Combined Arrhenius-Merz Law Describing Domain Relaxation in Type-II Multiferroics.

Electric fields were applied to multiferroic TbMnO_{3} single crystals to control the chiral domains, and the domain relaxation was studied over 8 decades in time by means of polarized neutron scattering. A surprisingly simple combination of an activation law and the Merz law describes the relaxation times in a wide range of electric field and temperature with just two parameters, an activation-field constant and a characteristic time representing the fastest possible inversion. Over the large part of field and temperature values corresponding to almost 6 orders of magnitude in time, multiferroic domain inversion is thus dominated by a single process, the domain wall motion. Only when approaching the multiferroic transition other mechanisms yield an accelerated inversion.

Uterine fluid proteome changes during diapause and resumption of embryo development in roe deer (Capreolus capreolus).

The uterine microenvironment during pre-implantation presents a pro-survival milieu and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid-diapause and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organising tree algorithm: (1) gradual reduction over development; (2) stable abundance during diapause, followed by a sharp rise at elongation; and (3) gradual increase over development. Proteins in the different clusters were subjected to gene ontology analysis. 'Cellular detoxification' in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2 and 'cortical cytoskeleton', 'regulation of substrate adhesion-dependent cell spreading' and 'melanosome' were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause.

Characterization of a non-human primate model for the study of testicular peritubular cells-comparison with human testicular peritubular cells.

STUDY QUESTION: Are monkey testicular peritubular cells (MKTPCs) from the common marmoset monkey (Callithrix jacchus) a suitable translational model for the study of human testicular peritubular cells (HTPCs)? SUMMARY ANSWER: MKTPCs can be isolated and propagated in vitro, retain characteristic markers for testicular peritubular cells and their proteome strongly (correlation coefficient of 0.78) overlaps with the proteome of HTPCs. WHAT IS KNOWN ALREADY: Smooth-muscle-like peritubular cells form the wall of seminiferous tubules, transport sperm, are immunologically active, secrete a plethora of factors and may contribute to the spermatogonial stem cell niche. Mechanistic studies are hampered by heterogeneity of human samples. STUDY DESIGN, SIZE, DURATION: We established a culture method for MKTPCs and characterized these cells from six young adult animals (2-3 years). To examine whether they qualify as a translational model we also examined HTPCs from seven men and compared the proteomes of both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used explant cultures to obtain MKTPCs, which express smooth muscle markers (calponin (CNN1), smooth muscle actin (ACTA2)), lack FSH-receptors (FSHR) and LH-receptors (LHCGR), but possess androgen receptors (AR). MKTPCs can be passaged at least up to eight times, without discernable phenotypic changes. Mass-spectrometry-based analyses of the MKTPC and HTPC proteomes were performed. MAIN RESULTS AND THE ROLE OF CHANCE: We established a method for isolation and cultivation of MKTPCs, and provide a comprehensive analysis of their protein repertoire. The results let us conclude that MKTPCs are suitable as a non-human primate model to study peritubular cell functions. LARGE SCALE DATA: List of identified proteins in MKTPCs by liquid chromatography-tandem mass spectrometry is accessible at the ProteomeXchange (identifier PXD009394). LIMITATIONS, REASON FOR CAUTION: This is an in vitro cellular non-human primate model used to provide a window into the role of these cells in the human testis. WIDER IMPLICATIONS OF THE FINDINGS: Previous studies with HTPCs from patients revealed a degree of heterogeneity, possibly due to age, lifestyle and medical history of the individual human donors. We anticipate that the new translational model, derived from young healthy non-human primates, may allow us to circumvent these issues and may lead to a better understanding of the role of peritubular cells. STUDY FUNDING AND COMPETION OF INTEREST(S): This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). The authors declare no competing financial interests.

Control of Chiral Magnetism Through Electric Fields in Multiferroic Compounds above the Long-Range Multiferroic Transition.

Polarized neutron scattering experiments reveal that type-II multiferroics allow for controlling the spin chirality by external electric fields even in the absence of long-range multiferroic order. In the two prototype compounds TbMnO_{3} and MnWO_{4}, chiral magnetism associated with soft overdamped electromagnons can be observed above the long-range multiferroic transition temperature T_{MF}, and it is possible to control it through an electric field. While MnWO_{4} exhibits chiral correlations only in a tiny temperature interval above T_{MF}, in TbMnO_{3} chiral magnetism can be observed over several kelvin up to the lock-in transition, which is well separated from T_{MF}.

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes.

STUDY QUESTION: Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)? SUMMARY ANSWER: Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance. WHAT IS KNOWN ALREADY: Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote. STUDY DESIGN, SIZE, DURATION: Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). MATERIAL AND METHODS: GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). MAIN RESULTS AND ROLE OF CHANCE: The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization. LIMITATION, REASONS FOR CAUTION: We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging. WIDER IMPLICATIONS OF THE FINDINGS: Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro. STUDY FUNDING/COMPETING INTERESTS: The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.

Measurement of alkaline phosphatase in canine seminal plasma--an update.

In dogs, diagnosis of incomplete ejaculation and azoospermia can be made by measuring the activity of the enzyme alkaline phosphatase (AP) in seminal plasma. However, even though upper cut-off value of 5000 IU/l is given in the literature, results by different assays may vary considerably. Furthermore, no data exist concerning the stability of the enzyme during storage of frozen seminal plasma, and no recommendations for pre-analytic dilutions can be found. During the present study, we compared results from a conventional large scale wet chemistry analyzer to a widely used dry chemistry point of care system (POC) and established a best practice for pre-analytical dilutions. Furthermore, stability of enzyme activities in seminal plasma during storage at -18 °C for 24 h was evaluated. The average activity of AP in the 2nd fraction of normal ejaculates measured by Reflotron® was 107,328 IU/l. After 24 h of frozen storage, activities did not differ significantly (96,844 IU / l, p > 0.05). Fresh and frozen samples were analysed in parallel by the POC and conventional chemistry analyser, and the results compared that did not reveal a significant difference (p > 0.05). A dilution of seminal plasma with physiologic saline 1:100 prior to analysis was sufficient for the qualitative information whether AP activity is below or above 5000 IU/l. Present data show that AP measurement by a POC dry chemistry system is sufficiently accurate in diluted seminal plasma for the diagnosis of azoospermia and that seminal plasma can be stored frozen for 24h before analysis.

Dynamic proteome signatures in gametes, embryos and their maternal environment.

Comprehensive molecular analysis at the level of proteins represents a technically demanding, but indispensable, task since several post-transcriptional regulation mechanisms disable a valid prediction of quantitative protein expression profiles from transcriptome analysis. In crucial steps of gamete and early embryo development, de novo transcription is silenced, meaning that almost all macromolecular events take place at the level of proteins. In this review, we describe selected examples of dynamic proteome signatures addressing capacitation of spermatozoa, in vitro maturation of oocytes, effect of oestrous cycle on oviduct epithelial cells and embryo-induced alterations to the maternal environment. We also present details of the experimental strategies applied and the experiments performed to verify quantitative proteomic data. Far from being comprehensive, examples were selected to cover several mammalian species as well as the most powerful proteomic techniques currently applied. To enable non-experts in the field of proteomics to appraise published proteomic data, our examples are preceded by a customised description of quantitative proteomic methods, covering 2D difference gel electrophoresis (2D-DIGE), nano-liquid chromatography combined with tandem mass spectrometry, and label-free as well as stable-isotope labelling strategies for mass spectrometry-based quantifications.

Does the etching strategy influence the bonding of universal adhesive systems to primary teeth? A systematic review and meta-analysis of in vitro studies.

PURPOSE: To assess through a systematic review and meta-analysis of in vitro studies, the influence of the etching strategy (etch-and-rinse versus self-etch) of universal adhesive systems on bonding to primary teeth. METHODS: A systematic search was carried out in PubMed, Web of Science and Scopus. In vitro studies that compared the bond strength of the etching strategies of universal adhesives to primary teeth were included. Pooled-effect estimates were derived from a random-effects model by comparing the standardized mean difference between the etching strategies (α < 0.05). The risk of bias and heterogeneity between studies were also assessed (Cochrane and I2 tests). RESULTS: Seven studies were included in the review and six in the meta-analyses. For dentin, the immediate bond strength was not influenced by the etching strategy regardless of sound (Z = 0.72, p = 0.47) or caries-affected (Z = 1.27, p = 0.21) substrate, nor after aging (Z = 0.24, p = 0.81). It was not possible to perform a meta-analysis for the enamel substrate. Most studies have a medium risk of bias. CONCLUSIONS: This systematic review and meta-analysis of in vitro studies provides evidence that universal adhesives can be used in both etching strategies in primary dentin. The evidence is currently insufficient about whether selective acid etching of primary enamel is necessary when universal adhesive systems are used.

Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Resonant inelastic x-ray incarnation of Young's double-slit experiment.

Young's archetypal double-slit experiment forms the basis for modern diffraction techniques: The elastic scattering of waves yields an interference pattern that captures the real-space structure. Here, we report on an inelastic incarnation of Young's experiment and demonstrate that resonant inelastic x-ray scattering (RIXS) measures interference patterns, which reveal the symmetry and character of electronic excited states in the same way as elastic scattering does for the ground state. A prototypical example is provided by the quasi-molecular electronic structure of insulating Ba3CeIr2O9 with structural Ir dimers and strong spin-orbit coupling. The double "slits" in this resonant experiment are the highly localized core levels of the two Ir atoms within a dimer. The clear double-slit-type sinusoidal interference patterns that we observe allow us to characterize the electronic excitations, demonstrating the power of RIXS interferometry to unravel the electronic structure of solids containing, e.g., dimers, trimers, ladders, or other superstructures.

Insights into the role of androgen receptor in human testicular peritubular cells.

Contractile smooth muscle-like peritubular cells build the wall of seminiferous tubules in men. They are crucial for sperm transport and complement the functions of Sertoli cells by secreting factors, including glial cell line-derived neurotrophic factor. Previous studies revealed that they also secrete the chemokine C-X-C motif chemokine ligand 12 (CXCL12), which has known roles in spermatogenesis. Peritubular cells express the androgen receptor (AR), which is retained in isolated human testicular peritubular cells. We aimed to explore AR-regulated functions in human testicular peritubular cells. Bearing in mind that infertile men often have high aromatase activity, which may lower intratesticular androgen concentrations, an animal model for male infertility was studied. These mice display an age-dependent loss in spermatogenesis due to high aromatase activity. Human testicular peritubular cells were exposed to dihydrotestosterone or the antiandrogen flutamide. We studied AR, smooth muscle cell markers, glial cell line-derived neurotrophic factor and 15 secreted factors previously identified, including CXCL12. We used qPCR, Western blotting, ELISA or selected reaction monitoring (SRM). In the animal model for male infertility, we employed qPCR and immunohistochemistry. Dihydrotestosterone increased AR and flutamide prevented these actions. The smooth muscle cell markers calponin and smooth muscle actin were likewise increased, while cell size or cellular proliferation was not changed. Dihydrotestosterone did not increase glial cell line-derived neurotrophic factor or CXCL12 secretion but increased levels of serine proteinase inhibitor (SERPIN) E1. The animal model for male infertility with high aromatase activity showed reduced numbers of AR-immunoreactive testicular peritubular cells, suggesting that altered androgen and/or oestrogen levels could influence AR-mediated responses in peritubular cells. Androgens act on human testicular peritubular cells to enhance AR levels, their contractile phenotype and to modulate the secretion of some secreted factors. This study suggests that some aspects of human peritubular cell functions are regulated by androgens.

Proteomic identification of turkey (Meleagris gallopavo) seminal plasma proteins.

SDS-PAGE combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 2-dimensional electrophoresis (2DE) combined with matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI TOF/TOF) were applied to characterize the turkey seminal plasma proteome. LC-MS/MS led to the identification of 175 proteins, which were classified according to their function and to corresponding biochemical pathways. Using 2DE and MALDI TOF/TOF, 34 different turkey seminal plasma proteins could be identified, of which 20 were found in more than one spot, indicating different proteoforms of these proteins. For validation, antibodies against turkey albumin and ovoinhibitor as well as sperm acrosin were used in 2DE Western blots experiments. The bioinformatic analysis of the results indicates that turkey seminal plasma proteins may be involved in regulation of lipid metabolism [liver X receptor/retinoid X receptor (LXR/RXR) activation and farnesoid X receptor/retinoid X receptor (FXR/RXR) activation pathways)], endocytic entry of proteins and lipids at the plasma membrane (clathrin-mediated endocytosis pathway), and defense against pathogens (acute phase response signaling pathway) and energy production (glycolysis and gluconeogenesis). Moreover, a comparative meta-analysis of seminal plasma proteomes from other species indicated the presence of proteins specific for avian reproduction, but distinct differences between turkey and chicken seminal plasma proteomes were detected. The results of our study provide basic knowledge of the protein composition of turkey seminal plasma highlighting important physiological pathways which may play crucial roles in the sperm environment after ejaculation. This knowledge can be the basis to further develop procedures improving the reproduction of farmed turkeys.

Piston-effect-induced thermal jets in near-critical fluids.

In very compressible fluids, such as fluids near their critical point, the bulk fluid is adiabatically thermalized by the expansion of a hot boundary layer. Thanks to this thermomechanical process (the so-called piston effect) the fluid velocity at the edge of the boundary layer can become very high when the heating power is concentrated in a fissure. Spectacular jets are then observed in SF6 and CO2. Data obtained under weightlessness (in order to remove convection) and data obtained under earth gravity are compared and analyzed. They emphasize the key role of the boundary layer expansion for thermal phenomena in compressible fluids and the hydrodynamic nature of the piston effect.

Muscle adenylate kinase in Duchenne muscular dystrophy.

On the basis of electrophoretic and enzyme inhibition studies it was postulated that an aberrant adenylate kinase occurs in muscle and serum of patients with Duchenne muscular dystrophy (Schirmer, R.H. and Thuma, E. (1972) Biochim. Biophys. Acta 268, 92-97; Hamada, M. et al. (1981) Biochim. Biophys. Acta 660, 227-237; Hamada et al. (1985) J. Biol. Chem. 260, 11595-11602). On the basis of the following results we conclude that Duchenne muscular dystrophy patients do not possess an unusual adenylate kinase isoenzyme. In muscle biopsies from five Duchenne patients, the electrophoretic mobility of adenylate kinase and the inhibition of the enzyme by P1, P5-di(adenosine-5')pentaphosphate (Ap5A) was normal. Because of the high SH-group content of the extracts from Duchenne muscle, high concentrations of Ellman's reagent were needed to inhibit adenylate kinase activity in these samples. In Duchenne plasma the adenylate kinase activity was elevated. Like in muscle specimens, the DTNB inhibition curves were shifted to higher reagent concentrations; this was due to a high SH-group content of Duchenne plasma when compared with normal plasma. With respect to inhibition by Ap5A and electrophoretic mobility, Duchenne adenylate kinase in Duchenne plasma behaved like normal muscle adenylate kinase in normal plasma. It was noted that normal muscle adenylate kinase changes its electrophoretic behaviour when mixed with normal or Duchenne plasma. This finding had been considered previously as evidence for the presence of an aberrant adenylate kinase in Duchenne plasma.

HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1 alpha and developmentally expressed in blood vessels.

Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Recently a bHLH-PAS protein, named trachealess (trl) was identified as a master regulator of tracheogenesis. Hypoxia-inducible factor, HIF-1 alpha, is a vertebrate relative of trl which is likely to be involved in growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. In the present study we describe mRNA cloning and mRNA expression pattern of mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha which is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role of this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis.

Functional genomics: tools for improving farm animal health and welfare.

The first genome sequence assemblies of farm animal species are now accessible through public domain databases, and further sequencing projects are in rapid progress. In addition, large collections of expressed sequences have been obtained, which will aid in constructing annotated transcript maps for many economically important species. Thus, the breeding of farm animals is entering the post-genome era. Functional genomics, defined as applying global experimental approaches to assess gene function, by using the information and reagents provided by structural genomics (i.e. mapping and sequencing), has become the focus of interest. Combining a holistic view of phenotypes at the molecular level with genetic marker data seems a particularly promising approach for improving health and welfare traits in farm animals. These traits are often difficult to define. They suffer from low heritabilities and a corresponding lack of genetic gain in conventional selection and breeding programmes. At the same time, genomic information from micro-organisms and parasites offers the potential for new vaccines and therapeutics. This review describes major functional genomics tools, lists genomic resources available for farm animals and discusses the prospects and challenges of functional genomics in improving the health and welfare of farm animals.

Human testicular peritubular cells secrete pigment epithelium-derived factor (PEDF), which may be responsible for the avascularity of the seminiferous tubules.

Male fertility depends on spermatogenesis, which takes place in the seminiferous tubules of the testis. This compartment is devoid of blood vessels, which are however found in the wall of the seminiferous tubules. Our proteomic study using cultured human testicular peritubular cells (HTPCs) i.e. the cells, which form this wall, revealed that they constitutively secrete pigment epithelium-derived factor, PEDF, which is known to exert anti-angiogenic actions. Immunohistochemistry supports its presence in vivo, in the human tubular wall. Co-culture studies and analysis of cell migration patterns showed that human endothelial cells (HUVECs) are repulsed by HTPCs. The factor involved is likely PEDF, as a PEDF-antiserum blocked the repulsing action. Thus testicular peritubular cells, via PEDF, may prevent vascularization of human seminiferous tubules. Dihydrotestosterone (DHT) increased PEDF (qPCR) in HTPCs, however PEDF expression in the testis of a non-human primate occurs before puberty. Thus PEDF could be involved in the establishment of the avascular nature of seminiferous tubules and after puberty androgens may further reinforce this feature. Testicular microvessels and blood flow are known to contribute to the spermatogonial stem cell niche. Hence HTPCs via control of testicular microvessels may contribute to the regulation of spermatogonial stem cells, as well.

Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

Myocardial infarction with angiographically normal coronary arteries.

To determine the prevalence rate and clinical and hemodynamic profile of patients with myocardial infarction (MI) with angiographically normal coronary arteries, we analyzed 3,403 consecutive angiograms performed within a 4.5-year period. Of these studies, 1,124 were performed following an acute MI. Through a computerized search, 12 patients were identified who had documented MI with normal or insignificant (< 30% stenosis in one epicardial vessel only) coronary disease. Q-wave MI developed in five patients (group A) and non-Q-wave MI developed in seven patients (group B). Group A patients were all men whereas group B patients were all women. Overall, group A patients were younger (p = 0.003), had a longer smoking history (p = 0.008), and a higher cardiac index (p = 0.005). In ten patients, areas of localized dyskinesia or hypokinesia were shown on left ventricular cineangiography. Mitral valve prolapse was present in four of the patients and varying degrees of mitral regurgitation were identified in another six. The prevalence rate of MI with angiographically normal coronary arteries was 1% in this study. This entity had a bimodal age and sex distribution: a younger age group, all men, with a stronger cigarette smoking history who had Q-wave MI vs an older age group, all women, and no significant association with cigarette smoking who developed non-Q-wave MI. A mean follow-up of 4 years demonstrated a favorable prognosis in both groups.

Right lower lobectomy for pulmonary infarction before orthotopic heart transplantation.

A 39-year-old potential heart transplant recipient had a right lower lobe infiltrate and on pulmonary angiography was found to have an embolous to the common basilar artery. This was successfully managed by a right lower lobectomy, after aggressive medical management failed. The patient was treated postoperatively with antibiotics and subsequently underwent orthotopic heart transplantation. At 1 year after transplant the patient has no evidence of cardiac or pulmonary insufficiency.