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At least 289 records · Page 16

Direct and rapid identification of T315I-Mutated BCR-ABL expressing leukemic cells using infrared microspectroscopy

Highlights: • BCR-ABL T315I mutation can be detected in single leukemic cells using infrared microspectroscopy. • The specificity of this spectral signature is demonstrated using Dox-inducible T315I-mutated BCR-ABL expressing cell lines. • Leukemic cells expressing T315I-mutated BCR-ABL exhibit a distinct transcriptome signature. • This technology could be applied to rapid detection of leukemic cells expressing T315I and to drug screening strategies. Despite the major success obtained by the use of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), resistances to therapies occur due to mutations in the ABL-kinase domain of the BCR-ABL oncogene. Amongst these mutations, the “gatekeeper” T315I is a major concern as it renders leukemic cells resistant to all licenced TKI except Ponatinib. We report here that Fourier transform infrared (FTIR) microspectroscopy is a powerful methodology allowing rapid and direct identification of a spectral signature in single cells expressing T315I-mutated BCR-ABL. The specificity of this spectral signature is confirmed using a Dox-inducible T315I-mutated BCR-ABL-expressing human UT-7 cells as well as in murine embryonic stem cells. Transcriptome analysis of UT-7 cells expressing BCR-ABL as compared to BCR-ABL T315I clearly identified a molecular signature which could be at the origin of the generation of metabolic changes giving rise to the spectral signature. Thus, these results suggest that this new methodology can be applied to the identification of leukemic cells harbouring the T315I mutation at the single cell level and could represent a novel early detection tool of mutant clones. It could also be applied to drug screening strategies to target T315I-mutated leukemic cells.

60 APPLIED LIFE SCIENCES↗

Single-cell profiling of Arabidopsis leaves to Pseudomonas syringae infection

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We expose Arabidopsis to Pseudomonas syringae or mock treatment and profile >11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from both treatments identifies distinct pathogen-responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection reveals a continuum of disease progression from an immune to a susceptible state. Confocal imaging of promoter-reporter lines for transcripts enriched in immune cell clusters shows expression surrounding substomatal cavities colonized or in close proximity to bacterial colonies, suggesting that cells within immune clusters represent sites of early pathogen invasion. Susceptibility clusters exhibit more general localization and are highly induced at later stages of infection. Overall, our work shows cellular heterogeneity within an infected leaf and provides insight into plant differential response to infection at a single-cell level.

59 BASIC BIOLOGICAL SCIENCES↗

Evidence for Divisome Localization Mechanisms Independent of the Min System and SlmA in Escherichia coli

Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. coli than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins.

59 BASIC BIOLOGICAL SCIENCES↗

Technoeconomic Analysis of Dye Sensitized Solar Cells (DSSCs) with WS 2 /Carbon Composite as Counter Electrode Material

Exploration of clean and renewable energy materials is necessary due to the coming energy crisis and environmental problems. Solar energy is one of the favorable energy sources because of the continuous energy reservoir and its affluence. Silicon-based solar devices are expensive due to their complicated production process, which limits this technology for urban and other commercial applications. Among the third generation of solar cells, Dye-Sensitized Solar Cells (DSSCs) have attracted widespread attention as potential cost-effective alternatives to silicon-based solar cells. In this paper, the commercializing potential of the DSSCs is investigated. A module is introduced where the materials, equipment, and distribution of direct manufacturing costs are calculated. The manufacturing costs and the Levelized Cost of Energy (LCOE) of these DSSCs for a system lifetime of 25 years were determined to be USD 22.40 per m 2 and USD 0.0438 per kWh and the module price of this technology is USD 0.18 per W and the total installed system cost is USD 0.88 per W in Kansas which suggest that this PV technology could challenge other leading PV technologies.

14 SOLAR ENERGY↗

Status Update on NETL Techno-Economic Analysis of Solid Oxide Cells

This is a presentation delivered at the Solid Oxide Fuel Cell Project Review Meeting portion of the 2023 FECM / NETL Spring R&D Project Review Meeting on April 19, 2023. The presentation highlights recently completed techno-economic analysis of solid oxide cell systems including a brief overview of pairing SOFC technology with energy storage, an analysis of the tradeoffs associated with reversible SOC technology versus paired SOFC/SOEC, and highlights of the recently released NGFC and IGFC pathway studies. A more detailed description of analysis conducted on hydrogen fueled SOFC systems is also included.

Hackett, Gregory↗

On the Efficacy of Repeat Voltage Holds for Conditioning and Calendar Life Testing of Graphite and Silicon Cells

Voltage-hold (V-hold) protocols have shown promise toward calendar lifetime analysis of cells with graphite (Gr) and silicon (Si) anodes. In this work, repeat V-holds are performed on Gr and Si cells paired with lithium iron phosphate cathodes to delineate their beneficial role in formation and conditioning. We find that V-hold at the top of charge supplements constant current cycling in conditioning the cell to higher capacities for both Gr and Si cells after the first V-hold. A reduced order model provides the irreversible capacity proportions of each V-hold. With each repeat V-hold, parasitic loss of lithium to the solid electrolyte interphase (SEI) decreases on both Gr and Si cells. Gr cells show the square-root-of-time capacity loss behavior within 200 h of V-hold, indicative of its fast relaxation and low impact of reference performance test cycles on the SEI growth. Lifetime estimates from repeat V-holds on Gr can reach years. Si exhibits longer transition times from kinetic to diffusion-limited SEI growth, evidenced by the 400 h and 200 h holds showing square-root-of-time and linear behavior, respectively. Lifetime predictions from repeat V-holds on Si only reach 1–2 months, highlighting its limitations. Recommended duration of V-holds for Si cells should be ≥400 h.

25 ENERGY STORAGE↗

Destructive Physical Analysis of Flight- and Ground-Tested Sodium-Sulfur Cells

Destructive physical analysis (DPA) was used to study the effects of microgravity on the sulfur electrode in sodium-sulfur cells. The cells examined in this work were provided by the Air Force Research Laboratory (AFRL) from their program on sodium-sulfur technology. The Naval Research Laboratory (NRL) provided electrical characterization of the flight-tested and ground-tested cells.

Wasz, Margot L.↗

Destructive physical analysis results of Ni/H2 cells cycled in LEO regime

Six 48-Ah individual pressure vessel (IPV) Ni/H2 cells containing 26 and 31 percent KOH electrolyte were life cycle tested in low Earth orbit. All three cells containing 31 percent KOH failed (3729, 4165, and 11,355 cycles), while those with 26 percent KOH were cycled over 14,000 times in the continuing test. Destructive physical analysis (DPA) of the failed cells included visual inspections, measurements of electrode thickness, scanning electron microscopy, chemical analysis, and measurements of nickel electrode capacity in an electrolyte flooded cell. The cycling failure was due to a decrease of nickel electrode capacity. As possible causes of the capacity decrease, researchers observed electrode expansion, rupture, and corrosion of the nickel electrode substrate, active material redistribution, and accumulation of electrochemically undischargeable active material with cycling.

Lim, Hong S.↗

Destructive physical analysis results of Ni/H2 cells cycled in LEO regime

Six 48-Ah individual pressure vessel (IPV) Ni/H2 cells containing 26 and 31 percent KOH electrolyte were life cycle tested in low earth orbit. All three cells containing 31 percent KOH failed (3729, 4165, and 11,355 cycles), while those with 26 percent KOH were cycled over 14,000 times in the continuing test. Destructive physical analysis (DPA) of the failed cells included visual inspections, measurements of electrode thickness, scanning electron microscopy, chemical analysis, and measurements of nickel electrode capacity in an electrolyte flooded cell. The cycling failure was due to a decrease of nickel electrode capacity. As possible causes of the capacity decrease, researchers observed electrode expansion, rupture, and corrosion of the nickel electrode substrate, active material redistribution, and accumulation of electrochemically undischargeable active material with cycling.

Lim, Hong S.↗

Silicon Solar Cell Process Development, Fabrication and Analysis

Ribbon to Ribbon (RTR) solar cells processed from polycrystalline feedstock showed maximum AMO efficiency of 5.6%. Solar cells from single crystalline feedstock showed slightly higher efficiency than those from polycrystalline feedstock, indicating maximum efficiency of about 6.6% with SiO AR coating. Single crystalline control cells gave 11-12% AMO efficiencies demonstrating that the poor performance of the RTR solar was due to the low quality of material itself. Dendritic web solar cells from the standard process showed maximum AMO efficiency of 9.8% while efficiency of control solar cells were around 11-12%. Web solar cells from back surface field (BSF) process indicated maximum AMO efficiency of 10.9%. Some improvement in open circuit voltage was noticed from the BSF process. Small light spot scanning experiments were carried out on the solar cells from Wacker Silso, EFG, RTR, and dendritic web ribbons. Photoresponse results provided information on localized cell performance and grain size in polycrystalline material, and agreed quite well with the cell performance data, such as efficiency, minority carrier diffusion length, and spectral response.

Yoo, H. I.↗

SINGLE: Atomic-resolution structure identification of nanocrystals by graphene liquid cell EM

Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. We developed a “one-particle Brownian 3D reconstruction method” based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2.5 ms) resolution and analyzed to obtain an ensemble of 3D reconstructions. Here, we introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo–electron microscopy. Our developments are made available through the open-source software package SINGLE.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Facile carrier-assisted targeted mass spectrometric approach for proteomic analysis of low numbers of mammalian cells

There is an unmet technical challenge for mass spectrometry (MS)-based proteomic analysis of single mammalian cells. Quantitative proteomic analysis of single cells has been previously achieved by antibody-based immunoassays but is limited by the availability of high-quality antibodies. Herein we report a facile targeted MS-based proteomics method, termed cPRISM-SRM (carrier-assisted high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected reaction monitoring), for reliable analysis of low numbers of mammalian cells. The method capitalizes on using “carrier protein” to assist processing of low numbers of cells with minimal loss, high-resolution PRISM separation for target peptide enrichment, and sensitive SRM for protein quantification. We have demonstrated that cPRISM-SRM has sufficient sensitivity to quantify proteins expressed at ≥200,000 copies per cell at the single-cell level and ≥3000 copies per cell in 100 mammalian cells. We envision that with further improvement cPRISM-SRM has the potential to move toward targeted MS-based single-cell proteomics.

59 BASIC BIOLOGICAL SCIENCES↗

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, in this paper, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally. Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.

59 BASIC BIOLOGICAL SCIENCES↗

Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells

Highlights: • Disulfiram and copper synergistically inhibit lung cancer cell proliferation. • Lung cancer cell colony formation ability is inhibited by Disulfiram/copper. • Disulfiram/copper increases the sensitivity of cisplatin to lung cancer cells. • Lung cancer stem cells are specifically targeted by Disulfiram/copper complex. - Abstract: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.

60 APPLIED LIFE SCIENCES↗

Growth and Remodeling in Blood Vessels Studied In Vivo With Fractal Analysis

Every cell in the human body must reside in close proximity to a blood vessel (within approximately 200 mm) because blood vessels provide the oxygen, metabolite, and fluid exchanges required for cellular existence. The growth and remodeling of blood vessels are required to support the normal physiology of embryonic development, reproductive biology, wound healing and adaptive remodeling to exercise, as well as abnormal tissue change in diseases such as cancer, diabetes, and coronary heart disease. Cardiovascular and hemodynamic (blood flow dynamics) alterations experienced by astronauts during long-term spaceflight, including orthostatic intolerance, fluid shifts in the body, and reduced numbers of red (erythrocyte) and white (immune) blood cells, are identified as risk factors of very high priority in the NASA task force report on risk reduction for human spaceflight, the "Critical Path Roadmap."

Parsons-Wingerter, Patricia A.↗

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2 - 2/Ins2 ). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.

59 BASIC BIOLOGICAL SCIENCES↗