Efficacy of host cell serine protease inhibitor MM3122 against SARS-CoV-2 for treatment and prevention of COVID-19.

We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and a therapeutic drug for the treatment of COVID-19 given intraperitoneally in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research, but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

Efficacy of Host Cell Serine Protease Inhibitor MM3122 against SARS-CoV-2 for Treatment and Prevention of COVID-19.

We have developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases including transmembrane protease serine 2 (TMPRSS2), matriptase and hepsin. TMPRSS2 is a membrane associated protease which is highly expressed in the upper and lower respiratory tract and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell receptor binding, entry, replication, and dissemination of new virion particles. We have previously shown that compound MM3122 exhibited sub nanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell-viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Further, we have evaluated MM3122 in a mouse model of COVID-19 and have demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion, and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in pro-inflammatory cytokines and chemokines production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2 infected mice. Therefore, MM3122 is a promising lead candidate small molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host-cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and therapeutic drug for the treatment of COVID-19 in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

Osteosarcoma of the wing in a sulfur-crested cockatoo.

A 26-year-old female sulfur-crested cockatoo (Cacatua galerita) was evaluated for vocalizing through the night and extending her right wing. Physical examination revealed a large, firm mass extending from the humerus to the distal aspect of the elbow. Computed tomography confirmed a large aggressive mass of the right distal humerus with a large soft tissue component, severe osteolysis, and adjacent periosteal proliferation. Fine-needle aspirates of the mass were most compatible with sarcoma, and osteosarcoma was prioritized. An unstained slide was treated with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase (NBT/BCIP) substrate for ALP detection and was strongly positive, confirming a diagnosis of osteosarcoma. A month later, the patient underwent wing amputation and arrested during recovery from anesthesia. Post-mortem examination and histopathology were consistent with osteosarcoma. This case report highlights a rare occurrence of osteosarcoma in a cockatoo as well as its cytologic and histologic features. Additionally, this report provides support for NBT/BCIP application in ALP-expressing tumors, a cytochemical stain that has been minimally investigated in avian species.

Investigation of Potassium Chloride for Euthanasia of Anesthetized African Clawed Frogs (Xenopus laevis).

Euthanasia is frequently performed in amphibians, but techniques are currently limited in number and variable in effectiveness. The current study examined the use of potassium chloride (KCl) for euthanasia of anesthetized African clawed frogs (Xenopus laevis). Twenty adult, female African clawed frogs were anesthetized by immersion in buffered tricaine methanesulfonate (MS-222) for 5 min beyond loss of righting reflex. Frogs were then randomly assigned to receive one of 4 treatments: KCl via intracardiac injection (10 mEq/kg; IC, n = 5), intracoelomic injection (100 mEq/kg; ICe, n = 5), immersion (4,500 mEq/L; IMS, n = 5), or no treatment (C, n = 5). After treatment, serial heart rate was measured via Doppler device until either the loss of Doppler sounds, a 60-min endpoint (IC, ICe, IMS), or recovery (C). Times to loss of righting reflex, loss of Doppler sounds, and/or recovery were recorded. Plasma potassium concentrations were measured immediately after Doppler sound cessation in frogs in IC (n = 1), ICe (n = 2), and IMS (n = 5). Injection failure occurred in 1 IC frog, and 1 Ice frog regained spontaneous movement 4 min after treatment administration. Data from these 2 frogs were not included in statistical analysis. Doppler sound cessation occurred in 4 of 4, 4 of 4, 0 of 5, and 0 of 5 frogs in IC, ICe, IMS, and C, respectively. Median (range) times to Doppler sound cessation in IC and ICe were 6 (0 to 16) s and 18 (10 to 25) min, respectively. Plasma potassium concentration was greater than 9.0 mmol/L in sampled frogs. Intracardiac KCl at 10 mEq/kg and intracoelomic KCl at 100 mEq/kg were effective for euthanasia of anesthetized African clawed frogs. Return to MS-222 solution after KCl administration may be warranted to prevent unintended, premature anesthetic recovery prior to death.

Retrospective analysis of histologic lesions in captive arachnids.

Invertebrates, including arachnids, are a common taxon in zoological collections. Invertebrate medicine and pathology are emerging subspecialties, but there is limited reference material or published resources describing histologic lesions in arachnids. Histopathology of 26 captive arachnids (20 spiders and 6 scorpions) from institutional collections was reviewed. Most animals were found dead with limited clinical signs. Tissues evaluated included body wall (cuticle and epidermis), skeletal muscle, book lungs, digestive tract (pharynx, esophagus, sucking stomach, midgut tube, midgut diverticula, and stercoral pocket), central and peripheral nervous system, heart, hemolymph vessels and sinuses, Malpighian tubules, coxal glands, and gonads. Inflammation was frequent (24/26, 92%), and seen in multiple organs (18/24, 75%) with the midgut diverticulum most commonly affected (14/24, 58%) followed by the book lungs (13/24 arachnids, 54%), and body wall (8/24 arachnids, 33%). Inflammation comprised hemocyte accumulation, hemocytic coagula, melanization, and nodulation. Infectious agents, including bacteria (11/26, 42%), fungi (10/26, 38%), and parasites (2/26, 8%), were seen within inflammatory aggregates. Coinfection with multiple infectious agents was common (6/24, 25%). No etiologic agent was identified in 7/24 (29%) cases with inflammatory lesions. Lesions suggestive of decreased nutritional status or increased metabolic rate included midgut diverticula atrophy in 11/26 (42%) animals and skeletal muscle atrophy in 6/26 (23%) animals. Atrophic lesions were seen in combination with infection (8/11, 73%), pregnancy (2/11, 18%), male sex (2/11, 18%), or without other lesions (1/11, 9%). Other suspected contributors to death included dysecdysis-associated trauma (2/26, 8%) and uterine intussusception (1/26, 4%). No animals had neoplasia.

Necrotizing Hepatitis Associated with Clostridium perfringens in Broiler Chicks.

In this retrospective study we describe unusual cases of clostridial hepatitis associated with high mortality in young broiler chicks. Eleven cases of necrotizing hepatitis in broiler chicks from four companies were submitted to the Poultry Diagnostic and Research Center or the Georgia Poultry Laboratory Network between 2017 and 2020. In most flocks, increased 3-day mortality was followed by an elevated 7-day mortality. Gross lesions included green to dark brown discoloration of the liver, congested lungs, serosanguineous fluid in the caudoventral aspect of the abdomen, and emphysema in the yolk sacs. In birds older than a week of age, disease with neurologic signs became evident and consisted of tremors, stargazing, and incoordination. Histopathologic evaluation revealed multifocal to coalescing fibrinoheterophilic and necrotizing hepatitis associated with gram-positive, long, rod-shaped bacteria. Formalin-fixed liver samples from six cases out of eight cases tested were positive for Clostridium perfringens by immunohistochemistry. Liver samples from two cases were culture positive for Clostridium spp., and C. perfringens was isolated from one sample. Toxinotyping by PCR performed in seven samples revealed the presence of the genes that code for alpha toxin phospholipase C (cpa or plc) and necrotic enteritis toxin B-like (netB) in six samples and as well as C. perfringens large cytotoxin (tpeL) in one sample. Broiler breeders are the suspected source of the infection, and testing revealed C. perfringens in hatchery samples and among broiler breeder flocks. Antimicrobial therapy was coupled with enhanced sanitation at the farm and hatchery in that company, markedly decreasing the mortality and clinical signs. This is the first comprehensive evaluation of clostridial necrotizing hepatitis in newly hatched chicks, and the second ever reported in the literature.

Hepatitis necrotizante asociada con Clostridium perfringens en pollos de engorde En este estudio retrospectivo se describen casos inusuales de hepatitis clostridial asociados con una alta mortalidad en pollos de engorde jóvenes. Once casos de hepatitis necrotizante en pollos de engorde de cuatro empresas se enviaron al Centro de Investigación y Diagnóstico Avícola o a la Red de Laboratorios Avícolas del Estado Georgia entre los años 2017 y 2020. En la mayoría de las parvadas, el aumento de la mortalidad a los tres días fue seguido por una mortalidad elevada a los siete días. Las lesiones macroscópicas incluyeron coloración del hígado de verde a marrón oscuro, pulmones congestionados, líquido serosanguinolento en la cara caudoventral del abdomen y enfisema en los sacos vitelinos. En aves mayores de una semana de edad, la enfermedad con signos neurológicos se hizo evidente y consistía en temblores, torticolis (aves como observando a las estrellas) y falta de coordinación. La evaluación histopatológica reveló hepatitis multifocal a fibrinoheterófila coalescente y necrotizante asociada con bacterias grampositivas largas en forma de bastón. Las muestras de hígado fijadas en formalina de seis casos de los ocho casos analizados dieron positivo para Clostridium perfringens por inmunohistoquímica. Las muestras de hígado de dos casos dieron positivo en cultivo para Clostridium spp., y se aisló C. perfringens de una muestra. La tipificación por el tipo de toxina mediante PCR realizado en siete muestras reveló la presencia de los genes que codifican la toxina alfa fosfolipasa C (cpa, plc) y la toxina de enteritis necrótica similar a la toxina B (netB) en seis muestras, así como la citotoxina grande de C. perfringens (tpeL) en una muestra. Se sospecha que las reproductoras de pollos de engorde son la fuente de la infección, y las pruebas revelaron C. perfringens en las muestras de las incubadoras y entre las parvadas de reproductoras de pollos de engorde. La terapia antimicrobiana se combinó con un saneamiento mejorado en la granja y en la incubadora de esa empresa, lo que redujo notablemente la mortalidad y los signos clínicos. Esta es la primera evaluación exhaustiva de la hepatitis necrosante por clostridios en pollitos recién nacidos y la segunda que se ha informado en la literatura.

Of Elephants and Other Mammals: A Comparative Review of Reproductive Tumors and Potential Impact on Conservation.

Reproductive tumors can impact conception, pregnancy, and birth in mammals. These impacts are well documented in humans, while data in other mammals are limited. An urgent need exists to understand the reproductive impact of these lesions in endangered species, because some endangered species have a documented high prevalence of reproductive tumors. This article documents that the prevalence of both benign and malignant neoplasia differs between African and Asian elephants, with Asian elephants more frequently diagnosed and negatively affected by both. The prevalence of these tumors across mammalian species is compared, and impact plus treatment options in human medicine are reviewed to inform decision making in elephants. Evidence suggests that reproductive tumors can negatively impact elephant conservation. Future studies that document reproductive outcomes, including the success of various treatment approaches in elephants with tumors will benefit conservation efforts.

A Multi-Institutional Collaboration to Understand Neoplasia, Treatment and Survival of Snakes.

This multi-institutional collaborative study of neoplasia in snakes reviewed medical records of snakes at each facility to determine species prevalence, survival, and methods of treatment. Complete species numbers of snakes were also collected at each facility. In total, 65 species, 133 snakes, and 149 unique neoplasias were included in this study. Affected species, age, sex, and their tumor prevalence, tumor type and location, metastasis, treatment, and survival data are reported. The highest species-specific tumor prevalence was in Common or Northern Watersnakes (Nerodia sipedon) (30.8%, n = 4 of 13), Eastern Diamond-Backed Rattlesnakes (Crotalus adamanteus) (26.3%, n = 5 of 19), and Timber rattlesnakes (Crotalus horridus) (22.7%, n = 5 of 22). Malignant tumors predominated (86.6%, n = 129 of 149) with soft tissue sarcomas being the most common (30.2%, n = 45 of 149). Snakes with malignant neoplasia, metastases, or indeterminate presence of metastases were statistically more likely to die from their neoplasms than snakes having either benign neoplasia or no diagnosed metastases (p < 0.05). Gender, taxonomic family, and species of those evaluated did not significantly affect the outcome of snakes with neoplasia. Only 27.1% (n = 36 of 133) of snakes received a reported form of treatment and, for those treated, surgical excision was the most common treatment modality. There was not a significant difference in outcome based on treatment; however, surgery and chemotherapy were associated with death from a cause other than their tumor.

Migration and Histologic Effects of Visible Implant Elastomer (VIE) and Passive Integrated Transponder (PIT) Tags in the Marine Toad (Rhinella marina).

Passive integrated transponder (PIT) and visible implant elastomer (VIE) tags are commonly used to identify reptiles, amphibians, and fish. Despite reports of good retention rates and little to no negative effect on survival time, migration remains a concern and histologic changes have not been widely evaluated. Fifty-six wild-caught marine toads (Rhinella marina) were marked with a PIT tag in the left caudal thigh and a VIE tag over the right gastrocnemius muscle prior to transport to the North Carolina Zoo. Fourteen toads were then humanely euthanized on day 9, 15, 32, and 62 for postmortem examination and histopathology which were compared to 10 control toads with no tags. All PIT tags were functional and 95% remained at the insertion site with minimal to no histologic changes. Externally, 48% of VIE tags were visible through the skin at the original site of injection under fluorescent or UV light. Upon gross examination of the tissues, VIE tags had an overall retention rate of 62% at the injection site, with similar retention rates across time points. Migrated VIE material was visible grossly and histologically in the kidneys of 98% of toads and along the right leg, proximally and distally, in 25% of toads. VIE material was also found sporadically in mesentery, colon, and free in the coelomic cavity. Histologically, VIE material in the skin was surrounded by minimal to mild granulomatous inflammation while in the kidney it was associated with dilation of the small vessels, edema, and granulomatous nephritis that progressed in severity over time. Based on these findings, the authors recommend the use of PIT tags over VIE tags for identification of adult anurans, when feasible.

Ductal Plate Malformations in Captive Snakes.

Ductal plate malformations are abnormalities in the liver that arise from inappropriate or incomplete remodeling of the embryologic ductal plate. Various types of ductal plate malformations are reported in the human and veterinary literature, most commonly affecting domestic mammalian species but also fish. We investigated the occurrence and described the histopathologic features of ductal plate malformations in captive snakes. Malformations were identified in 18 snakes: 10 colubrids, 6 vipers, and 2 boids. There was no sex predilection, and the mean age was 17 years. The majority of lesions were incidental with most snakes having one or more comorbidities, most commonly neoplasia or systemic inflammation, that resulted in natural death or euthanasia. Ductal plate malformations in all livers were broadly characterized by a well-demarcated nodule of irregular bile ducts embedded within a varying amount of fibrous stroma. Malformations were further categorized based on the amount of fibrous stroma and dilation of the bile ducts as von Meyenburg complexes, cystic liver disease, and/or an intermediate hybrid subtype representative of cysts arising within von Meyenburg complexes. Histochemical and immunohistochemical staining, including Gomori's trichome and pan-cytokeratin, respectively, were applied on select cases to confirm histologic features. Malignant transformation was not identified within this population.

STRONGYLOIDIASIS IN PINE BARRENS TREEFROGS (HYLA ANDERSONII).

Over a period of 4 mo, an entire collection of seven Pine Barrens treefrogs (Hyla andersonii) died or were euthanized after developing pallor, generalized edema, and coelomic effusion. Necropsy revealed large numbers of strongyloidid nematodes within the small intestines associated with a moderate mucosal hyperplasia. Strongyloides sp. parasitic females, representing a novel species, were isolated from the fixed intestinal tract. This case report represents the first full description of strongyloidiasis in a tree frog army and highlights the potential of Strongyloides spp. as a cause of rapid mortality events associated with protein-losing enteropathy in frogs.

Taxonomic Distribution of Neoplasia Among Non-Domestic Felid Species Under Managed Care.

As evidenced by numerous case reports from zoos, neoplasia in felids is common, but most reports are limited to Panthera species in North America or Europe. In order to obtain a wider epidemiologic understanding of neoplasia distribution, necropsy records at seven facilities (USA, Mexico, Colombia, Peru, and Brazil) were evaluated. In contrast to others, this study population (195 cases, 16 species), included many non-Panthera felids. Overall neoplasia prevalence was 28.2% (55/195). Panthera species had a higher prevalence of neoplasia than non-Panthera species (52.5%; vs. 13.0%). Lions (66.7%), jaguars (55.0%), and tigers (31.3%) had the highest species-specific prevalence of neoplasia. Neoplasms in Panthera species were more frequently malignant than in non-Panthera (86.1% vs. 55.6%). The systems most commonly affected were the reproductive, hematolymphoid, and respiratory. The range of management conditions and more varied genetic backgrounds support a robust taxonomic pattern and suggest that the reported propensity for neoplasia in jaguars may have a genetic basis at a taxonomic level higher than species, as lions and tigers also have high prevalence. Given the high prevalence of neoplasia and high likelihood of malignancy, routine medical exams in all nondomestic felids, but Panthera species in particular, should include thorough assessments of any clinical signs of neoplasia.

Pharmacokinetics of Transdermal Flunixin Meglumine Following a Single Dose in Marine Toads (Rhinella marina).

Transdermal administration is an important method of pharmacologic drug therapy in amphibians, made possible by their unique skin physiology and permeability. Despite this, there are relatively few studies that investigate transdermal pharmacokinetics in amphibians. The objective of this study was to investigate the pharmacokinetics of transdermal flunixin meglumine applied topically to marine toads (Rhinella marina). Twenty-one adult marine toads were administered flunixin meglumine (3.3 mg/kg) topically on their dorsum and randomized (n = 7/group) to blood collection at two timepoints from the following: 1, 2, 4, 8, 12, and 24 hours (h), using a sparse sampling protocol. Plasma was analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. Samples were analyzed individually and reported as a mean of the samples at each timepoint. The mean peak plasma concentration was 6.31 µg/ml, area under the curve was 29.37 µg-h/mL, and elimination half-life was 2.79 h. No adverse effects were noted in any animals. A subset of 12 animals were euthanized at serial timepoints and necropsied. Histopathology of skin and major organs revealed one minimal superficial lesion in a single toad potentially attributable to flunixin meglumine administration; otherwise, no other treatment-related lesions were observed in 11 of 12 toads. A single topical dose of transdermal flunixin meglumine was rapidly absorbed in marine toads in the current study, and peak plasma concentrations exceeded therapeutic ranges established in cattle with no significant pathologic findings.

Malignant hypertension and retinopathy in a western lowland gorilla (Gorilla gorilla gorilla).

BACKGROUND: A 34-year-old western lowland gorilla presented with peracute blindness. METHODS: Clinical evaluation, diagnostic imaging, laboratory analyses, blood pressure measurements, and necropsy were performed. RESULTS: The clinical and postmortem findings supported malignant hypertension. CONCLUSIONS: We describe a case of naturally occurring hypertensive encephalopathy and retinopathy in a gorilla.

Septicemia, endocarditis, and cerebral infarction due to Staphylococcus aureus in a harp seal (Phoca groenlandica).

An adult, wild-collected, male harp seal (Phoca groenlandica) was transferred from a rehabilitation center to a display facility because of unilateral phthisis bulbi and decreased use of the right forelimb, which precluded its release. In quarantine, the animal demonstrated limited use of the right forelimb, which acutely progressed to complete disuse of the limb accompanied by intermittent lethargy. One month after transfer, the animal was found dead on exhibit. Necropsy showed septic arthritis of the right scapulohumeral joint, valvular endocarditis with systemic bacterial thromboembolism, and infarction of the cerebrum and myocardium. Culture of the blood and affected joint space revealed Staphylococcus aureus. Bacterial polymerase chain reaction of formalin-fixed tissues from the heart and brain were also positive for S. aureus. Staphylococcus aureus infection should be considered as an additional cause of endocarditis and embolic encephalitis in seals.

Naturally transmitted herpesvirus papio-2 infection in a black and white colobus monkey.

CASE DESCRIPTION: A 6.5-year-old female eastern black and white colobus monkey (Colobus guereza) was evaluated after acute onset of ataxia and inappetence. CLINICAL FINDINGS: The monkey was ataxic and lethargic, but no other abnormalities were detected via physical examination, radiography, or clinicopathologic analyses. During the next 2 days, the monkey's clinical condition deteriorated, and its WBC count decreased dramatically. Cytologic examination of a CSF sample revealed marked lymphohistiocytic inflammation. TREATMENT AND OUTCOME: Despite supportive care, the monkey became apneic; after 20 hours of mechanical ventilation, fatal cardiac arrest occurred. At necropsy, numerous petechiae were detected within the white matter tracts of the brain; microscopic lesions of multifocal necrosis and hemorrhage with intranuclear inclusions identified in the brain and adrenal glands were consistent with an acute herpesvirus infection. A specific diagnosis of herpesvirus papio-2 (HVP-2) infection was made on the basis of results of serologic testing; PCR assay of tissue specimens; live virus isolation from the lungs; and immunohistochemical identification of the virus within brain, spinal cord, and adrenal gland lesions. Via phylogenetic tree analysis, the colobus HVP-2 isolate was grouped with neuroinvasive strains of the virus. The virus was most likely transmitted to the colobus monkey through toys shared with a nearby colony of baboons (the natural host of HVP-2). CLINICAL RELEVANCE: To the authors' knowledge, this is the first reported case of natural transmission of HVP-2 to a nonhost species. Infection with HVP-2 should be a differential diagnosis for acute encephalopathy in primate monkeys and humans, particularly following exposure to baboons.

Putative regulation of expression of members of the Ets variant 4 transcription factor family and their downstream targets in the rat epididymis.

Several genes expressed in the initial segment of the epididymis depend on factors from the testis that reach the epididymis via the luminal system. These include gamma-glutamyl transpeptidase mRNA IV (Ggt_pr4), steroid 5 alpha reductase (Srd5a1), glutathione peroxidase 5 (Gpx5), and cystatin-related epididymal spermatogenic (Cst8) genes. Promoter analyses indicated that these genes contain several ETS DNA-binding sites. Members of the polyomavirus enhancer activator 3 (ETV4) family bind to ETS sites on the promoter of target genes to regulate transcription. In this study, the role of ETV4 family members (ETV4, ETV5, ETV1) in the transcription of initial segment specific genes was evaluated. All three ETV4 family mRNAs are expressed in the principal cells of the initial segment and depend upon the presence of testicular luminal fluid factors. ETV4 protein was localized to principal cell nuclei and displayed the highest expression in the most proximal region of the initial segment. In addition, ETV4 protein levels were diminished after loss of testicular luminal fluid factors. A dominant-negative construct of ETV5 was in vivo electroporated into the initial segment to determine if ETV4 family members can regulate the transcription of testicular luminal fluid factor-regulated genes. Quantitative PCR indicated that 1 day postelectroporation, all three ETV4 family member mRNAs were significantly decreased. In addition, Ggt_pr4, Srd5a1, and Gpx5 mRNA levels were also significantly decreased. The data suggest that ETV4 family members regulate their own expression, and that they regulate transcription of a subset of genes that are dependent upon testicular luminal fluid factors.

CT findings of intracranial blastomycosis in a dog.

Computed tomography (CT) findings in a dog with intracranial blastomycosis were marked periventricular contrast enhancement of the lateral ventricles, the 3rd ventricle, and the mesencephalic aqueduct. The CT appearance correlated with the histopathologic findings, where severe ependymitis was present throughout the ventricular system and there was stenosis of the mesencephalic aqueduct due to an inflammatory infiltrate. CT is therefore recommended as a screening test for intracranial blastomycosis in dogs and also as an imaging modality for follow-up evaluation after treatment. This is particularly true in dogs with systemic or ocular blastomycosis, which appear to be at higher risk of developing CNS involvement.