Synchrotron-based X-ray Fluorescence Ghost Imaging

X-ray Fluorescence Ghost Imaging (XRF-GI) was recently demonstrated for x-ray lab sources. It has the potential to reduce acquisition time and deposited dose by choosing their trade-off with spatial resolution, while alleviating the focusing constraints of the probing beam. Here, we demonstrate the realization of synchrotron-based XRF-GI: We present both an adapted experimental setup and its corresponding required computational technique to process the data. This not only extends the above-mentioned advantages to synchrotron XRF imaging, it also presents new possibilities for developing strategies to improve precision in nano-scale imaging measurements

Analysis of the genetic basis of height in large Jewish nuclear families.

Despite intensive study, most of the specific genetic factors that contribute to variation in human height remain undiscovered. We conducted a family-based linkage study of height in a unique cohort of very large nuclear families from a founder (Jewish) population. This design allowed for increased power to detect linkage, compared to previous family-based studies. Loci we identified in discovery families could explain an estimated lower bound of 6% of the variance in height in validation families. We showed that these loci are not tagging known common variants associated with height. Rather, we suggest that the observed signals arise from variants with large effects that are rare globally but elevated in frequency in the Jewish population

STAT3 phosphorylation in APC: apoptotic cell co-cultures correlates with CD47 expression.

<p>The various cell types were treated as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075595#pone-0075595-g006" target="_blank">figure 6</a>. Monocytes were then co-cultured with either live or apoptotic cells for 2 hours or mixed without co-incubation as a control and then lysed. Cell extracts were separated on SDS-PAGE and anti-phosphorylated STAT3 immunoblotting (upper panels). Immunoblotting of STAT3 reveals relative amounts of protein in each lane (lower panels). (A) Staurosporine treated cells. (B) Anti-Fas treated fibroblasts. (C) γ-irradiation induced apoptotic cells. (D) H₂O₂-treated cells. Comparable results were obtained in four independent experiments.</p

Anti CD47 antibodies targeting CD47:SIRPα interaction slightly attenuates contact-dependent activation of STAT3.

<p>MCF-7 cells were treated with either anti-CD47 blocking mAb B6H12.2 or the anti-CD47 mAb 2D3, used as control. Monocytes were co-cultured with untreated or antibody-treated MCF-7. After 2 hours, cell extracts were subjected to SDS-PAGE and immunoblotting. Anti-phosphorylated STAT3 demonstrate induction of STAT3 (upper panels). Anti-STAT3 immunoblotting reveals relative amounts of protein in each lane (lower panels). Comparable results were obtained in three separate experiments.</p

The level of CD47 expression by apoptotic cells is dependent on the mode of apoptosis induction.

<p>Apoptosis was induced by various methods as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075595#s2" target="_blank">Materials and Methods</a> and CD47 surface expression was measured by flow cytometry. (A) MCF-7 cells were treated with staurosporine for 1.5 h and then washed and incubated for the indicated times. Time-dependent reduction in CD47 expression is shown. (B) CD47 expression in fibroblasts (upper panels) and HEK293 cells (lower panel) 24 h after induction of apoptosis with either staurosporine (left panels) or anti-FAS (right panel). (C) CD47 surface expression on MCF-7 cells (left panel), fibroblasts (middle panel) or HEK293 cells (right panel) 24 h after apoptosis induction with H₂O₂ (upper panels) or γ-irradiation (lower panels). Filled grey area: isotype control; Thick black line: untreated cells; Grey line: treated cells. Similar results were obtained in at least three experiments. (D) MFI results of three separate experiments as in A-C were normalized and are presented as relative to the level of CD47 expression by live untreated cells. White bar: γ-irradiation-treated cells; Black bar: H₂O₂-treated cells; Light grey bar: staurosporine-treated cells; Dark grey bar: anti-FAS-treated cells. Left panel: MCF-7 cells; Right panel: fibroblasts.</p

Targeting CD47 expression by shRNA abrogates STAT3 activation and restores T cell responses.

<p>HEK293 and MCF-7 cells were transfected with HuSH shRNA tRFP Cloning Vectors: clone 14: control plasmid; clone 15 plasmid containing scrambled shRNA sequence; clones 30–32: CD47 specific shRNAs. (A) Stable transfected cells were immunostained for surface CD47 expression. Numbers indicate the median fluorescent intensity (MFI) of CD47 expression in each clone. (B) The various MCF-7 transfectants were co-cultured with monocytes for 2 h (+). Control cell extracts were obtained by mixing cells and monocytes without co-culturing them together (-). Cell extracts were subjected to SDS-PAGE and immunoblotting of anti-phosphorylated STAT3 (upper panels). Anti-STAT3 immunoblotting reveals relative amounts of protein in each lane (lower panels). Comparable results were obtained in two separate experiments and similar results were obtained using HEK293 clones. (C) PBMC were incubated with or without the various HEK293 transfectants and were activated using anti-CD3 Ab (OKT3; 10 ng/ml). After 72 h, conditioned media were collected and analyzed for IFN-γ and IL-17 secretion. Left panel: to demonstrate the contact dependent inhibition, PBMC were co-cultured together or on opposite sides of a transwell and the ability of HEK293 cells to inhibit IFN-γ secretion was measured and is presented as percentage of inhibition. Right panels: The level of IFN-γ and IL-17 secreted by activated PBMC cultured in the absence or presence of the various 293 transfectants is presented. The data represent average of triplicate wells. Comparable results were obtained in two independent experiments.</p

Physical association between SIRPα STAT3 and phosphorylated-STAT3.

<p>(A) Monocytes were either co-cultured with MCF-7 (+), for 2 hours or mixed with MCF-7 without incubation (-) as a control and then lysed. Cell extracts were subjected to immunoprecipitation with anti- SIRPα antibodies and the precipitates were separated on SDS-PAGE. Anti-STAT3 immunoblotting demonstrates that STAT3 associates with SIRPα (lower panel) and anti-phosphorylated STAT3 immunoblotting demonstrates association of the activated form of STAT3 upon contact with MCF-7 (upper panel). (B) monocytes and MCF-7 cells were immunostained with anti- SIRPα antibodies and then analyzed by flow cytometry (left panel). The level of SIRPα and STAT3 expression in lysates prepared from monocytes and MCF7 (right upper panels); No STAT3 was detected in anti-SIRPα precipitates from the MCF7 lysates (right lower panel). Similar results were obtained in four separate experiments.</p

CD47 surface expression levels by various cell types and their corresponding ability to induce STAT3 phosphorylation in monocytes.

<p>(A) Different cell types were screened for surface CD47 expression using flow cytometric analysis. Grey histogram: isotype control. Black line: anti-CD47 staining. One representative experiment is shown. Comparable results were obtained in three independent experiments. (B) The average of three separate experiments is graphed as median fluorescence intensity (MFI) of CD47 staining. MFI was calculated by reducing the background isotype staining from the MFI value of anti-CD47 staining for each cell type. **- P≤0.001. (C) The various cell lines were co-cultured with monocytes for 2 h. Control cell extracts were obtained by incubating the cells and the monocytes separately and then mixing just before lysis. Cell extracts were subjected to SDS-PAGE and immunoblotting of anti-phosphorylated STAT3 (upper panels). Anti-STAT3 immunoblotting reveals relative amounts of protein in each lane (lower panels). Comparable results were obtained in at least two separate experiments for each cell type. Note: STAT3 phosphorylation in control lanes represents steady-state level of active STAT3 in the various cell lines. (D) Monocytes were either left untreated or treated with JSI-124 (10 µM) for 1 hour at 37°C, and then washed. Treated and untreated cells were then co-cultured with MCF7 and STAT3 phophorylation was determined as in B. (E) Monocytes were co-cultured with MCF7 cells for various time points and then lysed and analyzed as above. (F) Monocytes were co-cultured with MCF7 cells for 2 h in the absence or presence of 20 µM Actinomycin D, STAT3 phophorylation was determined as in B.</p

Synchrotron-based X-ray Fluorescence Ghost Imaging

International audienceX-ray Fluorescence Ghost Imaging (XRF-GI) was recently demonstrated for x-ray lab sources. It has the potential to reduce acquisition time and deposited dose by choosing their trade-off with spatial resolution, while alleviating the focusing constraints of the probing beam. Here, we demonstrate the realization of synchrotron-based XRF-GI: We present both an adapted experimental setup and its corresponding required computational technique to process the data. This not only extends the above-mentioned advantages to synchrotron XRF imaging, it also presents new possibilities for developing strategies to improve precision in nano-scale imaging measurements