Endoscopic sexing in turtles and tortoises: 467 cases (2007-2017).

BACKGROUND: This multi-institutional retrospective study evaluated the feasibility and safety of endoscopic sex identification in 467 turtles and tortoises, representing 10 species. METHODS: Medical records of turtles and tortoises that underwent endoscopic sex identification at the University of Georgia, New England Aquarium and Turtle Conservancy were reviewed for presurgical management, anaesthesia, endoscopic equipment and surgical techniques, endoscopic results and complications. RESULTS: The majority of animals weighed less than 200 g, were fasted and anaesthetised using an injectable combination of ketamine, dexmedetomidine and morphine or hydromorphone, supplemented by local lidocaine at the prefemoral site. Anaesthetic reversal using atipamezole alone or in combination with naloxone was routine. For uncomplicated procedures, mean total anaesthesia, surgery and recovery times were 22, 4 and 18 minutes, respectively. All animals were placed in lateral recumbency for a prefemoral endoscopic approach to the coelom using a rigid telescope and sterile fluid infusion to visualise the gonads. Sex identification was definitive in 99.4% (n = 464) of the animals. Iatrogenic bladder perforation was the most common complication (n = 5), which necessitated extended anaesthesia and surgical time for repair. Only a single anaesthetic-related death was reported, which was associated with human error and drug overdose. CONCLUSIONS: This is the first large-scale study to retrospectively evaluate endoscopic sex identification in multiple chelonian species. Results suggest that endoscopic sexing is a safe, accurate and practical means for sex identification in turtles and tortoises, and represents a valuable tool in their reproductive management.

Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme ß-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

7T MRI Predicts Amelioration of Neurodegeneration in the Brain after AAV Gene Therapy.

GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline ß-galactosidase (ß-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where ß-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and ß-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.

Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and ß subunits separately (TSD α + ß) injected at high (1.3 × 1013 vector genomes) or low (4.2 × 1012 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + ß sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + ß), and ganglioside clearance was most widespread in the TSD α + ß high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

How are PCORI-funded researchers engaging patients in research and what are the ethical implications?

BACKGROUND: Despite growing interest in patient engagement in research, there are few empirical investigations of the nature of engagement and its effects. This information is important, not only to inform practical decisions researchers and funders must make, but also to inform discussion of the ethical implications of engaging patients, which has received little attention to date. METHODS: The aim of this study was to characterize patient engagement in research funded by the Patient-Centered Outcomes Research Institute (PCORI) as a step toward enhancing current understanding of the nature and effects of engagement and prompting an in-depth consideration of the ethical implications of engaging patients in research. Qualitative interviews were conducted with 19 PCORI-funded principal investigators and with 33 patients engaged in 18 of the same 19 projects. RESULTS: Reasons cited for engaging patients included to enhance relevance and feasibility and to improve dissemination. While engagement occurred at different points during the research, patients were most commonly engaged in reviewing study materials and less commonly engaged at earlier points. Engagement varied by approach, frequency of interaction, and the extent to which patient input changed the research. Impacts of engagement included improving the relevance, feasibility, acceptability, and quality of the research. CONCLUSION: Our findings on the nature and impacts of engagement have importance not only for practical questions researchers, funders, and patients might raise, but also for several ethical considerations regarding patient engagement related to why patients are engaged, the kinds of patients engaged, when patients are engaged, and how patients are engaged. We discuss our findings in consideration of the main ethical issues they imply, including ethical rationales for engagement, justice-related concerns, and ethical concerns arising from when and how patients are engaged. As efforts to engage patients increase, this discussion provides insights that researchers, funders, and patients may find valuable.

Patient engagement in patient-centered outcomes research: challenges, facilitators and actions to strengthen the field.

AIM: To describe challenges to and facilitators of patient engagement to inform future strategies and suggested actions to strengthen engagement. METHODS: Interviews with 19 principal investigators of projects funded by the Patient-Centered Outcomes Research Institute and with 33 patients from 18 of the 19 projects. RESULTS: Facilitators included using existing resources, having clear goals, educating patients and treating patients respectfully. Logistical challenges included extra time and work, institutional barriers and difficulty having meetings. Substantive challenges to selecting, educating and engaging patients, and incorporating feedback were also reported. CONCLUSION: To bolster the infrastructure for engagement, we suggest funders, institutions and researchers focus on resources and training for researchers and patients, networks and programs to connect stakeholders and model policies.

Patient and Stakeholder Engagement in the PCORI Pilot Projects: Description and Lessons Learned.

BACKGROUND: Patients and healthcare stakeholders are increasingly becoming engaged in the planning and conduct of biomedical research. However, limited research characterizes this process or its impact. OBJECTIVE: We aimed to characterize patient and stakeholder engagement in the 50 Pilot Projects funded by the Patient-Centered Outcomes Research Institute (PCORI), and identify early contributions and lessons learned. DESIGN: A self-report instrument was completed by researchers between 6 and 12 months following project initiation. PARTICIPANTS: Forty-seven principal investigators or their designees (94 % response rate) participated in the study. MAIN MEASURES Self-report of types of stakeholders engaged, stages and levels of engagement, facilitators and barriers to engagement, lessons learned, and contributions from engagement were measured. KEY RESULTS: Most (83 %) reported engaging more than one stakeholder in their project. Among those, the most commonly reported groups were patients (90 %), clinicians (87 %), health system representatives (44 %), caregivers (41 %), and advocacy organizations (41 %). Stakeholders were commonly involved in topic solicitation, question development, study design, and data collection. Many projects engaged stakeholders in data analysis, results interpretation, and dissemination. Commonly reported contributions included changes to project methods, outcomes or goals; improvement of measurement tools; and interpretation of qualitative data. Investigators often identified communication and shared leadership strategies as "critically important" facilitators (53 and 44 % respectively); lack of stakeholder time was the most commonly reported challenge (46 %). Most challenges were only partially resolved. Early lessons learned included the importance of continuous and genuine partnerships, strategic selection of stakeholders, and accommodation of stakeholders' practical needs. CONCLUSIONS: PCORI Pilot Projects investigators report engaging a variety of stakeholders across many stages of research, with specific changes to their research attributed to engagement. This study identifies early lessons and barriers that should be addressed to facilitate engagement. While this research suggests potential impact of stakeholder engagement, systematic characterization and evaluation of engagement at multiple stages of research is needed to build the evidence base.

The effects of dissolved oxygen and triploidy on critical thermal maximum in brook charr (Salvelinus fontinalis).

This study examined the effect of dissolved oxygen (DO) level on critical thermal maximum (CTMax) in diploid and triploid brook charr (Salvelinus fontinalis) exposed to a temperature increase of 3°C/h. Because gas solubility is inversely proportional to temperature, DO declines during standard CTMax tests. With this treatment as a baseline, oxygen or nitrogen injection was used to provide three other DO conditions during CTMax tests: two hyperoxic (maintenance at initial 10mg/L and increase from 10mg/L at 2mg/L/h) and one hypoxic (decrease from 10mg/L at 2mg/L/h). Hyperoxia had no effect on temperature at CTMax or time taken to reach CTMax. Hypoxia, on the other hand, resulted in a significantly lower CTMax and shorter time to CTMax than under standard or hyperoxic conditions, with both indices affected by triploidy but not in a consistent fashion: in one experiment triploids had a lower CTMax and shorter time to CTMax than diploids and in a second experiment they had a higher CTMax and longer time to CTMax than diploids. Indices of the secondary stress response (plasma glucose and ions) during CTMax tests under hypoxia responded as would be predicted for an acute stress, with no difference between triploids and diploids.