Circumscribing Laser Cuts Attenuate Seizure Propagation in a Mouse Model of Focal Epilepsy.

In partial onset epilepsy, seizures arise focally in the brain and often propagate. Patients frequently become refractory to medical management, leaving neurosurgery, which can cause neurologic deficits, as a primary treatment. In the cortex, focal seizures spread through horizontal connections in layers II/III, suggesting that severing these connections can block seizures while preserving function. Focal neocortical epilepsy is induced in mice, sub-surface cuts are created surrounding the seizure focus using tightly-focused femtosecond laser pulses, and electrophysiological recordings are acquired at multiple locations for 3-12 months. Cuts reduced seizure frequency in most animals by 87%, and only 5% of remaining seizures propagated to the distant electrodes, compared to 80% in control animals. These cuts produced a modest decrease in cortical blood flow that recovered and left a ≈20-µm wide scar with minimal collateral damage. When placed over the motor cortex, cuts do not cause notable deficits in a skilled reaching task, suggesting they hold promise as a novel neurosurgical approach for intractable focal cortical epilepsy.

Evaluation of an ultrasound-guided freeze-core biopsy system for canine and feline brain tumors.

Objective: To determine if a single brain biopsy utilizing a freeze-core needle harvest system Cassi II under ultrasound guidance provides a diagnostic sample; to evaluate the technique's efficacy in procuring diagnostic samples in comparison with "open" surgical biopsies; and to describe intraoperative complications associated with the technique. Study design: Experimental clinical study. Animals: Seventeen dogs and four cats with magnetic resonance imaging (MRI) diagnoses of readily surgically accessible intracranial masses. Methods: Immediately prior to surgical biopsy (SB), freeze-core biopsy (FCB) sample was obtained from each patient under ultrasound guidance. Results: Histopathology results from single FCB samples were found to be in 100% agreement with the SB samples. Freezing artifact was minimal and did not interfere with histopathologic interpretation. There were no intraoperative complications specifically attributable to the use of the FCB system. Conclusion: Based on the results of this small experimental study, the FCB system is expected to safely yield diagnostic quality intracranial masses biopsy specimens. Clinical significance: This system has the potential of obtaining diagnostic biopsies of more deeply seated brain lesions (i.e., intra-axial tumors considered inaccessible or with large risks/difficulties by standard surgical means) which would provide a definitive diagnosis to guide appropriate therapy.

A Gamma-adapted subunit vaccine induces broadly neutralizing antibodies against SARS-CoV-2 variants and protects mice from infection.

In the context of continuous emergence of SARS-CoV-2 variants of concern (VOCs), one strategy to prevent the severe outcomes of COVID-19 is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD vaccine derived from the Gamma SARS-CoV-2 variant adjuvanted with Alum. The Gamma-adapted RBD vaccine is more immunogenic than the Ancestral RBD vaccine in terms of inducing broader neutralizing antibodies. The Gamma RBD presents more immunogenic B-cell restricted epitopes and induces a higher proportion of specific-B cells and plasmablasts than the Ancestral RBD version. The Gamma-adapted vaccine induces antigen specific T cell immune responses and confers protection against Ancestral and Omicron BA.5 SARS-CoV-2 challenge in mice. Moreover, the Gamma RBD vaccine induces higher and broader neutralizing antibody activity than homologous booster vaccination in mice previously primed with different SARS-CoV-2 vaccine platforms. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate.

Extreme plasticity of reproductive state in a female rodent.

Successful sexual reproduction relies on the coordination of multiple biological systems, yet traditional concepts of biological sex often ignore the natural plasticity in morphology and physiology underlying sex. Most female mammals develop a patent (i.e., opened) vaginal entrance (introitus) prenatally or postnatally before or during puberty, usually under the influence of estrogens, and remain patent for the remainder of their lifespan1. An exception is the southern African giant pouched rat (Cricetomys ansorgei), whose vaginal introitus remains sealed well into adulthood2. Here, we explore this phenomenon and report that the reproductive organs and the vaginal introitus can undergo astounding and reversible transformation. Non-patency is characterized by reduced uterine size and the presence of a sealed vaginal introitus. Furthermore, the female urine metabolome shows that patent and non-patent females profoundly differ in their urine content, a reflection of differences in physiology and metabolism. Surprisingly, patency state did not predict fecal estradiol or progesterone metabolite concentrations. Exploring the plasticity that exists in reproductive anatomy and physiology can uncover that traits long considered 'fixed' in adulthood can become plastic under specific evolutionary pressures. Moreover, the barriers to reproduction that such plasticity creates present unique challenges to maximizing reproductive potential.

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis.

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

Acute kidney injury in dogs following ingestion of cream of tartar and tamarinds and the connection to tartaric acid as the proposed toxic principle in grapes and raisins.

OBJECTIVE: To (1) describe exposure history, clinical signs, treatment, and diagnostic findings in 4 dogs following ingestion of tamarinds, and in 2 dogs following ingestion of cream of tartar, and (2) discuss tartaric acid, the common denominator, as the proposed toxic principle in tamarinds and grapes. SERIES SUMMARY: Reports in which dogs developed acute kidney injury following ingestion of cream of tartar or tamarinds were identified from the ASPCA Animal Poison Control Center electronic database. In these cases, decontamination was not performed, and treatments were delayed. Despite IV fluids and symptomatic and supportive care, 2 of the dogs became anuric and 1 became oliguric. Four dogs were euthanized, and the outcome is unknown for 2 of the dogs. Necropsies were performed on 3 of the dogs. Clinical signs, laboratory findings, and histopathologic lesions were similar to those reported in grape and raisin toxicosis. NEW OR UNIQUE INFORMATION PROVIDED: Acute kidney injury may develop following ingestion of cream of tartar or tamarinds in dogs. Connecting these reports with findings in grape and raisin toxicosis and the sensitivity to tartaric acid in dogs, tartaric acid is identified as the likely toxic component in grapes and tamarinds.

Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis.

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3', 5'-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

Myocardial protein aggregates in pet guinea pigs.

A retrospective study of guinea pigs submitted for necropsy revealed intracytoplasmic inclusions in the cardiomyocytes of 26 of 30 animals. The inclusions were found with approximately the same frequency in male and female guinea pigs and were slightly more common in older animals. In most cases, the animals did not have clinical signs or necropsy findings suggestive of heart failure, and the cause of death or reason for euthanasia was attributed to concurrent disease processes. However, the 4 guinea pigs with the highest inclusion body burden all had pulmonary edema, sometimes with intra-alveolar hemosiderin-laden macrophages, suggestive of heart failure. The inclusions were found in both the left and right ventricular myocardium, mainly in the papillary muscles, but were most common in the right ventricular free wall. No inclusions were detected in the atrial myocardium or in skeletal muscle. The inclusions did not stain with Congo red or periodic acid-Schiff. Electron microscopy revealed dense aggregates of disorganized myofilaments and microtubules that displaced and compressed the adjacent organelles. By immunohistochemistry, there was some scattered immunoreactivity for desmin and actin at the periphery of the inclusions and punctate actin reactivity within the aggregates. The inclusions did not react with antibodies to ubiquitin or cardiac myosin, but were variably reactive for alpha B crystallin, a small heat shock chaperone protein. The inclusions were interpreted as evidence of impaired proteostasis.

Pharmacological and genetic perturbation establish SIRT5 as a promising target in breast cancer.

SIRT5 is a member of the sirtuin family of NAD+-dependent protein lysine deacylases implicated in a variety of physiological processes. SIRT5 removes negatively charged malonyl, succinyl, and glutaryl groups from lysine residues and thereby regulates multiple enzymes involved in cellular metabolism and other biological processes. SIRT5 is overexpressed in human breast cancers and other malignancies, but little is known about the therapeutic potential of SIRT5 inhibition for treating cancer. Here we report that genetic SIRT5 disruption in breast cancer cell lines and mouse models caused increased succinylation of IDH2 and other metabolic enzymes, increased oxidative stress, and impaired transformation and tumorigenesis. We, therefore, developed potent, selective, and cell-permeable small-molecule SIRT5 inhibitors. SIRT5 inhibition suppressed the transformed properties of cultured breast cancer cells and significantly reduced mammary tumor growth in vivo, in both genetically engineered and xenotransplant mouse models. Considering that Sirt5 knockout mice are generally normal, with only mild phenotypes observed, these data establish SIRT5 as a promising target for treating breast cancer. The new SIRT5 inhibitors provide useful probes for future investigations of SIRT5 and an avenue for targeting SIRT5 as a therapeutic strategy.

Malunion of an In Utero Metacarpal Fracture in a Standardbred Mare Presenting for Dystocia.

In utero fracture and malunion of long bones is a rare condition in horses. Most foals with in utero fractures are aborted, and the identification of a fetal in utero fracture in a mare with dystocia has not been reported. A 7-year-old multiparous Standardbred mare presented to a referral center for correction of dystocia. Assisted vaginal delivery and controlled vaginal delivery attempts were unsuccessful mainly because of contracted tendons impeding mutation. As the foal was alive, a cesarean section was elected. The foal was delivered but ultimately euthanized because of the congenital abnormalities. Computed tomography of the right forelimb of the foal along with gross examination and histologic evaluation of the right metacarpus revealed the malunion of a previous in utero fracture. While a few cases have been reported of in utero fracture, many of these were in abortuses and not in fetuses at term, making this case a new presentation and potential etiology for dystocia.

Research-Relevant Conditions and Pathology of Laboratory Mice, Rats, Gerbils, Guinea Pigs, Hamsters, Naked Mole Rats, and Rabbits.

Animals are valuable resources in biomedical research in investigations of biological processes, disease pathogenesis, therapeutic interventions, safety, toxicity, and carcinogenicity. Interpretation of data from animals requires knowledge not only of the processes or diseases (pathophysiology) under study but also recognition of spontaneous conditions and background lesions (pathology) that can influence or confound the study results. Species, strain/stock, sex, age, anatomy, physiology, spontaneous diseases (noninfectious and infectious), and neoplasia impact experimental results and interpretation as well as animal welfare. This review and the references selected aim to provide a pathology resource for researchers, pathologists, and veterinary personnel who strive to achieve research rigor and validity and must understand the spectrum of "normal" and expected conditions to accurately identify research-relevant experimental phenotypes as well as unusual illness, pathology, or other conditions that can compromise studies involving laboratory mice, rats, gerbils, guinea pigs, hamsters, naked mole rats, and rabbits.

Equine Stereotaxtic Population Average Brain Atlas With Neuroanatomic Correlation.

There is growing interest in the horse for behavioral, neuroanatomic and neuroscientific research due to its large and complex brain, cognitive abilities and long lifespan making it neurologically interesting and a potential large animal model for several neuropsychological diseases. Magnetic resonance imaging (MRI) is a powerful neuroscientific research tool that can be performed in vivo, with adapted equine facilities, or ex-vivo in the research setting. The brain atlas is a fundamental resource for neuroimaging research, and have been created for a multitude animal models, however, none currently exist for the equine brain. In this study, we document the creation of a high-resolution stereotaxic population average brain atlas of the equine. The atlas was generated from nine unfixed equine cadaver brains imaged within 4 h of euthanasia in a 3-tesla MRI. The atlas was generated using linear and non-linear registration methods and quality assessed using signal and contrast to noise calculations. Tissue segmentation maps (TSMs) for white matter (WM), gray matter (GM) and cerebrospinal fluid (CSF), were generated and manually segmented anatomic priors created for multiple subcortical brain structures. The resulting atlas was validated and correlated to gross anatomical specimens and is made freely available at as an online resource for researchers (https://doi.org/10.7298/cyrs-7b51.2). The mean volume metrics for the whole brain, GM and WM for the included subjects were documented and the effect of age and laterality assessed. Alterations in brain volume in relation to age were identified, though these variables were not found to be significantly correlated. All subjects had higher whole brain, GM and WM volumes on the right side, consistent with the well documented right forebrain dominance of horses. This atlas provides an important tool for automated processing in equine and translational neuroimaging research.

Sex-specific hepatic lipid and bile acid metabolism alterations in Fancd2-deficient mice following dietary challenge.

Defects in the Fanconi anemia (FA) DNA damage-response pathway result in genomic instability, developmental defects, hematopoietic failure, cancer predisposition, and metabolic disorders. The endogenous sources of damage contributing to FA phenotypes and the links between FA and metabolic disease remain poorly understood. Here, using mice lacking the Fancd2 gene, encoding a central FA pathway component, we investigated whether the FA pathway protects against metabolic challenges. Fancd2-/- and wildtype (WT) mice were fed a standard diet (SD), a diet enriched in fat, cholesterol, and cholic acid (Paigen diet), or a diet enriched in lipid alone (high-fat diet (HFD)). Fancd2-/- mice developed hepatobiliary disease and exhibited decreased survival when fed a Paigen diet but not a HFD. Male Paigen diet-fed mice lacking Fancd2 had significant biliary hyperplasia, increased serum bile acid concentration, and increased hepatic pathology. In contrast, female mice were similarly impacted by Paigen diet feeding regardless of Fancd2 status. Upon Paigen diet challenge, male Fancd2-/- mice had altered expression of genes encoding hepatic bile acid transporters and cholesterol and fatty acid metabolism proteins, including Scp2/x, Abcg5/8, Abca1, Ldlr, Srebf1, and Scd-1 Untargeted lipidomic profiling in liver tissue revealed 132 lipid species, including sphingolipids, glycerophospholipids, and glycerolipids, that differed significantly in abundance depending on Fancd2 status in male mice. We conclude that the FA pathway has sex-specific impacts on hepatic lipid and bile acid metabolism, findings that expand the known functions of the FA pathway and may provide mechanistic insight into the metabolic disease predisposition in individuals with FA.

Histological, electrophysiological and clinical effects of thermal radiofrequency therapy of the saphenous nerve and pulsed radiofrequency therapy of the sciatic nerve in dogs.

OBJECTIVE: Thermal radiofrequency (TRF) of the saphenous nerve (a sensory nerve) combined with pulsed radiofrequency (PRF) of the sciatic nerve (a sensory and motor nerve) might relieve intractable stifle osteoarthritis (OA) pain in dogs. The objective was to determine if saphenous nerve TRF induces Wallerian degeneration and if sciatic nerve PRF induces degeneration or dysfunction. STUDY DESIGN: Blinded, controlled, randomized, preclinical study. ANIMALS: A group of six intact, female Beagle dogs aged 14-16 months. METHODS: In each dog, one pelvic limb was assigned randomly to the control group and the other to the treatment group. Dogs were anesthetized and, using ultrasonography, radiofrequency electrodes were positioned adjacent to saphenous and sciatic nerves bilaterally; TRF and PRF were performed only in the treatment limb. Motor nerve conduction velocity (MNCV) was measured in both sciatic nerves 2 weeks later, and the dogs were euthanized. Hematoxylin and eosin-stained sections of saphenous and sciatic nerves were examined using light microscopy. Degeneration and inflammation were scored 0 (none) to 3 (severe). A one-tailed, paired Wilcoxon signed-rank test was used to test for differences in scores and MNCV between control and treatment nerves. RESULTS: Degeneration and inflammation scores were higher in treatment saphenous nerves in 5/6 dogs [83%; 95% confidence interval (CI), 36%, 99%]; however, after Bonferroni correction only degeneration score was higher (p = 0.0313). Degeneration, inflammation or decreased MNCV were not observed in sciatic nerves (each outcome: 0/6 nerves, 0%; 95% CI, 0%, 48%). No dogs experienced postprocedural pain or neurological deficits. CONCLUSIONS AND CLINICAL RELEVANCE: The degeneration in TRF-treated saphenous nerves appears sufficient to impair transmission. Sciatic nerve PRF did not cause degeneration with attendant motor deficits, consistent with a proposed neuromodulatory mechanism. A clinical trial is needed to confirm the combined techniques produce analgesia without motor deficits in dogs with stifle OA.

Physical confinement induces malignant transformation in mammary epithelial cells.

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

In Vivo Anticancer Activity of a Rhenium(I) Tricarbonyl Complex.

The rhenium(I) complex fac-[Re(CO)3(2,9-dimethyl-1,10-phenanthroline)(OH2)]+ (1) was previously shown to exhibit potent in vitro anticancer activity in a manner distinct from conventional platinum-based drugs (J. Am. Chem. Soc. 2017, 139, 14302-14314). In this study, we report further efforts to explore its aqueous speciation and antitumor activity. The cellular uptake of 1 was measured in A2780 and cisplatin-resistant A2780CP70 ovarian cancer cells by inductively coupled plasma mass spectrometry, revealing similar uptake efficiency in both cell lines. High accumulation in the mitochondria was observed, contradicting prior fluorescence microscopy studies. The luminescence of 1 is highly dependent on pH and coordination environment, making fluorescence microscopy somewhat unreliable for determining compound localization. The in vivo anticancer activity of 1 was evaluated in mice bearing patient-derived ovarian cancer tumor xenografts. These studies conclusively show that 1 is capable of inhibiting tumor growth, providing further credibility for the use of these compounds as anticancer agents.

Salinomycin decreases feline sarcoma and carcinoma cell viability when combined with doxorubicin.

BACKGROUND: Cancer is a significant health threat in cats. Chemoresistance is prevalent in solid tumors. The ionophore salinomycin has anti-cancer properties and may work synergistically with chemotherapeutics. The purpose of our study was to determine if salinomycin could decrease cancer cell viability when combined with doxorubicin in feline sarcoma and carcinoma cells. RESULTS: We established two new feline injection-site sarcoma cell lines, B4 and C10, and confirmed their tumorigenic potential in athymic nude mice. B4 was more resistant to doxorubicin than C10. Dose-dependent effects were not observed until 92 µM in B4 cells (p = 0.0006) vs. 9.2 µM (p = 0.0004) in C10 cells. Dose-dependent effects of salinomycin were observed at 15 µM in B4 cells (p = 0.025) and at 10 µM in C10 cells (p = 0.020). Doxorubicin plus 5 µM salinomycin decreased viability of B4 cells compared to either agent alone, but only at supra-pharmacological doxorubicin concentrations. However, doxorubicin plus 5 µM salinomycin decreased viability of C10 cells compared to either agent alone at doxorubicin concentrations that can be achieved in vivo (1.84 and 4.6 µM, p < 0.004). In SCCF1 cells, dose-dependent effects of doxorubicin and salinomycin were observed at 9.2 (p = 0.036) and 2.5 (p = 0.0049) µM, respectively. When doxorubicin was combined with either 1, 2.5, or 5 µM of salinomycin in SCCF1 cells, dose-dependent effects of doxorubicin were observed at 9.2 (p = 0.0021), 4.6 (p = 0.0042), and 1.84 (p = 0.0021) µM, respectively. Combination index calculations for doxorubicin plus 2.5 and 5 µM salinomycin in SCCF1 cells were 0.4 and 0.6, respectively. CONCLUSIONS: We have developed two new feline sarcoma cell lines that can be used to study chemoresistance. We observed that salinomycin may potentiate (C10 cells) or work synergistically (SCCF1 cells) with doxorubicin in certain feline cancer cells. Further research is indicated to understand the mechanism of action of salinomycin in feline cancer cells as well as potential tolerability and toxicity in normal feline tissues.