| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Dass, Randall A., et al.
(författare)
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Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I
- 2016
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Ingår i: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 12:8
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.
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| 3. |
- Hoshino, Ayuko, et al.
(författare)
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Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers
- 2020
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Ingår i: Cell. - : CELL PRESS. - 0092-8674 .- 1097-4172. ; 182:4, s. 1044-
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Tidskriftsartikel (refereegranskat)abstract
- There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n =151) and plasma-derived (n =120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
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| 4. |
- Prakash, Varsha, et al.
(författare)
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Ribosome biogenesis during cell cycle arrest fuels EMT in development and disease
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ER alpha) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.
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| 5. |
- Blösch, Günter, et al.
(författare)
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Twenty-three unsolved problems in hydrology (UPH) - a community perspective
- 2019
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Ingår i: Hydrological Sciences Journal. - : Informa UK Limited. - 0262-6667 .- 2150-3435. ; 64:10, s. 1141-1158
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Tidskriftsartikel (refereegranskat)abstract
- This paper is the outcome of a community initiative to identify major unsolved scientific problems in hydrology motivated by a need for stronger harmonisation of research efforts. The procedure involved a public consultation through online media, followed by two workshops through which a large number of potential science questions were collated, prioritised, and synthesised. In spite of the diversity of the participants (230 scientists in total), the process revealed much about community priorities and the state of our science: a preference for continuity in research questions rather than radical departures or redirections from past and current work. Questions remain focused on the process-based understanding of hydrological variability and causality at all space and time scales. Increased attention to environmental change drives a new emphasis on understanding how change propagates across interfaces within the hydrological system and across disciplinary boundaries. In particular, the expansion of the human footprint raises a new set of questions related to human interactions with nature and water cycle feedbacks in the context of complex water management problems. We hope that this reflection and synthesis of the 23 unsolved problems in hydrology will help guide research efforts for some years to come.
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| 6. |
- Kurylo, Chad M., et al.
(författare)
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Endogenous rRNA Sequence Variation Can Regulate Stress Response Gene Expression and Phenotype
- 2018
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Ingår i: Cell Reports. - : CELL PRESS. - 2211-1247. ; 25:1, s. 236-248.e6
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Tidskriftsartikel (refereegranskat)abstract
- Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, ReIA and ReIE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology.
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| 7. |
- Mutvei, Anders P., et al.
(författare)
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Rap1-GTPases control mTORC1 activity by coordinating lysosome organization with amino acid availability
- 2020
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The kinase mTOR complex 1 (mTORC1) promotes cellular growth and is frequently dysregulated in cancers. In response to nutrients, mTORC1 is activated on lysosomes by Rag and Rheb guanosine triphosphatases (GTPases) and drives biosynthetic processes. How limitations in nutrients suppress mTORC1 activity remains poorly understood. We find that when amino acids are limited, the Rap1-GTPases confine lysosomes to the perinuclear region and reduce lysosome abundance, which suppresses mTORC1 signaling. Rap1 activation, which is independent of known amino acid signaling factors, limits the lysosomal surface available for mTORC1 activation. Conversely, Rap1 depletion expands the lysosome population, which markedly increases association between mTORC1 and its lysosome-borne activators, leading to mTORC1 hyperactivity. Taken together, we establish Rap1 as a critical coordinator of the lysosomal system, and propose that aberrant changes in lysosomal surface availability can impact mTORC1 signaling output.
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| 8. |
- Parks, Matthew M., et al.
(författare)
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Implications of sequence variation on the evolution of rRNA
- 2019
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Ingår i: Chromosome Research. - : Springer. - 0967-3849 .- 1573-6849. ; 27:1-2, s. 89-93
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Forskningsöversikt (refereegranskat)abstract
- The evolution of the multi-copy family of ribosomal RNA (rRNA) genes is unique in regard to its genetics and genome evolution. Paradoxically, rRNA genes are highly homogenized within and between individuals, yet they are globally distinct between species. Here, we discuss the implications for models of rRNA gene evolution in light of our recent discoveries that ribosomes bearing rRNA sequence variants can affect gene expression and physiology and that intra-individual rRNA alleles exhibit both context- and tissue-specific expression.
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| 9. |
- Parks, Matthew M., et al.
(författare)
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Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression
- 2018
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Ingår i: Science Advances. - : AMER ASSOC ADVANCEMENT SCIENCE. - 2375-2548. ; 4:2
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Tidskriftsartikel (refereegranskat)abstract
- The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosomes molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra-and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease.
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| 10. |
- Johard, Helena, et al.
(författare)
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HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment
- 2020
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Ingår i: Molecular Cancer Research. - 1541-7786 .- 1557-3125. ; 18:10, s. 1522-1533
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Tidskriftsartikel (refereegranskat)abstract
- Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition.
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| 11. |
- Maier, Jürgen, et al.
(författare)
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This time it's different? : Effects of the Eurovision Debate on young citizens and its consequence for EU democracy - evidence from a quasi-experiment in 24 countries
- 2018
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Ingår i: Journal of European Public Policy. - : Informa UK Limited. - 1350-1763 .- 1466-4429. ; 25:4, s. 606-629
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Tidskriftsartikel (refereegranskat)abstract
- For the very first time in EU history, the 2014 EP elections provided citizens with the opportunity to influence the nomination of the Commission President by casting a vote for the main Europarties’ ‘lead candidates’. By subjecting the position of the Commission President to an open political contest, many experts have formulated the expectation that heightened political competition would strengthen the weak electoral connection between EU citizens and EU legislators, which some consider a root cause for the EU’s lack of public support. In particular, this contest was on display in the so-called ‘Eurovision Debate’, a televised debate between the main contenders for the Commission President broadcasted live across Europe. Drawing on a quasi-experimental study conducted in 24 EU countries, we find that debate exposure led to increased cognitive and political involvement and EU support among young citizens. Unfortunately, the debate has only reached a very small audience.
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| 12. |
- Niklasson, Mia, et al.
(författare)
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Membrane-Depolarizing Channel Blockers Induce Selective Glioma Cell Death by Impairing Nutrient Transport and Unfolded Protein/Amino Acid Responses
- 2017
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Ingår i: Cancer Research. - : AMER ASSOC CANCER RESEARCH. - 0008-5472 .- 1538-7445. ; 77:7, s. 1741-1752
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Tidskriftsartikel (refereegranskat)abstract
- Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstreamsignaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas.
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| 13. |
- Nilchian, Azadeh, et al.
(författare)
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CXADR-Mediated Formation of an AKT Inhibitory Signalosome at Tight Junctions Controls Epithelial-Mesenchymal Plasticity in Breast Cancer.
- 2019
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Ingår i: Cancer Research. - : American Association for Cancer Research. - 0008-5472 .- 1538-7445. ; 79:1, s. 47-60
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Tidskriftsartikel (refereegranskat)abstract
- Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial-mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie- and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyper-activation of AKT and sensitized cells to TGF-β1-induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKT-inhibitory signalosome at TJ and regulates epithelial-mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer.
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| 14. |
- Rios-Fuller, Tiffany J., et al.
(författare)
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Translation Regulation by eIF2 α Phosphorylation and mTORC1 Signaling Pathways in Non-Communicable Diseases (NCDs)
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:15
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Forskningsöversikt (refereegranskat)abstract
- Non-communicable diseases (NCDs) are medical conditions that, by definition, are non-infectious and non-transmissible among people. Much of current NCDs are generally due to genetic, behavioral, and metabolic risk factors that often include excessive alcohol consumption, smoking, obesity, and untreated elevated blood pressure, and share many common signal transduction pathways. Alterations in cell and physiological signaling and transcriptional control pathways have been well studied in several human NCDs, but these same pathways also regulate expression and function of the protein synthetic machinery and mRNA translation which have been less well investigated. Alterations in expression of specific translation factors, and disruption of canonical mRNA translational regulation, both contribute to the pathology of many NCDs. The two most common pathological alterations that contribute to NCDs discussed in this review will be the regulation of eukaryotic initiation factor 2 (eIF2) by the integrated stress response (ISR) and the mammalian target of rapamycin complex 1 (mTORC1) pathways. Both pathways integrally connect mRNA translation activity to external and internal physiological stimuli. Here, we review the role of ISR control of eIF2 activity and mTORC1 control of cap-mediated mRNA translation in some common NCDs, including Alzheimer's disease, Parkinson's disease, stroke, diabetes mellitus, liver cirrhosis, chronic obstructive pulmonary disease (COPD), and cardiac diseases. Our goal is to provide insights that further the understanding as to the important role of translational regulation in the pathogenesis of these diseases.
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| 15. |
- Strbkova, Lenka, et al.
(författare)
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Automated interpretation of time-lapse quantitative phase image by machine learning to study cellular dynamics during epithelial-mesenchymal transition
- 2020
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Ingår i: Journal of Biomedical Optics. - : SPIE - The International Society for Optics and Photonics. - 1083-3668 .- 1560-2281. ; 25:8
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Tidskriftsartikel (refereegranskat)abstract
- Significance: Machine learning is increasingly being applied to the classification of microscopic data. In order to detect some complex and dynamic cellular processes, time-resolved live-cell imaging might be necessary. Incorporating the temporal information into the classification process may allow for a better and more specific classification.Aim: We propose a methodology for cell classification based on the time-lapse quantitative phase images (QPIs) gained by digital holographic microscopy (DHM) with the goal of increasing performance of classification of dynamic cellular processes.Approach: The methodology was demonstrated by studying epithelial-mesenchymal transition (EMT) which entails major and distinct time-dependent morphological changes. The time-lapse QPIs of EMT were obtained over a 48-h period and specific novel features representing the dynamic cell behavior were extracted. The two distinct end-state phenotypes were classified by several supervised machine learning algorithms and the results were compared with the classification performed on single-time-point images.Results: In comparison to the single-time-point approach, our data suggest the incorporation of temporal information into the classification of cell phenotypes during EMT improves performance by nearly 9% in terms of accuracy, and further indicate the potential of DHM to monitor cellular morphological changes.Conclusions: Proposed approach based on the time-lapse images gained by DHM could improve the monitoring of live cell behavior in an automated fashion and could be further developed into a tool for high-throughput automated analysis of unique cell behavior.
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