The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.

Proximity-dependent biotin labeling (BioID) may identify new targets for cancers driven by difficult-to-drug oncogenes such as Ras. Therefore, BioID was used with wild-type (WT) and oncogenic mutant (MT) H-, K-, and N-Ras, identifying known interactors, including Raf and PI3K, as well as a common set of 130 novel proteins proximal to all Ras isoforms. A CRISPR screen of these proteins for Ras dependence identified mTOR, which was also found proximal to MT Ras in human tumors. Oncogenic Ras directly bound two mTOR complex 2 (mTORC2) components, mTOR and MAPKAP1, to promote mTORC2 kinase activity at the plasma membrane. mTORC2 enabled the Ras pro-proliferative cell cycle transcriptional program, and perturbing the Ras-mTORC2 interaction impaired Ras-dependent neoplasia in vivo. Combining proximity-dependent proteomics with CRISPR screening identified a new set of functional Ras-associated proteins, defined mTORC2 as a new direct Ras effector, and offers a strategy for finding new proteins that cooperate with dominant oncogenes.

Burkholderia pseudomallei isolates in 2 pet iguanas, California, USA.

Burkholderia pseudomallei, the causative agent of melioidosis, was isolated from abscesses of 2 pet green iguanas in California, USA. The international trade in iguanas may contribute to importation of this pathogen into countries where it is not endemic and put persons exposed to these animals at risk for infection.

FasTag: Automatic text classification of unstructured medical narratives.

Unstructured clinical narratives are continuously being recorded as part of delivery of care in electronic health records, and dedicated tagging staff spend considerable effort manually assigning clinical codes for billing purposes. Despite these efforts, however, label availability and accuracy are both suboptimal. In this retrospective study, we aimed to automate the assignment of top-level International Classification of Diseases version 9 (ICD-9) codes to clinical records from human and veterinary data stores using minimal manual labor and feature curation. Automating top-level annotations could in turn enable rapid cohort identification, especially in a veterinary setting. To this end, we trained long short-term memory (LSTM) recurrent neural networks (RNNs) on 52,722 human and 89,591 veterinary records. We investigated the accuracy of both separate-domain and combined-domain models and probed model portability. We established relevant baseline classification performances by training Decision Trees (DT) and Random Forests (RF). We also investigated whether transforming the data using MetaMap Lite, a clinical natural language processing tool, affected classification performance. We showed that the LSTM-RNNs accurately classify veterinary and human text narratives into top-level categories with an average weighted macro F1 score of 0.74 and 0.68 respectively. In the "neoplasia" category, the model trained on veterinary data had a high validation accuracy in veterinary data and moderate accuracy in human data, with F1 scores of 0.91 and 0.70 respectively. Our LSTM method scored slightly higher than that of the DT and RF models. The use of LSTM-RNN models represents a scalable structure that could prove useful in cohort identification for comparative oncology studies. Digitization of human and veterinary health information will continue to be a reality, particularly in the form of unstructured narratives. Our approach is a step forward for these two domains to learn from and inform one another.

Evaluation of indirect blood pressure monitoring in awake and anesthetized red-tailed hawks (Buteo jamaicensis): effects of cuff size, cuff placement, and monitoring equipment.

OBJECTIVE: To compare Doppler and oscillometric methods of indirect arterial blood pressure (IBP) with direct arterial measurements in anesthetized and awake red-tailed hawks. STUDY DESIGN: Prospective, randomized, blinded study. ANIMALS: Six, sex unknown, adult red-tailed hawks. METHODS: Birds were anesthetized and IBP measurements were obtained by oscillometry (IBP-O) and Doppler (IBP-D) on the pectoral and pelvic limbs using three cuffs of different width based on limb circumference: cuff 1 (20-30% of circumference), cuff 2 (30-40%), and cuff 3 (40-50%). Direct arterial pressure measurements were obtained from the contralateral superficial ulnar artery. Indirect blood pressure measurements were compared to direct systolic arterial pressure (SAP) and mean arterial pressure (MAP) during normotension and induced states of hypotension and hypertension. Measurements were also obtained in awake, restrained birds. Three-way anova, linear regression and Bland-Altman analyses were used to evaluate the IBP-D data. Results are reported as mean bias (95% confidence intervals). RESULTS: The IBP-O monitor reported errors during 54% of the measurements. Indirect blood pressure Doppler measurements were most accurate with cuff 3 and were comparable to MAP with a bias of 2 (-9, 13 mmHg). However, this cuff consistently underestimated SAP with a bias of 33 (19, 48 mmHg). Variability in the readings within and among birds was high. There was no significant difference between sites of cuff placement. Awake birds had SAP, MAP and diastolic arterial pressure that were 56, 43, and 38 mmHg higher than anesthetized birds. CONCLUSIONS AND CLINICAL RELEVANCE: Indirect blood pressure (oscillometric) measurements were unreliable in red-tailed hawks. Indirect blood pressure (Doppler) measurements were closer to MAP measurements than SAP measurements. There was slightly better agreement with the use of cuff 3 on either the pectoral or pelvic limbs. Awake, restrained birds have significantly higher arterial pressures than those under sevoflurane anesthesia.

What is your diagnosis? Blood smear from an injured red-tailed hawk.

An injured juvenile red-tailed hawk (Buteo jamaicensis) was evaluated at the Veterinary Medical Teaching Hospital at the University of California, Davis. The hawk was quiet, alert, and emaciated, and had a closed comminuted, mid-diaphyseal ulnar fracture. CBC results included heterophilia with a left shift, monocytosis, and increased plasma fibrinogen concentration. The blood smear included rare heterophils containing small, dark blue inclusions approximately 1-2 mum in diameter that ranged from round to coccobacillary in shape and formed variably shaped aggregates; the morphology of the inclusions was suspicious for Chlamydophila or Ehrlichia spp. pathogens. The hawk died, and histopathologic examination of tissues obtained at necropsy found severe multifocal histiocytic and heterophilic splenitis in addition to chronic hepatitis, myocarditis and epicarditis, meningoencephalitis, and airsacculitis. Using immunohistochemistry the presence of Chlamydia/Chlamydophila spp. antigen within multiple tissues was confirmed. Chlamydophila psittaci DNA was demonstrated in whole blood and fresh splenic tissue via real-time PCR. Direct fluorescent antibody staining of air-dried blood smears was positive in rare leukocytes for Chlamydia/Chlamydophila spp. antigen, and immunocytochemical staining of blood smears for Chlamydia/Chlamydophila spp. antigen was focally positive in rare heterophils. These findings may represent the first reported diagnosis of natural avian C. psittaci infection by visualization of organisms in peripheral blood heterophils. Immunocytochemical evaluation of blood smears was valuable in confirming the diagnosis and may be a useful antemortem test to discriminate between bacteria and other inclusions within heterophils.

Lacrimal cystadenoma in a Chinese box turtle (Cuora flavomarginata).

An adult male Chinese box turtle (Cuora flavomarginata) presented to the Avian and Exotic Pet Service of the Animal Medical Center for periorbital swelling of the right eye. The swelling had failed to respond to nutritional supplementation and parenteral administration of vitamin A. What had initially presented as periorbital swelling developed into a growth ventral to the globe that impeded vision and was frequently traumatized by forelimb movements of the turtle. Twenty-six months after initial presentation, the turtle was anesthetized and the bulk of the mass was surgically removed. Histopathologic examination determined the mass to be a benign lacrimal cystadenoma.

Clinical features, treatment, and outcomes of cutaneous and oral squamous cell carcinoma in avian species.

OBJECTIVE To determine the clinical features, treatment, and outcomes of treatment for oral and cutaneous squamous cell carcinoma (SCC) in avian species. DESIGN Retrospective case series with nested cohort study. ANIMALS 87 client-owned birds of various species with histologically confirmed SCC of the skin or oral cavity. PROCEDURES Clinicians entered case information through an online survey tool. Data were collected regarding patient signalment, concurrent conditions, treatments, adverse effects, and clinical outcomes. Relationships were examined between complete excision and partial or complete response. Survival analysis was performed to compare outcomes among groupings of therapeutic approaches. RESULTS Only 7 of 64 (11%) birds for which full outcome data were available had complete remission of SCC; 53 (83%) had progressive disease, were euthanized, or died of the disease. The unadjusted OR for partial or complete response following complete tumor excision (vs other treatment approaches) was 6.9 (95% confidence interval [CI], 1.8 to 25.8). Risk of death was 62% lower (hazard ratio, 0.38; 95% CI, 0.19 to 0.77) for birds that underwent complete excision versus conservative treatment. Median survival time from initial evaluation for birds receiving complete excision was 628 days (95% CI, 210 to 1,008 days), compared with 171 days (95% CI, 89 to 286 days) for birds receiving monitoring with or without conservative treatment. Birds receiving any other additional treatment had a median survival time of 357 days (95% CI, 143 to 562 days). CONCLUSIONS AND CLINICAL RELEVANCE For birds with SCC, complete excision was the only treatment approach significantly associated with complete or partial response and increased survival time.

IQGAP1 is an oncogenic target in canine melanoma.

Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.

The use of altrenogest to control aggression in a male Grant's Zebra (Equus burchelli boehmi).

A male Grant's Zebra (Equus burchelli boehmi) housed with two mares at the Indianapolis Zoo had a 9-yr history of intermittent aggressive behavior toward mares and other animals. Periods of separation allowed the mares time to heal after sustaining superficial bite wounds. On 26 March 2003, the male (890293) was started on altrenogest at a dosage of 19.8 mg orally once daily to allow reintroduction. The dosage was doubled (40 mg once a day) because of a perceived lack of response. Reintroduction to the mares occurred on 17 May 2003 with no signs of aggression noted. Treatment was reduced to 19.8 mg orally once a day and then discontinued. Altrenogest was restarted at 39.5 mg orally once a day because of the planned introduction of a new mare. There have been no major aggressive displays at this dosage of altrenogest and the dosage has recently been reduced following successful introduction of a new mare.

IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors.

Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer. MAPK scaffolds, such as IQ motif-containing GTPase activating protein 1 (IQGAP1), assemble pathway kinases to affect signal transmission, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer.

Calculation of body surface area via computed tomography-guided modeling in domestic rabbits (Oryctolagus cuniculus).

OBJECTIVE: To optimize the use of CT-guided modeling for the calculation of body surface area (BSA) in domestic rabbits (Oryctolagus cuniculus). Animals-12 domestic rabbits. PROCEDURES: Adult rabbits (body weight, 1 to > 4 kg) that were client-owned animals undergoing CT for disease diagnosis or deceased laboratory animals donated from other research projects were scanned with a CT scanner. Images were transferred to a radiation therapy planning software program. Image slices were captured as contiguous slices at 100 kVp and 100 mA and processed to 0.1-cm-thick sections. The length of each contoured slice was summed to calculate a final BSA measurement. Nonlinear regression analysis was then used to derive an equation for the calculation of BSA in rabbits. RESULTS: The constant calculated by use of this method was 9.9 (range, 9.59 to 10). The R(2) for the goodness of fit was 0.9332. The equation that best described BSA as a function of body weight for domestic rabbits with this method was as follows: BSA = (9.9 × [body weight {in grams}](2/3))/10,000. CONCLUSIONS AND CLINICAL RELEVANCE: The BSA calculated via the CT-guided method yielded results similar to those obtained with equations for other similarly sized mammals and verified the use of such equations for rabbits. Additionally, this technique can be used for species that lack equations for the accurate calculation of BSA.

An unusual presentation of canine distemper virus infection in a domestic ferret (Mustela putorius furo).

A 4.5-year-old, male castrated ferret was examined with a 27-day history of severe pruritus, generalized erythema and scaling. Skin scrapings and a trichogram were negative for mites and dermatophyte organisms. A fungal culture of hair samples was negative. The ferret was treated presumptively for scabies and secondary bacterial and yeast infection with selamectin, enrofloxacin, fluconazole, diphenhydramine and a miconazole-chlorhexidine shampoo. The ferret showed mild improvement in clinical signs over the subsequent 3 weeks, but was inappetent and required supportive feeding and subcutaneous fluids by the owner. The ferret was then examined on an emergency basis at the end of 3 weeks (53 days following initial signs of illness) for severe blood loss from a haematoma over the interscapular region, hypotension and shock. The owners elected euthanasia due to a poor prognosis and deteriorating condition. On post-mortem examination intraepithelial canine distemper viral inclusions were identified systemically, and abundant canine distemper virus antigen was identified with immunohistochemical staining. It is important to note the prolonged course of disease along with the absence of respiratory and neurological signs because this differs from the classic presentation of canine distemper virus infection in ferrets. Canine distemper virus should remain a clinical suspicion for ferrets with skin lesions that do not respond to appropriate therapy, even in animals that were previously vaccinated.