Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells

The ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell‐based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen‐activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full‐term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells

TMED2 binding restricts SMO to the ER and Golgi compartments

Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein–coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER–Golgi leading to HH signaling activation.ISSN:1544-9173ISSN:1545-788

The effect of juice clarification, static or rotary fermentation and fining on ochratoxin A in wine

Semillon and Shiraz grapes containing ochratoxin A (OA) were produced by inoculation of bunches on the vine with Aspergillus carbonarius. Small scale vinification of these grapes was performed and the OA content at various stages was analysed. Semillon must was treated with pectolytic enzymes and the juice after pressing was fined with bentonite. Neither pectolytic enzymes nor bentonite influenced the OA content of the clarified juice. Shiraz must was vinified by either static or rotary fermentation. No differences in the OA content of wine after pressing were noted. OA was concentrated in juice lees and gross lees. However, additional juice or wine recovered from lees after centrifugation did not display increased OA contamination compared to racked juice or wine. Addition of bentonite at 2.5 g/L to a Semillon wine containing 56 mg grape-derived protein/L and ca. 8 μg OA/kg resulted in a 67% reduction in OA concentration. Potassium caseinate showed some effect, whereas gelatin, isinglass and PVPP did not significantly diminish OA in white wine compared to wine settled without fining agents. Addition of yeast hulls at 5.0 g/L to a Shiraz wine containing ca. 5 μg OA/kg yielded a 43% reduction. Gelatin was also slightly effective. Storage of wine for 10-14 months resulted in a decrease in OA concentration of 22% and 29% for white and red wines, respectively.Su-Lin l. Leong, Ailsa D. Hocking and Eileen S. Scot

The Chemokine Decoy Receptor D6 Prevents Excessive Inflammation and Adverse Ventricular Remodeling After Myocardial Infarction

OBJECTIVE: Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and overwhelming infiltration of innate immune cells has been shown to promote adverse remodeling and cardiac rupture. Recruitment of inflammatory cells in the ischemic heart depends highly on the family of CC-chemokines and their receptors. Here, we hypothesized that the chemokine decoy receptor D6, which specifically binds and scavenges inflammatory CC-chemokines, might limit inflammation and adverse cardiac remodeling after infarction. METHODS AND RESULTS: D6 was expressed in human and murine infarcted myocardium. In a murine model of myocardial infarction, D6 deficiency led to increased chemokine (C-C motif) ligand 2 and chemokine (C-C motif) ligand 3 levels in the ischemic heart. D6-deficient (D6(-/-)) infarcts displayed increased infiltration of pathogenic neutrophils and Ly6Chi monocytes, associated with strong matrix metalloproteinase-9 and matrix metalloproteinase-2 activities in the ischemic heart. D6(-/-) mice were cardiac rupture prone after myocardial infarction, and functional analysis revealed that D6(-/-) hearts had features of adverse remodeling with left ventricle dilation and reduced ejection fraction. Bone marrow chimera experiments showed that leukocyte-borne D6 had no role in this setting, and that leukocyte-specific chemokine (C-C motif) receptor 2 deficiency rescued the adverse phenotype observed in D6(-/-) mice. CONCLUSIONS: We show for the first time that the chemokine decoy receptor D6 limits CC-chemokine-dependent pathogenic inflammation and is required for adequate cardiac remodeling after myocardial infarction