Dietary choline, but not L-carnitine, increases circulating lipid and lipoprotein concentrations, without affecting body composition, energy expenditure or respiratory quotient in lean and obese male cats during weight maintenance.

Introduction: Due to the involvement in one-carbon metabolism and lipid mobilization, choline and L-carnitine supplementation have been recommended to minimize hepatic lipid accumulation and support fat oxidation, respectively. This study investigated the lipotropic benefits of choline or L-carnitine supplementation in lean and obese cats maintaining body weight (BW). Methods: Lean [n = 9; body condition score (BCS): 4-5/9] and obese (n = 9; BCS: 8-9/9) adult male neutered colony cats were used in a replicated 3 x 3 complete Latin square design. Treatments included choline (378 mg/kg BW0.67), L-carnitine (200 mg/kg BW) and control (no supplement). Treatments were supplemented to the food for 6 weeks each, with a 2-week washout between treatments. Cats were fed once daily to maintenance energy requirements, and BW and BCS were assessed weekly. Fasted blood collection, indirect calorimetry, and dual-energy X-ray absorptiometry occurred at the end of each treatment period. Serum was analyzed for cholesterol (CHOL), high-density lipoprotein CHOL (HDL-C), triglycerides (TAG), non-esterified fatty acids (NEFA), glucose, creatinine (CREAT), urea, alkaline phosphatase (ALP) and alanine aminotransferase (ALT). Very low-density lipoprotein CHOL (VLDL) and low-density lipoprotein CHOL (LDL-C) were calculated. Data were analyzed using proc GLIMMIX, with group and period as random effects, and treatment, body condition, and their interaction as fixed effects, followed by a Tukey's post-hoc test when significance occurred. Results: Cats supplemented choline had lower food intake (P = 0.025). Treatment did not change BW, BCS and body composition (P > 0.05). Obese cats had greater ALP, TAG, and VLDL, and lower HDL-C compared to lean cats (P < 0.05). Choline resulted in greater CHOL, HDL-C, LDL-C and ALT (P < 0.05). L-carnitine resulted in lower CREAT (P = 0.010). Following the post-hoc test, differences between treatment means were not present for ALP (P = 0.042). No differences were found for glucose, urea or NEFA (P > 0.05). Obese cats had a lower fed respiratory quotient (RQ), regardless of treatment (P = 0.045). Treatment did not affect fed or fasted RQ and energy expenditure (P > 0.05). Discussion: Choline appeared to increase circulating lipid and lipoprotein concentrations regardless of body condition, likely through enhanced lipid mobilization and hepatic elimination. Neither dietary choline or L-carnitine altered body composition or energy metabolism in the lean or obese cats, as compared to control.

Student Veterinarian Perceptions of Community-Based Primary Care Veterinary Clinics in Indigenous Communities in Southern Ontario, Canada.

Community-based primary care veterinary clinics represent an opportunity to benefit multiple populations. Student veterinarians are afforded the opportunity to build technical and non-technical professional skills, while underserved communities are provided with access to companion animal care. The Ontario Veterinary College (OVC), as with many other veterinary colleges across Canada and the United States, has hosted community-based primary care veterinary clinics, including in local Indigenous communities. As these clinics continue and grow, it is critical to evaluate their operation to ensure that they align with community goals and values, adequately support student learning, and do not perpetuate racism and implicit bias. The objective of this study was to explore the perceptions of student veterinarians who had volunteered at community-based primary care veterinary clinics in First Nations communities in southern Ontario, Canada. We used an online survey that consisted of multiple choice and short answer questions focused on motivating factors for involvement, supports available before and during the clinics, exposure to Indigenous cultures, and the challenges and rewards associated with volunteering. Forty-one student veterinarians from OVC completed the survey in January 2020. Most students were motivated to volunteer to make a positive difference in the lives of people and animals and improve their clinical skills. In general, respondents felt adequately prepared for and supported during their experiences but did recommend additional pre-departure instructions on roles and responsibilities. Participants were ambivalent as to whether they had learned about Indigenous culture, with several strongly recommending more cultural sensitivity training. Most students found their experiences rewarding because of the gratitude expressed by clients and the feeling that they had made a difference. We reflect on the potential benefits and challenges of community-based primary care veterinary clinics in light of student responses.

Serum metabolomic analysis of the dose-response effect of dietary choline in overweight male cats fed at maintenance energy requirements.

Choline participates in methyl group metabolism and has been recognized for its roles in lipid metabolism, hepatic health and muscle function in various species. Data regarding the impacts of choline on feline metabolic pathways are scarce. The present study investigated how choline intake affects the metabolomic profile of overweight cats fed at maintenance energy. Overweight (n = 14; body condition score:6-8/9) male adult cats were supplemented with five doses of choline in a 5x5 Latin Square design. Cats received a daily dose of choline on extruded food (3620 mg choline/kg diet) for three weeks at maintenance energy requirements (130 kcal/kgBW0.4). Doses were based on body weight (BW) and the daily recommended allowance (RA) for choline for adult cats (63 mg/kg BW0.67). Treatment groups included: Control (no additional choline, 1.2 x NRC RA, 77 mg/kg BW0.67), 2 x NRC RA (126 mg/kg BW0.67), 4 x NRC RA (252 mg/kg BW0.67), 6 x RA (378 mg/kg BW0.67), and 8 x NRC RA (504 mg/kg BW0.67). Serum was collected after an overnight fast at the end of each treatment period and analyzed for metabolomic parameters through nuclear magnetic resonance (NMR) spectroscopy and direct infusion mass spectrometry (DI-MS). Data were analyzed using GLIMMIX, with group and period as random effects, and dose as the fixed effect. Choline up to 8 x NRC RA was well-tolerated. Choline at 6 and 8 x NRC RA resulted in greater concentrations of amino acids and one-carbon metabolites (P < 0.05) betaine, dimethylglycine and methionine. Choline at 6 x NRC RA also resulted in greater phosphatidylcholine and sphingomyelin concentrations (P < 0.05). Supplemental dietary choline may be beneficial for maintaining hepatic health in overweight cats, as it may increase hepatic fat mobilization and methyl donor status. Choline may also improve lean muscle mass in cats. More research is needed to quantify how choline impacts body composition.

Dose-response relationship between dietary choline and serum lipid profile, energy expenditure, and respiratory quotient in overweight adult cats fed at maintenance energy requirements.

Choline is an essential nutrient linked to hepatic lipid metabolism in many animal species, including cats. The current study investigated the serum lipid profiles, serum liver enzymes, respiratory quotients, and energy expenditures of overweight cats fed maintenance diets, in response to graded doses of supplemental dietary choline. Overweight (body condition score [BCS]: ≥6/9) adult male neutered cats (n = 14) were supplemented with five choline chloride doses for 3-wk periods, in a 5 × 5 Latin square design. Doses were based on individual body weight (BW) and the daily recommended allowance (RA) for choline (63 mg/kg BW0.67) according to the National Research Council. Doses were control (no additional choline: 1.2 × RA, 77 mg/kg BW0.67), 2 × RA (126 mg/kg BW0.67), 4 × RA (252 mg/kg BW0.67), 6 × RA (378 mg/kg BW0.67), and 8 × RA (504 mg/kg BW0.67). Choline was top-dressed over the commercial extruded cat food (3,620 mg choline/kg diet), fed once a day at maintenance energy requirements (130 kcal/kgBW0.4). Body weight and BCS were assessed weekly. Fasted blood samples were taken and indirect calorimetry was performed at the end of each 3-wk period. Serum was analyzed for cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides, non-esterified fatty acids, glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), and alanine aminotransferase. Very low-density lipoprotein cholesterol (VLDL) and low-density lipoprotein cholesterol were calculated. Data were analyzed via SAS using proc GLIMMIX, with group and period as the random effects, and treatment as the fixed effect. Statistical significance was considered at P < 0.05. Body weight and BCS did not change (P > 0.05). Serum cholesterol, HDL-C, triglycerides, and VLDL increased with 6 × RA (P < 0.05). Serum ALP decreased with 8 × RA (P = 0.004). Choline at 4 × and 6 × RA decreased serum BUN (P = 0.006). Fed or fasted respiratory quotient and energy expenditure did not differ among dietary choline doses (P > 0.05). These results suggest that dietary choline supplementation at 6 × RA may increase hepatic fat mobilization through increased lipoprotein transport and beneficially support hepatic health in overweight cats. Future studies that combine these results with existing knowledge of feline weight loss and hepatic lipidosis are warranted.

Choline is an essential nutrient important for lipid metabolism in the liver of many mammals. In the present study, fourteen overweight cats had their commercial extruded cat food top-dressed with different amounts of choline chloride supplement. The amounts of choline were based on the individual body weights and the published recommended allowance (RA) for dietary choline intake in adult cats. The choline treatments were control (no additional choline added, 1.2 × RA), 2 × RA, 4 × RA, 6 × RA, and 8 × RA. The cats were separated into five groups. Each group received the choline treatments once daily for 3 wk per treatment. Choline at 6 × RA increased serum cholesterol, triglycerides, and lipoproteins. There were no significant differences in respiratory quotient or energy expenditure with choline intake. The results of this study suggest that choline at 6 × RA increases the transport of lipids from the liver. This may be beneficial in supporting liver health in overweight cats. Future studies should investigate supplementing choline to cats undergoing weight loss and those at risk of developing fatty liver.

Nrf2-mediated induction of Cyp2a5 partially protects against reductive endoplasmic reticulum stress in mouse hepatocytes.

Cytochrome P450 2a5 (Cyp2a5) is distinct from other P450 enzymes in that it is induced in the endoplasmic reticulum (ER) of mouse hepatocytes in conditions that are injurious to the liver. These conditions cause ER stress eventually resulting in apoptosis if not rectified. We previously showed that mouse hepatic Cyp2a5 is induced during reductive ER stress caused by the intramolecular disulfide form of dithiothreitol, trans-4,5-dihydroxy-1,2-dithiane (DTTox), and that overexpression of Cyp2a5 provides partial protection against apoptosis due to bilirubin (BR), a compound known to cause ER stress. The purpose of this study was to investigate the mechanism of Cyp2a5 gene regulation by DTTox and to determine if Cyp2a5 plays a cytoprotective role during reductive ER stress. Exposure to DTTox (10 mM) and another reductive ER stressor, 2-mercaptoethanol (1 mM), for 48 h markedly increased Cyp2a5 protein levels in primary mouse hepatocytes. In addition, DTTox transactivated Cyp2a5 via a mechanism involving the transcription factor nuclear factor-(erythroid-derived 2)-like 2 (Nrf2). Expression of the BR-conjugating enzyme, UDP glucuronosyl transferase 1A1 (UGT1A1) was also increased after DTTox treatment, however, this was reduced by Cyp2a5 overexpression. Hemin, a porphyrin inducer of Cyp2a5, induced mRNA splicing of X-box binding protein 1 (XBP-1), a transcription factor involved in the ER stress response, however, this was also reduced by Cyp2a5 overexpression. Finally, overexpression of Cyp2a5 partially blocked DTTox-mediated caspase-3 cleavage in Hepa 1-6 cells suggesting a cytoprotective role during ER stress. These findings demonstrate that Nrf2-mediated induction of Cyp2a5 in a reducing ER environment provides partial protection against ER stress-induced apoptosis by decreasing XBP-1 mRNA splicing and caspase-3 cleavage.

Regulation of Cytochrome P450 2a5 by Artemisia capillaris and 6,7-Dimethylesculetin in Mouse Hepatocytes.

Jaundice is a potentially fatal condition resulting from elevated serum bilirubin levels. For centuries, herbal remedies containing Artemisia capillaris Thunb. including the compound 6,7-dimethylesculetin (DE) have been used in Asia to prevent and treat jaundice in neonates. DE activates an important regulator of bilirubin metabolism, the constitutive androstane receptor (CAR), and increases bilirubin clearance. In addition, murine cytochrome P450 2a5 (Cyp2a5) is known to be involved in the oxidative metabolism of bilirubin. Moreover, treatment of mice with phenobarbital, a known inducer of both CAR and Cyp2a5, increases expression of Cyp2a5 suggesting a potential relationship between CAR and Cyp2a5 expression. The aim of this study is to investigate the influence of Artemisia capillaris and DE on the expression and regulatory control of Cyp2a5 and the potential involvement of CAR. Treatment of mouse hepatocytes in primary culture with DE (50 µM) significant increased Cyp2a5 mRNA and protein levels. In mice, Artemisia capillaris and DE treatment also increased levels of hepatic Cyp2a5 protein. Luciferase reporter assays showed that CAR increases Cyp2a5 gene transcription through a CAR response element in the Cyp2a5 gene promoter. Moreover, DE caused nuclear translocation of CAR in primary mouse hepatocytes and increased Cyp2a5 transcription in the presence of CAR. These results identify a potential CAR-mediated mechanism by which DE regulates Cyp2a5 gene expression and suggests that DE may enhance bilirubin clearance by increasing Cyp2a5 levels. Understanding this process could provide an opportunity for the development of novel therapies for neonatal and other forms of jaundice.

Serum Lipid, Amino Acid and Acylcarnitine Profiles of Obese Cats Supplemented with Dietary Choline and Fed to Maintenance Energy Requirements.

Obesity is a health concern for domestic cats. Obesity and severe energy restriction predispose cats to feline hepatic lipidosis. As choline is linked to lipid metabolism, we hypothesized that dietary choline supplementation would assist in reducing hepatic fat through increased lipoprotein transport and fatty acid oxidation. Twelve obese cats (body condition score [BCS] ≥ 8/9) were split into two groups. Cats were fed a control (n = 6; 4587 mg choline/kg dry matter [DM]) or a high choline diet (n = 6; 18,957 mg choline/kg DM) for 5 weeks, for adult maintenance. On days 0 and 35, fasted blood was collected, and the body composition was assessed. Serum lipoprotein and biochemistry profiles, plasma amino acids and plasma acylcarnitines were analyzed. The body weight, BCS and body composition were unaffected (p > 0.05). Choline increased the serum cholesterol, triacylglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol and plasma methionine (p < 0.05) and decreased the serum blood urea nitrogen and alkaline phosphatase (p < 0.05). Choline also reduced the plasma acylcarnitine to free carnitine ratio (p = 0.006). Choline may assist in eliminating hepatic fat through increased fat mobilization and enhanced methionine recycling.

Detection of Mycobacterium avium Subspecies paratuberculosis (MAP) Microorganisms Using Antigenic MAP Cell Envelope Proteins.

Cell envelope proteins from Mycobacterium avium subspecies paratuberculosis (MAP) that are antigenically distinct from closely related mycobacterial species are potentially useful for Johne's Disease (JD) diagnosis. We evaluated the potential of ELISAs, based on six antigenically distinct recombinant MAP cell envelope proteins (SdhA, FadE25_2, FadE3_2, Mkl, DesA2, and hypothetical protein MAP1233) as well as an extract of MAP total cell envelope proteins, to detect antibodies against MAP in the sera of infected cattle. The sensitivity (Se) and specificity (Sp) of an ELISA based on MAP total cell envelope proteins, when analyzing 153 bovine serum samples, was 75 and 96%, respectively. Analysis of the same samples, using a commercial serum ELISA resulted in a Se of 56% and Sp of 99%. Results of ELISA analysis using plates coated with recombinant cell envelope proteins ranged from a highest Se of 94% and a lowest Sp of 79% for Sdh A to a lowest Se of 67% and a highest Sp of 95% for hypothetical protein MAP1233. Using polyclonal antibodies to MAP total cell envelope proteins, immunohistochemical analysis of intestinal and lymph node tissues from JD-positive cattle detected MAP organisms whereas antibodies to recombinant proteins did not. Finally, polyclonal antibodies to MAP total cell envelope protein and to recombinant SdhA, FadE25_2, and DesA2 proteins immunomagnetically separated MAP microorganisms spiked in PBS. These results suggest that antigenically distinct MAP cell envelope proteins and antibodies to these proteins may have potential to detect MAP infection in dairy cattle.

Proteomic 2D-DIGE Analysis of Milk Whey from Dairy Cows with Staphylococcus aureus Mastitis Reveals Overexpression of Host Defense Proteins.

Bovine mastitis remains a primary focus of dairy cattle disease research due to its considerable negative economic impact on the dairy industry. Subclinical mastitis (SCM), commonly caused by Staphylococcus aureus, lacks overt clinical signs and the diagnosis is based on bacteriological culture and somatic cell counts of milk, both of which have limitations. The main objective of this study was to identify, characterize and quantify the differential abundance of milk whey proteins from cows with S. aureus SCM compared to whey from healthy cows. Using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with liquid chromatography and tandem mass spectrometry, 28 high-abundant proteins were detected in whey from mastitic milk, 9 of which had host defense functions. These included acute phase proteins involved in innate immunity and antimicrobial functions (e.g., serotransferrin, complement C3, fibrinogen gamma-B chain and cathepsin B), and proteins associated with the immune response to pathogens (e.g., polymeric immunoglobulin receptor-like protein, MHC class I antigen and beta-2-microglobulin). These results provide a unique 2D map of the modulated milk proteome during S. aureus mastitis. The broader importance is that the identified proteins, particularly those with host-defense biological functions, represent potential candidate biomarkers of subclinical mastitis in dairy cows.

The Essential Oils and Eucalyptol From Artemisia vulgaris L. Prevent Acetaminophen-Induced Liver Injury by Activating Nrf2-Keap1 and Enhancing APAP Clearance Through Non-Toxic Metabolic Pathway.

Artemisia has long been used in traditional medicine and as a food source for different functions in eastern Asia. Artemisia vulgaris L. (AV) is a species of the genus Artemisia. Essential oils (EOs) were extracted from AV by subcritical butane extraction. EO contents were detected by electronic nose and headspace solid-phase microextraction coupled with gas chromatography (HS-SPME-GC-MS). To investigate the hepatoprotective effects, mice subjected to liver injury were treated intragastrically with EOs or eucalyptol for 3 days. Acetaminophen (APAP) alone caused severe liver injury characterized by significantly increased serum AST and ALT levels, ROS and hepatic malondialdehyde (MDA), as well as liver superoxide dismutase (SOD) and catalase (CAT) depletions. EOs significantly attenuated APAP-induced liver damages. Further study confirmed that eucalyptol is an inhibitor of Keap1, the affinity K D of eucalyptol and Keap1 was 1.42 × 10-5, which increased the Nrf2 translocation from the cytoplasm into the mitochondria. The activated Nrf2 increased the mRNA expression of uridine diphosphate glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also inhibiting CYP2E1 activities. Thus, the activated Nrf2 suppressed toxic intermediate formation, promoting APAP hepatic non-toxicity, whereby APAP was metabolized into APAP-gluc and APAP-sulf. Collectively, APAP non-toxic metabolism was accelerated by eucalyptol in protecting the liver against APAP-induced injury, indicating eucalyptol or EOs from AV potentials as a natural source of hepatoprotective agent.

An update on Mycobacterium avium subspecies paratuberculosis antigens and their role in the diagnosis of Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Johne's disease (JD) or paratuberculosis. Diagnosis of MAP infection by measuring host cell-mediated and humoral immune responses has been a major focus in MAP research. For this purpose, several MAP antigens such as secreted protein, cell envelope protein, cell-mediated immune and lipoprotein antigens have been identified and tested to measure their diagnostic utility with varying degree of success. Identifying the optimal antigen or antigen combinations for diagnosis of infected animals is hindered by the complex nature of the disease, prolonged subclinical infection, the differential expression of antigens and scarcity of well characterized MAP-specific epitopes making selection of a single MAP antigen very difficult. Thus, multiplexing of antigens with larger scale and longitudinal studies may lead to development of cost-effective next generation serodiagnostics. This mini review focuses on the role of different MAP antigens in the diagnosis of JD.

Mass spectrometry data from identification of host-defense related proteins using label-free quantitative proteomic analysis of milk whey from cows with Staphylococcus aureus subclinical mastitis.

This dataset is associated with our research article 'Identification of host-defence related proteins using label-free quantitative proteomic analysis of milk whey from cows with Staphylococcus aureus Subclinical mastitis' published in International Journal of Molecular Sciences. Milk samples were collected from cows suffering from S. aureus-associated subclinical mastitis and the proteins abundance were identified in comparison with samples collected from the control cows using liquid chromatography-mass spectrometry (LC-MS)-based label free proteomics analysis. Following the MS measurements, the raw spectra were processed using MaxQuant-Andromeda software and the protein identification was carried out through a search against Uniprot FASTA files of the Bos taurus reference proteome. Perseus software analysis was applied for computation of protein abundance. The raw file presented in this DiB was deposition at PRIDE repository in the PRIDE repository with the dataset identifier PXD007516.

Echinacoside Increases Sperm Quantity in Rats by Targeting the Hypothalamic Androgen Receptor.

Male infertility is a major health issue with an estimated prevalence of 4.2% of male infertility worldwide. Our early work demonstrated that Cistanche extracts protect against sperm damage in mice and that echinacoside (ECH) is one of the major active components. Here we report an essential role for ECH, a natural product that reverses or protects against oligoasthenospermia in rats. ECH was assayed by HPLC, the quantity and quality of sperm was evaluated and hormone levels were determined by radioimmunosorbent assay. ECH reduced levels of androgen receptor (AR) and key steroidogenic-related genes as determined by Western blot and qPCR analysis. The interaction between ECH and AR were evaluated by indirect ELISA and molecular docking. The results show that ECH combined with hypothalamic AR in the pocket of Met-894 and Val-713 to inhibit transfer of AR from the cytoplasm to nuclei in the hypothalamus. While negative feedback of sex hormone regulation was inhibited, positive feedback was stimulated to increase the secretion of luteinizing hormone and testosterone subsequently enhancing the quantity of sperm. Taken together, these data demonstrate that ECH blocks AR activity in the hypothalamus to increase the quantity of sperm and protect against oligoasthenospermia in rats.

Identification of antigenic proteins from Mycobacterium avium subspecies paratuberculosis cell envelope by comparative proteomic analysis.

Johne's disease (JD) is a contagious, chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The aim of this study was to identify antigenic proteins from the MAP cell envelope (i.e. cell wall and cytoplasmic membranes) by comparing MAP, M. avium subsp. hominissuis (MAH) and M. smegmatis (MS) cell envelope protein profiles using a proteomic approach. Composite two-dimensional (2D) difference gel electrophoresis images revealed 13 spots present only in the image of the MAP cell envelope proteins. Using serum from MAP-infected cattle, immunoblot analysis of 2D gels revealed that proteins in the 13 spots were antigenic. These proteins were identified by liquid chromatography tandem mass spectrometry as products of the following genes: sdhA, fadE25_2, mkl, citA, gapdh, fadE3_2, moxR1, mmp, purC, mdh, atpG, fbpB and desA2 as well as two proteins without gene names identified as transcriptional regulator (MAP0035) protein and hypothetical protein (MAP1233). Protein functions ranged from energy generation, cell wall biosynthesis, protein maturation, bacterial replication and invasion of epithelial cells, functions considered essential to MAP virulence and intracellular survival. Five MAP cell envelope proteins, i.e. SdhA, FadE25_2, FadE3_2, MAP0035 and DesA2 were recombinantly expressed, three of which, i.e. SdhA, FadE25_2 and DesA2, were of sufficient purity and yield to generate polyclonal antibodies. Immunoblot analysis revealed antibodies reacted specifically to the respective MAP cell envelope proteins with minimal cross-reactivity with MAH and MS cell envelope proteins. Identification and characterization of MAP-specific proteins and antibodies to those proteins may be useful in developing new diagnostic tests for JD diagnosis.

Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis.

Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

Assessment of platelet function in healthy cats in response to commonly prescribed antiplatelet drugs using three point-of-care platelet function tests.

Objectives The objective was to determine if decreased platelet function could be detected after treatment with aspirin and/or clopidogrel in healthy cats using three point-of-care platelet function tests that evaluate platelet function by different methods: Multiplate (by impedance), Platelet Function Analyzer 100 (by mechanical aperture closure) and Plateletworks (by platelet counting). Methods Thirty-six healthy cats were randomly assigned to receive one of three oral treatments over an 8 day period: (1) aspirin 5 mg q72h; (2) aspirin 20.25 mg q72h; or (3) clopidogrel 18.75 mg q24h. Cats treated with 5 and 20.25 mg aspirin also received clopidogrel on days 4-8. Platelet aggregation in response to adenosine diphosphate and collagen ± arachidonic acid was assessed on days 1 (baseline), 4 and 8. Aspirin and clopidogrel metabolites were measured by high-performance liquid chromatography. Platelet function in response to treatment was analyzed by ANCOVA, linear regression and Spearman correlation. Results The only solitary aspirin effect was detected using Plateletworks with collagen in cats treated with 20.25 mg. The only effect detected by Multiplate was using arachidonic acid in cats treated with both aspirin 20.25 mg and clopidogrel. All clopidogrel treatment effects were detected by Platelet Function Analyzer 100, Plateletworks (adenosine diphosphate) and Plateletworks (collagen). Drug metabolites were present in all cats, but concentrations were minimally correlated to platelet function test results. Conclusions and relevance Platelet Function Analyzer 100 and Plateletworks using adenosine diphosphate ± collagen agonists may be used to detect decreased platelet function in response to clopidogrel treatment. Either aspirin is not as effective an antiplatelet drug as clopidogrel, or the tests used were not optimal to measure aspirin effect. Cats with heart disease are commonly prescribed antiplatelet drugs to decrease the risk of aortic thromboembolism. Platelet Function Analyzer 100 and Plateletworks may be useful for confirming clopidogrel treatment in these cats.

Cistanche tubulosa ethanol extract mediates rat sex hormone levels by induction of testicular steroidgenic enzymes.

CONTEXT: Plants of the genus Cistanche Hoffmg. et Link (Orobanchaceae) are usually used as ethno-medicine in Eastern Asia. Pharmacology studies have shown that Cistanche possesses an androgen-like effect; however, the exact mechanism is unclear. OBJECTIVE: The present study determines the effect of ethanol extract of Cistanche tubulosa (Schenk) R. Wight stem (CTE) on hormone levels and testicular steroidogenic enzymes in rats. MATERIALS AND METHODS: Phenylethanoid glycoside content of CTE was detected by UV spectrophotometry. Rats were fed with different doses of CTE (0.2, 0.4, and 0.8 g/kg) by intragastric administration for 20 d. Sperm parameters were measured by staining and counting method. The level of progesterone and testosterone in serum was quantified by radioimmunoassay. The expression levels of cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase/17, 20-lyase (CYP17A1), and a liver metabolic enzyme (CYP3A4) in the microsome were assessed by immunohistochemical staining or/and western blot analysis. RESULTS: The study illustrates that the administration of CTE (0.4 and 0.8 g/kg) increased sperm count (2.3- and 2.7-folds) and sperm motility (1.3- and 1.4-folds) and decreased the abnormal sperm (0.76- and 0.6-folds). The serum level of progesterone and testosterone in rats was also increased by CTE administration (p < 0.05). Results of immunohistochemistry and western blot analysis confirmed that the expression of CYP11A1, CYP17A1, and CYP3A4 was enhanced by CTE (p < 0.05). It was also found that high-dose of CTE can cause mild hepatic edema. CONCLUSION: Our results suggest that the increase in sex hormone levels could be mediated by the induction of testicular steroidogenic enzymes.

Assessment of platelet function in healthy sedated cats using three whole blood platelet function tests.

The objectives of this study were to establish feline references intervals for 3 commercial whole blood platelet function test analyzer systems: Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), Platelet Function Analyzer-100 (PF: Siemens Canada, Mississauga, Ontario, Canada), and Plateletworks Combo-25 kit (PW; Helena Laboratories, Beaumont, TX). Venipuncture was performed on 55 healthy sedated cats, and platelet aggregation in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA; MP only) was assessed using citrated blood. For the MP analyzer, median (95% confidence intervals [CIs]) area under curve (Units) for ADP, COL, and AA agonists were 87 (11-176), 81 (32-129), and 91 (59-129), respectively. For the PF analyzer, median (95% CIs) closure time, using COL-ADP cartridges, was 69 (46-89) sec. For the PW assay, median (95% CIs) percent aggregations for ADP and COL agonists were 71 (18-92) and 49 (9-96), respectively, using impedance hematology analyzer platelet counts, and 94 (25-98) and 68 (14-119), respectively, using flow cytometry hematology analyzer platelet counts. There were low correlations between the PF analyzer (COL-ADP cartridge) and MP analyzer (COL agonist; ρ = 0.11), and between the PF analyzer (COL-ADP cartridge) and PW assay (COL agonist using impedance platelet counts; ρ = 0.14). The PW assay percent aggregations using impedance and flow cytometric platelet counts were correlated for both ADP (ρ = 0.64) and COL (ρ = 0.64) agonists. Platelet function testing using these tests are feasible in cats, but 95% CIs are wide, so single results may be difficult to interpret. Platelet counting by impedance or flow cytometry may be used for the PW assay but are not interchangeable.

The day/night proteome in the murine heart.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

Cytochrome P450 2A5 and bilirubin: mechanisms of gene regulation and cytoprotection.

Bilirubin (BR) has recently been identified as the first endogenous substrate for cytochrome P450 2A5 (CYP2A5) and it has been suggested that CYP2A5 plays a major role in BR clearance as an alternative mechanism to BR conjugation by uridine-diphosphate glucuronyltransferase 1A1. This study investigated the mechanisms of Cyp2a5 gene regulation by BR and the cytoprotective role of CYP2A5 in BR hepatotoxicity. BR induced CYP2A5 expression at the mRNA and protein levels in a dose-dependent manner in primary mouse hepatocytes. BR treatment also caused nuclear translocation of Nuclear factor-E2 p45-related factor 2 (Nrf2) in hepatocytes. In reporter assays, BR treatment of primary hepatocytes transfected with a Cyp2a5 promoter-luciferase reporter construct resulted in a 2-fold induction of Cyp2a5 reporter activity. Furthermore, cotransfection of the hepatocytes with a Nrf2 expression vector without BR treatment resulted in an increase in Cyp2a5 reporter activity of approximately 2-fold and BR treatment of Nrf2 cotransfectants further increased reporter activity by 4-fold. In addition, site-directed mutation of the ARE in the reporter construct completely abolished both the BR- and Nrf2-mediated increases in reporter activity. The cytoprotective role of CYP2A5 against BR-mediated apoptosis was also examined in Hepa 1-6 cells that lack endogenous CYP2A5. Transient overexpression of CYP2A5 partially blocked BR-induced caspase-3 cleavage in Hepa 1-6 cells. Furthermore, in vitro degradation of BR was increased by microsomes from Hepa 1-6 cells overexpressing CYP2A5 compared to control cells transfected with an empty vector. Collectively, these results suggest that Nrf2-mediated CYP2A5 transactivation in response to BR may provide an additional mechanism for adaptive cytoprotection against BR hepatotoxicity.