The Maestro (Mro) Gene Is Dispensable for Normal Sexual Development and Fertility in Mice

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function

Minor Abnormalities of Testis Development in Mice Lacking the Gene Encoding the MAPK Signalling Component, MAP3K1

In mammals, the Y chromosome is a dominant male determinant, causing the bipotential gonad to develop as a testis. Recently, cases of familial and spontaneous 46,XY disorders of sex development (DSD) have been attributed to mutations in the human gene encoding mitogen-activated protein kinase kinase kinase 1, MAP3K1, a component of the mitogen-activated protein kinase (MAPK) signal transduction pathway. In individuals harbouring heterozygous mutations in MAP3K1, dysregulation of MAPK signalling was observed in lymphoblastoid cell lines, suggesting a causal role for these mutations in disrupting XY sexual development. Mice lacking the cognate gene, Map3k1, are viable and exhibit the eyes open at birth (EOB) phenotype on a mixed genetic background, but on the C57BL/6J genetic background most mice die at around 14.5 dpc due to a failure of erythropoiesis in the fetal liver. However, no systematic examination of sexual development in Map3k1-deficient mice has been described, an omission that is especially relevant in the case of C57BL/6J, a genetic background that is sensitized to disruptions to testis determination. Here, we report that on a mixed genetic background mice lacking Map3k1 are fertile and exhibit no overt abnormalities of testis development. On C57BL/6J, significant non-viability is observed with very few animals surviving to adulthood. However, an examination of development in Map3k1-deficient XY embryos on this genetic background revealed no significant defects in testis determination, although minor abnormalities were observed, including an increase in gonadal length. Based on these observations, we conclude that MAP3K1 is not required for mouse testis determination. We discuss the significance of these data for the functional interpretation of sex-reversing MAP3K1 mutations in humans

A Novel Mouse Fgfr2 Mutant, Hobbyhorse (hob), Exhibits Complete XY Gonadal Sex Reversal

The secreted molecule fibroblast growth factor 9 (FGF9) plays a critical role in testis determination in the mouse. In embryonic gonadal somatic cells it is required for maintenance of SOX9 expression, a key determinant of Sertoli cell fate. Conditional gene targeting studies have identified FGFR2 as the main gonadal receptor for FGF9 during sex determination. However, such studies can be complicated by inefficient and variable deletion of floxed alleles, depending on the choice of Cre deleter strain. Here, we report a novel, constitutive allele of Fgfr2, hobbyhorse (hob), which was identified in an ENU-based forward genetic screen for novel testis-determining loci. Fgr2hob is caused by a C to T mutation in the invariant exon 7, resulting in a polypeptide with a mis-sense mutation at position 263 (Pro263Ser) in the third extracellular immunoglobulin-like domain of FGFR2. Mutant homozygous embryos show severe limb and lung defects and, when on the sensitised C57BL/6J (B6) genetic background, undergo complete XY gonadal sex reversal associated with failure to maintain expression of Sox9. Genetic crosses employing a null mutant of Fgfr2 suggest that Fgr2hob is a hypomorphic allele, affecting both the FGFR2b and FGFR2c splice isoforms of the receptor. We exploited the consistent phenotype of this constitutive mutant by analysing MAPK signalling at the sex-determining stage of gonad development, but no significant abnormalities in mutant embryos were detected

A quantitative systems pharmacology approach, incorporating a novel liver model, for predicting pharmacokinetic drug-drug interactions

All pharmaceutical companies are required to assess pharmacokinetic drug-drug interactions (DDIs) of new chemical entities (NCEs) and mathematical prediction helps to select the best NCE candidate with regard to adverse effects resulting from a DDI before any costly clinical studies. Most current models assume that the liver is a homogeneous organ where the majority of the metabolism occurs. However, the circulatory system of the liver has a complex hierarchical geometry which distributes xenobiotics throughout the organ. Nevertheless, the lobule (liver unit), located at the end of each branch, is composed of many sinusoids where the blood flow can vary and therefore creates heterogeneity (e.g. drug concentration, enzyme level). A liver model was constructed by describing the geometry of a lobule, where the blood velocity increases toward the central vein, and by modeling the exchange mechanisms between the blood and hepatocytes. Moreover, the three major DDI mechanisms of metabolic enzymes; competitive inhibition, mechanism based inhibition and induction, were accounted for with an undefined number of drugs and/or enzymes. The liver model was incorporated into a physiological-based pharmacokinetic (PBPK) model and simulations produced, that in turn were compared to ten clinical results. The liver model generated a hierarchy of 5 sinusoidal levels and estimated a blood volume of 283 mL and a cell density of 193 × 106 cells/g in the liver. The overall PBPK model predicted the pharmacokinetics of midazolam and the magnitude of the clinical DDI with perpetrator drug(s) including spatial and temporal enzyme levels changes. The model presented herein may reduce costs and the use of laboratory animals and give the opportunity to explore different clinical scenarios, which reduce the risk of adverse events, prior to costly human clinical studies

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

The boygirl (byg) mouse mutant reveals that MAP3K4-mediated signaling is necessary for normal SRY expression and testis specification in the developing mouse gonad

Expression of the Salmonella Spp. Virulence Factor SifA in Yeast Alters Rho1 Activity on Peroxisomes

SifA is a virulence protein required for assembly and tubulation of a modified phagosome that promotes Salmonella replication. We show that SifA expressed in yeast induces membrane invagination during peroxisome proliferation and requires functional Rho1p. This is consistent with SifA ability to interact with RhoA and the fact that it is a GEF structural homologue

The preparation of HEMA-MPC films for ocular drug delivery

There is a need to prolong drug residence time using a biocompatible formulation in the subconjunctival space after surgery to treat glaucoma. Drug releasing discs were prepared with 2-(hydroxyethyl)methacrylate (HEMA) and 2-methacryloyl-oxyethyl phosphorylcholine (MPC). The ratio of bound water (Wb) to free water (Wf) ratio increased from 1:0.3 to 1:6.8 with increasing MPC (0 to 50%, w/w). The optimal balance between water content, SR and mechanical strength were obtained with 10% MPC (w/w) hydrogels. Water-alcohol mixtures were examined to facilitate loading of poorly soluble drugs, and they showed greater hydrogel swelling than either water or alcohol alone. The SR was 1.2 ± 0.02 and 3.3 ± 0.1 for water and water:ethanol (1:1) respectively. HEMA-MPC (10%) discs were loaded with dexamethasone using either water:ethanol (1:1) or methanol alone. Drug release was examined in an outflow rig model that mimics the subconjunctival space in the eye. Dexamethasone loading increased from 0.3 to 1.9 mg/disc when the solvent was changed from water:ethanol (1:1) to methanol with the dexamethasone half-life (t½) increasing from 1.9 to 9.7 days respectively. These encouraging results indicate that HEMA-MPC hydrogels have the potential to sustain the residence time of a drug in the subconjunctival space of the eye

Factors influencing gastrointestinal tract and microbiota immune interaction in preterm infants

The role of microbial colonization is indispensable for keeping a balanced immune response in life. However, the events that regulate the establishment of the microbiota, their timing, and the way in which they interact with the host are not yet fully understood. Factors such as gestational age, mode of delivery, environment, hygienic measures, and diet influence the establishment of microbiota in the perinatal period. Environmental microbes constitute the most important group of exogenous stimuli in this critical time frame. However, the settlement of a stable gut microbiota in preterm infants is delayed compared to term infants. Preterm infants have an immature gastrointestinal tract and immune system which predisposes to infectious morbidity. Neonatal microbial dynamics and alterations in early gut microbiota may precede and/or predispose to diseases such as necrotizing enterocolitis (NEC), late-onset sepsis or others. During this critical period, nutrition is the principal contributor for immunological and metabolic development, and microbiological programming. Breast milk is a known source of molecules that act synergistically to protect the gut barrier and enhance the maturation of the gut-related immune response. Host-microbe interactions in preterm infants and the protective role of diet focused on breast milk impact are beginning to be unveiled.M.C. acknowledges a “Rio Hortega” Research Fellowship Grant (CM13/0017) and M.V. acknowledges grants PI11/0313 and RD12/0026/0012 (Red SAMID) from the Instituto Carlos III (Spanish Ministry of Economy and Competitivity). M.C.C. and G.P-M. were supported by the grant AGL2013-47420-R from the Spanish Ministry of Science and Innovation.Peer reviewe