed to simply evidence-based practice, across professions and health services and to establish accreditable, health promotion practice standards for physical therapy.

An Evidence-Based Case Study of Unilateral Shin Splints: Do Red Flags Function in Paediatric Osteosarcoma?

Physiotherapists use red flags to screen for serious pathology. Paediatric osteosarcoma is a rare disease, occurring predominantly in the area of the knee and shoulder, and it is not always included by physiotherapists on a differential diagnosis list. Traditional red flags do not always correspond to the initial signs and symptoms of osteosarcoma. Physiotherapists should routinely palpate along the length of the bone to detect a potential mass. The detection of a mass or symptoms that do not follow the expected course indicates the need for reassessment and possibly referral for further investigation.

Les physiothérapeutes utilisent des drapeaux rouges pour dépister les pathologies graves. L'ostéosarcome pédiatrique est une maladie rare qui atteint principalement la région du genou et celle de l'épaule et que les physiothérapeutes n'incluent pas toujours dans une liste de diagnostic différentiel. Les drapeaux rouges traditionnels ne correspondent pas toujours aux signes et symptômes initiaux de l'ostéosarcome. Les physiothérapeutes devraient procéder de façon routinière à une palpation le long de l'os afin de détecter la présence possible d'une masse. La détection d'une masse ou des symptômes qui ne suivent pas l'évolution prévue indiquent qu'il faut réévaluer le problème et référer peut-être le patient pour un examen plus poussé.

Comparison of the Eragen Multi-Code Respiratory Virus Panel with conventional viral testing and real-time multiplex PCR assays for detection of respiratory viruses.

High-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing.

Characterization of dendritic cells generated in vivo by an E. coli derived chimeric dual receptor agonist.

BACKGROUND: Progenipoietin-4 (ProGP-4) is an E. coli derived chimeric growth factor that activates the human Flt3 and G-CSF receptors. ProGP-4 possesses cross-species activity and treatment of mice with ProGP-4 results in increases in the number of WBC and Class II+/CD11c+ cells in both spleen and peripheral blood. Herein, we report morphologic, phenotypic and functional evaluation of Class II+/CD11c+ cells generated by in vivo administration of ProGP-4. MATERIAL/METHODS: C57BL/6 mice were injected daily with ProGP for 7 to 18 days. Leukocytes from spleen and peripheral blood were analyzed by flow cytometry to enumerate and characterize changes in DC populations. Spleens from ProGP treated mice were evaluated by immunocytochemistry and enriched CD11c+ populations were functionally assessed in a mixed lymphocyte assay and in an antigen dependent CTL assay. RESULTS: Administration of this dual receptor agonist to mice resulted in dose-dependent increases in the numbers of total white blood cells and Class II+/CD11c+ cells in spleen and peripheral blood. CD11c+ cells from ProGP-4 treated mice co-expressed DEC205 and also expressed CD80, CD86 and CD40, albeit at lower levels as compared to Class II+/CD11c+ cells from untreated animals. Despite lower co-stimulatory molecule expression, ProGP-4-generated Class II+/CD11c+ cells stimulated proliferation of allogeneic T cells and an antigen-specific T cell hybridoma as efficiently as bone marrow derived dendritic cells from untreated mice. CONCLUSIONS: The data presented in this report highlight the ability of E. coli derived ProGP-4 to expand large numbers of functional DC in the peripheral blood and lymphoid organs in vivo using a rodent model of hematopoiesis. E. coli derived chimeric receptor agonists such as ProGP-4 may enable further investigations of immunotherapeutic approaches to the treatment of diseases such as cancer and autoimmunity.

Mating system instability in Schiedea menziesii (Caryophyllaceae).

We investigated inbreeding depression and selfing in hermaphroditic Schiedea menziesii to assess the stability of the breeding system. A combination of high selfing rates and strong inbreeding depression suggests that the mating system is unstable. The population-level selfing rate measured in three years ranged considerably from 0.425 (SE = 0.138) to 0.704 (0.048); family measures of selfing rate varied from zero to one in all three years. Inbreeding coefficients did not differ from zero, suggesting that inbred plants do not survive to reproduction in the field. Average inbreeding depression measured in two greenhouse experiments was 0.608-0.870, with values for individual plants ranging from -0.170 to 0.940. The magnitude of inbreeding depression expressed at different life-history stages depended on experimental conditions. When plants were grown during the winter, inbreeding depression was expressed at early and late life-history stages. When plants were grown during the summer, inbreeding depression was detected for germination but not for later life-history stages. Inbreeding depression for vegetative and inflorescence biomass was also measured using field-collected seeds where cross status was assigned using genotypes determined electrophoretically. We did not detect a relation between inbreeding depression and the selfing rate at the level of the individual plant. We saw no evidence for intrafloral selfing, suggesting that the evolution of increased selfing through autogamy is unlikely, despite high selfing rates. A more likely outcome of breeding system instability is the evolution of gynodioecy, which occurs in species of Schiedea closely related to S. menziesii. Females have been detected in progeny of S. menziesii that have been raised in the greenhouse. In the absence of biotic pollen vectors, the failure of these females to establish in the natural population may result from the absence of adaptations for wind pollination.