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  • Ten years of human metapneumovirus research. 22074934

    Described for the first time in 2001, human metapneumovirus (hMPV) has become one of the main viral pathogens responsible for acute respiratory tract infections in children but also in the elderly and immuno-compromised patients. The pathogen most closely related to hMPV is human respiratory syncytial virus (hRSV), the most common cause of bronchiolitis and pneumonia in young children. hMPV has been classified into two main viral groups A and B and has a seasonal distribution in temperate countries with most cases occurring in winter and spring. Given the difficulties encountered in culturing hMPV in vitro, diagnosis is generally achieved using real-time polymerase chain reaction. Like other Paramyxoviridae, hMPV has a negative-sense single-stranded RNA genome that includes 8 genes coding for 9 different proteins. The genomic organization and functions of surface attachment and fusion glycoproteins are relatively similar to those of hRSV. Although many groups have studied the viral life cycle of hMPV, many questions remain unanswered concerning the exact roles of the viral proteins in the attachment, fusion and replication of hMPV. To date, there remains no approved modality to combat hMPV infections. The majority of treatments that have been tested on hMPV have already demonstrated activity against hRSV infections. Some innovative approaches based on RNA interference and on fusion inhibitors have shown efficacy in vitro and in animal studies and could be beneficial in treating human hMPV disease. Difficulties faced inducing a durable immune response represent the biggest challenge in the development of an effective hMPV vaccine. Several strategies, such as the use of live-attenuated viruses generated by reverse genetics or recombinant proteins, have been tested in animals with encouraging results.
    Document Type:
    Reference
    Product Catalog Number:
    MAB338
    Product Catalog Name:
    Anti-Ciliary Neurotrophic Factor Antibody, clone 4-68

  • Runx2 disruption promotes immortalization and confers resistance to oncogene-induced senescence in primary murine fibroblasts. 18056452

    The Runx genes play paradoxical roles in cancer where they can function either as dominant oncogenes or tumor suppressors according to context. We now show that the ability to induce premature senescence in primary murine embryonic fibroblasts (MEF) is a common feature of all three Runx genes. However, ectopic Runx-induced senescence contrasts with Ras oncogene-induced senescence, as it occurs directly and lacks the hallmarks of proliferative stress. Moreover, a fundamental role for Runx function in the senescence program is indicated by the effects of Runx2 disruption, which renders MEFs prone to spontaneous immortalization and confers an early growth advantage that is resistant to stress-induced growth arrest. Runx2(-/-) cells are refractory to H-Ras(V12)-induced premature senescence, despite the activation of a cascade of growth inhibitors and senescence markers, and are permissive for oncogenic transformation. The aberrant behavior of Runx2(-/-) cells is associated with signaling defects and elevated expression of S-G(2)-M cyclins and their associated cyclin dependent kinase activities that may override the effects of growth inhibitory signals. Coupling of stress responses to the cell cycle represents a novel facet of Runx tumor suppressor function and provides a rationale for the lineage-specific effects of loss of Runx function in cancer.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3682

  • Generation of p53 target database via integration of microarray and global p53 DNA-binding site analysis. 15220520

    The completion of the human genome sequence and availability of cDNA microarray technology provide new approaches to explore global cellular regulatory mechanisms. Here we present a strategy to identify genes regulated by specific transcription factors in the human genome, and apply it to p53. We first collected promoters or introns of all genes available using two methods: GenBank annotation and a computationally derived transcript map. The FindPatterns program is then used to search sequences in regulatory regions that match the p53 DNA-binding consensus sequence, resulting in the p53 Target Database. This database collects human genes that have at least one p53 DNA-binding sequence in their regulatory region. cDNA microarray was also used to identify genes that respond to p53 at a genomic scale. Integration of the microarray data and the p53 Target Database should greatly enrich direct p53 target genes. Taqman analysis and quantitative chromatin immunoprecipitation analysis are used to validate the in silico prediction and microarray data. Enrichment factor analysis is used to demonstrate that in silico prediction greatly enriches for genes that are transcriptionally regulated by p53 and assists us to identify other signaling pathways that are potentially connected to p53. The approaches can be extended to other transcription factors. The methods shown here illustrate a novel approach to the analysis of global gene regulatory networks through the integration of human genomic sequence information and genome-wide gene expression analysis.
    Document Type:
    Reference
    Product Catalog Number:
    16-157
    Product Catalog Name:
    Protein A Agarose/Salmon Sperm DNA, 2.5 mL

  • Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. 24086725

    Recombinant Adeno-associated virus vectors (rAAV) are widely used for gene delivery and multiple naturally occurring serotypes have been harnessed to target cells in different tissues and organs including the brain. Here, we provide a detailed and quantitative analysis of the transduction profiles of rAAV vectors based on six of the most commonly used serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, AAV9) that allows systematic comparison and selection of the optimal vector for a specific application. In our studies we observed marked differences among serotypes in the efficiency to transduce three different brain regions namely the striatum, hippocampus and neocortex of the mouse. Despite the fact that the analyzed serotypes have the general ability to transduce all major cell types in the brain (neurons, microglia, astrocytes and oligodendrocytes), the expression level of a reporter gene driven from a ubiquitous promoter varies significantly for specific cell type / serotype combinations. For example, rAAV8 is particularly efficient to drive transgene expression in astrocytes while rAAV9 appears well suited for the transduction of cortical neurons. Interestingly, we demonstrate selective retrograde transport of rAAV5 along axons projecting from the ventral part of the entorhinal cortex to the dentate gyrus. Furthermore, we show that self-complementing rAAV can be used to significantly decrease the time required for the onset of transgene expression in the mouse brain.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking. 24646489

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a "do-it-yourself" basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1553
    Product Catalog Name:
    Anti-Titin Antibody, clone 9B9

  • In vitro osteogenic differentiation is affected in Wiedemann-Rautenstrauch-Syndrome (WRS). 16097434

    BACKGROUND: Wiedemann-Rautenstrauch (neonatal progeroid) syndrome (WRS) is a rare autosomal recessive condition, with the characteristic appearance of premature aging already present at birth and other typical features (hypotrichosis, macrocephaly, mental retardation, aged face, generalized lipoatrophy, abnormal tooth status, osteopenia and other skeletal abnormalities). To date, there are no data about the differentiation capacity of WRS progenitor cells available in the literature. PATIENTS AND METHODS: To elucidate the osteoblastic and chondroblastic regeneration potential in WRS, a progenitor cell culture system was used. Bone marrow-derived stem cells of a 16-year-old WRS patient were cultivated and stimulated by dexamethasone, ascorbic acid and beta-glycerolphosphate (DAG) over 21 days. Immunocytochemical stainings of CD34, CD45, CD105, osteocalcin, osteopontin and collagen II served for a quantitative evaluation of the differentiated cells. The results were compared to bone marrow-derived stem cells of a healthy female volunteer donor. RESULTS: It was shown, for the first time, that WRS cells showed a highly significant lower in vitro response to osteoblastic differentiation stimulus. Furthermore, significantly fewer chondrocytes and hematopoietic cells were induced in WRS progenitors compared to the control group. CONCLUSION: Our data suggest a lack of cellular differentiation capacity in WRS patients, which may be responsible for the clinical appearance and symptoms of this rare disorder.
    Document Type:
    Reference
    Product Catalog Number:
    AB1870
    Product Catalog Name:
    Anti-Osteopontin Antibody

  • Studying protein degradation pathways in vivo using a cranial window-based approach. 21187150

    Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction in the ubiquitin-proteasome system (UPS) specifically has been implicated in several important neurodegenerative diseases including Alzheimer's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis. Research in this area has been limited by the fact that many inhibitors of the UPS given systemically do not cross the blood-brain barrier (BBB) in appreciable levels. This limits the ability to easily test in vivo specific hypotheses generated in reduced systems, like brain slice or dissociated cell culture, about whether the UPS may degrade a particular protein of interest. Although several techniques including intracerebral application via direct syringe injection, catheter-pump systems and drug-eluting beads are available to introduce BBB-impermeant drugs into brain they each have certain limitations and new approaches could provide further insights into this problem. In order to test the role of the UPS in protein degradation in vivo we have developed a strategy to treat mouse cortex with the UPS inhibitor clasto-lactacystin beta-lactone (CLBL) via a "cranial window" and recover the treated tissue for immunoblot analysis. This approach can be used in several different cranial window configurations including single window and double hemi-window arrangements that are tailored for different applications. We have also developed two different strategies for recovering treated cortical tissue including a vibratome/laser capture microscopy (LCM)-based and a vibratome only-based approach, each with its own specific advantages. We have documented UPS inhibition greater than 600μm deep into the cortex with this strategy. This set of techniques in the living mammalian brain is complementary to previously developed approaches and extends the repertoire of tools that can be used to the study protein degradation pathways relevant to neurodegenerative disease.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Cell cycle kinetics and immunohistochemical characterization of dissociated fetal neocortical cultures: evidence that differentiated neurons have mitotic capacity. 10915906

    Neurons in the neocortex (regardless of their developmental state) are considered to be post-mitotic and incapable of dividing. We used dissociated primary cultures derived from the neocortices of 16-day-old fetuses to test the counter-hypothesis, that is, differentiating neocortical neurons can divide. The cultured cells experienced considerable cell death, yet the number of viable cells remained relatively constant over the first 5 days in vitro. The implication was that the cultures contained proliferating cells. This was confirmed with a [(3)H]thymidine ([3H]dT) incorporation study and cumulative bromodeoxyuridine labeling. In fact, over 1/4 of the cells were cycling and the length of the cell cycle was 20.0 h; kinetics which mirror those of the developing cortex in vivo. This population of proliferating cells was eliminated by 48 h treatment with fluorodeoxyuridine. Immunohistochemical procedures determined that most cultured cells (>/=90%) expressed proteins associated with differentiating or mature neurons, e.g., neurofilament (NF) 200 and isoforms of microtubule-associated protein (MAP) 2. Markers for immature neurons (e.g., nestin) were expressed by 10% of the cells. In contrast, markers for glia and their precursors were expressed by /=2% of the population. Double-labeling with [3H]dT and a neural-specific antibody showed that cells expressing an antigen for immature neurons constituted most of the proliferating cells, however, a considerable number of [3H]dT-labeled cells expressed markers for differentiating neurons (e.g., NF200 and MAP2). Thus, differentiating neocortical neurons can be mitotically active and it appears that differentiating neurons are derived from both the ventricular and subventricular proliferative zones.
    Document Type:
    Reference
    Product Catalog Number:
    MAB314

  • Epstein-Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation. 24097438

    Epstein-Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Scaled-up production of mammalian neural precursor cell aggregates in computer-controlled suspension bioreactors. 16489624

    The clinical use of neural precursor cells (NPCs) for the treatment of neurological diseases, such as Parkinson's disease and Huntington's disease, requires overcoming the scarcity of these cells through controlled expansion. The main objective of the present study was to develop a large-scale computer-controlled bioprocess for the expansion of mammalian NPCs in suspension culture by scaling up existing reactor protocols. In order to support the oxygen demands of the maximum cell densities achieved, the volumetric mass transfer coefficient was kept above 1.10/h while scaling-up from small-scale 125 mL vessels to large-scale 500 mL bioreactors. In addition, the maximum shear stress at the impeller tip was maintained between 0.30 and 0.75 Pa to reduce damage to the cells. The resulting large-scale bioprocess achieved maximum viable cell densities of 1.2 x 10(6) cells/mL and a batch multiplication ratio of 9.1. Moreover, the process successfully maintained the NPC characteristics observed in small-scale studies.
    Document Type:
    Reference
    Product Catalog Number:
    MAB345
    Product Catalog Name:
    Anti-O4 Antibody, clone 81