Transgene design

Transgenics are powerful mouse models to understand the biological functions of genes. This chapter gives a short overview of the requirements and considerations in designing a transgene. In addition, potential important choices that have to be made in advance for the successful designing and generating a transgenic mouse model are discussed. Methods for DNA purification for microinjection are also provided in this chapter.</p

Nature versus nurture in the spectrum of rheumatic diseases: Classification of spondyloarthritis as autoimmune or autoinflammatory

Abstract Spondyloarthritides (SpA) include inflammatory joint diseases with various clinical phenotypes that may also include the axial skeleton and/or entheses. SpA include psoriatic arthritis, reactive arthritis, enteropathic arthritis and ankylosing spondylitis; the latter is frequently associated with extra-articular manifestations, such as uveitis, psoriasis, and inflammatory bowel disease. SpA are associated with the HLA-B27 allele and recognize T cells as key pathogenetic players. In contrast to other rheumatic diseases, SpA affect women and men equally and are not associated with detectable serum autoantibodies. In addition, but opposite to rheumatoid arthritis, SpA are responsive to treatment regimens including IL-23 or IL-17-targeting biologics, yet are virtually unresponsive to steroid treatment. Based on these differences with prototypical autoimmune diseases, such as rheumatoid arthritis or connective tissue diseases, SpA may be better classified among autoinflammatory diseases, with a predominant innate immunity involvement. This would rank SpA closer to gouty arthritis and periodic fevers in the spectrum of rheumatic diseases, as opposed to autoimmune-predominant diseases. We herein provide available literature on risk factors associated with SpA in support of this hypothesis with a specific focus on genetic and environmental factors

Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences

A hydrogenosome with pyruvate formate-lyase: Anaerobic chytrid fungi use an alternative route for pyruvate catabolism

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes ex

Activation of NF-κB/p65 Facilitates Early Chondrogenic Differentiation during Endochondral Ossification

BACKGROUND: NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling. METHODOLOGY/PRINCIPAL FINDINGS: The role of NF-κB/p65 in early chondrogenesis was investigated in different in vitro, ex vivo and in vivo endochondral models: ATDC5 cells, hBMSCs, chicken periosteal explants and growth plates of 6 weeks old mice. NF-κB/p65 activation was manipulated using pharmacological inhibitors, RNAi and activating agents. Gene expression and protein expression analysis, and (immuno)histochemical stainings were employed to determine the role of NF-κB/p65 in the chondrogenic phase of endochondral development. Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-κB/p65. NF-κB/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways. CONCLUSIONS/SIGNIFICANCE: The presented data demonstrate that NF-κB/p65 signaling, as well as its intensity and timing, represents one of the transcriptional regulatory mechanisms of the chondrogenic developmental program of chondroprogenitor cells during endochondral ossification. Importantly, these results provide novel possibilities to improve the success of cartilage and bone regenerative techniques

The interaction between AMPK beta 2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

11 páginas, 7 figuras.AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at threonine-148 (Thr-148), which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. Here, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid analyses and co-immunoprecipitation of the overexpressed proteins in HEK293T cells revealed that both AMPKβ1 and β2 wild-type (WT) isoforms bind to R6. The AMPKβ/R6 interaction was stronger with the muscle-specific β2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced AMPKβ2/R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous to increased AMPKβ2 Thr-148 autophosphorylation. Such model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.DN is recipient of a VIDI-Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ALW Grant no. 864.10.007). This work has further been supported by grants from the Spanish Ministry of Education and Science SAF2014-54604-C3-1-R and a grant from Generalitat Valenciana (PrometeoII/2014/029) to PS.Peer reviewe

Hypoxia promotes stem cell phenotypes and poor prognosis through epigenetic regulation of DICER

MicroRNAs are small regulatory RNAs that post-transcriptionally control gene expression. Reduced expression of DICER, the enzyme involved in microRNA processing, is frequently observed in cancer and is associated with poor clinical outcome in various malignancies. Yet the underlying mechanisms are not well understood. Here, we identify tumor hypoxia as a regulator of DICER expression in large cohorts of breast cancer patients. We show that DICER expression is suppressed by hypoxia through an epigenetic mechanism that involves inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B and results in silencing of the DICER promoter. Subsequently, reduced miRNA processing leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and ultimately results in the acquisition of stem cell phenotypes in human mammary epithelial cells. Our study uncovers a previously unknown relationship between oxygen-sensitive epigenetic regulators, miRNA biogenesis and tumor stem cell phenotypes that may underlie poor outcome in breast cancer

Neoadjuvant chemoradiotherapy plus surgery versus active surveillance for oesophageal cancer: A stepped-wedge cluster randomised trial

Background: Neoadjuvant chemoradiotherapy (nCRT) plus surgery is a standard treatment for locally advanced oesophageal cancer. With this treatment, 29% of patients have a pathologically complete response in the resection specimen. This provides the rationale for investigating an active surveillance approach. The aim of this study is to assess the (cost-)effectiveness of active surveillance vs. standard oesophagectomy after nCRT for oesophageal cancer. Methods: This is a phase-III multi-centre, stepped-wedge cluster randomised controlled trial. A total of 300 patients with clinically complete response (cCR, i.e. no local or disseminated disease proven by histology) after nCRT will be randomised to show non-inferiority of active surveillance to standard oesophagectomy (non-inferiority margin 15%, intra-correlation coefficient 0.02, power 80%, 2-sided α 0.05, 12% drop-out). Patients will undergo a first clinical response evaluation (CRE-I) 4-6 weeks after nCRT, consisting of endoscopy with bite-on-bite biopsies of the primary tumour site and other suspected lesions. Clinically complete responders will undergo a second CRE (CRE-II), 6-8 weeks after CRE-I. CRE-II will include 18F-FDG-PET-CT, followed by endoscopy with bite-on-bite biopsies and ultra-endosonography plus fine needle aspiration of suspected lymph nodes and/or PET- positive lesions. Patients with cCR at CRE-II will be assigned to oesophagectomy (first phase) or active surveillance (second phase of the study). The duration of the first phase is determined randomly over the 12 centres, i.e., stepped-wedge cluster design. Patients in the active surveillance arm will undergo diagnostic evaluations similar to CRE-II at 6/9/12/16/20/24/30/36/48 and 60 months after nCRT. In this arm, oesophagectomy will be offered only to patients in whom locoregional regrowth is highly suspected or proven, without distant dissemination. The main study parameter is overall survival; secondary endpoints include percentage of patients who do not undergo surgery, quality of life, clinical irresectability (cT4b) rate, radical resection rate, postoperative complications, progression-free survival, distant dissemination rate, and cost-effectiveness. We hypothesise that active surveillance leads to non-inferior survival, improved quality of life and a reduction in costs, compared to standard oesophagectomy. Discussion: If active surveillance and surgery as needed after nCRT leads to non-inferior survival compared to standard oesophagectomy, this organ-sparing approach can be implemented as a standard of care