Contributions of in situ hybridization of sRNA to the study on spatio-temporal gene expression in Hevea brasiliensis

Since the dawn of the genomics era, much research has focused on functional studies of genes of interest. In situ hybridization is a method that can be used to precisely localize the expression of a gene in tissues and cells. This article describes how the method has been adapted to the analysis of Hevea tissues. Initially, the conventional method of digoxigenin detection with NBT/BCIP revealed the expression of strongly expressed genes in tissues of different differentiation intensity. A new digoxigenin detection method using Alexa488 fluorochrome-labelled antibodies has been used to detect the expression of more weakly expressed genes. This method, combined with observation under a confocal microscope, has enabled very precise localization of expression. Some examples of in situ hybridization use are described for Hevea gene expression in somatic plantlets and shoot bark: the uidA gene in callus and transgenic somatic plantlets, the HEV2.1 gene encoding hevein and the ACO-H5 gene involved in ethylene metabolism. Cell imaging methods therefore open up fundamental prospects for studying the different molecular mechanisms involved in some agronomic traits of Hevea. (Résumé d'auteur

Examination of Acid-Fast Bacilli in Sputum Using Modified Light Microscope with Homemade Light Emitting Diode Additional Attachment

Typical clinical symptoms and chest X-ray is a marker of Tuberculosis (TB) sufferers. However, the diagnosis of TB in adults should be supported by microscopic examination. Currently, Bacilli microscopic examination of acid-fast bacilli (AFB) in sputum by Ziehl-Neelsen (ZN) coloring is the most widely used. However, for reasons of convenience, especially for laboratories with a considerable amount of smear samples, and due to higher sensitivity compared with ZN staining, the World Health Organization (WHO) has recommended the use of auramine-O-staining (fluorochrome  staining), which is visualized by light emitting diode (LED) fluorescence microscopy. The aim of this study was to evaluate the performance of modified light microscope with homemade LED additional attachment for examination of AFB in sputum using auramine-O-staining method. We compared the sensitivity and specificity of 2 kinds of AFB in sputum methods: ZN and fluorochrome, using culture on Lowenstein-Jensen media as the gold standard. The results showed auramine-O-staining gives more proportion of positive findings (81%) compared to the ZN method (70%). These results demonstrated that the sensitivity of auramine-O-staining was higher than ZN, however it gives more potential false positive results than ZN. The sensitivity of auramine-O-staining in detecting AFB in sputum was 100% while the specificity was 88%

Antibodies for immunolabeling by light and electron microscopy : not for the faint hearted

Reliable antibodies represent crucial tools in the arsenal of the cell biologist and using them to localize antigens for immunocytochemistry is one of their most important applications. However, antibody-antigen interactions are much more complex and unpredictable than suggested by the old 'lock and key' analogy, and the goal of trying to prove that an antibody is specific is far more difficult than is generally appreciated. Here, we discuss the problems associated with the very complicated issue of trying to establish that an antibody (and the results obtained with it) is specific for the immunolabeling approaches used in light or electron microscopy. We discuss the increasing awareness that significant numbers of commercial antibodies are often not up to the quality required. We provide guidelines for choosing and testing antibodies in immuno-EM. Finally, we describe how quantitative EM methods can be used to identify reproducible patterns of antibody labeling and also extract specific labeling distributions.Peer reviewe

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity

Immunostaining of skeletal tissues

Peer reviewedPreprin

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Gene deficiency in activating Fcγ receptors influences the macrophage phenotypic balance and reduces atherosclerosis in mice

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis

Regulation of CD4+NKG2D+ Th1 cells in patients with metastatic melanoma treated with sorafenib : role of IL-15Rα and NKG2D triggering

Beyond cancer-cell intrinsic factors, the immune status of the host has a prognostic impact on patients with cancer and influences the effects of conventional chemotherapies. Metastatic melanoma is intrinsically immunogenic, thereby facilitating the search for immune biomarkers of clinical responses to cytotoxic agents. Here, we show that a multi-tyrosine kinase inhibitor, sorafenib, upregulates interleukin (IL)-15Rα in vitro and in vivo in patients with melanoma, and in conjunction with natural killer (NK) group 2D (NKG2D) ligands, contributes to the Th1 polarization and accumulation of peripheral CD4+NKG2D+ T cells. Hence, the increase of blood CD4+NKG2D+ T cells after two cycles of sorafenib (combined with temozolomide) was associated with prolonged survival in a prospective phase I/II trial enrolling 63 patients with metastatic melanoma who did not receive vemurafenib nor immune checkpoint-blocking antibodies. In contrast, in metastatic melanoma patients treated with classical treatment modalities, this CD4+NKG2D+ subset failed to correlate with prognosis. These findings indicate that sorafenib may be used as an "adjuvant" molecule capable of inducing or restoring IL-15Rα/IL-15 in tumors expressing MHCclass I-related chain A/B (MICA/B) and on circulating monocytes of responding patients, hereby contributing to the bioactivity of NKG2D+ Th1 cells.peer-reviewe