An ES Cell System for Rapid, Spatial and Temporal Analysis of Gene Function in vitro and in vivo

We describe a versatile genetic system for rapid analysis of mammalian gene function. In this, loss of reporter activity in a novel embryonic stem (ES) cell line enables rapid identification of targeting to the ubiquitously expressed Rosa26 locus. Subsequent regulation of gene activity is governed by a dual regulatory strategy utilizing two drugs, Tamoxifen and Doxycycline. To illustrate this approach, a dominant allele of Smoothened was introduced into this cell line, enabling regulated activation of Hedgehog signaling. By coupling Cre-loxP dependent activation with tetracycline dependent transcription in a single allele, we established a conditional method to control Smoothened activity and neural progenitor specification in differentiating ES cells in vitro and in chimeric embryos in vivo When crossed to an appropriate Cre driver strain, gene activity can also be temporally regulated within a specific cell lineage. This platform will facilitate rapid analysis of gene function in the mouse.Molecular and Cellular BiologyStem Cell and Regenerative Biolog

Guided self-help cognitive behavioural therapy for depression in primary care : a randomised controlled trial

Peer reviewedPublisher PD

A non-apoptotic role for caspase-9 in muscle differentiation

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits caspase-9 activation, had the same effect on muscle differentiation as knockdown of caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of caspase-9 activation during muscle differentiation.</jats:p

Seeing the wood for the trees: towards improved quantification of glial cells in central nervous system tissue

The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue. It follows from our examination of the utility of Fiji ImageJ thresholding and binarization algorithms. Describing how we identified the maximum intensity projection as the best of six tested for two dimensional (2D)-rendering of three-dimensional (3D) images derived from a series of z-stacked micrographs, the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein (GFAP), microglial ionized calcium binding adapter molecule 1 (IBA1) and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices. The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections, is also described. Fiji’s BioVoxxel plugin allowed categorisation of algorithms according to their sensitivity, specificity accuracy and relative quality. The Percentile algorithm was deemed best for quantifying levels of GFAP, the Li algorithm was best when quantifying IBA expression, while the Otsu algorithm was optimum for Olig2 staining, albeit with over-quantification of oligodendrocyte number when compared to a stereological approach. Also, GFAP and IBA expression in 3,3′-diaminobenzidine (DAB)/haematoxylin-stained cerebellar tissue was best quantified with Default, Isodata and Moments algorithms. The workflow presented in [Figure 1] could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue

UPR Induction Prevents Iron Accumulation and Oligodendrocyte Loss in ex vivo Cultured Hippocampal Slices

The accumulation of iron within the brain occurs in many chronic disorders including Alzheimer’s and Parkinson’s disease and multiple sclerosis. Outside the CNS, a link between levels of iron and the unfolded protein response has already been established. To determine if such a relationship operates in within the brain, we used our ex vivo hippocampal slice-based model of iron accumulation. Ferrocene addition caused accumulation of iron within slices and loss of oligodendrocytes, an effect that was partially inhibited when ferrocene and ER stressor tunicamycin (Tm) were added together. An upward trend (not found to be statistically significant) in the expression of UPR transcripts in response to ferrocene was demonstrated using real-time PCR, while a significant upregulation of mRNA for B cell immunoglobulin-binding protein (BiP) and C/EBP homologous binding protein (CHOP) occurred following exposure to Tm. In silico analysis revealed consensus DNA-binding sequences for UPR-associated transcription factors within the promoter regions of eight iron-regulatory genes. In addition, dual-staining for CHOP and oligodendrocyte transcription factor 2 (OLIG2) or Ionized calcium binding adaptor molecule 1 (Iba1) showed nuclear expression of CHOP in some oligodendrocyte-lineage cells in response to Tm or Tm+ferrocene, but CHOP was rarely found in microglia. Co-expression of UPR-associated activated transcription factor 6 (ATF6) was detected in the nuclei of some oligodendrocyte-lineage cells exposed to Tm alone, or to Tm and ferrocene, but rarely in microglia. These data highlight the therapeutic potential of targeting UPR-associated proteins when developing novel treatments for chronic brain disorders that are affected by dysregulated iron

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

Introduction: A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia. Methods: Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-alpha-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated. Results: The second generation lentiviral vector rHIV-pWPT-EF1-alpha-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression. Conclusions: Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies

Characterization of a mutant polyoma that expresses in F9 embryonal carcinoma cells: Morphology, tumorigenicity, and restriction enzyme analysis

A mutant polyoma virus (TT340), which replicates in F9 embryonal carcinoma (EC) cells and contains 2500 base pairs (bp) of additional DNA located in the early noncoding region of the genome, was analyzed to determine the DNA origin of the mutant insertion. Two fragments, representing repeated units of the 2500-bp insert, were isolated from TT340, labeled, and hybridized to the parental wild-type viral DNA. A BglI 500-bp unit, of which there are approximately five copies within the 2500-bp insert, contains sequences homologous to regions on the early and late side of the viral origin of replication. A HpaII 400-bp repeated fragment shows homology to sequences on the early side with little hybridization to the late side. Removal of the 2500-bp insert results in the loss of infectivity on F9 EC cells but not on 3T6 or mouse embryo fibroblasts. Insertion of the BglI 500-bp repeat element into wild-type DNA at the BglI site allows replication of the constructed virus in F9 cells. The mutant virions were tumorigenic in newborn Syrian hamsters and the morphology of the virus was that of wild-type as assayed by electron microscopy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26615/1/0000156.pd

Neuroepithelial Body Microenvironment Is a Niche for a Distinct Subset of Clara-Like Precursors in the Developing Airways

Clara cells of mammalian airways have multiple functions and are morphologically heterogeneous. Although Notch signaling is essential for the development of these cells, it is unclear how Notch influences Clara cell specification and if diversity is established among Clara cell precursors. Here we identify expression of the secretoglobin Scgb3a2 and Notch activation as early events in a program of secretory cell fate determination in developing murine airways. We show that Scgb3a2 expression in vivo is Notch-dependent at early stages and ectopically induced by constitutive Notch1 activation, and also that in vitro Notch signaling together with the pan-airway transcription factor Ttf1 (Nkx2.1) synergistically regulate secretoglobin gene transcription. Furthermore, we identified a subpopulation of secretory precursors juxtaposed to presumptive neuroepithelial bodies (NEBs), distinguished by their strong Scgb3a2 and uroplakin 3a (Upk3a) signals and reduced Ccsp (Scgb1a1) expression. Genetic ablation of Ascl1 prevented NEB formation and selectively interfered with the formation of this subpopulation of cells. Lineage labeling of Upk3a-expressing cells during development showed that these cells remain largely uncommitted during embryonic development and contribute to Clara and ciliated cells in the adult lung. Together, our findings suggest a role for Notch in the induction of a Clara cell-specific program of gene expression, and reveals that the NEB microenvironment in the developing airways is a niche for a distinct subset of Clara-like precursors.Molecular and Cellular Biolog

Current Status of Nutrition Training in Graduate Medical Education From a Survey of Residency Program Directors

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142009/1/jpen0095.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142009/2/jpen0095-sup-0001.pd

Efficiency in education

Education is important at national, local and individual levels. Its benefits accrue both to society and to individuals, and as such provision of education in many countries is paid for at least in part from the public purse. With competing demands for government funding, it is important for education to be provided as efficiently as possible. Efficiency occurs when outputs from education (such as test results or value added) are produced at the lowest level of resource (be that financial or, for example, the innate ability of students). This special issue is devoted to the topic of efficiency in education, and is well-timed given that governments around the world struggle with public finances in the wake of the global financial crisis of 2008. In this paper, we explore and provide an overview of the themes of the special issue and introduce the papers contained therein