Assessing susceptibility for polycyclic aromatic hydrocarbon toxicity in an in vitro 3D respiratory model for asthma.

There is increased emphasis on understanding cumulative risk from the combined effects of chemical and non-chemical stressors as it relates to public health. Recent animal studies have identified pulmonary inflammation as a possible modifier and risk factor for chemical toxicity in the lung after exposure to inhaled pollutants; however, little is known about specific interactions and potential mechanisms of action. In this study, primary human bronchial epithelial cells (HBEC) cultured in 3D at the air-liquid interface (ALI) are utilized as a physiologically relevant model to evaluate the effects of inflammation on toxicity of polycyclic aromatic hydrocarbons (PAHs), a class of contaminants generated from incomplete combustion of fossil fuels. Normal HBEC were differentiated in the presence of IL-13 for 14 days to induce a profibrotic phenotype similar to asthma. Fully differentiated normal and IL-13 phenotype HBEC were treated with benzo[a]pyrene (BAP; 1-40 µg/mL) or 1% DMSO/PBS vehicle at the ALI for 48 h. Cells were evaluated for cytotoxicity, barrier integrity, and transcriptional biomarkers of chemical metabolism and inflammation by quantitative PCR. Cells with the IL-13 phenotype treated with BAP result in significantly (p < 0.05) decreased barrier integrity, less than 50% compared to normal cells. The effect of BAP in the IL-13 phenotype was more apparent when evaluating transcriptional biomarkers of barrier integrity in addition to markers of mucus production, goblet cell hyperplasia, type 2 asthmatic inflammation and chemical metabolism, which all resulted in dose-dependent changes (p < 0.05) in the presence of BAP. Additionally, RNA sequencing data showed that the HBEC with the IL-13 phenotype may have increased potential for uncontrolled proliferation and decreased capacity for immune response after BAP exposure compared to normal phenotype HBEC. These data are the first to evaluate the role of combined environmental factors associated with inflammation from pre-existing disease and PAH exposure on pulmonary toxicity in a physiologically relevant human in vitro model.

Identification and Characterization of a Small Molecule Bcl-2 Functional Converter.

Cancer cells exploit the expression of anti-apoptotic protein Bcl-2 to evade apoptosis and develop resistance to therapeutics. High levels of Bcl-2 leads to sequestration of pro-apoptotic proteins causing the apoptotic machinery to halt. In this study, we report discovery of a small molecule, BFC1108 (5-chloro-N-(2-ethoxyphenyl)-2-[(4-methoxybenzyol)amino]benzamide), which targets Bcl-2 and converts it into a pro-apoptotic protein. The apoptotic effect of BFC1108 is not inhibited, but rather potentiated, by Bcl-2 overexpression. BFC1108 induces a conformational change in Bcl-2, resulting in the exposure of its BH3 domain both in vitro and in vivo. BFC1108 suppresses the growth of triple-negative breast cancer xenografts with high Bcl-2 expression and inhibits breast cancer lung metastasis. This study demonstrates a novel approach to targeting Bcl-2 using BFC1108, a small molecule Bcl-2 functional converter that effectively induces apoptosis in Bcl-2-expressing cancers. SIGNIFICANCE: We report the identification of a small molecule that exposes the Bcl-2 killer conformation and induces death in Bcl-2-expressing cancer cells. Selective targeting of Bcl-2 and elimination of cancer cells expressing Bcl-2 opens up new therapeutic avenues.

Time course of western diet (WD) induced nonalcoholic steatohepatitis (NASH) in female and male Ldlr-/- mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a global health problem. Identification of factors contributing to the onset and progression of NAFLD have the potential to direct novel strategies to combat NAFLD. METHODS: We examined the time course of western diet (WD)-induced NAFLD and its progression to nonalcoholic steatohepatitis (NASH) in age-matched female and male Ldlr-/- mice, with time-points at 1, 4, 8, 20 and 40 weeks on the WD. Controls included Ldlr-/- mice maintained on a purified low-fat diet (LFD) for 1 and 40 weeks. The approach included quantitation of anthropometric, plasma and liver markers of disease, plus hepatic histology, lipids, oxylipins, gene expression and selected metabolites. RESULTS: One week of feeding the WD caused a significant reduction in hepatic essential fatty acids (EFAs: 18:2, ω6, 18:3, ω3) which preceded the decline in many C20-22 ω3 and ω6 polyunsaturated fatty acids (PUFA) and PUFA-derived oxylipins after 4 weeks on the WD. In addition, expression of hepatic inflammation markers (CD40, CD44, Mcp1, Nlrp3, TLR2, TLR4, Trem2) increased significantly in both female & male mice after one week on the WD. These markers continued to increase over the 40-week WD feeding study. WD effects on hepatic EFA and inflammation preceded all significant WD-induced changes in body weight, insulin resistance (HOMA-IR), oxidative stress status (GSH/GSSG ratio) and histological and gene expression markers of macrosteatosis, extracellular matrix remodeling and fibrosis. CONCLUSIONS: Our findings establish that feeding Ldlr-/- mice the WD rapidly lowered hepatic EFAs and induced key inflammatory markers linked to NASH. Since EFAs have an established role in inflammation and hepatic inflammation plays a major role in NASH, we suggest that early clinical assessment of EFA status and correcting EFA deficiencies may be useful in reducing NASH severity.

Association of equine gammaherpesvirus-5 with facial lymphohistiocytic interface dermatitis in seven adult horses from the United States.

Equine herpesvirus-5 (EHV-5) is commonly found in healthy asymptomatic horses worldwide. Although a cause-and-effect relationship has not been thoroughly determined, this virus has been associated with several disease conditions including equine multinodular pulmonary fibrosis (EMPF) and 1 case of interface dermatitis. The authors searched the New York State Animal Health Diagnostic Center database for cases of equine interface dermatitis between 2007 and 2022. Ten cases were identified and scrutinized for viral inclusion bodies which were present in 5 of 10 cases. Two similar cases with interface dermatitis and viral inclusion bodies, which were not part of a retrospective search, were from the Oregon Veterinary Diagnostic Laboratory. The authors describe a total of 7 horses with dermatitis characterized by crusted, alopecic, non-pruritic, non-painful, irregular to annular areas over the face, most commonly the muzzle, for up to several years duration. Histologically, there was a CD3+ T lymphocyte-dominated lymphohistiocytic interface dermatitis with hydropic degeneration, apoptotic keratinocytes, and pigmentary incontinence. Keratinocytes within the upper stratum spinosum and stratum granulosum had glassy pale basophilic intranuclear inclusion bodies consistent with herpesvirus. The presence of EHV-5 was confirmed by quantitative polymerase chain reaction (qPCR) and in situ hybridization in 7 horses and by electron microscopy in 1 horse. One horse later developed EMPF and was euthanized. EHV-5 was not detected with qPCR from 5 control horses and 5 horses with interface dermatitis without histologic evidence of viral inclusion bodies. These are the first cases of facial interface dermatitis associated with EHV-5 reported in the United States.

Early transcriptome changes associated with western diet induced NASH in Ldlr-/- mice points to activation of hepatic macrophages and an acute phase response.

Background: Nonalcoholic fatty liver disease (NAFLD) is a global health problem. Identifying early gene indicators contributing to the onset and progression of NAFLD has the potential to develop novel targets for early therapeutic intervention. We report on the early and late transcriptomic signatures of western diet (WD)-induced nonalcoholic steatohepatitis (NASH) in female and male Ldlr-/- mice, with time-points at 1 week and 40 weeks on the WD. Control Ldlr-/- mice were maintained on a low-fat diet (LFD) for 1 and 40 weeks. Methods: The approach included quantitation of anthropometric and hepatic histology markers of disease as well as the hepatic transcriptome. Results: Only mice fed the WD for 40 weeks revealed evidence of NASH, i.e., hepatic steatosis and fibrosis. RNASeq transcriptome analysis, however, revealed multiple cell-specific changes in gene expression after 1 week that persisted to 40 weeks on the WD. These early markers of disease include induction of acute phase response (Saa1-2, Orm2), fibrosis (Col1A1, Col1A2, TGFß) and NASH associated macrophage (NAM, i.e., Trem2 high, Mmp12 low). We also noted the induction of transcripts associated with metabolic syndrome, including Mmp12, Trem2, Gpnmb, Lgals3 and Lpl. Finally, 1 week of WD feeding was sufficient to significantly induce TNFα, a cytokine involved in both hepatic and systemic inflammation. Conclusion: This study revealed early onset changes in the hepatic transcriptome that develop well before any anthropometric or histological evidence of NALFD or NASH and pointed to cell-specific targeting for the prevention of disease progression.

CT features of malignant tubular genital tract tumors in seven goats.

Neoplasia of the tubular genital tract in goats, while rarely described, is most commonly reported as uterine adenocarcinoma, leiomyoma, or leiomyosarcoma. In this retrospective, single-center, case series, medical records were searched for goats with a computed tomography (CT) diagnosis of tubular genital mass and a definitive histologic (surgical biopsy or necropsy) diagnosis of malignant neoplasia. Data recorded from CT images were presence of peritoneal/retroperitoneal fluid, urinary tract obstruction, abdominal lymphadenomegaly, additional abdominal nodules/masses, and pulmonary nodules. For masses, maximum cross-sectional area, contrast enhancement, and uterine luminal fluid accumulation were also recorded. Seven goats met the inclusion criteria (leiomyosarcoma n = 5, adenocarcinoma n = 2). Both goats with adenocarcinoma had upper urinary tract obstruction, moderate to severe regional lymphadenopathy, peritoneal fluid, and peritoneal or hepatic nodules/masses; one goat with adenocarcinoma was discharged and subsequently euthanized, and the other had palliative mass debulking and was lost to follow up. Goats with leiomyosarcoma had infrequent, mild peritoneal fluid and mild sublumbar lymphadenopathy. Of the goats with leiomyosarcoma, two were euthanized at or near the time of CT imaging, two were euthanized at the time of surgery due to perceived mass non-resectability, and one had mass regression approximately four months post ovariohysterectomy but was subsequently lost to follow up. Five goats had pulmonary nodules, three of which had pathologic confirmation (pulmonary metastasis in a single patient with adenocarcinoma, and lungworm granulomas in two goats with leiomyosarcoma). Severe sublumbar lymphadenopathy and obstructive uropathy were sequelae in the two caprine patients with genital adenocarcinoma, and in none with leiomyosarcoma.

Evaluating the impact of organic chromium with flax seed in broiler diets: effects on production performance, breast muscle pathology, and meat quality aspects.

The study investigated the impact of organic chromium (Cr) and flax seed supplementation on live performance, carcass yield, muscle lipid profile, histopathological aspects, and meat quality parameters in broilers. Ninety (n = 90), day-old Cobb chicks were fed a corn-soybean meal-based diet containing 0% flax seed (Control), 10% flax seed (Diet 1) and Diet 1 + 0.05% organic Cr (Diet 2). The experiment was a completely randomized design and chicks were placed in 6 pens with 5 chicks per each pen. Pen was the experimental unit for production performance and bird collected from each pen was considered as experimental unit for all other analysis. On d 43, 45, and 48, one bird per pen were euthanized. A one-way ANOVA was performed with diet as the main factor and significance was set at P < 0.05. Significant differences between each treatment were analyzed by GLM Lean Square Method and Tukey's Honestly Significant Difference test. Weight gain and feed:gain was determined at d (1-11), (12-21), and (22-42). For all response variables, the effects among dietary treatments were compared using ANOVA separately using SAS 9.4. P -values were considered significant at ≤0.05. At d 22, Diet 1 and Diet 2 birds had lower BW and feed:gain than Control (P < 0.05). At d 42, Diet 2 birds were higher in BW with improved feed:gain when compared to Diet 1 (P < 0.05) but were not different from Control (P > 0.05). The overall weight gain was higher in Diet 2 and Control compared to Diet 1 (P < 0.05) and overall feed:gain was the highest in Control than the experimental diets (P < 0.05). Histopathological changes in breast muscle including floccular/vacuolar degeneration, fibrosis, lipidosis, interstitial inflammation, and muscle lysis were less pronounced in Diet 1 compared to Diet 2 (P < 0.05). Breast muscle total fat and cholesterol was lower in Diet 1 compared to Control (P < 0.05). Diet 1 and Diet 2 increased (>2-5 fold) total and long chain (≥20C) n-3 fatty acids (FA) in the breast muscle (P < 0.05) compared to Control. Lipid peroxidation products measured as thiobarbituric acid reactive substances were lower in the breast muscle of Diet 1 and Diet 2 compared to Control (P < 0.05). Phospholipid n-3 FA molecular species in phosphatidylcholine (PC) 36:5, 38:6, and phosphatidylethanolamine (PE) 36:5 were higher in breast muscle of Diet 1 than Control (P < 0.05) and was not different from Diet 2 (P > 0.05). A decrease in n-6 FA species (36:4 and 38:4) was observed in PC and PE of Diet 1and Diet 2 compared to Control (P < 0.05). Drip loss values were reduced in Diet 1 and Diet 2 versus Control (P < 0.05). As consumer demand for n-3 FA-rich poultry products are on the rise, Cr may serve as a feed supplement that could be used in broilers fed flax seed-containing diets for enriching edible tissues with n-3 FA, while enhancing production performance.

Effects of capture on acute and long-term reflex impairment, survival, and health of a deepwater fish: Shortspine thornyhead (Sebastolobus alaskanus).

Shortspine thornyhead (Sebastolobus alaskanus) are a benthic, deepwater species in the family Scorpaenidae. They have been tagged annually in Alaska since 1992, but have a low tag return rate of 1.6%. This may be at least partially attributed to post-release mortality related to capture. In this study, 21 shortspine thornyhead were caught on bottom hook-and-line longline gear and immediately given reflex tests. Eighteen were transported to the laboratory and held for 10-42 days, given reflex tests again, and then given postmortem examinations, including histopathology of tissues; three were given postmortem examinations after reflex tests on the vessel. There were no histological findings that could be directly linked to capture and holding; however, there were occurrences of myxozoan (protozoa) and metazoan (nematode) parasites, sometimes associated with minor inflammation. The vibration response reflex was found in only 24% of fish on deck and in 56% of fish after holding in the laboratory. The vestibular-ocular response was present in 47% of fish on deck and 89% of fish in the laboratory. A fish's ability to right itself was successful on deck in 43% of fish (an additional 19% responded slowly) and 100% in the laboratory. Some reflex impairments may be permanent or may take more than days or weeks to improve. Reflex responses to other tests, the tail grab, gag, and operculum flare, were 95-100% successful on deck and later in the laboratory. A lack of reflexes may increase the risk of predation after release and may affect other behaviors related to survival and productivity.

Loss of the aryl hydrocarbon receptor increases tumorigenesis in p53-deficient mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cell fate via activation of a diverse set of genes. There are conflicting reports describing the role of AhR in cancer. AhR-knockout mice do not develop tumors spontaneously, yet the AhR can act as a tumor suppressor in certain contexts. Loss of tumor suppression by p53 is common in human cancer. To investigate AhR function in the absence of p53, we generated mice lacking both AhR and p53. Mice deficient for AhR and p53 had shortened lifespan, increased tumorigenesis, and an altered tumor spectrum relative to control mice lacking only p53. In addition, knockout of both AhR and p53 resulted in reduced embryonic survival and neonatal fitness. We also examined the consequences of loss of AhR in p53-heterozygous mice and observed a significantly reduced lifespan and enhanced tumor burden. These findings reveal an important role for the AhR as a tumor suppressor in the absence of p53 signaling and support the development of anti-cancer therapeutics that would promote the tumor suppressive actions of the AhR.

What Is Your Diagnosis?

In collaboration with the American College of Veterinary Radiology.

Tetrahydroxanthohumol, a xanthohumol derivative, attenuates high-fat diet-induced hepatic steatosis by antagonizing PPARγ.

We previously reported xanthohumol (XN), and its synthetic derivative tetrahydro-XN (TXN), attenuates high-fat diet (HFD)-induced obesity and metabolic syndrome in C57Bl/6J mice. The objective of the current study was to determine the effect of XN and TXN on lipid accumulation in the liver. Non-supplemented mice were unable to adapt their caloric intake to 60% HFD, resulting in obesity and hepatic steatosis; however, TXN reduced weight gain and decreased hepatic steatosis. Liver transcriptomics indicated that TXN might antagonize lipogenic PPARγ actions in vivo. XN and TXN inhibited rosiglitazone-induced 3T3-L1 cell differentiation concomitant with decreased expression of lipogenesis-related genes. A peroxisome proliferator activated receptor gamma (PPARγ) competitive binding assay showed that XN and TXN bind to PPARγ with an IC50 similar to pioglitazone and 8-10 times stronger than oleate. Molecular docking simulations demonstrated that XN and TXN bind in the PPARγ ligand-binding domain pocket. Our findings are consistent with XN and TXN acting as antagonists of PPARγ.

Targeting Peritumoral Lesions Identified by Computed Tomography and Magnetic Resonance Imaging in Feline Injection-Site Sarcomas for Microscopic Examination.

Peritumoral lesions identified during in vivo imaging of feline injection-site sarcoma (FISS) are frequently interpreted as neoplastic. We recently showed that most peritumoral imaging-identified lesions (PTIILs) in FISS are non-neoplastic. In this article, we describe a protocol to target PTIIL for microscopic examination and report on the protocol's performance. Ten client-owned cats with FISS were prospectively enrolled. A fiducial marker sutured onto the skin, centered on the palpable mass, served as reference point throughout the study. Each FISS and surrounding tissue was imaged in vivo by dual phase computed tomography angiography and multiple magnetic resonance imaging pulse sequences and each PTIIL documented. Subgross measurements obtained during trimming aided localization and identification of PTIIL during microscopy. Histologic findings were categorized by descending clinical relevance: neoplastic, equivocal, non-neoplastic, within normal limits (WNL). Based on in vivo imaging resolution limits, histologic findings were ≥3 mm in at least one dimension and ≥3 mm apart. Surgical margins served as control tissue for PTIILs. Eighty-one of 87 PTIIL were examined histologically; 13 were neoplastic, 16 equivocal, and 28 non-neoplastic; 24 had no identified histologic correlate. Two neoplastic and 10 equivocal findings were located outside of PTIILs but none of them were located in sections of surgical margins. Computation of a simple confusion matrix yielded fair sensitivity (70.4%) and low specificity (59.7%) for prediction of PTIIL by histologic findings. After combining instances of normal microanatomy with non-neoplastic histologic findings, specificity increased (85.1%) and sensitivity decreased (35.8%). The protocol is a blueprint for targeting PTIIL for microscopic examination but may benefit from further refinement.

Docosahexaenoic acid (22:6 n-3)-rich microalgae along with methionine supplementation in broiler chickens: effects on production performance, breast muscle quality attributes, lipid profile, and incidence of white striping and myopathy.

The effect of docosahexaenoic acid (DHA, 22:6 n-3)-rich microalgae and methionine (Met) supplementation on production performance, incidence of breast muscle white striping (WS), and pathology, lipid profile, and meat quality aspects in broiler chickens was investigated. The hypothesis tested was that feeding Met and n-3 fatty acid (FA)-rich diet enhances muscle n-3 FA content and meat quality while attenuating breast muscle WS and myopathy in broiler chickens. One hundred and forty four (n = 144) 10-day-old Cornish cross chicks were fed a corn-soybean meal-based diet containing 0% microalgae (control), 2% microalgae (diet 1), and diet 1 + 100% more National Research Council requirement of Met (diet 2) up to day 42 of growth. All diets were isocaloric and isonitrogenous. The chicks were kept in 6 pens with 8 chicks per replicate pen. Feed consumption and feed efficiency were calculated on day 21 and 42. On day 43, 3 chicks per pen (n = 18/treatment) were euthanized. The breast muscle (pectoralis major) was visually scored for muscle WS (1 = no striping, 2 = mild, 3 = severe) and was subjected to histopathology. Breast muscle lipid profile (total lipids, FA composition, cholesterol, lipid oxidation products), quality (moisture, color, drip loss, shear force, cook loss, pH), and chemical characterization (protein, minerals) were recorded. A one-way analysis of variance was carried out with diet as the main factor and significance was set at P < 0.05. The incidence of muscle WS was lower (P < 0.02) for control vs. diet 2 and a trend for reduction in WS was observed in birds fed diet 1 vs. control (P = 0.09). Histopathological changes consisted of floccular or vacuolar degeneration, fibrosis, lipidosis, interstitial inflammation, and lysis of fibers, and were minimal in diet 2 when compared to control (P < 0.05). The total lipid content was lowest in birds fed diet 1 (P < 0.05). Total n-3 and total long chain (≥20C) n-3 FA were highest in the breast muscle of diet 2 birds (P < 0.05). Muscle drip loss and shear force were highest in diet 2 (P < 0.05). Meat color (a∗, redness) was reduced (P < 0.05) and a trend for reduction in b∗ (yellowness) was observed in diet 2 (P = 0.07). No effect of diet on body weight gain, feed efficiency, breast muscle yield, pH, moisture, lipid oxidation products, cook loss, minerals (Ca, P, Mg, Na), cholesterol, or protein content was observed (P > 0.05). The results demonstrated a significant effect of DHA-rich microalgae along with Met supplementation in reducing the incidence of breast muscle striping and myopathy, while enriching meat with n-3 FA. However, inclusion of Met in microalgae-based diets could influence meat tenderness and color.

Mast Cell Tumors and Histiocytomas in Domestic Goats and Diagnostic Utility of CD117/c-Kit and Iba1 Immunohistochemistry.

Cutaneous round cell tumors in goats present a diagnostic challenge. In this article, we provide a description of caprine cutaneous mast cell tumors (MCT) and histiocytomas, and report on the validation of anti-human antibodies to CD117/KIT and Iba1 by immunohistochemistry on a range of caprine tissues. Cells immunolabeled for CD117/KIT included resident mast cells in normal lung and skin, interstitial cells of Cajal (intestine), and neuronal cell bodies (brain). Cells immunolabeled for Iba1 included resident macrophages in many tissues including normal lung, dendritic cells (hemolymphatic tissues), Kupffer cells, and microglia. Of 5 cutaneous MCT, only one had metachromasia of cytoplasmic granules; however, neoplastic cells of all 5 MCT had positive immunolabeling for CD117/KIT. The CD117/KIT immunolabeling pattern was predominately focal paranuclear in 3 cases, and cytoplasmic or membranous in 1 case each. Two histiocytomas were identified and had strong positive immunolabeling for Iba1 but not CD117/KIT. All 7 cutaneous round cell tumors described herein occurred in goats less than 4 years of age; the 2 cutaneous histiocytomas were in goats less than 14 months of age. Neither of the cutaneous histiocytomas recurred within 24 months of surgical removal.

Inter-Institutional Collaboration for the Development of a Local Peer Observation Process to Enhance Teaching.

Local peer observation of teaching is considered an important mechanism for instructors to improve the quality and effectiveness of their teaching, but there is an absence of uniformity to establish a best practice for this process in veterinary curricula. The Regional Teaching Academy (RTA) of the Consortium of Western Colleges of Veterinary Medicine is comprised of educational advocates from five western veterinary colleges with a common goal of enhancing the quality and effectiveness of education in veterinary medical curricula. Members of the RTA recognized this deficit in best practices for local peer observation (LPO) and formed a working group called "Local Peer Observation of Teaching." The goal was to meet a critical need for the enhancement of individual teaching skills by using a scholarly approach to develop robust methods for peer observation of teaching. Two rubric-based instruments were developed: one for large-group/didactic settings, and the second for small-group/clinical settings. Each is accompanied by pre- and post-observation worksheets which are considered instrumental to success. Results of a qualitative survey of instrument users' experiences are shared. Both observers and observees view the experiential learning from faculty peer colleagues very positively and the meaningful feedback is appreciated and incorporated by observees. Suggestions for implementation of the peer observation process are discussed, considering strengths and challenges. The purpose of this article is to describe in depth, the development process and output of the efforts of the Local Peer Observation of Teaching working group as a potential best practice guideline for peer observation.

Pan-tissue transcriptome analysis of long noncoding RNAs in the American beaver Castor canadensis.

BACKGROUND: Long noncoding RNAs (lncRNAs) have roles in gene regulation, epigenetics, and molecular scaffolding and it is hypothesized that they underlie some mammalian evolutionary adaptations. However, for many mammalian species, the absence of a genome assembly precludes the comprehensive identification of lncRNAs. The genome of the American beaver (Castor canadensis) has recently been sequenced, setting the stage for the systematic identification of beaver lncRNAs and the characterization of their expression in various tissues. The objective of this study was to discover and profile polyadenylated lncRNAs in the beaver using high-throughput short-read sequencing of RNA from sixteen beaver tissues and to annotate the resulting lncRNAs based on their potential for orthology with known lncRNAs in other species. RESULTS: Using de novo transcriptome assembly, we found 9528 potential lncRNA contigs and 187 high-confidence lncRNA contigs. Of the high-confidence lncRNA contigs, 147 have no known orthologs (and thus are putative novel lncRNAs) and 40 have mammalian orthologs. The novel lncRNAs mapped to the Oregon State University (OSU) reference beaver genome with greater than 90% sequence identity. While the novel lncRNAs were on average shorter than their annotated counterparts, they were similar to the annotated lncRNAs in terms of the relationships between contig length and minimum free energy (MFE) and between coverage and contig length. We identified beaver orthologs of known lncRNAs such as XIST, MEG3, TINCR, and NIPBL-DT. We profiled the expression of the 187 high-confidence lncRNAs across 16 beaver tissues (whole blood, brain, lung, liver, heart, stomach, intestine, skeletal muscle, kidney, spleen, ovary, placenta, castor gland, tail, toe-webbing, and tongue) and identified both tissue-specific and ubiquitous lncRNAs. CONCLUSIONS: To our knowledge this is the first report of systematic identification of lncRNAs and their expression atlas in beaver. LncRNAs-both novel and those with known orthologs-are expressed in each of the beaver tissues that we analyzed. For some beaver lncRNAs with known orthologs, the tissue-specific expression patterns were phylogenetically conserved. The lncRNA sequence data files and raw sequence files are available via the web supplement and the NCBI Sequence Read Archive, respectively.

Deregulating MYC in a model of HER2+ breast cancer mimics human intertumoral heterogeneity.

The c-MYC (MYC) oncoprotein is often overexpressed in human breast cancer; however, its role in driving disease phenotypes is poorly understood. Here, we investigate the role of MYC in HER2+ disease, examining the relationship between HER2 expression and MYC phosphorylation in HER2+ patient tumors and characterizing the functional effects of deregulating MYC expression in the murine NeuNT model of amplified-HER2 breast cancer. Deregulated MYC alone was not tumorigenic, but coexpression with NeuNT resulted in increased MYC Ser62 phosphorylation and accelerated tumorigenesis. The resulting tumors were metastatic and associated with decreased survival compared with NeuNT alone. MYC;NeuNT tumors had increased intertumoral heterogeneity including a subtype of tumors not observed in NeuNT tumors, which showed distinct metaplastic histology and worse survival. The distinct subtypes of MYC;NeuNT tumors match existing subtypes of amplified-HER2, estrogen receptor-negative human tumors by molecular expression, identifying the preclinical utility of this murine model to interrogate subtype-specific differences in amplified-HER2 breast cancer. We show that these subtypes have differential sensitivity to clinical HER2/EGFR-targeted therapeutics, but small-molecule activators of PP2A, the phosphatase that regulates MYC Ser62 phosphorylation, circumvents these subtype-specific differences and ubiquitously suppresses tumor growth, demonstrating the therapeutic utility of this approach in targeting deregulated MYC breast cancers.

miRWoods: Enhanced precursor detection and stacked random forests for the sensitive detection of microRNAs.

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome.