Targeting AP-1 transcription factors by CRISPR in the prostate.

Prostate cancer is the second most diagnosed cancer in men. It is a slow progressing cancer, but when the disease reaches an advanced stage, treatment options are limited. Sequencing analyses of cancer samples have identified genes that can potentially drive disease progression. We implemented the CRISPR/Cas9 technology to simultaneously manipulate multiple genes in the murine prostate and thus to functionally test putative cancer driver genes in vivo. The activating protein-1 (AP-1) transcription factor is associated with many different cancer types, with the proto-oncogenes JUN and FOS being the two most intensely studied subunits. We analyzed expression of FOS and JUNB in human prostate cancer datasets and observed decreased expression in advanced stages. By applying CRISPR/Cas9 technology, the role of these two transcription factors in prostate cancer progression was functionally tested. Our data revealed that loss of either JunB or Fos in the context of Pten loss drives prostate cancer progression to invasive disease. Furthermore, loss of Fos increases Jun expression, and CRISPR inactivation of Jun in this context decreases cell proliferation. Overall, these in vivo studies reveal that JunB and Fos exhibit a tumor suppressor function by repressing invasive disease, whereas Jun is oncogenic and increases cell proliferation. This demonstrates that AP-1 factors are implicated in prostate cancer progression at different stages and display a dual function as tumor suppressor and as an oncogene in cancer progression.

Resveratrol protects quail hepatocytes against heat stress: modulation of the Nrf2 transcription factor and heat shock proteins.

In the present study, the effects of dietary resveratrol on the induction of heat shock proteins, transcription factors and antioxidative enzyme system in liver of quails under heat stress were investigated. A total of 180 (55-day-old) female Japanese quails (Coturnix coturnix japonica) were reared either at 22 °C for 24 h/day (thermoneutral, TN) or 34 °C for 8 h/day (heat stress, HS; 09:00-17:00 hours) for 12 weeks. Birds in both environments were randomly fed one of three diets: basal diet and basal diet added with either 200 or 400 mg of resveratrol per kg of diet. The results showed that exposure to high ambient temperature induced decreases in feed intake, egg production, and hepatic superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities but increases in hepatic malondialdehyde (MDA) concentrations (p < 0.001). Liver Hsp70, Hsp90 and NF-κB expression was greater while Nrf2 expression was lower for quails reared under the heat stress than for those reared under the TN environment (p < 0.0001). There were linear increases in feed intake, egg production, hepatic SOD, CAT, and GSH-Px activities as well as Nrf2 expression, but linear decreases in hepatic MDA concentrations and Hsp70, Hsp90, and NF-κB expressions with increasing supplemental resveratrol level (p < 0.0001). Two-way treatment interactions revealed that the degree of restorations in all response variables was more notable under the high ambient temperature than that of the TN environment as dietary resveratrol concentration was increased. The results of the present study suggest that supplemental resveratrol reduces oxidative stress in heat-stressed quails through modulating the hepatic heat shock proteins and nuclear transcription factors.

Risk factors of colic in horses in Austria.

A hospital-based case study was conducted at the University of Veterinary Medicine Vienna for a 1-year period. The purpose of this study was to determine possible alterable and non-alterable risk factors of equine colic in Austria. The investigated parameters were obtained from hospital medical records (individual factors, duty of the horse, deworming, change in diet and water intake), from questionnaires (feed intake, watering types, housing and pasture practices) and from http://www.orf.at (weather-related factors). Moreover, 221 collected feed samples were investigated through hygienic quality sensory evaluation and mould, yeast and bacteria presumptive samples were confirmed by microbiological investigation. Variables that were observed from the hospital medical records and found to be significantly associated with colic in a univariate analysis were included in multivariate analysis and the only remaining risk factor was decreased water consumption (p < 0.001, OR = 5.025). Consequently, a total of 2743 horses and 366 cases of colic were observed during the study period. The risk factors for colic in Austria were identified for the first time in this study and these increased risk factors were decreased water consumption, high amount of concentrate intake (p = 0.037), low hygienic quality of hay (p = 0.027) and high temperature on the arrival date (p = 0.003). Results suggest that the occurrence of colic may not stop, but may decrease with better feed management practices in Austria.

Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa.

UNLABELLED: The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca(2+)) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris-citrate-fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 microg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF-EY). In egg yolks and the TCF-EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca(2+) by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF-EY. One half of each TCF- and TCF-EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 degrees C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. RESULTS: Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca(2+) concentrations in egg yolks were 524.8+/-131.4 ng/g, 13.9+/-2.03 mg/g and 1.27+/-0.17 mg/g, respectively. In the TCF-EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1mg/g. In TCF-semen, at D1, motility and viability were significantly higher than in TCF-EY-samples (p<0.05), however at D4, no significant differences were detectable. Further, in TCF-semen, percentages of spermatozoa with intact membranes decreased significantly (p<0.05) and capacitated spermatozoa increased (p<0.05), which was not seen in TCF-EY-samples. In all samples, low percentages of AR were detected and after 4 days, the highest value of AR in TCF-EY-samples was 5.3% on average, as detected by flow cytometry. We therefore conclude that progesterone from egg yolk in routine extenders does not substantially influence semen longevity or AR of canine semen during cold-storage for 4 days. In contrary, egg yolk seems to prevent a significant increase in capacitated spermatozoa.

Evacuation of sand from the equine intestine with mineral oil, with and without psyllium.

The aim of this study was to determine the evacuation of sand from the equine intestine after a double treatment with psyllium and mineral oil or mineral oil only. A crossover study was conducted. Twelve healthy horses were fed 1 kg sand once a day for 5 days. Subsequently, these horses were divided into two groups: A and B. From day 6-10, both groups were treated with 2 l of mineral oil once a day and group B received an additional 0.5 kg of psyllium twice a day. The trial was repeated after 2 weeks with treatment crossover of groups A and B. The horses were housed sand free and 1.8 kg hay/100 kg body weight was offered to meet the maintenance energy requirement. Prior to the sand administration, faeces were collected from each horse for 3 days and the crude ash was determined to establish a baseline output of ash. There was no difference between the baseline crude ash output of the first and second treatment. From day 6-10, faeces were collected daily and the fresh weight and the dry matter and the crude ash contents were determined. For administration, sand or psyllium was mixed with 1 l of Irish mash (concentrate mixed with water), respectively, and mineral oil was administered via a nasogastric tube. All horses showed higher crude ash excretion when treated with psyllium and mineral oil compared with the mineral oil administration only. On the second, third and fourth day of the treatment, the difference was significant. Faeces crude ash weight corrected for baseline crude ash output while treated with psyllium plus oil and oil solely, reached a mean of 51.0 (SD 20.5) and 26.1 (SD 17.7) % of the administered sand mass, respectively. The results of this trial show that the ash output differed highly between the horses. Nevertheless, all horses showed a higher total ash output within the 5 days treatment period when the psyllium semen and mineral oil were used for the treatment than when treated with mineral oil solely.

Dioxin residues in the edible tissue of finishing pigs after dioxin feeding.

The aim of this study was to determine the contamination of finishing pigs with polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs) after feeding either uncontaminated feed or feed contaminated with 0.75, 2 or 4 ng/kg toxic equivalents (TEQ; calculated by multiplying individual congener concentrations by congener-specific toxicity equivalency factors). The feed was mixed with pure substances of PCDD/Fs to get the intended contamination. Five groups of six piglets each were fed contaminated feed, one group of five piglets served as control. One group was fed contaminated feed (4 ng TEQ/kg) only for the rearing period (6 weeks), and another group for the first 8 weeks of the fattening period (4 ng TEQ/kg feed). The other groups received the contaminated feed during the 12-weeks fattening period. After slaughtering, the edible parts of the belly, loin and fore-end were collected and homogenized. The samples of group 2 and 4a were investigated uncooked as well as roasted. Fattening yield and feed conversion (kg feed/kg weight gain) of the animals of all groups were in the normal range (final weight 109.7 kg; feed conversion 2.55-2.69 kg). The PCDD/F-content in 1 kg fat of the belly, loin and fore-end in relation to the intake was between 0.016 (4 ng TEQ/kg feed for a 6-weeks rearing period) and 1.39% (fore-end; 2 ng TEQ/kg feed for 12-weeks fattening period). There was a decrease in dioxin residues after a 12-weeks period but not after a 4-weeks period of feeding an uncontaminated feed. When feed contaminated with 0.75, 2 and 4 ng TEQ/kg was given for a 12-weeks fattening period, the residue concentrations of PCDD/F-TEQ in 1 kg belly was 0.455, 1.07 and 1.55 ng, in 1 kg fore-end 0.04 ng, 0.32 ng and 0.34 ng and in 1 kg loin 0.015 ng, 0.07 ng and 0.30 ng respectively. Roasting had no influence on the dioxin-residues. The residues per g belly fat exceed the maximum limits for dioxin in food of 0.6 pg WHO-PCDD/F-TEQ/g fat (EC Recommendations 2002/201/EC), when feed containing 0.75 ng PCDD/F-TEQ/kg is given for 12 weeks. When feed containing 0.4 ng TEQ (maximum content; EC recommendations 2002/201/EC) is given for 12 weeks, approximately 0.55 pg TEQ/g fat can be expected in the food. This value is within the action level of 0.6 pg/g fat of porks. In conclusion, the results of the study allow prediction of dioxin residues in the edible tissue of pork, if the feed contamination is known and the amount of feed intake can be estimated.

Feeding laying hens seal blubber oil: effects on egg yolk incorporation, stereospecific distribution of omega-3 fatty acids, and sensory aspects.

Seventy-two 26-wk-old Single Comb White Leghorn laying hens were randomly assigned to 36 cages (2 per cage) in a 3-orthogonal 4 x 4 latin square, with the fourth row suppressed, to assess the effect of feeding refined seal blubber oil (SBO, containing 22.2% omega-3 fatty acids) on the fatty acid composition and position in the egg yolk lipids. The experiment was conducted over a period of 9 wk. Eggs were collected and numbered, and the weights were recorded for each week and cage. Eggs collected at wk 5 and 9 were used for total lipid, lipid class, fatty acid, and positional analyses. Sensory evaluation was carried out on eggs collected at wk 6 and 7. Feeding SBO at 1.25% led to an increase (P < 0.0001) in the long-chain omega-3 polyunsaturated fatty acids (LCn3PUFA) and a concomitant decrease (P < 0.0001) in arachidonic acid (ARA) in the egg yolk lipids. Yet this amount of SBO in the diet had no effect (P > 0.1) on the sensory attributes of the egg and on production parameters such as egg weight, number of eggs laid, and feed intake (P > 0.05). When feeding SBO in amounts higher than 1.25% proportionately, a plateau effect of the LCn3PUFA content of the eggs was observed. This appears to be because the PUFA content in the sn-2 position of the phospholipids cannot exceed a certain amount. When this amount is reached, the LCn3PUFA will be increasingly stored in triglycerides. The results presented here clearly indicate how eggs can be produced with optimized composition of LCn3PUFA without affecting (P > 0.1) the sensory properties of the eggs. The procedures elaborated herein provide directly applicable consequences for the food industry.

In vitro quality assessment of two tropical shrub legumes in relation to their extractable tannins contents.

The in vitro dry matter digestibility (IVDMD) of the two leguminous tree fodders Gliricidia sepium and Calliandra calothyrsus which differ in their tannin content was examined by the rumen simulation technique. Extractable condensed tannin (CT) concentrations ranged from 0.57% in G. sepium to 5.05% in C. calothyrsus using the butanol-HCl extraction. On the basis of their respective CT contents, G. sepium was classified as containing traces of CT, whereas C. calothyrsus had medium amounts of CT. Polyethylene glycol (PEG) was added at different concentrations (0, 1.5, 5 and 10 g/100 g plant material substrate) to assess the effect of tannins on IVDMD. The IVDMD was higher for G. sepium (range: 60-65%) than for C. calothyrsus (39.5-53.5%). In vitro gas production and IVDMD increased with increased PEG concentrations especially for C. calothyrsus. A non-significant response to increasing PEG concentrations for IVDMD of G. sepium confirms PEG binding with the tannins. On the basis of these results, it is assumed that G. sepium has a higher nutritive value than C. calothyrsus. Good relationship between PEG binding and the improvement of IVDMD confirms the usefulness of this technique for improving the nutritive value of tanniniferous tropical browses.

Dioxin residues in the edible tissue of broiler chicken.

The aim of this study was to determine the contamination of broiler chicken with polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs) after feeding either uncontaminated feed or feed contaminated with 1, 2 or 4 ng/kg toxic equivalents (TEQ). The feed was mixed with pure substances of PCDD/Fs to get the intended contamination. Ten groups of seven 1-day-old chickens each were housed in special cages for broiler fattening. The fattening period lasted for 6 weeks. The contaminated feed was given for either 2, 4, or 6 weeks, one group received uncontaminated feed (control group). After slaughtering the edible parts of the chickens breast and leg including the skin were homogenized. Body weight gain and feed conversion (kg feed/kg body weight gain) were in the normal range (final weight 1.98+/-0.07 kg; feed conversion 1.74+/-0.03). One kilogram edible tissue contained an average of 21.2+/-4.1% of the total TEQ-intake in all groups. The PCDD/Fs residues in the edible tissues significantly correlated with the amount of PCDD/Fs-intake (r=0.99; Pearson correlation). There was no decrease in dioxin residues (% of total PCDD/Fs intake) after a 2 or 4 weeks withdrawal period. The results of this trial predict that a threshold value of 2 pg/g chicken fat can be met if the PCDD/Fs content in chicken feed is not higher than 0.4 ng/kg.

Influence of environmental humidity and dietary protein on pyramidal growth of carapaces in African spurred tortoises (Geochelone sulcata).

The carapaces of captive-raised tortoises (terrestrial chelonians of the zoological family Testudinidae, often develop pyramidal-shaped osseous growth centrally within the horny plates. With very few exceptions (e.g. Geochelone elegans, Psammobates sp.), this conical growth pattern is considered to be pathologic. This very common defect is believed to be an important indicator of the quality of captive tortoise management. This study was designed to examine the effect of dietary protein level and environmental humidity on the degree of pyramidal growth in the carapaces. Fifty recently hatched African spurred tortoises (G. sulcata) were raised for 5 months under artificial conditions of varying environmental humidity and dietary protein content (14% vs. 19% vs. 30% crude protein in dry matter). Humps of the carapaces that developed and blood values of calcium, phosphorus and haematocrit were measured and compared among groups. Dry environmental conditions (24.3-57.8% and 30.6-74.8% relative humidity) produced taller humps than humid conditions (45-99% relative humidity). Hump formation differed significantly (p < or = 0.001) between these three groups kept under different humidity conditions. Variable dietary protein had a minor, positive impact on this pathological formation of humps (pyramidal growth syndrome, PGS). Analysis of blood (calcium, phosphorus and haematocrit) offered no further explanation as to the development of the humps.