Functional and multi-omics signatures of mitapivat efficacy upon activation of pyruvate kinase in red blood cells from patients with sickle cell disease.

Mitapivat, a pyruvate kinase activator, shows great potential as a sickle cell disease (SCD)-modifying therapy. The safety and efficacy of mitapivat as a long-term maintenance therapy are currently being evaluated in two open-label studies. Here we applied a comprehensive multi-omics approach to investigate the impact of activating pyruvate kinase on red blood cells (RBC) from 15 SCD patients. HbSS patients were enrolled in one of the open-label, extended studies (NCT04610866). Leukodepleted RBC obtained from fresh whole blood at baseline, prior to drug initiation, and at longitudinal timepoints over the course of the study were processed for multi-omics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBC. Mitapivat decreased 2,3-diphosphoglycerate levels, increased adenosine triphosphate levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of treatment, with minimal changes in reticulocyte counts. In the first 6 months of treatment there were also transient elevations of lysophosphatidylcholines and oxylipins with depletion of free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBC identified benefits for glycolysis, as well as activation of the Lands cycle.

Retrospective evaluation of postoperative joint immobilization using a temporary calcaneotibial screw for medial or lateral tarsocrural joint instability in dogs.

OBJECTIVE: To evaluate the use of a temporary calcaneotibial screw (CTS) to immobilize medial or lateral tarsocrural joint instability (TCI) in dogs. STUDY DESIGN: Retrospective study. ANIMALS: Twelve dogs (including five active working farm dogs) with TCI. METHODS: Medical records (January 2015-June 2023) were retrospectively reviewed for cases of TCI managed surgically including temporary joint immobilization using a CTS and external coaptation. Clinical data consisted of medical records and an online survey completed by the owner. RESULTS: Surgical techniques to address TCI included primary ligamentous repair, synthetic ligament reconstruction, or malleolar fracture repair. Immobilization with a CTS was employed for 6-8 weeks postoperatively. The online survey was completed for 10 dogs. All dogs exhibited good-to-excellent functional outcomes at the follow-up (median, 31 months; range, 4-66). All working farm dogs (5) were able to return to normal or substantial levels of their work. Four distinct complications were reported in three dogs including one CTS breakage and three bandage-related soft-tissue injuries. CONCLUSION: This retrospective study represents the first report of employing a temporary CTS for TCI in dogs. CLINICAL SIGNIFICANCE: A temporary CTS was effective in immobilizing the tarsocrural joint for dogs with TCI and the postoperative complication rate in this study was relatively low. A CTS screw and external coaptation is a viable alternative to previously reported methods of tarsocrural joint stabilization.

Accuracy of Lumbosacral Pedicle Screw Placement in Dogs: A Novel 3D Printed Patient-Specific Drill Guide versus Freehand Technique in Novice and Expert Surgeons.

OBJECTIVE: The aim of this study was to compare the accuracy of pedicle screw placement at the canine lumbosacral junction using a novel unilateral three-dimensional printed patient-specific guide (3D-PSG) versus a freehand drilling technique. Additionally, accuracy of screw placement between a novice and an experienced surgeon was determined. STUDY DESIGN: Preoperative computed tomography images from 20 lumbosacral cadaveric specimens were used to design a novel unilateral 3D-PSG for the L7 and sacral vertebrae which was printed in acryl-nitrile butadiene styrene plastic. A novice and an expert surgeon each placed 3.5mm cortical screws in 10 cadavers; on the left using the unilateral 3D-PSG and by the freehand (anatomic landmark) technique on the right. RESULTS: Sixty screws were placed using the unilateral 3D-PSG and 60 using the freehand technique. There was no statistical difference in accuracy for the comparison between methods performed by the expert (p = 0.679) and novice (p = 0.761) surgeon, nor between an expert and novice surgeon overall (p = 0.923). Unexpectedly, the use of a unilateral 3D-PSG increased variability for the expert surgeon in our study (p = 0.0314). CONCLUSION: Using a novel unilateral 3D-PSG did not improve the accuracy of screw placement for lumbosacral stabilization by a novice surgeon compared with an expert surgeon in lumbar spine surgery. This may reflect a suboptimal PSG design.

Translatability and transferability of in silico models: Context of use switching to predict the effects of environmental chemicals on the immune system.

Immunotoxicity hazard identification of chemicals aims to evaluate the potential for unintended effects of chemical exposure on the immune system. Perfluorinated alkylate substances (PFAS), such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are persistent, globally disseminated environmental contaminants known to be immunotoxic. Elevated PFAS exposure is associated with lower antibody responses to vaccinations in children and in adults. In addition, some studies have reported a correlation between PFAS levels in the body and lower resistance to disease, in other words an increased risk of infections or cancers. In this context, modelling and simulation platforms could be used to simulate the human immune system with the aim to evaluate the adverse effects that immunotoxicants may have. Here, we show the conditions under which a mathematical model developed for one purpose and application (e.g., in the pharmaceutical domain) can be successfully translated and transferred to another (e.g., in the chemicals domain) without undergoing significant adaptation. In particular, we demonstrate that the Universal Immune System Simulator was able to simulate the effects of PFAS on the immune system, introducing entities and new interactions that are biologically involved in the phenomenon. This also revealed a potentially exploitable pathway for assessing immunotoxicity through a computational model.

Integration of data across toxicity endpoints for improved safety assessment of chemicals: the example of carcinogenicity assessment.

In view of the need to enhance the assessment of consumer products called for in the EU Chemicals Strategy for Sustainability, we developed a methodology for evaluating hazard by combining information across different systemic toxicity endpoints and integrating the information with new approach methodologies. This integrates mechanistic information with a view to avoiding redundant in vivo studies, minimising reliance on apical endpoint tests and ultimately devising efficient testing strategies. Here, we present the application of our methodology to carcinogenicity assessment, mapping the available information from toxicity test methods across endpoints to the key characteristics of carcinogens. Test methods are deconstructed to allow the information they provide to be organised in a systematic way, enabling the description of the toxicity mechanisms leading to the adverse outcome. This integrated approach provides a flexible and resource-efficient means of fully exploiting test methods for which test guidelines are available to fulfil regulatory requirements for systemic toxicity assessment as well as identifying where new methods can be integrated.

A retrospective evaluation of complications associated with forkless tibial tuberosity advancement performed in primary care practice.

OBJECTIVE: To report postoperative complications associated with forkless tibial tuberosity advancement (TTA) performed in primary care veterinary practice and to compare results with previous publications. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Three hundred seventy-four forkless TTAs in 329 dogs performed by six nonspecialist veterinarians. METHODS: Medical records of dogs treated with a standard forkless TTA (2013-2016) and with at least 12 months of postoperative follow-up were reviewed. Complications recorded by the referring practice or the operating veterinarian were classified as minor (medically treated) or major (surgically treated). RESULTS: Complications occurred in 57 of 374 (15.2%) TTAs; 28 (7.5%) complications were major, and 29 (7.7%) complications were minor. Postliminary meniscal injuries were documented in 12 of 374 (3.2%) TTAs (12/57 major complications) and were more common when the ratio of cage size to bodyweight was ≤0.25 (P = .019). Mean TTA (cage size) was greater in this population than what has been previously reported for a lower median bodyweight. CONCLUSION: The incidence of major complications was low and within the range previously reported for TTA in referral practice after adjusting for study design. The magnitude of advancement was greater, and the incidence of postliminary meniscal injury was lower than what has been previously reported, after accounting for dogs that had a preliminary meniscal injury or medial meniscal release. CLINICAL SIGNIFICANCE: Forkless TTA may be successfully performed by experienced veterinarians in primary care practice with a low rate of complications. The incidence of postliminary meniscal injury may be reduced by a greater degree of advancement of the tibial tuberosity.

Making better use of toxicity studies for human health by extrapolating across endpoints.

To develop and evaluate scientifically robust and innovative approaches for the safety assessment of chemicals across multiple regulatory sectors, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) has started a project to explore how to better use the available information, including that from existing animal studies. The aim is to minimize reliance on in vivo testing to avoid redundancy and to facilitate the integration of novel non-animal methods in the regulatory setting with the ultimate goal of designing sustainable testing strategies. In this thought-starter paper, we present a number of examples to illustrate and trigger further discussions within the scientific and regulatory communities on ways to extrapolate useful information for predicting toxicity from one toxicity endpoint to another or across endpoints based on mechanistic information.

Carcinogenicity assessment: Addressing the challenges of cancer and chemicals in the environment.

Cancer is a key public health concern, being the second leading cause of worldwide morbidity and mortality after cardiovascular diseases. At the global level, cancer prevalence, incidence and mortality rates are increasing. These trends are not fully explained by a growing and ageing population: with marked regional and socioeconomic disparities, lifestyle factors, the resources dedicated to preventive medicine, and the occupational and environmental control of hazardous chemicals all playing a role. While it is difficult to establish the contribution of chemical exposure to the societal burden of cancer, a number of measures can be taken to better assess the carcinogenic properties of chemicals and manage their risks. This paper discusses how these measures can be informed not only by the traditional data streams of regulatory toxicology, but also by using new toxicological assessment methods, along with indicators of public health status based on biomonitoring. These diverse evidence streams have the potential to form the basis of an integrated and more effective approach to cancer prevention.

Computer-Assisted Surgery Using 3D Printed Saw Guides for Acute Correction of Antebrachial Angular Limb Deformities in Dogs.

OBJECTIVE: The aim of this study was to report the use of custom saw guides produced using computed tomographic imaging (CT), computer simulation and three-dimensional (3D) printing to aid surgical correction of antebrachial deformities in six dogs. MATERIALS AND METHODS: Antebrachial limb deformities in four small, and two large, breed dogs (seven limbs) were surgically corrected by a radial closing wedge ostectomy and ulnar osteotomy. The location and orientation of the wedge ostectomy were determined using CT data, computer-assisted planning and production of a saw guide in plastic using a 3D printer. At surgery, the guide was clamped to the surface of the radius and used to direct the oscillating saw blade. The resultant ostectomy was closed and stabilized with a bone plate. RESULTS: Five limbs healed without complications. One limb was re-operated due to a poorly resolved rotational component of the deformity. One limb required additional stabilisation with external fixation due to screw loosening. The owners of five dogs completed a Canine Orthopedic Index survey at a follow-up period of 37 to 81 months. The median preoperative score was 3.5 and the median postoperative score was 1, representing an overall positive effect of surgery. Radiographically, 5/7 limbs were corrected in the frontal plane (2/7 were under-corrected). Similarly, 5/7 limbs were corrected in the sagittal plane, and 2/7 were over-corrected in the sagittal place. CONCLUSIONS: Computer-aided design and rapid prototyping technologies can be used to create saw guides to simplify one-stage corrective osteotomies of the antebrachium using internal fixation in dogs. Despite the encouraging results, accurate correction of rotational deformity was problematic and this aspect requires further development.

Peripheral arteriovenous fistula manifesting as antebrachial dermatopathy in a cat.

CASE DESCRIPTION A 13-year-old neutered male Abyssinian cat with a 4-month history of right forelimb edema and multifocal crusting lesions at the distal aspect of the antebrachium was referred to a veterinary teaching hospital for evaluation. Extensive hemorrhage from the lesions had been observed after self-grooming, and findings on histologic examination of a skin biopsy sample prior to referral were consistent with atypical dermal hemodynamics and inflammation. CLINICAL FINDINGS Diffuse pitting edema and multifocal, 3- to 4-mm-diameter sanguineous crusting lesions affecting the antebrachium were observed distal to a pulsatile subcutaneous mass in the right elbow joint region that had a palpable thrill and auscultable bruit. No systemic abnormalities were detected. TREATMENT AND OUTCOME Contrast-enhanced CT angiography with 3-D reconstruction identified an arteriovenous fistula with a large aberrant vessel coursing distally. Surgical ligation of an arterialized vein distal to the fistula without en bloc resection led to resolution of all clinical signs. The vascular anomaly was no longer patent when diagnostic imaging was repeated 5 months after surgery. CLINICAL RELEVANCE Acquired arteriovenous fistulas can lead to bleeding skin lesions affecting the antebrachium in cats. Surgical ligation of an aberrant reverse-shunting vein distal to the fistula successfully resolved clinical signs in the cat of this report and may warrant investigation as a treatment option in cats with this condition.

The Site of Bone Marrow Acquisition Affects the Myeloid to Erythroid Ratio in Apparently Healthy Dogs.

Bone marrow (BM) cytology and histopathology are complementary tools used to investigate hematological diseases. The purpose of this study was to determine if there are site-dependent differences in the diagnostic quality, myeloid to erythroid ratio (MER), and discordant findings in samples from different sites in the same dog. Eighteen apparently healthy dogs were used in the study. The sequence of sample acquisition was randomized according to a Latin square, and samples for BM cytology and histology were collected from both humeri and both ilial crests immediately after death. Board-certified clinical and anatomical pathologists read the cytology and histology, respectively. The data were analyzed using a mixed-effect model. The site of BM acquisition did not affect BM sample quality. The rate of discordant clinical findings between sites was 0.05 (95% confidence interval, 0.01-0.13). In general, by cytology, the MERs were slightly but significantly greater in samples from the ilial crests than from the humeri ( P = .01). The measured MER for histology was nearly twice that for cytology for all sites ( P < .001). In conclusion, there was a low-rate, site-dependent discordance in diagnostic findings in BM samples and differences in MER between the ilial crest and the humerus. A similar study is justified in sick dogs with hematological disease to determine the effect of sampling site on discordant findings between sites.

Evaluation of the rostral projection of the sacral lamina as a component of degenerative lumbosacral stenosis in German shepherd dogs.

OBJECTIVE: To determine the association between a greater rostral projection of the sacral lamina and clinical signs of cauda equina syndrome (CES) in German shepherd dogs (GSD) with presumptive degenerative lumbosacral disease (DLSS). STUDY DESIGN: Retrospective cohort study. SAMPLE POPULATION: One hundred forty-three GSD (125 police dogs and 18 pet dogs) presenting for either CES or prebreeding evaluation. Fifty-five were classified as affected by CES and diagnosed with DLSS, and 88 were classified as unaffected on the basis of clinical and imaging findings. METHODS: The position of the rostral edge of the sacral lamina was measured from radiographs and/or computed tomography (CT) scans. This position was compared between affected and unaffected dogs. In dogs that underwent both radiography and CT scanning, the agreement between sacral lamina localization using each imaging modality was determined. Owners/handlers were contacted to determine whether dogs subsequently developed clinical signs compatible with CES at a mean of 29 months (unaffected). RESULTS: The sacral lamina did not extend as far rostrally in affected dogs, compared to unaffected dogs (P = .04). Among the 88 dogs unaffected by CES at initial evaluation, 2 developed clinical signs consistent with CES at follow-up. CONCLUSION: Rostral projection of the sacral lamina, previously proposed as a potential risk factor in dogs with CES due to lumbosacral degeneration, was not associated with a diagnosis of DLSS in this study; the opposite was true. CLINICAL SIGNIFICANCE: Rostral projection of the sacral lamina may not be a predisposing factor in the development of CES due to DLSS in GSD.

Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity.

Kidney cancer, one of the ten most prevalent malignancies in the world, has exhibited increased incidence over the last decade. The most common subtype is "clear cell" renal cell carcinoma (ccRCC), which features consistent metabolic abnormalities, such as highly elevated glycogen and lipid deposition. By integrating metabolomics, genomic, and transcriptomic data, we determined that enzymes in multiple metabolic pathways are universally depleted in human ccRCC tumors, which are otherwise genetically heterogeneous. Notably, the expression of key urea cycle enzymes, including arginase 2 (ARG2) and argininosuccinate synthase 1 (ASS1), is strongly repressed in ccRCC. Reduced ARG2 activity promotes ccRCC tumor growth through at least two distinct mechanisms: conserving the critical biosynthetic cofactor pyridoxal phosphate and avoiding toxic polyamine accumulation. Pharmacological approaches to restore urea cycle enzyme expression would greatly expand treatment strategies for ccRCC patients, where current therapies only benefit a subset of those afflicted with renal cancer.

Medium-Term Outcome and CT Assessment of Lateral Foraminotomy at the Lumbosacral Junction in Dogs with Degenerative Lumbosacral Stenosis.

OBJECTIVE: This article aims to report the medium-term clinical outcome and assess persistence of enlargement of the lumbosacral lateral intervertebral neurovascular foramen using computed tomography (CT) volumetric analysis in dogs following lateral foraminotomy. MATERIALS: Six dogs that underwent lumbosacral lateral foraminotomy on one or both sides were evaluated with CT prior to, immediately postoperatively (n = 2) and at 12 to 44 months of follow-up. Five out of six dogs had successful clinical outcomes with alleviation of pain and increased levels of activity, according to subjective assessment. Immediate postoperative CT volumetric analysis of the lateral intervertebral neurovascular foramina in two dogs indicated a 650 to 800% increase in volume in extension achieved by foraminotomy (four foramens). At subsequent follow-up, bone regrowth had occurred with reduction in foraminal volume, though in both dogs foraminal volume remained higher than preoperative values. Follow-up CT at a median of 24 months postoperatively indicated a mean 335% increase in volume of the lumbosacral lateral intervertebral neurovascular foramina in extension compared with the preoperative foraminal volume. The follow-up volume was substantially greater than the presurgical volume in four out of six dogs. CLINICAL SIGNIFICANCE: In this limited case series, lateral foraminotomy achieved persistent enlargement of the lumbosacral lateral intervertebral neurovascular foramen in the medium term, but osseous regrowth at the site was demonstrated which may limit the effectiveness of lateral foraminotomy in the longer term. One of two working dogs had recurrent clinical signs that necessitated further surgery.

Clinical outcomes of patient-specific porous titanium endoprostheses in dogs with tumors of the mandible, radius, or tibia: 12 cases (2013-2016).

OBJECTIVE To characterize the processes involved in and outcomes achieved with custom-designed patient-specific implants to provide functional replacement of skeletal structures in dogs with tumors of the mandible, radius, or tibia. DESIGN Prospective case series. ANIMALS 6 dogs with mandibular tumors, 5 with tumors of the distal aspect of the radius, and 1 with a tumor in the distal aspect of the tibia treated from June 2013 to September 2016 at 3 referral centers. PROCEDURES After tumor staging, implants were designed from patients' CT scans by means of various computer-aided design applications and printed by means of selective laser melting in titanium-6 aluminum-4 vanadium alloy. A cutting jig was created in thermoplastic to ensure each osteotomy was performed as planned. Following ostectomy, the implant was secured into the defect with screws of appropriate size and length. RESULTS Initial return to normal clinical function was good to excellent for 11 of the 12 dogs. However, major complications resulted in revision of the implant or amputation of the limb in 5 dogs, and at least 3 of these complications were considered a consequence of faulty implant design or manufacturing. Infection developed in 2 dogs and was successfully treated in 1 dog. The longest-surviving dog maintained good limb function for 2 years. CONCLUSIONS AND CLINICAL RELEVANCE This is the largest reported series of dogs managed with customized 3-D-printed titanium implants. The 3-D printing allowed complex and patient-specific 3-D geometries to be fabricated, enabling function-sparing treatment of bone cancer affecting multiple anatomic sites.

Adrenocortical carcinoma and succinate dehydrogenase gene mutations: an observational case series.

OBJECTIVE: Germline loss-of-function mutations in succinate dehydrogenase (SDHx) genes results in rare tumor syndromes that include pheochromocytoma, paraganglioma, and others. Here we report a case series of patients with adrenocortical carcinoma (ACC) that harbor SDHx mutations. PATIENTS AND RESULTS: We report four unrelated patients with ACC and SDHx mutations. All cases presented with Cushing syndrome and large adrenal masses that were confirmed to be ACC on pathology. All four ACC specimens were found to have truncating mutations in either SDHC or SDHA, while cases 1, 2 and 3 also had the mutations confirmed in the germline: Case 1: SDHC c.397C > T, pR133X; Case 2: SDHC c.43C > T, p.R15X; Case 3: SDHA c.91C > T, p.R31X; Case 4: SDHA c.1258C > T, p.Q420X. Notably, Case 1 had a father and daughter who both harbored the same SDHC germline mutation, and the father had a paraganglioma and renal cell carcinoma. A combination of next generation sequencing, and/or immunohistochemistry, and/or mass spectroscopy was used to determine whether there was loss of heterozygosity and/or loss of SDH protein expression or function within the ACC. Potential evidence of loss of heterozygosity was observed only in Case 2. CONCLUSIONS: We observed truncating mutations in SDHA or SDHC in the ACC and/or germline of four unrelated patients. Given how statistically improbable the concurrence of ACC and pathogenic germline SDHx mutations is expected to be, these observations raise the question whether ACC may be a rare manifestation of SDHx mutation syndromes. Further studies are needed to investigate the possible role of SDH deficiency in ACC pathogenesis.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling.

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

Effect of dorsal laminectomy and dorsal annulectomy with partial lumbosacral discectomy on the volume of the lateral intervertebral neuroforamina in dogs when the lumbosacral junction is extended.

OBJECTIVE: To determine the effect of dorsal annulectomy and partial discectomy on the volume of the lumbosacral lateral intervertebral neurovascular foramina (intervertebral foramina) in canine cadavers during extension of the lumbosacral junction. STUDY DESIGN: Ex vivo experiment. SAMPLE POPULATION: Lumbosacral specimens from 10 large breed dogs euthanatized for reasons unrelated to lumbosacral disease. METHODS: The lumbosacral specimens were clamped in a wooden jig and scanned using computed tomography (CT) with the lumbosacral junction in a neutral position and loaded in extension using a tensioning device. The 3-dimensional volumes of the lumbosacral intervertebral neurovascular foramina were measured and the extent of any disc degeneration was determined from the CT data. A limited dorsal laminectomy of S1 and a dorsal LS annulectomy and partial discectomy were then performed. The lumbosacral specimens were remounted into the jig and loaded into extension at the same tension and were re-scanned. Measurements of intervertebral foraminal volume were then repeated. RESULTS: The mean volume of the lumbosacral foramina (n = 20) was 381 mm3 in neutral (unloaded) positioning and 137 mm3 when loaded in extension. Following dorsal annulectomy, the mean volume was significantly reduced by a mean of 28% to 98 mm3 (P < .01). The foraminal volume was reduced in 19/20 lumbosacral foramen, with the post-annulectomy volume ranging from 31% to 97% of the pre-annulectomy volume (3%-69% reduction). CONCLUSIONS: This study suggests that a dorsal annulectomy with partial discectomy may induce further dynamic collapse of the lumbosacral articulation in the dog.

Computed tomographic evaluation of dynamic alteration of the canine lumbosacral intervertebral neurovascular foramina.

OBJECTIVE: To develop a computed tomographic (CT) method to measure the volume of the lumbosacral intervertebral neurovascular foramina (IVF) in dogs, and determine the effect of the range of motion of the lumbosacral (LS) junction on this measurement in German shepherd dogs (GSDs) with degenerative lumbosacral stenosis (DLSS) compared to unaffected controls. STUDY DESIGN: In vivo analysis and retrospective case series. SAMPLE POPULATION: Twenty-four working Police GSDs, 12 diagnosed with DLSS and 12 unaffected by DLSS were compared to 10 Greyhounds without DLSS. METHODS: Three-dimensional renderings of CT data were used to measure the lumbosacral foraminal volume of dogs positioned in dorsal recumbency with the LS junction alternately positioned in extension, neutral position, and flexion. RESULTS: Volumetric analysis of the IVF was found repeatable for the extended and neutral positions (interclass correlation coefficient of 0.89 and 0.8, respectively). The mean lumbosacral IVF volume was decreased by 74% between LS flexion and extension in Greyhounds, compared to 79 and 85% reductions in GSDs unaffected and affected by DLSS, respectively. The lumbosacral IVF volume was decreased by 23% when comparing extended to neutral LS positions in Greyhounds, 29% in unaffected GSDs, and 31% in affected GSDs. IVF volumes were smaller in affected GSDs compared to unaffected GSDs (P < .05) and Greyhounds (P < .01). CONCLUSIONS: Positioning the LS junction in full extension decreases the volume of the lumbosacral IVF. This dynamic narrowing was more pronounced in GSDs with signs of DLSS than in GSDs not overtly affected by DLSS.

The application of molecular modelling in the safety assessment of chemicals: A case study on ligand-dependent PPARγ dysregulation.

The aim of this paper was to provide a proof of concept demonstrating that molecular modelling methodologies can be employed as a part of an integrated strategy to support toxicity prediction consistent with the mode of action/adverse outcome pathway (MoA/AOP) framework. To illustrate the role of molecular modelling in predictive toxicology, a case study was undertaken in which molecular modelling methodologies were employed to predict the activation of the peroxisome proliferator-activated nuclear receptor γ (PPARγ) as a potential molecular initiating event (MIE) for liver steatosis. A stepwise procedure combining different in silico approaches (virtual screening based on docking and pharmacophore filtering, and molecular field analysis) was developed to screen for PPARγ full agonists and to predict their transactivation activity (EC50). The performance metrics of the classification model to predict PPARγ full agonists were balanced accuracy=81%, sensitivity=85% and specificity=76%. The 3D QSAR model developed to predict EC50 of PPARγ full agonists had the following statistical parameters: q2cv=0.610, Nopt=7, SEPcv=0.505, r2pr=0.552. To support the linkage of PPARγ agonism predictions to prosteatotic potential, molecular modelling was combined with independently performed mechanistic mining of available in vivo toxicity data followed by ToxPrint chemotypes analysis. The approaches investigated demonstrated a potential to predict the MIE, to facilitate the process of MoA/AOP elaboration, to increase the scientific confidence in AOP, and to become a basis for 3D chemotype development.