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Significance: Label-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to 75 frames / s ). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining. Aim: We aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images. Approach: Here, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz., M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability. Results: We validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves 912.3 lp / mm , and our analysis scheme accurately retrieves the phase with a high correlation coefficient ( â¼ 0.99 ), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be 0.55 µ m for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings. Conclusions: Label-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects.
Assuntos
Algoritmos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Linhagem Celular Tumoral , Microscopia de Contraste de Fase/métodos , Células MCF-7 , Microscopia de Fluorescência/métodosRESUMO
Approximately 75% of breast cancers are estrogen receptor alpha-positive (ERα+), and targeting ERα directly with ERα antagonists/degraders or indirectly with aromatase inhibitors is a successful therapeutic strategy. However, such treatments are rarely curative and development of resistance is universal. We recently reported ErSO, a compound that induces ERα-dependent cancer cell death through a mechanism distinct from clinically approved ERα drugs, via hyperactivation of the anticipatory unfolded protein response. ErSO has remarkable tumor-eradicative activity in multiple ERα+ tumor models. While ErSO has promise as a new drug, it has effects on ERα-negative (ERα-) cells in certain contexts. Herein, we construct modified versions of ErSO and identify variants with enhanced differential activity between ERα+ and ERα- cells. We report ErSO-DFP, a compound that maintains antitumor efficacy, has enhanced selectivity for ERα+ cancer cells, and is well tolerated in rodents. ErSO-DFP and related compounds represent an intriguing new class for the treatment of ERα+ cancers.
Assuntos
Neoplasias da Mama , Receptor alfa de Estrogênio , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Receptores de Estrogênio/metabolismo , Resposta a Proteínas não DobradasRESUMO
[This corrects the article DOI: 10.1021/acscentsci.2c00598.].
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Genomic studies and experiments with permeability-deficient strains have revealed a variety of biological targets that can be engaged to kill Gram-negative bacteria. However, the formidable outer membrane and promiscuous efflux pumps of these pathogens prevent many candidate antibiotics from reaching these targets. One such promising target is the enzyme FabI, which catalyzes the rate-determining step in bacterial fatty acid biosynthesis. Notably, FabI inhibitors have advanced to clinical trials for Staphylococcus aureus infections but not for infections caused by Gram-negative bacteria. Here, we synthesize a suite of FabI inhibitors whose structures fit permeation rules for Gram-negative bacteria and leverage activity against a challenging panel of Gram-negative clinical isolates as a filter for advancement. The compound to emerge, called fabimycin, has impressive activity against >200 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and does not kill commensal bacteria. X-ray structures of fabimycin in complex with FabI provide molecular insights into the inhibition. Fabimycin demonstrates activity in multiple mouse models of infection caused by Gram-negative bacteria, including a challenging urinary tract infection model. Fabimycin has translational promise, and its discovery provides additional evidence that antibiotics can be systematically modified to accumulate in Gram-negative bacteria and kill these problematic pathogens.
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Zampanolide and dactylolide are microtubule-stabilizing polyketides possessing potent cytotoxicity towards a variety of cancer cell lines. Using our understanding of the conformational preferences of the macrolide core in both natural products, we hypothesized that analogues lacking the C17-methyl group would maintain the necessary conformation for bioactivity while reducing the number of synthetic manipulations necessary for their synthesis. Analogues 3, 4 and 5 were prepared via total synthesis, and their conformational preferences were determined through computational and high-field NMR studies. While no observable activities were present in dactylolide analogues 3 and 4, zampanolide analogue 5 exhibited sub-micromolar cytotoxicity. Herein, we describe these efforts towards understanding the structure- and conformation-activity relationships of dactylolide and zampanolide.