Optimal sparsity allows reliable system-aware restoration of fluorescence microscopy images

Incluye: artículo, material suplementario, videos y software.Fluorescence microscopy is one of the most indispensable and informative driving forces for biological research, but the extent of observable biological phenomena is essentially determined by the content and quality of the acquired images. To address the different noise sources that can degrade these images, we introduce an algorithm for multiscale image restoration through optimally sparse representation (MIRO). MIRO is a deterministic framework that models the acquisition process and uses pixelwise noise correction to improve image quality. Our study demonstrates that this approach yields a remarkable restoration of the fluorescence signal for a wide range of microscopy systems, regardless of the detector used (e.g., electron-multiplying charge-coupled device, scientific complementary metal-oxide semiconductor, or photomultiplier tube). MIRO improves current imaging capabilities, enabling fast, low-light optical microscopy, accurate image analysis, and robust machine intelligence when integrated with deep neural networks. This expands the range of biological knowledge that can be obtained from fluorescence microscopy.We acknowledge the support of the National Institutes of Health grants R35GM124846 (to S.J.) and R01AA028527 (to C.X.), the National Science Foundation grants BIO2145235 and EFMA1830941 (to S.J.), and Marvin H. and Nita S. Floyd Research Fund (to S.J.). This research project was supported, in part, by the Emory University Integrated Cellular Imaging Microscopy Core and by PHS Grant UL1TR000454 from the Clinical and Translational Science Award Program, National Institutes of Health, and National Center for Advancing Translational Sciences.S

Properties of Immature Myeloid Progenitors with Nitric-Oxide-Dependent Immunosuppressive Activity Isolated from Bone Marrow of Tumor-Free Mice

<div><p>Myeloid derived suppressor cells (MDSCs) from tumor-bearing mice are important negative regulators of anti-cancer immune responses, but the role for immature myeloid cells (IMCs) in non-tumor-bearing mice in the regulation of immune responses are poorly described. We studied the immune-suppressive activity of IMCs from the bone marrow (BM) of C57Bl/6 mice and the mechanism(s) by which they inhibit T–cell activation and proliferation. IMCs, isolated from BM by high-speed FACS, inhibited mitogen-induced proliferation of CD4⁺ and CD8⁺ T-cells <i>in vitro</i>. Cell-to-cell contact of T-cells with viable IMCs was required for suppression. Neither neutralizing antibodies to TGFβ1, nor genetic disruption of indolamine 2,3-dioxygenase, abrogated IMC-mediated suppressive activity. In contrast, suppression of T-cell proliferation was absent in cultures containing IMCs from interferon-γ (IFN-γ) receptor KO mice or T-cells from IFN-γ KO mice (on the C57Bl/6 background). The addition of NO inhibitors to co-cultures of T-cells and IMC significantly reduced the suppressive activity of IMCs. IFN-γ signaling between T-cells and IMCs induced paracrine Nitric Oxide (NO) release in culture, and the degree of inhibition of T-cell proliferation was proportional to NO levels. The suppressive activity of IMCs from the bone marrow of tumor-free mice was comparable with MDSCs from BALB/c bearing mice 4T1 mammary tumors. These results indicate that IMCs have a role in regulating T-cell activation and proliferation in the BM microenvironment.</p></div

T-cell inhibition is mediated by an IFN-γ/NO pathway.

<p>Supernatants were collected after 4–5 days of co culturing Dynabead-activated T-cells with CD11b⁺GR-1⁺ immature myeloid cells sorted from BM. A) NO concentration in the supernatants of wild-type T-cells with and without wild-type IMCs (<i>p</i> = 0.0033). B) NO concentration in the supernatants of combinations of wild- type T-cells with IMCs, wild type T-cells with IFN-γ receptor KO IMCs, and IFN-γ KO T-cells with wild type IMCs (black bars from left to right). White bars show NO concentrations in supernatant of IFN-γ (50 ng/ml) treated IMCs cultures FACS-purified from wild type, IFN-γ receptor KO, and IFN-γ KO BM. Data represent mean and SD of four experiments. C) Inhibition of Dynabead-induced proliferation of CD4⁺ T-cells with mix of both subunits CD11b⁺GR-1^hi and GR-1^low in the presence and absence of NO inhibitors <i>p</i> = 0.0419. D) Correlation of NO production and inhibition of proliferation in co-cultures containing different ratios of CD11b⁺GR-1⁺ IMCs: T-cells. Solid and dashed lines represent best-fit correlation of NO concentration with inhibition of proliferation of CD4⁺ and CD8⁺ T-cells. (* Indicates <i>p<0.05</i>, ** indicates <i>p<0.001</i>).</p

Expression of surface molecules on BM-derived CD11b⁺GR-1⁺ IMC subsets.

<p>Flow cytometry analysis of cell surface marker expression on lineage (−) CD11b⁺GR-1^hi and CD11b⁺GR-1^low/int IMC subsets was performed as described in the Methods section. Histograms represent expression of the indicated markers on viable CD11b⁺GR-1⁺ cells (open dashed histograms) compared with gated isotype control (filed gray histograms). Data represent of average of frequencies (± SD) from replicate samples. B) Logarithmic mean fluorescence index (MFI) of three experiments for both subsets of CD11b⁺GR-1^hi and CD11b⁺GR^−/low/int IMCs respectively (B & C) ordered by marker from the greatest to the least mean MFI.</p

Suppressor activity of bone marrow myeloid cell subsets.

<p>Single cell suspensions of bone marrow were prepared from C57BL/6 mice and stained with anti-CD11b APC-Cy, anti GR-1 FITC, and lineage PE cocktail. Lineage (+), lineage (−), CD11b⁺GR-1^hi and CD11b⁺GR-1^low/int BM populations were isolated by FACS. A) Pre sort gates (upper panels). Reanalysis of sorted populations (middle panels). Lower panels represent morphology of Giemsa stained cells (63× magnification). B) CFSE fluorescence histograms of viable 7-AAD (−) MACs purified T-cells co-cultured with different CD11b⁺ GR-1⁺/splenocyte ratios (left panel). C) Comparison of the percentage inhibition of proliferation of T-cells co-cultured with CD11b⁺GR-1^hi, CD11b^hiGR-1^low/int and lineage (−) CD11b⁺ cells (ratio1/1). Bars represent the mean values ± SD of two experiments. D) Comparison of the potency of sorted BM fractions of IMCs (mix of both CD11b⁺GR-^1hi, CD11b^hiGR-1^low/int) on percentage of undivided CFSE labeled T-cells following 5 days co culture in the presence of anti-CD3/CD28 beads, and IL-2. The legend shows the ratio of sorted IMC: T-cells with the size of the symbol representing the relative numbers of IMCs in the culture. <i>P</i> value<0.05 represent significant difference for both percentage of undivided CD4⁺ and CD8⁺ T-cells between lineage positive with lineage negative and CD11b⁺ GR-1⁺ IMCs at (IMC: T ratios of 1 and 0.5). Data from a single experiment, representative of 4 individual experiments, is shown.</p

Conjugated monoclonal antibodies.

<p>Conjugated monoclonal antibodies.</p

Co culture of Dynabead activated T-cells with CD11b⁺ GR-1⁺ IMC induced T-cell apoptosis.

<p>Left panel: frequencies of 7-AAD (+)/Annexin V (+) CD4⁺ and CD8⁺ T-cells cultured in the absence (filled symbols) or presence of CD11b⁺GR-1⁺ IMCs (empty symbols), as determined by flow cytometry. Right panel: Viability of T-cells and the T-cell lymphoblastic cell line, LBRM, cultured in the presence and absence of CD11b⁺ sorted IMCs Trypan blue staining was performed after 4 days of culture (<i>p</i> = 0.0088). Data represent of mean values (± SD) of three experiments.</p

Partially abrogation of IMC-suppression by IL-4 neutralizing antibody and IFN-γ KO IMCs.

<p>A) Inhibition of Dynabead-induced proliferation of T-cells in co-culture with CD11b⁺GR-1⁺ IMCs was measured in the presence of neutralizing antibodies to TGFβ <i>(p>0.05)</i>, IL-4 (<i>p</i> = 0.0103), and IL-10 (<i>p>0.05</i>). B) Inhibition of proliferation of T-cells in co-culture with CD11b⁺GR-1⁺ IMCs isolated from wild-type mice (controls) or knock out mice for IDO <i>(p>0.05)</i>, and IFN-γ receptor (<i>p</i> = 0.0358). Additional combination used wild-type IMCs co cultured with IFN-γ KO T-cells. Data represent the mean and SD of three experiments.</p

Carfilzomib Treatment Causes Molecular and Functional Alterations of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Background Anticancer therapies have significantly improved patient outcomes; however, cardiac side effects from cancer therapies remain a significant challenge. Cardiotoxicity following treatment with proteasome inhibitors such as carfilzomib is known in clinical settings, but the underlying mechanisms have not been fully elucidated. Methods and Results Using human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) as a cell model for drug‐induced cytotoxicity in combination with traction force microscopy, functional assessments, high‐throughput imaging, and comprehensive omic analyses, we examined the molecular mechanisms involved in structural and functional alterations induced by carfilzomib in hiPSC‐CMs. Following the treatment of hiPSC‐CMs with carfilzomib at 0.01 to 10 µmol/L, we observed a concentration‐dependent increase in carfilzomib‐induced toxicity and corresponding morphological, structural, and functional changes. Carfilzomib treatment reduced mitochondrial membrane potential, ATP production, and mitochondrial oxidative respiration and increased mitochondrial oxidative stress. In addition, carfilzomib treatment affected contractility of hiPSC‐CMs in 3‐dimensional microtissues. At a single cell level, carfilzomib treatment impaired Ca2+ transients and reduced integrin‐mediated traction forces as detected by piconewton tension sensors. Transcriptomic and proteomic analyses revealed that carfilzomib treatment downregulated the expression of genes involved in extracellular matrices, integrin complex, and cardiac contraction, and upregulated stress responsive proteins including heat shock proteins. Conclusions Carfilzomib treatment causes deleterious changes in cellular and functional characteristics of hiPSC‐CMs. Insights into these changes could be gained from the changes in the expression of genes and proteins identified from our omic analyses