Cell Morphology Properties Research Articles

OpenDEP: An Open-Source Platform for Dielectrophoresis Spectra Acquisition and Analysis.

Dielectrophoretic (DEP) cell separation, which utilizes electric fields to selectively manipulate and separate cells based on their electrical properties, has emerged as a cutting-edge label-free technique. DEP separation techniques rely on differences in the electrical and morphological properties of cells, which can be obtained by a thorough analysis of DEP spectra. This article presents a novel platform, named OpenDEP, for acquiring and processing DEP spectra of suspended cells. The platform consists of lab-on-a-chip and open-source software that enables the determination of DEP spectra and electric parameters. The performance of OpenDEP was validated by comparing the results obtained using this platform with the results obtained using a commercially available device, 3DEP from DEPtech. The lab-on-a-chip design features two indium tin oxide-coated slides with a specific geometry, forming a chamber where cells are exposed to an inhomogeneous alternating electric field with different frequencies, and microscopic images of cell distributions are acquired. A custom-built software written in the Python programing language was developed to convert the acquired images into DEP spectra, allowing for the estimation of membrane and cytoplasm conductivities and permittivities. The platform was validated using two cell lines, DC3F and NIH 3T3. The OpenDEP platform offers several advantages, including easy manufacturing, statistically robust computations due to large cell population analysis, and a closed environment for sterile work. Furthermore, continuous observation using any microscope allows for integration with other techniques.

ВЛИЯНИЕ ВНЕШНИХ ФАКТОРОВ НА СВОЙСТВА ДРОЖЖЕЙ CANDIDA TROPICALIS ПРИ УТИЛИЗАЦИИ СПИРТОВОЙ БАРДЫ

To prevent environmental pollution from distillery waste, enterprises are required to carry out complete processing of stillage and / or dispose of it at treatment facilities, but it is not possible to fully utilize distillery stillage due to the lack of necessary strains of microorganisms. Therefore, in the processing of distillery stillage, the choice of a strain of microorganisms with sufficient biological activity is of particular relevance. For the first time, biotechnological utilization by the Candida tropicalis yeast strain AP-31-KBP Y-4883 was used for the processing of distillery stillage. Yeast activity was increased by two external factors: physical (UV radiation) and chemical (19-mycosaminyl nystatinolide) exposure. A rational concentration of 19-mycosaminyl-nystatinolide (500 U) was established at which Candida tropicalis colonies reached their maximum size. The efficiency of vinasse disposal was assessed by the accumulation of amine nitrogen in an aqueous solution after vinasse hydrolysis, the amount of which was determined by the formal titration method. Yeast inoculum, obtained after exposure to external factors, was introduced into a nutrient medium based on vinasse, and aerobic treatment was carried out for 72 hours. The morphological properties of cells and their number were controlled by optical and atomic force microscopy, the results of which are consistent with each other and with the biological activity of yeast. The greatest changes in the morphology of the cell surface are noted after chemical exposure: compared with the initial sample, the average cell height increased by 0.24 µm, and the surface area increased by 1.25 times. An increase in the degree of utilization of alcohol stillage by yeast was shown in the series “Candida tropicalis strain AP-31-KBP Y-4883 without treatment → UV-treated strain → antibiotic-treated strain”: indicators of biological activity increased by 4.3% (the proportion of cells saturated with glycogen ) and by 6.67% (amine nitrogen content); biomass yield increased by 1.7%. Increments of indicators are calculated in the transition from the original sample to the treated antibiotic. The assessment of the environmental hazard of bard samples was carried out using biotesting. It is shown that the efficiency of vinasse utilization increases after its treatment with yeast, which was previously exposed to UV radiation and an antibiotic. These samples were characterized by the highest germination of seeds relative to bards not treated with yeast.

Nanoscale insight into biochemical changes in cervical cancer cells exposed to adaptogenic drug

This paper describes for the first time the application of atomic force microscopy-based infrared spectroscopy (AFM-IR) to evaluate cellular response to adaptogen, based on an in vitro model of cervical cancer. HeLa cervical cells were exposed to different concentrations of withaferin A, a very promising anti-cancer adaptogenic substance. AFM-IR approach was used to image single cells post-adaptogen treatment and to track subtle biochemical changes in cells at the nanoscale level. Partial least squares (PLS) regression was applied to build predictive models that allowed for the identification of spectral markers of adaptogen-induced alterations Spectroscopic studies were enriched with fluorescence staining to determine whether the adaptogen affects cell morphology. The results showed that with the increase in the concentration of adaptogen, changes in the cell nucleus and the actin cytoskeleton become more and more significant. It has been demonstrated that the AFM-IR technique can successfully study the cellular response to the anti-cancer agent at the single-cell level with nanoscale spatial resolution. On the basis of the promising findings presented in this paper, it is possible to conclude that withaferin A has great potential in inhibiting the proliferation of cervical cancer cells in a dose-dependent manner. It has been found that both the increase in the concentration of withaferin A and the increase in incubation time with the adaptogen resulted in a decrease in the intensity of the bands assigned to nucleic acids. This may be due to DNA condensation, internuclear cleavage, or degradation during apoptosis. The findings also suggest changes in the secondary structure of proteins that may be a consequence of disruption of the actin cytoskeleton, progressive apoptosis, or significant biochemical changes. Furthermore, noticeable changes were also observed in the bands originating from lipids vibrations, and an increased share of the band near 2920 cm−1, considered an important marker of apoptosis, was noted. The metabolism of carbohydrates in cells also changes under the influence of the adaptogen. AFM-IR provides nanoscale insight into the structural and morphological properties of cells after drug treatment and is an indisputable milestone in the development of new anti-cancer approaches.

Stemness potency and structural characteristics of thyroid cancer cell lines.

Thyroid cancer is the most frequent type of endocrine malignancy. Thyroid carcinomas are derived from the follicular epithelium and classified as papillary (PTC) (85%), follicular (FTC) (12%), and anaplastic (ATC) (<3%). Thyroid cancer could arise from thyroid cancer stem-like cells (CSCs). CSCs are cancer cells that feature stem-like properties. Kruppel-like factor (KLF4) and Stage-spesific embryonic antigen 1 (SSEA-1) are types of stem cell markers. Filamentous actin (F-actin) is an essential part of the cellular cytoskeleton. The purpose of this study was to evaluate the stem cell potency and the spatial distribution of the cytoskeletal element F-actin in PTC, FTC, and ATC cell lines. Normal thyroid cell line (NTC) Nthy-ori-3-1, PTC cell line BCPAP, FTC cell line FTC-133 and ATC cell line 8505c were stained with SSEA-1 and KLF4 for stem cell potency and F-actin for cytoskeleton. The morphological properties of cells were assessed by a scanning electron microscope (SEM) and elemental ratios were compared with EDS. PTCs had greater percentages of SSEA-1 and KLF4 protein intensity (0.32% and 0.49%, respectively) than NTCs. ATCs had a greater proportion of KLF4 expression (0.8%) than NTCs. NTCs and FTCs had increased F-actin intensity across the cell, but PTCs had the lowest among these four cell lines. NTCs and PTCs, as well as NTCs and FTCs, have statistically identical aspect ratios and round values. These values, however, were statistically different in ATCs. The study of stem cell markers and the cytoskeletal element F-actin in cancer and normal thyroid cell lines may assist in the identification of new therapeutic targets and contribute in the understanding of treatment resistance mechanisms.

A multiscale framework for evaluating three-dimensional cell mechanics in fibril-reinforced poroelastic tissues with anatomical cell distribution – Analysis of chondrocyte deformation behavior in mechanically loaded articular cartilage

Characterization of the mechanical environment of cells in collagenous biological tissues during different daily activities is crucial for understanding the role of mechanics on cell biosynthesis and tissue health. However, current imaging methods are limited in characterizing very fast deformations of cells. This could be achieved with computational multiscale modeling, but current models accommodating collagen fibril networks and poroelastic ground matrix have included only a single cell. In this study, a workflow was developed for generating a three-dimensional multiscale model with imaging-based anatomical cell distributions and their micro-environment (pericellular and extracellular matrix). Fibril-reinforced poroelastic material models with (FRPES) and without (FRPE) swelling were implemented into the model and simulations were performed for evaluating cell deformations before and after experimental loading conducted for rabbit knee joint cartilage. We observed that the cells experienced considerably different deformation based on their location in all models. Both FRPE and FRPES models were able to predict the trends in the changes in cell deformations, although the average and median magnitudes differed between the model predictions and experiments. However, the FRPES model results were generally closer to the experimental results. Current findings suggest that morphological properties of cells are affected by the variations in the tissue properties between the samples and variations within the sample caused by the measurement geometry, local structure and composition. Thus, it would be important to consider the anatomical distribution and location of multiple cells along with the structure of fibril networks if cell deformation metrics are evaluated in collagenous tissues.

Cytokit: a single-cell analysis toolkit for high dimensional fluorescent microscopy imaging

BackgroundMultiplexed in-situ fluorescent imaging offers several advantages over single-cell assays that do not preserve the spatial characteristics of biological samples. This spatial information, in addition to morphological properties and extensive intracellular or surface marker profiling, comprise promising avenues for rapid advancements in the understanding of disease progression and diagnosis. As protocols for conducting such imaging experiments continue to improve, it is the intent of this study to provide and validate software for processing the large quantity of associated data in kind.ResultsCytokit offers (i) an end-to-end, GPU-accelerated image processing pipeline; (ii) efficient input/output (I/O) strategies for operations specific to high dimensional microscopy; and (iii) an interactive user interface for cross filtering of spatial, graphical, expression, and morphological cell properties within the 100+ GB image datasets common to multiplexed immunofluorescence. Image processing operations supported in Cytokit are generally sourced from existing deep learning models or are at least in part adapted from open source packages to run in a single or multi-GPU environment. The efficacy of these operations is demonstrated through several imaging experiments that pair Cytokit results with those from an independent but comparable assay. A further validation also demonstrates that previously published results can be reproduced from a publicly available multiplexed image dataset.ConclusionCytokit is a collection of open source tools for quantifying and analyzing properties of individual cells in large fluorescent microscopy datasets that are often, but not necessarily, generated from multiplexed antibody labeling protocols over many fields of view or time periods. This project is best suited to bioinformaticians or other technical users that wish to analyze such data in a batch-oriented, high-throughput setting. All source code, documentation, and data generated for this article are available under the Apache License 2.0 at https://github.com/hammerlab/cytokit.

Cytoarchitectonically-driven MRI atlas of nonhuman primate hippocampus: Preservation of subfield volumes in aging.

Identification of primate hippocampal subfields in vivo using structural MRI imaging relies on variable anatomical guidelines, signal intensity differences, and heuristics to differentiate between regions (Yushkevich et al., 2015a). Thus, a clear anatomically-driven basis for subfield demarcation is lacking. Recent work, however, has begun to develop methods to use ex vivo histology or ex vivo MRI (Adler et al., 2014; Iglesias et al., 2015) that have the potential to inform subfield demarcations of in vivo images. For optimal results, however, ex vivo and in vivo images should ideally be matched within the same healthy brains, with the goal to develop a neuroanatomically-driven basis for in vivo structural MRI images. Here, we address this issue in young and aging rhesus macaques (young n = 5 and old n = 5) using ex vivo Nissl-stained sections in which we identified the dentate gyrus, CA3, CA2, CA1, subiculum, presubiculum, and parasubiculum guided by morphological cell properties (30 μm thick sections spaced at 240 μm intervals and imaged at 161 nm/pixel). The histologically identified boundaries were merged with in vivo structural MRIs (0.625 × 0.625 × 1 mm) from the same subjects via iterative rigid and diffeomorphic registration resulting in probabilistic atlases of young and old rhesus macaques. Our results indicate stability in hippocampal subfield volumes over an age range of 13 to 32 years, consistent with previous results showing preserved whole hippocampal volume in aged macaques (Shamy et al., 2006). Together, our methods provide a novel approach for identifying hippocampal subfields in non-human primates and a potential 'ground truth' for more accurate identification of hippocampal subfield boundaries on in vivo MRIs. This could, in turn, have applications in humans where accurately identifying hippocampal subfields in vivo is a critical research goal.

Multiparameter mechanical and morphometric screening of cells.

We introduce a label-free method to rapidly phenotype and classify cells purely based on physical properties. We extract 15 biophysical parameters from cells as they deform in a microfluidic stretching flow field via high-speed microscopy and apply machine-learning approaches to discriminate different cell types and states. When employing the full 15 dimensional dataset, the technique robustly classifies individual cells based on their pluripotency, with accuracy above 95%. Rheological and morphological properties of cells while deforming were critical for this classification. We also show the application of this method in accurate classifying cells based on their viability, drug screening and detecting populations of malignant cells in mixed samples. We show that some of the extracted parameters are not linearly independent, and in fact we reach maximum classification accuracy by using only a subset of parameters. However, the informative subsets could vary depending on cell types in the sample. This work shows the utility of an assay purely based on intrinsic biophysical properties of cells to identify changes in cell state. In addition to a label-free alternative to flow cytometry in certain applications, this work, also can provide novel intracellular metrics that would not be feasible with labeled approaches (i.e. flow cytometry).

Molecular Biology of Glucocorticoid Signaling.

Well-defined as signaling hormones for the programming of cell type-specific and context-dependent gene expression signatures, glucocorticoids control experience-driven allostasis. One unifying model is that glucocorticoids help maintaining the integrity and plasticity of cellular networks in changing environments through the mobilization of cellular energy stores, profiling of gene expression, and changes in the electrical and morphological properties of cells. The nucleus is their primary site of action, yet recent discoveries point to additional gene transcription-independent functions at the plasma membrane of neuronal synapses. Glucocorticoids are secreted factors that reflect intrinsically the changes coming from the external world, temporally and regionally, during development and adulthood. In this review, we will enumerate the properties and signaling attributes of glucocorticoids and their receptors that characterize them as allostatic modulators. The molecular mechanisms used to support their role at the synapse will be highlighted.

Quantitative analysis of spiny neurons in the adult human caudate nucleus: can it confirm the current qualitative cell classification?

The caudate nucleus, as a part of the striatum (neostriatum or dorsal striatum), is involved in the control of cognitive, motor and limbic functions. The majority of the caudate nucleus cells are projection spiny neurons, whose activity is determined by excitatory inputs from the cortex, thalamus, globus pallidus and brainstem. A qualitative analysis of human caudate nucleus neurons involves the description of the structure and features of cells, and accordingly, their classification into an appropriate type. The aim of this study is to determine the justification of the current qualitative classification of spiny neurons in the precommissural head of the human caudate nucleus by quantifying morphological properties of neurons. After the qualitative analysis of microscopic images of the Golgi-impregnated caudate nucleus neurons, five morphological properties of cells were measured/quantified. In terms of the dendritic field area, caudate nucleus neurons were divided into two subgroups: small and large neurons. In our sample of 251 projection nerve cells, 58.17 % (146) were small and 41.83 % (105) were large neurons. These data show that suggested groups of spiny neurons in the human caudate nucleus differ in their morphology. Since the structure and function of cells are closely correlated, it is possible that these morphologically different types of neurons may represent different functional groups.

The cytotoxicity and anticancer mechanisms of alterporriol L, a marine bianthraquinone, against MCF-7 human breast cancer cells

Alterporriol L, a new bianthraquinone derivative, was isolated from a marine fungus Alternaria sp. ZJ9-6B. The cytotoxic activity and anticancer mechanisms of alterporriol L towards breast cancer cells lines were detected using MTT assay, immunofluorescence, and flow cytometry. Simultaneously, the changes in morphological properties of cells were detected before and after treatment with alterporriol L by atomic force microscope (AFM) at a nanometer scale. MTT assay showed that alterporriol L could effectively inhibit the growth and proliferation, and there was a dose-dependent manner of cell death. Moreover, the alterporriol L could induce cancer cell apoptosis or necrosis. Furthermore, the reactive oxygen species, mitochondrial membrane potential, and cytosolic free calcium level were changed after treatment with alterporriol L, suggesting that alterporriol L played vital roles in breast cancer cells through destroying the mitochondrial. And all these alterations are in accord with changes of morphology detected by AFM, which suggested that the AFM is a useful tool to detect the morphological changes of the cancer cells.

Long-term HIV-1 infection of neural progenitor populations

HIV can reside in the brain for many years. While astrocytes are known to tolerate long-term HIV infection, the potential of other neural cell types to harbour HIV is unclear. To investigate whether HIV can persist in neural progenitor cell populations. A multipotent human neural stem cell line (HNSC.100) was used to compare HIV infection in neural progenitor and astrocyte cell populations. Expression of cellular genes/proteins was analysed by real-time reverse transcriptase PCR, Western blot, immunocytochemistry and flow cytometry. Morphological properties of cells were measured by quantitative fluorescent image analysis. Virus release by cells exposed to HIV-1IIIB was monitored by enzyme-linked immunosorbent assay for Gag. Proviral copy numbers were determined by real-time PCR and early HIV transcripts by reverse transcriptase PCR. Rev activity was determined with a fluorescent-based reporter assay. Progenitor populations differed from astrocyte populations by showing much lower glial fibrillary acidic protein (GFAP) production, higher cell-surface expression of the CXCR4 chemokine receptor, higher Rev activity and distinct cell morphologies. HIV-exposed progenitor cultures released moderate amounts of virus for over 2 months and continued to display cell-associated HIV markers (proviral DNA, early HIV transcripts) during the entire observation period (115 days). Differentiation of HIV-infected progenitor cells to astrocytes was associated with transient activation of virus production. Long-term HIV infection of progenitor populations led to upregulation of GFAP and changes in cell morphology. These studies suggest that neural progenitor populations can contribute to the reservoir for HIV in the brain and undergo changes as a consequence of HIV persistence.

Hippocampal Heterotopia Lack Functional Kv4.2 Potassium Channels in the Methylazoxymethanol Model of Cortical Malformations and Epilepsy

Human cortical malformations often result in severe forms of epilepsy. Although the morphological properties of cells within these malformations are well characterized, very little is known about the function of these cells. In rats, prenatal methylazoxymethanol (MAM) exposure produces distinct nodules of disorganized pyramidal-like neurons (e.g., nodular heterotopia) and loss of lamination in cortical and hippocampal structures. Hippocampal nodular heterotopias are prone to hyperexcitability and may contribute to the increased seizure susceptibility observed in these animals. Here we demonstrate that heterotopic pyramidal neurons in the hippocampus fail to express a potassium channel subunit corresponding to the fast, transient A-type current. In situ hybridization and immunohistochemical analysis revealed markedly reduced expression of Kv4.2 (A-type) channel subunits in heterotopic cell regions of the hippocampus of MAM-exposed rats. Patch-clamp recordings from visualized heterotopic neurons indicated a lack of fast, transient (I(A))-type potassium current and hyperexcitable firing. A-type currents were observed on normotopic pyramidal neurons in MAM-exposed rats and on interneurons, CA1 pyramidal neurons, and cortical layer V-VI pyramidal neurons in saline-treated control rats. Changes in A-current were not associated with an alteration in the function or expression of delayed, rectifier (Kv2.1) potassium channels on heterotopic cells. We conclude that heterotopic neurons lack functional A-type Kv4.2 potassium channels and that this abnormality could contribute to the increased excitability and decreased seizure thresholds associated with brain malformations in MAM-exposed rats.

The study of dielectrophoresis of cells using the optical technique of measuring

The present report gives the main results obtained in the studies of dielectrophoresis of cellular suspensions by the optical technique of registration. The description of this technique and its comparison with the earlier developed microscopic techniques of cell dielectrophoresis is given. It is shown that the frequency dependences of the orientation and pearl-chain-formation effect of cells in the electric field and the effect of their dielectrophoretic precipitation on the electrodes, called orientation and dielectrophoretic spectra, respectively, depend on the conductivity and pH of suspension as well as on the adsorption of substances on the cell surface. The conclusion is advanced that the method of cellular dielectrophoresis is a promising one for the analysis of electrical and morphological properties of cells and for the study of the influence of physical and chemical factors on the living cells.