Delayed puberty, gonadotropin abnormalities and subfertility in male Padi2/Padi4 double knockout mice.

BACKGROUND: Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. METHODS: Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice along with wild-type FVB/NJ (WT) and both strains of SKO mice for a range of reproductive defects. RESULTS: Controlled breeding trials found that male DKO mice appeared to take longer to have their first litter than WT controls. This tendency was maintained when these mice were mated to either DKO or WT females. Additionally, unsexed 2-day old DKO pups and male DKO weanlings both weighed significantly less than their WT counterparts, took significantly longer than WT males to reach puberty, and had consistently lower serum testosterone levels. Furthermore, 90-day old adult DKO males had smaller testes than WT males with increased rates of germ cell apoptosis. CONCLUSIONS: The Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.

Chromatin run-on sequencing analysis finds that ECM remodeling plays an important role in canine hemangiosarcoma pathogenesis.

BACKGROUND: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. RESULTS: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions. CONCLUSION: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.

Safety of Strontium Chloride as a Skeletal Marking Agent for Pacific Salmon.

The purpose of this study was to evaluate the biological effects associated with administering strontium chloride as a marking agent to age-0 Chinook Salmon Oncorhynchus tshawytscha fry. Fish were held in a 0× (0 mg/L), 1× (3,000 mg/L; current standard dosage), 3× (9,000 mg/L), or 5× (15,000 mg/L) solution of strontium chloride for 72 h (three times the standard duration of 24 h). The mortality among fish in the 5× strontium chloride exposure group was significantly higher than that observed in the other groups. A dose-related effect on general fish behavior and on feeding behavior was observed. Fish in all test tanks appeared to feed to satiation, except for fish in the 5× tanks during days 2 and 3. Fish in all other test tanks behaved normally. No dose-related effect on fish growth was detected. Histopathological evaluations showed that fish in the 5× exposure group had a significantly higher number of gill lesions than the 0× group. Our mortality, behavioral, and histological assessments suggested that juvenile Chinook Salmon could be safely immersed for three consecutive days in a 9,000-mg/L solution of strontium chloride. This finding potentially expands the present 1,000-3,000-mg/L dosage and 24-h holding period that can be used to mark juvenile fish with strontium chloride solutions. The research also provides necessary target animal safety data for U.S. Food and Drug Administration approval of strontium chloride as an alternative marking method that is suitable for fish with a short holding time. Received February 19, 2017; accepted July 16, 2017.

Coastal water bacteriophages infect various sets of Vibrio parahaemolyticus sequence types.

Introduction: Gastrointestinal illnesses associated with the consumption of shellfish contaminated with Vibrio parahaemolyticus have a negative impact on the shellfish industry due to recalls and loss of consumer confidence in products. This bacterial pathogen is very diverse and specific sequence types (STs), ST631 and ST36, have emerged as prevalent causes of Vibrio foodborne disease outbreaks in the US, though other STs have been implicated in sporadic cases. We investigated whether bacteriophages could be used as a proxy to monitor for the presence of distinct V. parahaemolyticus STs in coastal waters. Methods: For this purpose, bacteriophages infecting V. parahaemolyticus were isolated from water samples collected on the Northeast Atlantic coast. The isolated phages were tested against a collection of 29 V. parahaemolyticus isolates representing 18 STs, including six clonal complexes (CC). Four distinct phages were identified based on their ability to infect different sets of V. parahaemolyticus isolates. Results and Discussion: Overall, the 29 bacterial isolates segregated into one of eight patterns of susceptibility, ranging from resistance to all four phages to susceptibility to any number of phages. STs represented by more than one bacterial isolate segregated within the same pattern of susceptibility except for one V. parahaemolyticus ST. Other patterns of susceptibility included exclusively clinical isolates represented by distinct STs. Overall, this study suggests that phages populating coastal waters could be exploited to monitor for the presence of V. parahaemolyticus STs known to cause foodborne outbreaks.

Histologic and molecular characterization of Edwardsiella piscicida infection in largemouth bass (Micropterus salmoides).

The genus Edwardsiella is composed of a diverse group of facultative anaerobic, gram-negative bacteria that can produce disease in a wide variety of hosts, including birds, reptiles, mammals, and fish. Our report describes the isolation and identification of Edwardsiella piscicida associated with chronic mortality events in 2 separate captive largemouth bass (Micropterus salmoides) populations in New York and Florida. Wet-mount biopsies of skin mucus, gill, kidney, and spleen from several affected largemouth bass contained significant numbers of motile bacteria. Histologic examination revealed multifocal areas of necrosis scattered throughout the heart, liver, anterior kidney, posterior kidney, and spleen. Many of the necrotic foci were encapsulated or replaced by discrete granulomas and associated with colonies of gram-negative bacteria. Initial phenotypic and matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis against existing spectral databases of recovered isolates identified these bacteria as Edwardsiella tarda Subsequent molecular analysis using repetitive sequence mediated and species-specific PCR, as well as 16S rRNA, rpoB, and gyrB sequences, classified these isolates as E. piscicida As a newly designated taxon, E. piscicida should be considered as a differential for multiorgan necrosis and granulomas in largemouth bass.

PAD2 overexpression in transgenic mice promotes spontaneous skin neoplasia.

Peptidylarginine deiminase 2 (PAD2/PADI2) has been implicated in various inflammatory diseases and, more recently, cancer. The goal of this study was to test the hypothesis that PAD2 promotes oncogenesis using a transgenic mouse model. We found that about 37% of transgenic mice overexpressing human FLAG-PAD2 downstream of the MMTV-LTR promoter develop spontaneous neoplastic skin lesions. Molecular and histopathologic analyses of the resulting lesions find that they contain increased levels of markers for invasion, inflammation, and epithelial-to-mesenchymal transition (EMT) and that a subset of the lesions progress to invasive squamous cell carcinoma (SCC). We then stably overexpressed FLAG-PAD2 in the human SCC cell line, A431, and found that the PAD2-overexpressing cells were more tumorigenic in vitro and also contained elevated levels of markers for inflammation and EMT. Collectively, these studies provide the first genetic evidence that PAD2 functions as an oncogene and suggest that PAD2 may promote tumor progression by enhancing inflammation within the tumor microenvironment.