Browsing by Subject "IMAGE-ANALYSIS"

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  • Barmaki, Samineh; Jokinen, Ville; Obermaier, Daniela; Blokhina, Daria; Korhonen, Matti; Ras, Robin H. A.; Vuola, Jyrki; Franssila, Sami; Kankuri, Esko (2018)
    Physiological oxygen levels within the tissue microenvironment are usually lower than 14%, in stem cell niches these levels can be as low as 0-1%. In cell cultures, such low oxygen levels are usually mimicked by altering the global culture environment either by O-2 removal (vacuum or oxygen absorption) or by N-2 supplementation for O-2 replacement. To generate a targeted cellular hypoxic microenvironment under ambient atmospheric conditions, we characterised the ability of the dissolved oxygen-depleting sodium sulfite to generate an in-liquid oxygen sink. We utilised a microfluidic design to place the cultured cells in the vertical oxygen gradient and to physically separate the cells from the liquid. We demonstrate generation of a chemical in-liquid oxygen sink that modifies the surrounding O-2 concentrations. O-2 level control in the sink-generated hypoxia gradient is achievable by varying the thickness of the polydimethylsiloxane membrane. We show that intracellular hypoxia and hypoxia response element-dependent signalling is instigated in cells exposed to the microfluidic in-liquid O-2 sink-generated hypoxia gradient. Moreover, we show that microfluidic flow controls site-specific microenvironmental kinetics of the chemical O-2 sink reaction, which enables generation of intermittent hypoxia/re-oxygenation cycles. The microfluidic O-2 sink chip targets hypoxia to the cell culture microenvironment exposed to the microfluidic channel architecture solely by depleting O-2 while other sites in the same culture well remain unaffected. Thus, responses of both hypoxic and bystander cells can be characterised. Moreover, control of microfluidic flow enables generation of intermittent hypoxia or hypoxia/re-oxygenation cycles. (C) 2018 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
  • Hlushchenko, Iryna; Khanal, Pushpa; Abouelezz, Amr; Paavilainen, Ville O.; Hotulainen, Pirta (2018)
    Many actin cytoskeleton-regulating proteins control dendritic spine morphology and density, which are cellular features often altered in autism spectrum disorder (ASD). Recent studies using animal models show that autism-related behavior can be rescued by either manipulating actin regulators or by reversing dendritic spine density or morphology. Based on these studies, the actin cytoskeleton is a potential target pathway for developing new ASD treatments. Thus, it is important to understand how different ASD-associated actin regulators contribute to the regulation of dendritic spines and how ASD-associated mutations modulate this regulation. For this study, we selected five genes encoding different actin-regulating proteins and induced ASD-associated de novo missense mutations in these proteins. We assessed the functionality of the wild-type and mutated proteins by analyzing their subcellular localization, and by analyzing the dendritic spine phenotypes induced by the expression of these proteins. As the imbalance between excitation and inhibition has been suggested to have a central role in ASD, we additionally evaluated the density, size and subcellular localization of inhibitory synapses. Common for all the proteins studied was the enrichment in dendritic spines. ASD-associated mutations induced changes in the localization of alpha-actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, which localized more to dendritic spines. Among the wild-type proteins studied, only alpha-actinin-4 expression caused a significant change in dendritic spine morphology by increasing the mushroom spine density and decreasing thin spine density. We hypothesized that mutations associated with ASD shift dendritic spine morphology from mushroom to thin spines. An M554V mutation in alpha-actinin-4 (ACTN4) resulted in the expected shift in dendritic spine morphology by increasing the density of thin spines. In addition, we observed a trend toward higher thin spine density withmutations inmyosin IXb and SWAP-70. Myosin IIb and myosin IXb expression increased the proportion of inhibitory synapses in spines. The expression of mutated myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) induced variable changes in inhibitory synapses.
  • Hultsch, Susanne; Kankainen, Matti; Paavolainen, Lassi; Kovanen, Ruusu-Maaria; Ikonen, Elina; Kangaspeska, Sara; Pietiäinen, Vilja; Kallioniemi, Olli (2018)
    Background: Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment. Methods: In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort Results: We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib. Conclusion: Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype.
  • Hurskainen, Pekka; Adhikari, Hari; Siljander, Mika; Pellikka, Petri; Hemp, Andreas (2019)
    Classifying land use/land cover (LULC) with sufficient accuracy in heterogeneous landscapes is challenging using only satellite imagery. To improve classification accuracy inclusion of features from auxiliary geospatial datasets in classification models is applied since 1980s. However, the method is mostly limited to pixel-based classifications, and the coverage, accuracy and resolution of free and open-access auxiliary datasets have been poor until recent years. We evaluated how recent global coverage open-access geospatial datasets improve object-based LULC classification accuracy compared to using only spectral and texture features from satellite images. We applied feature sets topography, population, soil, canopy cover, distance to watercourses and spectral-temporal metrics from Landsat-8 time series on the southern foothills and savanna of Mt. Kilimanjaro, Tanzania, where the landscape is characterized by heterogeneous and fragmented mosaic of disturbed savanna vegetation, croplands, and settlements. The classification was based on image objects (groups of spectrally similar pixels) derived from segmentation of four Formosat-2 scenes with 8m spatial resolution using 1370 ground reference points for training, validation, and for defining 17 LULC classes. We built six Random Forest classification models with different sets of object features in each. The baseline model having only spectral and texture features was compared with five other models supplemented with auxiliary features. Inclusion of auxiliary features significantly improved classification overall accuracy (OA). The baseline model gave a median OA of 60.7%, but auxiliary features in other models increased median OA between 6.1 and 16.5 percentage points. The best OA was achieved with a model including all features of which elevation was the most important auxiliary feature followed by Enhanced Vegetation Index temporal range and slope degree. Applying object-based classification to geospatial information on topography, soil, settlement patterns and vegetation phenology, the discriminatory potential of challenging LULC classes can be significantly improved. We demonstrated this for the first time, and the technique shows good potential for improving LULC mapping across a multitude of fragmented landscapes worldwide.
  • Jokinen, Hanna; Goncalves, Nicolau; Vigario, Ricardo; Lipsanen, Jari; Fazekas, Franz; Schmidt, Reinhold; Barkhof, Frederik; Madureira, Sofia; Verdelho, Ana; Inzitari, Domenico; Pantoni, Leonardo; Erkinjuntti, Timo; LADIS Study Grp (2015)
    White matter lesions (WML) are the main brain imaging surrogate of cerebral small-vessel disease. A new MRI tissue segmentation method, based on a discriminative clustering approach without explicit model based added prior, detects partial WML volumes, likely representing very early-stage changes in normal-appearing brain tissue. This study investigated how the different stages of WML, from a "pre-visible" stage to fully developed lesions, predict future cognitive decline. MRI scans of 78 subjects, aged 65-84 years, from the Leukoaraiosis and Disability (LADIS) study were analyzed using a self supervised multispectral segmentation algorithm to identify tissue types and partial VVML volumes. Each lesion voxel was classified as having a small (33%), intermediate (66%), or high (100%) proportion of lesion tissue. The subjects were evaluated with detailed clinical and neuropsychological assessments at baseline and at three annual follow-up visits. We found that voxels with small partial WML predicted lower executive function compound scores at baseline, and steeper decline of executive scores in follow-up, independently of the demographics and the conventionally estimated hyperintensity volume on fluid-attenuated inversion recovery images. The intermediate and fully developed lesions were related to impairments in multiple cognitive domains including executive functions, processing speed, memory, and global cognitive function. In conclusion, early-stage partial WML, still too faint to be clearly detectable on conventional MRI, already predict executive dysfunction and progressive cognitive decline regardless of the conventionally evaluated WML load. These findings advance early recognition of small vessel disease and incipient vascular cognitive impairment.
  • Hynninen, Ville; Hietala, Sami; McKee, Jason R.; Murtomäki, Lasse; Rojas, Orlando J.; Ikkala, Olli; Nonappa, [No Value] (2018)
    We show that composite hydrogels comprising methyl cellulose (MC) and cellulose nanocrystal (CNC) colloidal rods display a reversible and enhanced rheological storage modulus and optical birefringence upon heating, i.e., inverse thermoreversibility. Dynamic rheology, quantitative polarized optical microscopy, isothermal titration calorimetry (ITC), circular dichroism (CD), and scanning and transmission electron microscopy (SEM and TEM) were used for characterization. The concentration of CNCs in aqueous media was varied up to 3.5 wt % (i.e, keeping the concentration below the critical aq concentration) while maintaining the MC aq concentration at 1.0 wt %. At 20 degrees C, MC/CNC underwent gelation upon passing the CNC concentration of 1.5 wt %. At this point, the storage modulus (G') reached a plateau, and the birefringence underwent a stepwise increase, thus suggesting a percolative phenomenon. The storage modulus (G') of the composite gels was an order of magnitude higher at 60 degrees C compared to that at 20 degrees C. ITC results suggested that, at 60 degrees C, the CNC rods were entropically driven to interact with MC chains, which according to recent studies collapse at this temperature into ring-like, colloidal-scale persistent fibrils with hollow cross-sections. Consequently, the tendency of the MC to form more persistent aggregates promotes the interactions between the CNC chiral aggregates towards enhanced storage modulus and birefringence. At room temperature, ITC shows enthalpic binding between CNCs and MC with the latter comprising aqueous, molecularly dispersed polymer chains that lead to looser and less birefringent material. TEM, SEM, and CD indicate CNC chiral fragments within a MC/CNC composite gel. Thus, MC/CNC hybrid networks offer materials with tunable rheological properties and access to liquid crystalline properties at low CNC concentrations.
  • Redchuk, Taras A.; Karasev, Maksim M.; Omelina, Evgeniya S.; Verkhusha, Vladislav V. (2018)
    Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner, QPAS1, is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested the functionality of two BphP1-QPAS1-based optogenetic toolsan NIR- and blue-light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA)in several cell types, including cortical neurons. We found that the performance of these optogenetic tools often relied on physiological properties of a specific cell type, such as nuclear transport, which could limit the applicability of the blue-light-sensitive component of iRIS. In contrast, the NIR-light-sensing component of iRIS performed well in all tested cell types. The TA system showed the best performance in cervical cancer (HeLa), bone cancer (U-2 OS), and human embryonic kidney (HEK-293) cells. The small size of the QPAS1 component allowed the design of adeno-associated virus (AAV) particles, which were applied to deliver the TA system to neurons.
  • Redchuk, Taras A.; Kaberniuk, Andrii A.; Verkhusha, Vladislav V. (2018)
    Near-infrared (NIR, 740-780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1-QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1-QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1-QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3-5 d. Use of NIR light-controlled optogenetic systems should advance basic and biomedical research.
  • De Santis, Ilaria; Tasnadi, Ervin; Horvath, Peter; Bevilacqua, Alessandro; Piccinini, Filippo (2019)
    Featured Application The main goal of this work is to provide an overview of open-source tools available for researchers interested in estimating the volume of 3D multicellular aggregates (e.g., spheroids, organoids), besides introducing a new version of the Reconstruction and Visualization from Multiple Sections (ReViMS) tool ( Abstract The volume is one of the most relevant features that define the treatment of an in vivo tumour. When using cancer 3D in vitro models in pre-clinical studies, it becomes important to evaluate the macroscopic effects of drugs and radiotherapy treatments. Depending on the nature of the 3D in vitro model used, different open-source solutions can be used for measuring the volume by starting from microscope-acquired images. In this work, we introduced several open-source tools today available for estimating the volume of 3D multicellular aggregates (e.g., spheroids, organoids), also giving hints for defining the best software by analysing characteristics of 3D in vitro models and limits of the tools. Finally, using several cancer organoids imaged by a fluorescent microscope, we compared volume estimations obtained with different tools, besides presenting a new version of the Reconstruction and Visualization from Multiple Sections (ReViMS version 2.0) tool. This work aims to be the reference for researchers interested in estimating the volume of 3D multicellular aggregates through an open-source tool.
  • Piccinini, Filippo; Balassa, Tamas; Carbonaro, Antonella; Diosdi, Akos; Toth, Timea; Moshkov, Nikita; Tasnadi, Ervin A.; Horvath, Peter (2020)
    Today, we are fully immersed into the era of 3D biology. It has been extensively demonstrated that 3D models: (a) better mimic the physiology of human tissues; (b) can effectively replace animal models; (c) often provide more reliable results than 2D ones. Accordingly, anti-cancer drug screenings and toxicology studies based on multicellular 3D biological models, the so-called "-oids" (e.g. spheroids, tumoroids, organoids), are blooming in the literature. However, the complex nature of these systems limit the manual quantitative analyses of single cells' behaviour in the culture. Accordingly, the demand for advanced software tools that are able to perform phenotypic analysis is fundamental. In this work, we describe the freely accessible tools that are currently available for biologists and researchers interested in analysing the effects of drugs/treatments on 3D multicellular-oids at a single-cell resolution level. In addition, using publicly available nuclear stained datasets we quantitatively compare the segmentation performance of 9 specific tools. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.
  • Pezzini, Francesco; Bettinetti, Laura; Di Leva, Francesca; Bianchi, Marzia; Zoratti, Elisa; Carrozzo, Rosalba; Santorelli, Filippo M.; Delledonne, Massimo; Lalowski, Maciej; Simonati, Alessandro (2017)
    Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.
  • Almangush, Alhadi; Pirinen, Matti; Heikkinen, Ilkka; Mäkitie, Antti A.; Salo, Tuula; Leivo, Ilmo (2018)
    Background: Tumour budding has been reported as a promising prognostic marker in many cancers. This meta-analysis assessed the prognostic value of tumour budding in oral squamous cell carcinoma (OSCC). Methods: We searched OvidMedline, PubMed, Scopus and Web of Science for articles that studied tumour budding in OSCC. We used reporting recommendations for tumour marker (REMARK) criteria to evaluate the quality of studies eligible for meta-analysis. Results: A total of 16 studies evaluated the prognostic value of tumour budding in OSCC. The meta-analysis showed that tumour budding was significantly associated with lymph node metastasis (odds ratio = 7.08, 95% CI = 1.75-28.73), disease-free survival (hazard ratio = 1.83, 95% CI = 1.34-2.50) and overall survival (hazard ratio = 1.88, 95% CI = 1.25-2.82). Conclusions: Tumour budding is a simple and reliable prognostic marker for OSCC. Evaluation of tumour budding could facilitate personalised management of OSCC.
  • Lehtimaki, Jaakko I.; Fenix, Aidan M.; Kotila, Tommi M.; Balistreri, Giuseppe; Paavolainen, Lassi; Varjosalo, Markku; Burnette, Dylan T.; Lappalainen, Pekka (2017)
    Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.