Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.

Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.

An HTLV-1 envelope mRNA vaccine is immunogenic and protective in New Zealand rabbits.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma, a severe and fatal CD4+ T-cell malignancy. Additionally, HTLV-1 can lead to a chronic progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Unfortunately, the prognosis for HTLV-1-related diseases is generally poor, and effective treatment options are limited. In this study, we designed and synthesized a codon optimized HTLV-1 envelope (Env) mRNA encapsulated in a lipid nanoparticle (LNP) and evaluated its efficacy as a vaccine candidate in an established rabbit model of HTLV-1 infection and persistence. Immunization regimens included a prime/boost protocol using Env mRNA-LNP or control green fluorescent protein (GFP) mRNA-LNP. After immunization, rabbits were challenged by intravenous injection with irradiated HTLV-1 producing cells. Three rabbits were partially protected and three rabbits were completely protected against HTLV-1 challenge. These rabbits were then rechallenged 15 weeks later, and two rabbits maintained sterilizing immunity. In Env mRNA-LNP immunized rabbits, proviral load and viral gene expression were significantly lower. After viral challenge in the Env mRNA-LNP vaccinated rabbits, an increase in both CD4+/IFN-γ+ and CD8+/IFN-γ+ T-cells was detected when stimulating with overlapping Env peptides. Env mRNA-LNP elicited a detectable anti-Env antibody response after prime/boost vaccination in all animals and significantly higher levels of neutralizing antibody activity. Neutralizing antibody activity was correlated with a reduction in proviral load. These findings hold promise for the development of preventive strategies and therapeutic interventions against HTLV-1 infection and its associated diseases.IMPORTANCEmRNA vaccine technology has proven to be a viable approach for effectively triggering immune responses that protect against or limit viral infections and disease. In our study, we synthesized a codon optimized human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) mRNA that can be delivered in a lipid nanoparticle (LNP) vaccine approach. The HTLV-1 Env mRNA-LNP produced protective immune responses against viral challenge in a preclinical rabbit model. HTLV-1 is primarily transmitted through direct cell-to-cell contact, and the protection offered by mRNA vaccines in our rabbit model could have significant implications for optimizing the development of other viral vaccine candidates. This is particularly important in addressing the challenge of enhancing protection against infections that rely on cell-to-cell transmission.

Protein Kinase Signaling Networks Driven by Oncogenic Gq/11 in Uveal Melanoma Identified by Phosphoproteomic and Bioinformatic Analyses.

Metastatic uveal melanoma (UM) patients typically survive only 2 to 3 years because effective therapy does not yet exist. Here, to facilitate the discovery of therapeutic targets in UM, we have identified protein kinase signaling mechanisms elicited by the drivers in 90% of UM tumors: mutant constitutively active G protein α-subunits encoded by GNAQ (Gq) or GNA11 (G11). We used the highly specific Gq/11 inhibitor FR900359 (FR) to elucidate signaling networks that drive proliferation, metabolic reprogramming, and dedifferentiation of UM cells. We determined the effects of FR on the proteome and phosphoproteome of UM cells as indicated by bioinformatic analyses with CausalPath and site-specific gene set enrichment analysis. We found that inhibition of oncogenic Gq/11 caused deactivation of PKC, Erk, and the cyclin-dependent kinases CDK1 and CDK2 that drive proliferation. Inhibition of oncogenic Gq/11 in UM cells with low metastatic risk relieved inhibitory phosphorylation of polycomb-repressive complex subunits that regulate melanocytic redifferentiation. Site-specific gene set enrichment analysis, unsupervised analysis, and functional studies indicated that mTORC1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 drive metabolic reprogramming in UM cells. Together, these results identified protein kinase signaling networks driven by oncogenic Gq/11 that regulate critical aspects of UM cell biology and provide targets for therapeutic investigation.

Amplatzer Vascular Plug closure of an iatrogenic interatrial tunnel-type communication.

A 50-year-old woman who had atrial septal defect surgery at 11 months old underwent ascending aortic aneurysm resection and two attempts at closure of a residual atrial septal defect. Post-operatively, she had severe cyanosis. She was referred to our centre where a transesophageal echocardiogram and cardiac catheterisation showed an iatrogenic interatrial tunnel-type communication that was closed with an Amplatzer Vascular Plug.

A model for diabetic foot ulcer clinical trials on advanced therapies.

DECLARATION OF INTEREST: TS is a consultant for Inotec AMD Ltd., UK. The authors have no other conflicts of interest to declare.

A double-blind trial comparing an antimicrobial combination to standard care in hard-to-heal wounds.

OBJECTIVE: Excessive numbers of bacteria in hard-to-heal wounds impede wound healing. Numerous topical antiseptics have demonstrated effectiveness in benchtop studies; however, few clinical studies have demonstrated efficacy in the target population: patients with hard-to-heal wounds. This study addressed the clinical efficacy of a novel antibiofilm cleanser and gel in reducing bacterial load and improving wound outcomes. METHOD: Hard-to-heal wounds were photographed, measured and evaluated for bacterial load using fluorescence imaging weekly for four weeks. The target ulcers were randomised to be cleaned and treated with either a synergistic antibiofilm cleanser and antibiofilm gel with standard of care (AMC-AMG + SoC) or normal saline wash and an amorphous gel with standard of care (NSS-HG + SoC). RESULTS: A Chi-squared test of independence determined that the relationship between the treatment and the patient reaching 40% percentage area reduction (PAR) in four weeks was not significant (χ2(1, n=54)=0.73; p=0.39 at a significance level of 0.05); however, there was a strong trend favouring the antibiofilm cleanser and gel. A significant reduction (p<0.05) in bacterial load was observed in the antibiofilm group. CONCLUSION: This randomised controlled double-blind proof-of-concept study suggests that the performance of antibiofilm agents in vivo is comparable to that in vitro studies.

Developing and Retaining Homecare Nurses Through Employer-Based Tuition Assistance Programs: A Mixed Methods Study.

PURPOSE: This study describes how an employer-based tuition-assistance program for homecare workers at one Canadian homecare organization enabled nursing career advancement and retention. DESIGN: A convergent parallel mixed-methods design. METHODS: We reviewed existing administrative data and concurrently conducted semi-structured interviews. Descriptive statistics were used on quantitative data and qualitative data was analyzed using thematic analysis. A joint data display was developed to integrate findings from both quantitative and qualitative data together. FINDINGS: Tuition assistance reduced financial barriers to career advancement; 83% of recipients remained with their employer for at least 1-year post-studies but only 29% experienced career advancement. Psychosocial supports, career navigation and coaching to ease the licensing and role transition processes were identified as opportunities to support learners. CONCLUSION: Employer-based tuition assistance programs are impactful in helping to develop skilled employees. Practical enhancements to further support career transitions may maximize retention to address urgent homecare staffing challenges. CLINICAL EVIDENCE: Employer-based tuition assistance can be a useful strategy to support nursing career growth and staff retention.

The 3' Untranslated Regions of Ebola Virus mRNAs Contain AU-Rich Elements Involved in Posttranscriptional Stabilization and Decay.

The 3' untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3' UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3' UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3' UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3' UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity.

Single-cell RNA sequencing reveals unique monocyte-derived interstitial macrophage subsets during lipopolysaccharide-induced acute lung inflammation.

Interstitial macrophages (IMs) reside in the lung tissue surrounding key structures including airways, vessels, and alveoli. Recent work has described IM heterogeneity during homeostasis, however, there are limited data on IMs during inflammation. We sought to characterize IM origin, subsets, and transcriptomic profiles during homeostasis and lipopolysaccharide (LPS) induced acute lung inflammation. During homeostasis, we used three complementary methods, spectral flow cytometry, single-cell RNA-sequencing, and gene regulatory network enrichment, to demonstrate that IMs can be divided into two core subsets distinguished by surface and transcriptional expression of folate receptor ß (Folr2/FRß). These subsets inhabited distinct niches within the lung interstitium. Within FRß+ IMs we identified a subpopulation marked by coexpression of LYVE1. During acute LPS-induced inflammation, lung IM numbers expand. Lineage tracing revealed IM expansion was due to recruitment of monocyte-derived IMs. At the peak of inflammation, recruited IMs were comprised two unique subsets defined by expression of genes associated with interferon signaling and glycolytic pathways. As recruited IMs matured, they adopted the overall transcriptional state of FRß- resident IMs but retained expression in several origin-specific genes, such as IL-1ß. FRß+ IMs were of near-pure resident origin. Taken together our data show that during LPS-induced inflammation, there are distinct populations of IMs that likely have unique functions. FRΒ+ IMs comprise a stable, resident population, whereas FRß- ΙΜs represent a mixed population of resident and recruited IMs.

Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies.

BACKGROUND: Idiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels and muscle fibres, chronic weakness and fatigue. While complement-mediated destruction of capillary endothelia is implicated in paediatric and adult dermatomyositis, the complex diversity of complement C4 in IIM pathology was unknown. METHODS: We elucidated the gene copy number (GCN) variations of total C4, C4A and C4B, long and short genes in 1644 Caucasian patients with IIM, plus 3526 matched healthy controls using real-time PCR or Southern blot analyses. Plasma complement levels were determined by single radial immunodiffusion. RESULTS: The large study populations helped establish the distribution patterns of various C4 GCN groups. Low GCNs of C4T (C4T=2+3) and C4A deficiency (C4A=0+1) were strongly correlated with increased risk of IIM with OR equalled to 2.58 (2.28-2.91), p=5.0×10-53 for C4T, and 2.82 (2.48-3.21), p=7.0×10-57 for C4A deficiency. Contingency and regression analyses showed that among patients with C4A deficiency, the presence of HLA-DR3 became insignificant as a risk factor in IIM except for inclusion body myositis (IBM), by which 98.2% had HLA-DR3 with an OR of 11.02 (1.44-84.4). Intragroup analyses of patients with IIM for C4 protein levels and IIM-related autoantibodies showed that those with anti-Jo-1 or with anti-PM/Scl had significantly lower C4 plasma concentrations than those without these autoantibodies. CONCLUSIONS: C4A deficiency is relevant in dermatomyositis, HLA-DRB1*03 is important in IBM and both C4A deficiency and HLA-DRB1*03 contribute interactively to risk of polymyositis.

Whole versus hole: enabling community nurses to implement holistic wound care.

OBJECTIVE: To improve wound-related quality of life (QoL) in clients with hard-to-heal wounds in their lower limbs and to increase referrals to multidisciplinary teams in the management of care for these clients. METHOD: This was a quality improvement project with a two-group pretest-posttest interventional evaluation design. We implemented a package of interventions including the WounDS app, education related to wound care, and client engagement through a QoL self-assessment. Wound-related QoL was measured using the Cardiff Wound Impact Schedule and referrals to the multidisciplinary team were tracked through chart audits. We explored nurses' experiences with the interventions through semi-structured interviews. RESULTS: Clients' average ratings for 'wellbeing', 'physical symptoms and daily living', and 'overall QoL' improved by 27%, 38% and 54%, respectively. The number of referrals increased by 78% post intervention. Nurses described the interventions as effective strategies that motivated them to implement a holistic approach to care. CONCLUSION: The project was successful in creating a culture shift to practice holistic wound care. This package of interventions (WounDS app, education and client self-assessment of QoL) led to improvements in the QoL of clients with hard-to-heal wounds. Further studies are needed to generalise the findings. Strategies for sustainability include forming a champion group and providing the education and decision supports based on nurses' educational needs assessment.

Family perspectives on gaps in health care for people with Down syndrome.

Patients with Down syndrome (DS) have significant specialized healthcare needs. Our objective was to understand what families of patients with DS perceive to be the most pressing gaps in health care, barriers to attendance at a DS specialty clinic, and what they thought a specialty healthcare clinic for people with DS ought to include as part of the clinical package. A qualitative survey was distributed nationally through the online platform SurveyMonkey. We divided respondents into two groups: those who attended a DS specialty clinic (n = 141) and those who did not (n = 100). Data were cleaned and analyzed in RStudio 3.6.3. Results demonstrate that families value mental health services, therapies (e.g., physical therapy, occupational therapy, and speech therapy), developmental specialists, dietitians, and educational advocates. Lack of clear advertisement, especially within low-income communities, a lack of awareness of DS specialty clinics, and travel time to clinics constituted significant barriers to care. These findings are arguably of benefit to those who direct DS specialty clinics because they offer direction for resource allocation in a time of increasing healthcare costs and financial scrutiny.

Are drug targets with genetic support twice as likely to be approved? Revised estimates of the impact of genetic support for drug mechanisms on the probability of drug approval.

Despite strong vetting for disease activity, only 10% of candidate new molecular entities in early stage clinical trials are eventually approved. Analyzing historical pipeline data, Nelson et al. 2015 (Nat. Genet.) concluded pipeline drug targets with human genetic evidence of disease association are twice as likely to lead to approved drugs. Taking advantage of recent clinical development advances and rapid growth in GWAS datasets, we extend the original work using updated data, test whether genetic evidence predicts future successes and introduce statistical models adjusting for target and indication-level properties. Our work confirms drugs with genetically supported targets were more likely to be successful in Phases II and III. When causal genes are clear (Mendelian traits and GWAS associations linked to coding variants), we find the use of human genetic evidence increases approval by greater than two-fold, and, for Mendelian associations, the positive association holds prospectively. Our findings suggest investments into genomics and genetics are likely to be beneficial to companies deploying this strategy.

Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer.

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.

Create and Sustain a Culture of Curiosity: A Case Study of a Home Healthcare Organization in Toronto.

Increased integration in the healthcare sector requires traditionally non-research-based organizations to contribute to evidence-based decision making as equal partners. This requires a culture and infrastructure that support structured inquiry to improve best practice, quality and safety. We present the roadmap used by one home healthcare organization to create an embedded research unit to drive this transformation. The use of a relevant model and a framework provided structure to guide and sustain the process. We expect that the core strategies and combination of frameworks should be transferrable to others wishing to contribute meaningfully to evidence-based health system transformation.

Estimating colocalization probability from limited summary statistics.

BACKGROUND: Colocalization is a statistical method used in genetics to determine whether the same variant is causal for multiple phenotypes, for example, complex traits and gene expression. It provides stronger mechanistic evidence than shared significance, which can be produced through separate causal variants in linkage disequilibrium. Current colocalization methods require full summary statistics for both traits, limiting their use with the majority of reported GWAS associations (e.g. GWAS Catalog). We propose a new approximation to the popular coloc method that can be applied when limited summary statistics are available. Our method (POint EstiMation of Colocalization, POEMColoc) imputes missing summary statistics for one or both traits using LD structure in a reference panel, and performs colocalization using the imputed summary statistics. RESULTS: We evaluate the performance of POEMColoc using real (UK Biobank phenotypes and GTEx eQTL) and simulated datasets. We show good correlation between posterior probabilities of colocalization computed from imputed and observed datasets and similar accuracy in simulation. We evaluate scenarios that might reduce performance and show that multiple independent causal variants in a region and imputation from a limited subset of typed variants have a larger effect while mismatched ancestry in the reference panel has a modest effect. Further, we find that POEMColoc is a better approximation of coloc when the imputed association statistics are from a well powered study (e.g., relatively larger sample size or effect size). Applying POEMColoc to estimate colocalization of GWAS Catalog entries and GTEx eQTL, we find evidence for colocalization of 150,000 trait-gene-tissue triplets. CONCLUSIONS: We find that colocalization analysis performed with full summary statistics can be closely approximated when only the summary statistics of the top SNP are available for one or both traits. When applied to the full GWAS Catalog and GTEx eQTL, we find that colocalized trait-gene pairs are enriched in tissues relevant to disease etiology and for matches to approved drug mechanisms. POEMColoc R package is available at https://github.com/AbbVie-ComputationalGenomics/POEMColoc .

Tolerogenic nanoparticles restore the antitumor activity of recombinant immunotoxins by mitigating immunogenicity.

Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.

Low-Dose Methotrexate Prevents Primary and Secondary Humoral Immune Responses and Induces Immune Tolerance to a Recombinant Immunotoxin.

Recombinant immunotoxins (RITs) are chimeric proteins being developed for cancer treatment. They are composed of an Ab fragment that targets a cancer Ag and a cytotoxic portion of Pseudomonas exotoxin A. They are effective for patients with hematologic malignancies with defective immunity, but their efficacy against solid tumors is limited by anti-drug Ab (ADA) responses in immune-competent patients. Pre-existing Abs or immune memory owing to previous toxin exposure represent additional hurdles because they induce rapid and strong ADA responses. Here, we evaluated the efficacy of methotrexate (MTX) to prevent ADA formation against the mesothelin-targeting RIT LMB-100 in naive mice and in mice with pre-existing Abs. We found that low-dose MTX combined with LMB-100 completely suppressed the formation of ADAs in a dose- and frequency-dependent manner. Suppression of the immune response restored blood levels of LMB-100 and prevented its neutralization. Furthermore, combination of MTX with LMB-100 did not compromise the immune response against a second Ag given after stopping MTX, indicating specific immune tolerance. Adoptive transfer of splenocytes suppressed Ab responses to LMB-100 in recipient mice, indicating a durable immune tolerance. We conclude that combination of MTX and LMB-100 is effective at preventing immune responses in a durable, Ag-specific manner. We propose combining low-dose MTX in immune-competent cancer patients receiving RIT therapy to prevent immunogenicity. This approach could be applied to other immunogenic therapeutic agents and to proteins for which there is pre-existing immunity.

Serum prednisolone levels as a marker of oral corticosteroid adherence in severe asthma.

BACKGROUND: Severe asthma is a complex heterogeneous disease typically requiring advanced therapies. Underlying the treatment of all asthma, however, is the consistent recommendation across international guidelines to ensure that adherence to therapy is adequate. Currently, there is no consensus on an objective marker of adherence. METHODS: We performed a prospective observational study of 17 participants taking oral prednisolone using serum prednisolone levels as a marker of adherence, and sputum eosinophilia as a marker of control of type 2 airway inflammation. Based on these biomarkers, we classified participants into a non-adherent and an adherent cohort, and further stratified by the presence of ongoing sputum eosinophilia. RESULTS: We identified 3 non-adherent participants and 14 who were adherent, based on their serum prednisolone levels. Stratification using sputum eosinophil counts identified one participant as having ongoing sputum eosinophilia in the setting of non-adherence, while six were identified as steroid resistant with ongoing sputum eosinophilia despite adherence to oral prednisolone therapy. CONCLUSION: Serum prednisolone can be used an objective marker of adherence in those patients with severe asthma taking daily oral prednisolone. In combination with sputum eosinophil counts, a steroid resistant cohort can be distinguished from one with ongoing inflammation in the setting of non-adherence. This information can then be used by clinicians to differentiate the optimal next steps for treatment in these specific populations. TRIAL REGISTRATION: Participants were recruited as part of the Markers of Inflammation in the Management of Severe Asthma (MIMOSA) study, trial registration ACTRN12616001015437 , 02 August 2016.

Hepatic CT attenuation differs in three species of freshwater turtles and hepatic Hounsfield units increase with folliculogenesis in wild Blanding's turtles (Emydoidea blandingii).

Freshwater turtle species are suffering from anthropocentric-caused population declines, making preservation of professionally managed populations increasingly important. Turtles under professional care have an increased risk to develop hepatic lipidosis, potentially resulting in early death. Computed tomography can provide an antemortem screening for increased fatty liver composition. A goal of this prospective analytical cross-sectional study was to assess the hepatic attenuation measured as Hounsfield units (HU) in a wild population of a freshwater turtle species, and then compare hepatic HU to freshwater turtles under professional care. Ninety-five wild Blanding's turtles (BT; Emydoidea blandingii) as well as 10 Vietnamese Pond turtles (VPT; Mauremys annamensis) and six Northern Snake-Necked turtles (NSNT; Chelodina oblonga) under professional care underwent CT with quantification of hepatic HU. Hepatic HU were correlated to serum chemistry findings and the presence of follicles was recorded in BT. The mean (±SD) hepatic attenuation for 95 wild BT was 97.5 HU ±9.6. There were significant differences in hepatic attenuation among VPT, NSNT, and BT, with median HU values (range) of 5.39 HU (-6.45 to 61.50), 71.74 HU (59.44-94.49), and 95.43 HU (74.41-124.56), respectively. Aspartate aminotransferase (AST) values had a weak negative correlation to HU with a coefficient of -0.85 (P < .001). The hepatic attenuation was significantly higher for individuals undergoing folliculogenesis (P = .007). The decreased HU values were significantly negatively correlated with AST values. Findings supported the use of CT as an aid for guiding future management practices in freshwater turtles.