Differences in the stability of commonly used reference genes have been discovered between species and among different tissue types. These inconsistencies underscore the importance of validation and selection of an appropriate reference for normalization of gene expression to an endogenous control in RT-qPCR experiments. Three different reference transcripts (GAPDH, RPS19 and 18S rRNA) of 3 different functional categories were selected for evaluation during pre-implantation embryonic development and in spermatogonial cells in the domestic cat. Amplification efficiency was done by standard curve analysis with 5-fold template dilutions. For pre-implantation analysis, transcripts were assayed at 4 developmental stages: 2- to 6-cell (2-6C), 8- to 16-cell (8-16C), morula (M) and blastocyst (BL). Embryos were from pools of 4 to 10 in vitro matured and fertilized domestic cat embryos. For spermatogonia, isolates of 20000 to 30000 cells were assayed from single testis isolates following a collagenase and trypsin with DNase digestion and Percoll gradient separation. Total mRNA was isolated using the Cells-to-cDNA II Kit, with a minimum of 2 biological replicates for each sample type. The RNA quantitation was done by RiboGreen analysis and 47 ng of RNA was used for cDNA synthesis. Transcript abundance was detected in 2 technical replicates per sample by SYBR Green chemistry and data were analysed with NormFinder and one-way ANOVA. Each transcript showed varying levels of expression throughout development and among embryonic stages and in spermatogonial cells. The GADPH and 18S rRNA transcripts were expressed at higher levels in BL than in 2-6C and 8-16C, respectively. Contrarily, transcript levels of RSP19 were lower in BL compared with levels at 2-6C. Even though differences were observed among embryonic stages, analysis with NormFinder indicated that the most stable reference gene throughout early pre-implantation development was RPS19. When each stage was analysed separately, 18S rRNA was the most stable at 2-6C, whereas RPS19 was found to be the most stable at 8-16C, M and BL stages. In spermatogonial samples, GAPDH was the most stably expressed gene. Our results support the selection of an appropriate reference gene based on the needs of the experimental design. We conclude from these findings that when gene expression throughout the duration of early embryonic development is examined, RPS19 is the preferred selection. If gene expression is analysed within discrete time points of development only, it is appropriate to select 18S rRNA at the 2-6C stage and RPS19 for 8-16C, M and BL stage embryos. For spermatogonial samples, if comparing only different biological replicates, GAPDH should be selected for normalization of expression; however if the purpose of the experiment is to assay genes that are also expressed in BL, the most stably expressed reference gene is RPS19.

The black-footed cat (Felis nigripes), a diminutive spotted cat whose native habitat is arid grasslands in South Africa, Namibia and Botswana, is classified as vulnerable by the International Union for Conservation of Nature and is listed as CITES Appendix I. They are perhaps the rarest of the African cats and their status is threatened by habitat deterioration and poisoning from ingestion of baited carcasses intended for other species of cats. Here, we examined (1) ovarian response of black-footed cat females to exogenous gonadotropin stimulation,(2) in vitro production of embryos by IVF with cooled vs cryopreserved sperm and (3) in vivo developmental ability of in vitro-derived embryos. Six females, 1.5 to 2.5 years of age at first treatment, were administered a total of 3.0 to 3.6 IU of porcine FSH (IM; Sioux Biochemical Co., Sioux City, IA) daily over 4 days. On Day 5, 3.0 (n=12) or 5.0 (n=2) IU of porcine LH (IM; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24h later. One, two, three, or four LOR were done on 1, 3, 1 and 1 females, respectively (total=14 LOR). The average age at LOR was 3.3 years (range=1.5-7.5 years). Semen was obtained by electroejaculation of 3 males (1.5, 6.0, 7.5 years). Anaesthesia for LOR and electroejaculation was induced and maintained, after intubation, with 5 and 2.5% isoflurane, respectively. Sperm samples were used after storage at 4°C for 24h (TEST yolk, TY) or after cryopreservation (TY+6% glycerol). Luteal tissue was present on the ovaries at 4 of 5 LOR done during January to May as compared with none of 9 LOR done from June to December. Of 165 oocytes (mean=11.8) recovered, 38/54 (70%) and 50/106 (47%) underwent cleavage after IVF with cooled or cryopreserved sperm, respectively (P<0.01, chi-square). None of 5 oocytes cleaved after intracytoplasmic sperm injection with cryopreserved sperm. Procedures for in vitro embryo production were as described previously (Gómez et al. 2006 Theriogenology 66, 72-81; Pope et al. 2006 Theriogenology 66, 1518-1524). Four laparoscopic oviducal embryo transfer procedures were done on Day 1: 2 recipients received fresh Day 2 embryos (n=5, 8) and 2 recipients received embryos that had been cryopreserved on Day 1 (n=6) or 2 (n=8) at a slow, controlled rate in 1.4M of propylene glycol/0.125M of sucrose/10% dextran 70. Each recipient (1.75 to 4.5 years) had undergone LOR on Day 0 (5-19 oocytes recovered). Upon ultrasonographic examination on Day 50, a 2.3-year-old recipient of cryopreserved embryos was determined to be pregnant. She delivered 2 live male kittens, without assistance, on Day 69. When first examined at 15 days of age, the kittens weighed 156 and 198g. At 5 months, their weights were 1.62 and 1.81kg. The sperm sample used to produce the embryos (in 2005) that resulted in the births of kittens (in 2011) was collected from a male at the Henry Doorly Zoo, Omaha, NE (in 2003), extended and transported overnight at 4°C to New Orleans, LA, before cryopreservation. In summary, we have further demonstrated that assisted reproductive technology can be used for conservation of rare and vulnerable small felids.

Previously, we have shown that survival of cat sperm is maintained in both non-egg yolk, semi-defined extenders and in extenders with greatly reduced levels of egg yolk (2%). Usually, cryoprotectant is added to extended samples after gradual cooling to 4°C, but recent reports have shown that satisfactory sperm survival can be obtained after addition at 22°C. Here, our objectives were to examine sperm survival after (1) cryopreservation from 22°C vs after gradually cooling to 4°C or (2) cryopreservation in a completely defined extender without animal or plant proteins vs extender+2% egg yolk. Epididymides from local veterinary clinics were dissected in HEPES 199 medium (He199). The sperm suspension was filtered (40 μ), layered onto a density gradient column and centrifuged at 650×g for 20min. Then, the sperm pellet was resuspended in 1mL of He199 and centrifuged for 5min at 800×g and the subsequent pellet was extended in TEST Buffer with either 0%(0% EY) or 2% egg yolk (2% EY). Next, 0% EY samples were further split into 2 groups-either gradually cooled to 4°C before 12% glycerol (1:1) was added (4C-0%EY) or 12% glycerol (1:1) was added at 22°C without cooling (22C-0%EY). Control samples extended in 2% EY were cooled to 4°C before addition of 12% glycerol (1:1)(4C-2%EY). Samples were loaded into 0.25-mL straws and placed in a -80°C freezer for 20min before storage in LN2. Sperm samples were thawed in air (22°C) for 5s and immersed in a 60°C water bath for 5s. After a 7-step addition of He199, samples were centrifuged at 800×g for 5min and pellets resuspended in He199. Sperm samples were evaluated for motility (Mot; computer-assisted semen analysis, 37°C) at 0h (initial assessment), after cooling to 4°C (PC) and at 0-h (0-PT) and 3-h post-thaw (3-PT) incubation at 37°C. Membrane integrity (MI; SYBR 14-PI) and acrosomal status (AS; FITC-PNA) were analysed at the initial assessment, 0-PT and 3-PT. Results are shown in Table 1. At 4°C (PC), sperm extended in 0% EY and 2% EY maintained 92 and 91%, respectively, of their initial motility (66%). At 0-PT and 3-PT, motility in the 3 groups had decreased by >50% and >70%, respectively. Motility at 3-PT in the 22C-0%EY treatment was less than the other 2 treatments (P<0.05; 1-way ANOVA). At 0-PT, samples in the 4C-2%EY group had a higher membrane integrity value (P<0.05) than did the 22C-0%EY group, whereas that of the 4C-0%EY group was not different from the other 2 groups. However, at 3-PT, both groups cooled to 4°C before cryopreservation had higher membrane integrity values (P<0.05) than the group cryopreserved at 22°C. At 0-PT and 3-PT, the percentage of sperm with intact acrosomes ranged from 69%(4C-2%EY) to 59%(22C-0%EY) and from 55%(4C-2%EY) to 43%(22C-0%EY) of the initial value (89%), respectively. In summary, we demonstrated that cat epididymal sperm could be frozen successfully in a completely defined TEST-buffered extender. Furthermore, we confirmed that addition of cryoprotectant (i.e. glycerol) after gradual cooling to 4°C is beneficial to post-thaw survival.

The role of the total solids (TS) content on anaerobic digestion was investigated in batch reactors. A range of TS contents from 10% to 35% was evaluated, four replicates were performed. The total methane production slightly decreased with TS concentrations increasing from 10% to 25% TS. Two behaviors were observed at 30% TS: two replicates had similar performances to that at 25% TS; for the two other replicates, the methane production was inhibited as observed at 35% TS. This difference suggested that 30% TS content corresponded to a threshold of the solids content, above which methanogenesis was strongly inhibited. The Anaerobic Digestion Model No. 1 (ADM1) was used to describe the experimental data. The effects of hydrolysis step and liquid/gas mass transfer were particularly investigated. The simulations showed that mass transfer limitation could explain the low methane production at high TS, and that hydrolysis rate constants slightly decreased with increasing TS.

Menard C., Dumas C., Gillot N., Laurent L., Labarbe B., Ireland J.& Volatier J.-L.(2012) The French OQALI survey on dairy products: comparison of nutrient contents and other nutrition information on labels among types of brands. J Hum Nutr Diet. ABSTRACT: Background:  Depending on their spending power, consumers may choose foodstuffs from more or less expensive types of brands (national, retailer, economy-line retailer or discount brands). The present study, on dairy products, assesses the nutritional composition and the frequencies of labelled nutrition parameters, according to types of brands. Methods:  The 1646 most consumed dairy products were collected. Nutrient contents and other labelled nutrition parameters provided on the packaging (i.e. nutrition and health claims, nutrition guidelines such as guideline daily amounts, consumption advice and information on added vitamins and minerals) were captured in the French branded product composition database (OQALI). Results:  Significant differences between brands were found for energy, protein, fat, saturates, carbohydrate, sugars, dietary fibre, calcium and sodium, in four of six dairy groups studied, but not systematically. National brands and retailer brands provided more detailed nutrition labelling and more frequent nutrition claims than cheaper brands. More retailer brand products provided nutrition guidelines and consumption advice than the other branded products. National brand products more frequently contained added vitamins and minerals and more frequently bore health claims. Conclusions:  Nutrient contents of the cheaper brands of dairy products did not vary systematically from more expensive ones. However, national brands and retailer brands products provided more nutrition information on labels than the cheaper ones. There should be more detailed studies comparing different types of brands regarding labelling practices for nutrient contents and other nutrition information about foodstuffs to help prepare public health recommendations, adapted to all consumers, regardless of their income.

The most effective innovations in healthcare require the input of multidisciplinary teams working from white board to bedside. Innovations must ultimately deliver tangible results in the real world. The skills required at each stage of the development from drawing board to bench top and from the lab to the clinic may be entirely unrelated. The key results at each stage also vary depending on perspective; they may be acclaim and awards, sales and profits or improved clinical parameters. As teams are enlisted on a specific challenge they each focus primarily on their own key performance indicators. In this paper we report the deliberations at a workshop involving a variety of disciplines working in healthcare. The participants emphasised the need for clear agreement on three aspects: the outputs of the project including the financial and intellectual property rights; the risks, costs and benefits; and the timelines for completion. A lead organisation must broker and maintain relationships ideally facilitated by an experienced project manager. The greatest challenges were highlighted as: the return on investment for commercial partners; the timelines for academic outputs; and the potential for disruption of clinical practice routines.

INTRODUCTION: A variety of pharmaceuticals have been developed directed at mitigating the symptoms associated with benign prostatic hypertrophy (BPH) and have also been evaluated for their potential role in prevention and treatment of prostate cancer. One such agent is dutasteride , a non-selective inhibitor of 5α-reductase, an enzyme responsible for conversion of testosterone to a more potent androgen dihydrotestosterone (DHT). AREAS COVERED: This review will cover the safety profile of dutasteride when it is used in the treatment of prostate-related conditions, specifically looking at the pivotal clinical trials on this drug. EXPERT OPINION: Dutasteride has proved to be a safe and efficacious treatment for symptoms related to BPH. The primary safety concern relates to the increased incidence of high-grade prostate cancer seen in men treated with dutasteride in the setting of prostate cancer prevention. Dutasteride has a role as an adjunct in the treatment of prostate cancer; however, this is an area still under active investigation. It is not recommended for use in prostate cancer prevention given the increased risk of high-grade cancers.

The electrical performances of stabilized lipid monolayers on H-terminated silicon are reported for the first time. We show that these ultra-thin layers of only 2.7 nm thick present extremely low current leakage at high electric field and high breakdown voltage that both compare favorably with the best data reported on organic thin film dielectrics. We demonstrate a very unique property of autonomic self-healing of the layer at room temperature with the total recovery of its performances after electrical breakdown. The mechanisms involved in breakdown and self-healing are discussed.

Metal-dielectric transitions are important structures that can display a host of optical characteristics including excitation of plasmons. Metal-dielectric discontinuities can furthermore support plasmon excitation without a severe condition on the incident angle of the exciting photons. Using a semi-infinite thin gold film, we study surface plasmon (SP) excitation and the associated electromagnetic near-field distribution by recording the resulting plasmon interference patterns. In particular, we measure interference periods involving SPs at the scanable metal/air interface and the buried metal/glass one. Supported by optical near-field simulations and experiments, we demonstrate that the metal/glass surface plasmon is observable over a wide range of incident angles encompassing values above and below the critical incident angle. As a result, it is shown that scanning near-field microscopy can provide quantitative evaluation of the real part of the buried surface plasmon wavevector.

Abstract The black-footed cat (BFC; Felis nigripes), one of the smallest wild cats, is listed as threatened. Interspecies somatic cell nuclear transfer (Is-SCNT) offers the possibility of preserving endangered species. Development to term of interspecies BFC (Is-BFC) cloned embryos has not been obtained, possibly due to abnormal epigenetic reprogramming. Treatment of intraspecies cloned embryos with TSA improves nuclear reprogramming and in vitro and in vivo viability. In this study, we evaluated (1) whether covalent histone modifications differ between Is-BFC cloned embryos and their IVF counterparts,(2) the optimal TSA concentration and exposure times to modify the covalent histone patterns,(3) if TSA enhances in vitro and in vivo developmental competence of cloned embryos, and (4) expression of pluripotent genes. Results indicated that the covalent histone modifications of Is-BFC cloned embryos aberrantly differ from their DSH-IVF counterpart embryos. Aberrant epigenetic events may be due partially to the inability of the DSH cytoplasm to modify the restrictive epigenetic marks of the BFC nuclei after somatic cell nuclear transfer (SCNT). Incomplete remodeling of the histone H3K9me2 in Is-BFC cloned embryos possibly contributes to abnormal expression of pluripotent genes and low embryonic development. Treatment of Is-BFC cloned embryos with TSA remodeled the covalent pattern in H3K9ac and H3K9me2, resembling epigenetic patterns in IVF counterpart embryos, and resulted in activation of some pluripotent genes. However, genomic reprogramming of Is-BFC cloned blastocysts did not follow the same reprogramming pattern observed in DSH-IVF embryos, and in vitro and in vivo developmental competence was not enhanced.