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Contents It is now widely accepted in human medicine that prolactin ( PRL) and growth hormone ( GH) function in the mammary gland in an autocrine and paracrine manner in tumour formation. The aim of this study was to compare PRL and GH immunoactivity in canine mammary tumours submitted for histopathologic evaluation. Formalin-fixed specimens from spontaneously occurring mammary adenomas and adenocarcinomas from 24 female client-owned dogs were used. Information pertaining to the reproductive status of the patient at the time of mammary tumour diagnosis was obtained from each of the submitting veterinarians. Tissues were paraffin-embedded and sectioned (5 μm) onto charged slides. All slides were deparaffinized and rehydrated. Endogenous peroxidase activity was inactivated with 3% H2O2, and non-specific binding was blocked. Polyclonal rabbit antihuman PRL ( DAKO A0569) and GH antibody ( DAKO A0570) were applied at a 1:250 and 1:200 dilutions, respectively. A universal rabbit negative control ( DAKO N1699) was used. Slides were then reacted with anti-rabbit horseradish peroxidase followed by Nova Red Peroxidase substrate. Slides were counter-stained with haematoxylin, dehydrated and mounted. Tumour type and reproductive status at time of tumour diagnosis were compared individually between tumours that were negative or positive for PRL and GH using a two-tailed analysis of variance. Significance was defined as p < .05. There was no significant relationship between tumour type and PRL and GH presence. In addition, reproductive status at the time of tumour removal was found to be not significant. These results vary from previous reports in canine mammary tumours and warrant further investigation. [ABSTRACT FROM AUTHOR]

Chronic inflammation and nuclear factor-kappa B (NFκB) have been implicated in prostate cancer development; thus, dietary factors that inhibit NFκB may serve as effective chemo-preventative agents. Prostate cancer risk is significantly lower in Asian countries compared to the United States, which has prompted interest in the potential chemopreventative action of Asian dietary components such as soy and green tea. This study examined the effects of dietary soy and tea on NFκB activation and inflammation in vivo using a hormone-induced rat model for prostate cancer. Male Noble rats implanted with estradiol and testosterone were divided into 4 dietary groups: control, soy, tea, or soy+tea. NFκB activation and inflammatory cytokines were measured post implantation. The combination of soy and tea suppressed NFκB p50 binding activity and protein levels via induction of IκBα. Soy and tea also decreased prostate inflammatory infiltration, increased Bax/BcL2 ratio and decreased protein expression of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1β compared to control. Soy and tea attenuated prostate malignancy by decreasing prostate hyperplasia. These effects were not apparent in groups treated with soy or tea alone. The ongoing in vivo studies thus far suggest that combination of foods, such as soy and tea, may inhibit hormone-induced proinflammatory NFκB signals that contribute to prostate cancer development. [ABSTRACT FROM AUTHOR]

The B-cell lymphoma-2 (Bcl-2) family of proteins plays a vital role in tumorigenesis. Cancer cells utilize the expression of Bcl-2 to evade therapy and develop resistance. Bcl-2 overexpression also causes cancer cells to be more invasive and metastatic. About 80% of cancer deaths are due to metastases, and yet targeted therapies for metastatic cancers are scarce. We discovered a small molecule, BFC1103, which changes the conformation of Bcl-2 to convert the antiapoptotic protein to a proapoptotic protein. BFC1103-induced apoptosis is dependent on the expression levels of Bcl-2, with higher levels causing more apoptosis. BFC1103 suppressed the growth of breast cancer lung metastasis. BFC1103 has the potential for further optimization and development for clinical testing in metastatic cancers that express Bcl-2. This study demonstrates a new approach to target Bcl-2 using a small molecule, BFC1103, to suppress metastatic disease., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Valdez, Rivera, Chang, Pennington, Fischer, Löhr and Tilton.)

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., (© 2024 The Authors; Published by the American Association for Cancer Research.)

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., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Spooner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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, TGFβ ) 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Padiadpu, Spooner, Li, Newman, Löhr, Apperson, Dzutsev, Trinchieri, Shulzhenko, Morgun and Jump.)

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., (© 2022 American College of Veterinary Radiology.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

In collaboration with the American College of Veterinary Radiology.

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.

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.

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., (Copyright © 2020. Published by Elsevier Inc.)

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.

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.

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.

Computed tomographic angiography (CTA) and magnetic resonance imaging (MRI) have been described as methods for preoperative surgical planning in cats with feline injection site sarcomas (FISS), however, few published studies have compared these modalities. The objective of this retrospective, secondary analysis study was to determine if imaging features of FISS on CTA and MRI are predictive of neoplastic peritumoral projections. Archived data from a previous prospective study were retrieved for 10 cats with FISS. All cats had been evaluated in a single anesthetic episode with MRI and dual phase CT (CTA) imaging followed by surgical removal. Histopathological grading and targeted histopathology of imaging-identified peritumoral projections were performed. Two observers evaluated the CTA and MRI studies for FISS shape, margination, size, enhancement pattern, postcontrast uniformity, pre- and postcontrast margination, the number of muscles involved, mass mineralization, and bone lysis. Metal was present in the imaging field of three of 10 cats, resulting in one nondiagnostic MRI. Peritumoral projections were detected in all cats with both imaging modalities, and most were benign. At least one neoplastic peritumoral projection was detected in six cats using MRI, five cats using CTA, and three cats with both modalities. Higher grade FISS were larger than low grade using MRI, and FISS were larger using MRI. Other FISS imaging features using MRI and CTA were similar. Findings supported use of either MRI or CTA for detecting neoplastic peritumoral projections in cats with FISS. Authors recommend CTA for cats with known metallic objects in the scan field., (© 2019 American College of Veterinary Radiology.)

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., Competing Interests: The authors have declared that no competing interests exist.

Current assumption for assessing carcinogenic risk of polycyclic aromatic hydrocarbons (PAHs) is that they function through a common mechanism of action; however, recent studies demonstrate that PAHs can act through unique mechanisms potentially contributing to cancer outcomes in a non-additive manner. Using a primary human 3D bronchial epithelial culture (HBEC) model, we assessed potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from transcriptional profiling. Differentiated bronchial epithelial cells were treated with BAP (100-500 μg/ml), DBC (10 μg/ml), and coal tar extract (CTE 500-1500 μg/ml, SRM1597a) for 48 h and gene expression was measured by RNA sequencing or quantitative PCR. Comparison of BAP and DBC gene signatures showed that the majority of genes (~60%) were uniquely regulated by treatment, including signaling pathways for inflammation and DNA damage by DBC and processes for cell cycle, hypoxia and oxidative stress by BAP. Specifically, BAP upregulated targets of AhR, NRF2, and KLF4, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. Other processes were regulated in common by all PAH treatments, BAP, DBC and CTE, including downregulation of genes involved in cell adhesion and reduced functional measurements of barrier integrity. This work supports prior in vivo studies and demonstrates the utility of profiling short-term biosignatures in an organotypic 3D model to identify mechanisms linked to carcinogenic risk of PAHs in humans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We report herein Rhodococcus equi infection in an 11-y-old, male llama with a history of diarrhea and endoparasitism. Postmortem examination revealed granulomatous and ulcerative enteritis, pyogranulomatous mesenteric lymphadenitis, fibrinosuppurative peritonitis, and granulomatous hepatitis. Intralesional macrophages were laden with gram-positive cocci. Bacteriology identified R. equi , and cultures tested positive for R. equi choE and vapA genes by PCR. This case expands the reported spectrum of lesions associated with R. equi infections in llamas from pyogranulomatous bronchopneumonia and peripheral lymphadenitis to pyogranulomatous mesenteric lymphadenitis and enteritis. We also link a R. equi that is carrying the virulent-associated protein gene VapA to clinical disease in New World camelids.

Fixation and decalcification can alter protein structure in tissues, influencing the efficacy of primary antibodies routinely used in immunohistochemical (IHC) staining. Histologic examination of zebrafish requires both processes, making staining and analysis potentially challenging. Here, we investigated the effects of common fixation and decalcification protocols on IHC staining in zebrafish. We also identified zebrafish-reactive and -specific antibodies for use in research and diagnostics. For several of the antibodies, time spent in Dietrich's fixative containing 2% glacial acetic acid or 3.4% formaldehyde followed by decalcification with ethylenediaminetetraacetic acid (EDTA) significantly impacted IHC staining quality, particularly regarding staining intensity. Protocols utilizing shorter fixation times produced higher-quality stains. In addition, individual markers were variably affected by the type of fixative. Dietrich's fixative significantly reduced staining quality for the "neural" markers: glial fibrillar acidic protein, chromogranin A, S100. A negative time-dependent effect of fixation on staining quality was found for several antibodies: muscle actin (Dietrich's only), cytokeratin AE1/AE3, chromogranin, and S100. Neither decalcification protocol had a statistically significant negative time-dependent effect on staining quality. Based on our results, we suggest shorter fixation and decalcification protocols to best preserve IHC staining quality as well as recommend deliberate selection of the fixative used depending on the protein of interest.

Background: Feline injection-site sarcoma (FISS), an aggressive iatrogenic subcutaneous malignancy, is challenging to manage clinically and little is known about the molecular basis of its pathogenesis. Tumor transcriptome profiling has proved valuable for gaining insights into the molecular basis of cancers and for identifying new therapeutic targets. Here, we report the first study of the FISS transcriptome and the first cross-species comparison of the FISS transcriptome with those of anatomically similar soft-tissue sarcomas in dogs and humans., Methods: Using high-throughput short-read paired-end sequencing, we comparatively profiled FISS tumors vs. normal tissue samples as well as cultured FISS-derived cell lines vs. skin-derived fibroblasts. We analyzed the mRNA-seq data to compare cancer/normal gene expression level, identify biological processes and molecular pathways that are associated with the pathogenesis of FISS, and identify multimegabase genomic regions of potential somatic copy number alteration (SCNA) in FISS. We additionally conducted cross-species analyses to compare the transcriptome of FISS to those of soft-tissue sarcomas in dogs and humans, at the level of cancer/normal gene expression ratios., Results: We found: (1) substantial differential expression biases in feline orthologs of human oncogenes and tumor suppressor genes suggesting conserved functions in FISS; (2) a genomic region with recurrent SCNA in human sarcomas that is syntenic to a feline genomic region of probable SCNA in FISS; and (3) significant overlap of the pattern of transcriptional alterations in FISS with the patterns of transcriptional alterations in soft-tissue sarcomas in humans and in dogs. We demonstrated that a protein, BarH-like homeobox 1 (BARX1), has increased expression in FISS cells at the protein level. We identified 11 drugs and four target proteins as potential new therapies for FISS, and validated that one of them (GSK-1059615) inhibits growth of FISS-derived cells in vitro., Conclusions: (1) Window-based analysis of mRNA-seq data can uncover SCNAs. (2) The transcriptome of FISS-derived cells is highly consistent with that of FISS tumors. (3) FISS is highly similar to soft-tissue sarcomas in dogs and humans, at the level of gene expression. This work underscores the potential utility of comparative oncology in improving understanding and treatment of FISS.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, particularly in obese and type 2 diabetic individuals. NAFLD ranges in severity from benign steatosis to nonalcoholic steatohepatitis (NASH); and NASH can progress to cirrhosis, primary hepatocellular carcinoma (HCC) and liver failure. As such, NAFLD has emerged as a major public health concern. Herein, we used a lipidomic and transcriptomic approach to identify lipid markers associated with western diet (WD) induced NASH in female mice., Methods: Female mice (low-density lipoprotein receptor null (Ldlr -/-) were fed a reference or WD diet for 38 and 46 weeks. Transcriptomic and lipidomic approaches, coupled with statistical analyses, were used to identify associations between major NASH markers and transcriptomic & lipidomic markers., Results: The WD induced all major hallmarks of NASH in female Ldlr -/- mice, including steatosis (SFA, MUFA, MUFA-containing di- and triacylglycerols), inflammation (TNFα), oxidative stress (Ncf2), and fibrosis (Col1A). The WD also increased transcripts associated with membrane remodeling (LpCat), apoptosis & autophagy (Casp1, CtsS), hedgehog (Taz) & notch signaling (Hey1), epithelial-mesenchymal transition (S1004A) and cancer (Gpc3). WD feeding, however, suppressed the expression of the hedgehog inhibitory protein (Hhip), and enzymes involved in triglyceride catabolism (Tgh/Ces3, Ces1g), as well as the hepatic abundance of C18-22 PUFA-containing phosphoglycerolipids (GpCho, GpEtn, GpSer, GpIns). WD feeding also increased hepatic cyclooxygenase (Cox1 & 2) expression and pro-inflammatory ω6 PUFA-derived oxylipins (PGE2), as well as lipid markers of oxidative stress (8-iso-PGF2α). The WD suppressed the hepatic abundance of reparative oxylipins (19, 20-DiHDPA) as well as the expression of enzymes involved in fatty epoxide metabolism (Cyp2C, Ephx)., Conclusion: WD-induced NASH in female Ldlr -/- mice was characterized by a massive increase in hepatic neutral and membrane lipids containing SFA and MUFA and a loss of C18-22 PUFA-containing membrane lipids. Moreover, the WD increased hepatic pro-inflammatory oxylipins and suppressed the hepatic abundance of reparative oxylipins. Such global changes in the type and abundance of hepatic lipids likely contributes to tissue remodeling and NASH severity., Competing Interests: The authors have declared that no competing interests exist.

Knowledge of changes in the composition of microbial communities (microbiota) in tissues after death, over time, is critical to correctly interpret results of microbiologic testing from postmortem examinations. Limited information is available about postmortem changes of the microbiota and the associated microbial genes (microbiome) of internal organs in any species. We examined the effect of time and ambient temperature on the postmortem microbiome (thanatomicrobiome) of tissues typically sampled for microbiologic testing during autopsies. Twenty rabbits were euthanized and their bodies stored at 4°C or 20°C for 6 or 48 h. Ileum, cecum, kidney, and lung tissue were sampled. Bacterial DNA abundance was determined by RT-qPCR. Microbiome diversity was determined by 16S rRNA gene sequencing. By relative abundance of the microbiome composition, intestinal tissues were clearly separated from lungs and kidneys, which were similar to each other, over all times and temperatures. Only cecal thanatomicrobiomes had consistently high concentrations and consistent composition in all conditions. In lungs and kidneys, but not intestine, proteobacteria were highly abundant at specific times and temperatures. Thanatomicrobiome variation was not explained by minor subclinical lesions identified upon microscopic examination of tissues. Bacterial communities typically found in the intestine were not identified at extra-intestinal sites in the first 48 h at 4°C and only in small amounts at 20°C. However, changes in tissue-specific microbiomes during the postmortem interval should be considered when interpreting results of microbiologic testing.

Background: A novel equine parvovirus (EqPV-H) was recently discovered in the equine liver with Theiler's disease., Objectives: To determine the prevalence of EqPV-H infection in naturally occurring Theiler's disease cases and in-contact horses in the absence of historical equine biologic product administration., Animals: Ten cases of Theiler's disease from 6 separate properties were included in the study, based on the criteria of acute onset of clinical signs of liver failure with laboratory or histopathologic findings characteristic of Theiler's disease and no history of receiving an equine biologic product within the preceding 4 months. In addition, 37 in-contact horses from 4 of the 6 properties were screened for EqPV-H infection and hepatitis., Methods: In prospective case series, cases were diagnosed with Theiler's disease by the attending veterinarian and were tested for EqPV-H by PCR of liver or serum. In-contact horses were assessed via serum chemistry and PCR at the attending veterinarian's discretion. Hepatitis was defined as serum gamma-glutamyltransferase activity above reference interval. The association of EqPV-H with hepatitis was determined by Fisher's exact test., Results: Nine of 10 (90%) Theiler's disease cases and 54% of tested in-contact horses were EqPV-H positive. Hepatitis was significantly associated with EqPV-H infection (P = .036)., Conclusions and Clinical Importance: Although further study is required to identify EqPV-H as the causative agent of Theiler's disease, EqPV-H appears strongly associated with cases of fatal Theiler's disease and subclinical hepatitis in horses in contact with those cases. The prevalence of EqPV-H infection on affected properties can be high., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Although quantitative assessment of margins is recommended for describing excision of cutaneous malignancies, there is poor understanding of limitations associated with this technique. We described and quantified histologic artifacts in inked margins and determined the association between artifacts and variance in histologic tumor-free margin (HTFM) measurements based on a novel grading scheme applied to 50 sections of normal canine skin and 56 radial margins taken from 15 different canine mast cell tumors (MCTs). Three broad categories of artifact were 1) tissue deformation at inked edges, 2) ink-associated artifacts, and 3) sectioning-associated artifacts. The most common artifacts in MCT margins were ink-associated artifacts, specifically ink absent from an edge (mean prevalence: 50%) and inappropriate ink coloring (mean: 45%). The prevalence of other artifacts in MCT skin was 4-50%. In MCT margins, frequency-adjusted kappa statistics found fair or better inter-rater reliability for 9 of 10 artifacts; intra-rater reliability was moderate or better in 9 of 10 artifacts. Digital HTFM measurements by 5 blinded pathologists had a median standard deviation (SD) of 1.9 mm (interquartile range: 0.8-3.6 mm; range: 0-6.2 mm). Intraclass correlation coefficients demonstrated good inter-pathologist reliability in HTFM measurement (κ = 0.81). Spearman rank correlation coefficients found negligible correlation between artifacts and HTFM SDs ( r ≤ 0.3). These data confirm that although histologic artifacts commonly occur in inked margin specimens, artifacts are not meaningfully associated with variation in HTFM measurements. Investigators can use the grading scheme presented herein to identify artifacts associated with tissue processing.

Background: Diagnostic accuracy of the 3 mm laparoscopic cup biopsy forceps for collection of tissue samples from canine livers is unproven., Hypotheses/objectives: Compare sample surface area and portal triad count between 3 mm and 5 mm laparoscopic cup biopsies and compare the histologic diagnosis obtained by each instrument to a standard necropsy wedge. The hypothesis was that more portal triads and greater sample surface area would be found with the 5 mm samples and the laparoscopic instruments would not have significantly different levels of agreement with necropsy wedge diagnosis., Animals: Twenty-one client-owned dogs undergoing necropsy., Methods: Prospective ex vivo study. Three samples (3 mm, 5 mm, and wedge) were taken of 2 different hepatic divisions within 24 hours of death. Morphologic diagnosis, World Small Animal Veterinary Association histologic features, surface area, and portal triad numbers were compared among the 3 samples., Results: There were significantly more portal triads (mean 21.4 versus 13.8; P < .0001) and a higher surface area (20.3 mm 2 versus 11.5 mm 2 ; P < .0001) in the 5 mm samples compared to 3 mm samples. Kappa coefficients and percent agreement for histologic diagnosis as compared to the wedge biopsy were not significantly different between the 2 instrument sizes (κ = 0.383 and 0.436, respectively; 67% and 69%, respectively)., Conclusions and Clinical Importance: Despite yielding smaller sample sizes, the 3 mm laparoscopic cup biopsy has a similar level of histologic diagnostic accuracy to the 5 mm instrument., (Copyright © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

The laying hen ( Gallus domesticus ) is a robust animal model for epithelial ovarian cancer. The use of animal models is critical in identifying early disease markers and developing and testing chemotherapies. We describe the microscopic characteristics of the normally functioning laying hen ovary and proximal oviduct to establish baselines from which lesions associated with ovarian cancer can be more readily identified. Ovaries and oviducts were collected from 18-month-old laying hens ( n = 18) and fixed in 10% neutral buffered formalin. Hematoxylin- and eosin-stained sections were examined by light microscopy. Both post-ovulatory follicular regression and atresia of small follicles produce remnant clusters of vacuolated cells with no histological evidence that scar tissue persists. Infiltrates of heterophils are associated with atresia of small follicles, a relationship not previously documented in laying hen ovaries. Because these tissues can be mistaken for cancerous lesions, we present a detailed histological description of remnant Wolffian tissues in the laying hen ovary. Immunohistochemical staining for pancytokeratin produced a positive response in ovarian surface epithelium and staining for vimentin produced a positive response in granulosa cells of follicles. Epithelial cells lining glands of the remnant epoöphoron had a positive response to both pancytokeratin and vimentin, a result also observed in women., Competing Interests: The authors declare no conflict of interest.

Background: Understanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment. Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation., Methods: We generated a trigenic mouse model system (RXRα ep-/- | Tyr-NRAS Q61K | CDK4 R24C/R24C ) harboring an epidermal knockout of Retinoid X Receptor α (RXRα ep-/- ), combined with oncogenic NRAS Q61K (constitutively active RAS) and activated CDK4 R24C/R24C (constitutively active CDK4). Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRα ep-/- mice compared to the control mice with functional RXRα., Results: Here we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα. Melanomas from the trigenic RXRα ep-/- mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization. Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRα ep-/- mice. Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRα ep-/- mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment. Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRAS Q61K and CDK4 R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα., Conclusions: Our study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4 R24C/R24C and oncogenic NRAS Q61K .

Heterocyclic amines (HCAs) produced during high-temperature cooking have been studied extensively in terms of their genotoxic/genetic effects, but recent work has implicated epigenetic mechanisms involving non-coding RNAs. Colon tumors induced in the rat by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have altered microRNA (miRNA) signatures linked to dysregulated pluripotency factors, such as c-Myc and Krüppel-like factor 4 (KLF4). We tested the hypothesis that dysregulated miRNAs from PhIP-induced colon tumors would provide a "PhIP signature" for use in other target organs obtained from a 1-year carcinogenicity bioassay in the rat. Downstream targets that were corroborated in the rat were then investigated in human cancer datasets. The results confirmed that multiple let-7 family members were downregulated in PhIP-induced skin, colon, lung, small intestine, and Zymbal's gland tumors, and were associated with c-myc and Hmga2 upregulation. PhIP signature miRNAs with the profile mir-21 high /mir-126 low /mir-29c low /mir-215 low /mir-145 low were linked to reduced Klf4 levels in rat tumors, and in human pan-cancer and colorectal cancer. It remains to be determined whether this PhIP signature has predictive value, given that more than 20 different genotoxic HCAs are present in the human diet, plus other agents that likely induce or repress many of the same miRNAs. Future studies should define more precisely the miRNA signatures of other HCAs, and their possible value for human risk assessment.

Koi herpesvirus (KHV) is a highly pathogenic virus of common carp and koi. KHV becomes latent in recovered koi or exposed koi without symptoms, and the latent infection can reactivate under stress conditions. KHV reactivation from latency often occurs when water temperature rapidly rises above 17°C. Dissolved O 2 is lower at ≥17°C than at non-stress temperatures ≤15°C. To determine whether reduced dissolved O 2 level has a role in KHV reactivation during temperature stress, KHV reactivation was investigated in KHV latently infected koi (KHV + koi) under stress temperatures by maintaining dissolved O 2 consistent with the O 2 level at 15°C. There was no significant difference in the amount of reactivated virus between KHV + koi maintained with and without O 2 supplementation during temperature stress. Both handling and sampling were found to be stressful to koi and can contribute to KHV reactivation from latency. There was an increase in KHV genome within white blood cells (WBC) during KHV reactivation, which is about 3-4 fold higher than the amount of KHV genome detectable in WBC during the latency stage. At day 15 post-temperature stress (PTS), inflammation and necrosis were observed in multiple tissues, especially in the gills, eye, intestine, skin and kidney. KHV DNA was also detectable in multiple tissues on days 6, 9 and 15 PTS. Following day 3 PTS, the plasma cortisol levels were higher than that observed in koi before temperature stress, suggesting that KHV reactivation is associated with physiological stress in KHV + koi., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Breast cancer metastasizes to bone in the majority of patients with advanced disease. We investigated the effects of inadequate dietary calcium (Ca) on bone turnover, tumor growth, and bone response to tumor in tibia inoculated with 4T1 mammary carcinoma cells. Nine-month-old female Balb/c mice were placed on an adequate Ca (5 g/kg diet, n = 30) or low Ca (80 mg/kg diet, n = 31) diet for 14 days, then injected intratibially with 1,000 4T1 cells (transfected with luciferase for bioluminescence imaging), and sacrificed at 5, 10, or 21 days post-inoculation (n = 7-10 mice/group). Control mice (n = 6/group) were injected with carrier and sacrificed at 10 days post-inoculation. Tibiae with muscle intact were excised and evaluated by microcomputed tomography and histology. In vivo bioluminescent imaging revealed that 4T1 cells metastasized to lung. Therefore, lungs were removed for quantification of tumor. Mice fed low Ca exhibited higher bone turnover and higher tibial lesion scores than mice fed adequate Ca. Lesion severity, manifested as cortical osteolysis and periosteal woven bone formation, and tumor cell infiltration to muscle, increased with time, irrespective of diet. However, for most skeletal endpoints the rates of increase were greater in mice consuming low Ca compared to mice consuming adequate Ca. Infiltration of tumor cells into adjacent muscle, but not metastasis to lung, was also greater in mice consuming low Ca diet. The findings suggest that high bone turnover due to Ca insufficiency results in greater local mammary tumor cell growth, cortical osteolysis, woven bone formation, and invasion to muscle in mice.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) targets multiple organs for tumorigenesis in the rat, including the colon and the skin. PhIP-induced skin tumors were subjected to mutation screening, which identified genetic changes in Hras (7/40, 17.5%) and Tp53 (2/40, 5%), but not in Ctnnb1, a commonly mutated gene in PhIP-induced colon tumors. Despite the absence of Ctnnb1 mutations, β-catenin was overexpressed in nuclear and plasma membrane fractions from PhIP-induced skin tumors, coinciding with loss of p120-catenin from the plasma membrane, and the appearance of multiple p120-catenin-associated bands in the nuclear extracts. Real-time RT-PCR revealed that p120-catenin isoforms 1 and 4 were upregulated in PhIP-induced skin tumors, whereas p120-catenin isoform 3 was expressed uniformly, compared with adjacent normal-looking tissue. In human epidermoid carcinoma and colon cancer cells, transient transfection of p120-catenin isoform 1A enhanced the viability and cell invasion index, whereas transient transfection of p120-catenin isoform 4A increased cell viability and cell proliferation. Knockdown of p120-catenin revealed a corresponding reduction in the expression of β-catenin and a transcriptionally regulated target, Ccnd1/Cyclin D1. Co-immunoprecipitation experiments identified associations of β-catenin with p120-catenin isoforms in PhIP-induced skin tumors and human cancer cell lines. The results are discussed in the context of therapeutic strategies that might target different p120-catenin isoforms, providing an avenue to circumvent constitutively active β-catenin arising via distinct mechanisms in skin and colon cancer., (© 2017 Wiley Periodicals, Inc.)

Lymphoma has been described in individual cases for goats but not systematically characterized in a larger cohort. This study aimed to subtype caprine lymphoma based on topographic and subgross distribution, immunophenotype, and cellular morphology following the World Health Organization classification system for hematopoietic tumors in domestic animals. Fifteen caprine lymphoma cases were assessed with 6 submitted as biopsy and 9 for postmortem examination. Goats were predominantly young adult (median 3 years) and dwarf breeds (Pygmy and Pygora). The sexes were similarly represented. Nuclear size was measured relative to red blood cells (RBCs) and then adjusted for species-specific differences and designated small (<3× RBCs), intermediate (3-4× RBCs), or large (>4× RBCs). Using immunohistochemistry, 11 of 15 (73%) goats had T-cell lymphoma (TCL; CD3 positive, CD79α negative) and 4 of 15 (27%) had B-cell lymphoma (BCL; CD79α positive, CD3 negative). A multicentric distribution was most common. TCL generally involved the thoracic cavity and/or neck, suggestive of thymic origin or homing. TCLs were further classified as lymphoblastic lymphomas (3/11; 27%), large granular lymphocyte lymphoma (1/11; 9%), diffuse small lymphocytic lymphomas (3/11; 27%), or peripheral/mature T-cell lymphoma (PTCL) not otherwise specified (4/11 [36%], of which 3 were high grade and 1 intermediate grade). In 1 goat with PTCL, lymph nodes had either paracortical expansion or diffuse infiltrates suggesting transition from nodular to diffuse PTLC. BCLs were classified as diffuse large B-cell lymphoma (2/4; 50%) or B-cell lymphocytic lymphoma intermediate type (2/4; 50%). In contrast to dogs and horses, lymphomas in goats are predominantly TCL and frequently involve the mediastinum.

Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Long noncoding RNAs (lncRNAs) have emerged as important in cancer development and progression. The impact of diet on lncRNA expression is largely unknown. Sulforaphane (SFN), obtained from vegetables like broccoli, can prevent and suppress cancer formation. Here we tested the hypothesis that SFN attenuates the expression of cancer-associated lncRNAs. We analyzed whole-genome RNA-sequencing data of normal human prostate epithelial cells and prostate cancer cells treated with 15 μM SFN or dimethylsulfoxide. SFN significantly altered expression of ~100 lncRNAs in each cell type and normalized the expression of some lncRNAs that were differentially expressed in cancer cells. SFN-mediated alterations in lncRNA expression correlated with genes that regulate cell cycle, signal transduction and metabolism. LINC01116 was functionally investigated because it was overexpressed in several cancers, and was transcriptionally repressed after SFN treatment. Knockdown of LINC01116 with siRNA decreased proliferation of prostate cancer cells and significantly up-regulated several genes including GAPDH (regulates glycolysis), MAP1LC3B2 (autophagy) and H2AFY (chromatin structure). A four-fold decrease in the ability of the cancer cells to form colonies was found when the LINC01116 gene was disrupted through a CRISPR/CAS9 method, further supporting an oncogenic function for LINC01116 in PC-3 cells. We identified a novel isoform of LINC01116 and bioinformatically investigated the possibility that LINC01116 could interact with target genes via ssRNA:dsDNA triplexes. Our data reveal that chemicals from the diet can influence the expression of functionally important lncRNAs, and suggest a novel mechanism by which SFN may prevent and suppress prostate cancer., (Published by Elsevier Inc.)

Mycobacterium spp. infections are common in zebrafish kept in research facilities. These comorbidities can substantially modulate the responses of these fish to external and internal stimuli. Therefore, diagnostic tests to detect Mycobacterium spp. infections in zebrafish colonies prove essential. Here, we outline the development of quantitative simplex real-time PCR assays to detect the 3 Mycobacterium species most commonly identified in laboratory zebrafish. The assays targeted the heatshock protein 65 gene of M. marinum, M. chelonae, and M. haemophilum. The assays are both highly specific and sensitive for fresh-frozen samples and highly specific and moderately sensitive for formalin-fixed paraffin-embedded (FFPE) samples. Two sampling techniques for FFPE samples of sagittally sectioned zebrafish were evaluated. Both paraffin cores targeting granulomas containing bacteria and scrolls from the entire fish yielded DNA of equivalent quantity and purity. The diagnostic sensitivity of cores was superior to that of scrolls for M. chelonae and M. haemophilum but not M. marinum. The assays are cost-effective and ideally suited to diagnosing common Mycobacterium spp. infections in zebrafish.