Morphology Of Nuclei Research Articles

Anticancer activity of Lannea coromandelica on B16F10 melanoma cell line: an in vitro and molecular docking approach

IntroductionPhytochemical screening was conducted on various bark extracts of Lannea coromandelica to assess their anticancer property against the B16F10 melanoma cell line. The phytoconstituents that were previously identified were utilized in molecular docking studies against the human tyrosinase related protein 1 (TYRP1) as a target receptor in order to provide more evidence for anticancer property. MethodsBark powder was extracted by maceration method using distilled water and soxhlet extraction using ethanol. The preliminary phytochemical evaluation and determination of total phenolic and flavonoid content of both extracts were conducted using biochemical assays. The present study investigated the possible anticancer effects of ethanol and aqueous extracts on the B16F10 melanoma cell line using the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay and 4,6-diamidino-2-phenylindole (DAPI) labeling techniques. The present study employed molecular docking techniques to assess the binding interactions between phytoconstituents and the TYRP1 protein, utilizing AutoDock Vina module of PyRx 0.8 software. ResultsThe phytochemical analysis found flavonoids, steroids, terpenoids, tannins, phenolic compounds, saponins, anthraquinones, cardiac glycosides, and proteins. Ethanolic extract shown preferential cytotoxicity to B16F0 melanoma cell line in-vitro (IC50=9.69±0.68μg/ml), while aqueous extract exhibited IC50=75.49±5.95μg/ml. DAPI staining showed that treated cells had altered nucleus morphology, including apoptotic bodies. According to molecular docking investigations, Quercetin has the highest binding affinity (−9.6 Kcal/mol), followed by Catechin and Myricadiol. ConclusionThe current investigation has determined that L. coromandelica exhibits cytotoxic characteristic, as evidenced by the utilization of computer aided drug design models and in-vitro experimentation.

Microwave scattering properties of ice crystal particles during the melting process.

Ice crystal particles play an important role in the study of cloud resolution, climate models, and radiative forcing. During the melting process, significant changes occur in the microphysical properties of ice crystal particles, such as the ice phase state, morphology, and mixing state. This process further affects the scattering and radiation characteristics properties of ice crystal particles. In this study, we constructed a non-spherical and inhomogeneous particle model based on the melting process of ice crystal particles. The scattering properties of melting ice crystal particles under four selected microwave frequency bands (92 GHz, 220 GHz, 280 GHz, and 340 GHz) are investigated by using discrete dipole approximation (DDA) method. The influence of ice crystal content (ICC) and particle aspect ratio on the scattering properties of ice crystal particles under thin coating and medium coating conditions are emphasized. The results show that the melting process significantly affects the scattering properties of melting ice crystal particles in a frequency dependent manner. Additionally, even slight melting of ice crystal particles leads to drastic changes in their scattering properties. Furthermore, we found that the morphology of ice crystal nuclei has a significant impact on their scattering characteristics even at medium levels of melting degree. In summary, this study confirms that it is essential to consider morphology and inhomogeneous characteristics during the melting process for microwave detection of ice crystal particles. This research may have significant implications for studies related to detection and inversion techniques for ice crystal particles.

Active Galactic Nucleus Jet-inflated Bubbles as Possible Origin of Odd Radio Circles

Odd radio circles (ORCs) are newly discovered extragalactic radio objects with an unknown origin. In this work, we carry out three-dimensional cosmic-ray (CR) magnetohydrodynamic simulations using the FLASH code and predict the radio morphology of end-on active galactic nucleus (AGN) jet-inflated bubbles considering hadronic emission. We consider CR proton (CRp)-dominated jets as they tend to inflate oblate bubbles, promising to reproduce the large inferred sizes of the ORCs when viewed end-on. We find that powerful and long-duration CRp-dominated jets can create bubbles with similar sizes (∼300–600 kpc) and radio morphology (circular and edge-brightened) to the observed ORCs in low-mass (M vir ∼ 8 × 1012 − 8 × 1013 M ⊙) halos. Given the same amount of input jet energy, longer-duration (thus lower-power) jets tend to create larger bubbles since high-power jets generate strong shocks that carry away a significant portion of the jet energy. The edge-brightened feature of the observed ORCs is naturally reproduced due to efficient hadronic collisions at the interface between the bubbles and the ambient medium. We further discuss the radio luminosity, X-ray detectability, and the possible origin of such strong AGN jets in the context of galaxy evolution. We conclude that end-on CR-dominated AGN bubbles could be a plausible scenario for the formation of ORCs.

Microtubules Disruption Alters the Cellular Structures and Mechanics Depending on Underlying Chemical Cues.

The extracellular matrix determines cell morphology and stiffness by manipulating the cytoskeleton. The impacts of extracellular matrix cues, including the mechanical and topographical cues on microtubules and their role in biological behaviors, are previously studied. However, there is a lack of understanding about how microtubules (MTs)are affected by environmental chemical cues, such as extracellular matrix density. Specifically, it is crucial to understand the connection between cellular morphology and mechanics induced by chemical cues and the role of microtubules in these cellular responses. To address this, surfaces with high and low cRGD (cyclic Arginine-Glycine-Aspartic acid) peptide ligand densities are used. The cRGD is diluted with a bioinert ligand to prevent surface native cellular remodeling. The cellular morphology, actin, and microtubules differ on these surfaces. Confocal fluorescence microscopes and atomic force microscopy (AFM) are used to determine the structural and mechanical cellular responses with and without microtubules. Microtubules are vital as an intracellular scaffold in elongated morphology correlated with low cRGD compared to rounded morphology in high cRGD substrates. The contributions of MTs to nucleus morphology and cellular mechanics are based on the underlying cRGD densities. Finally, this study reveals a significant correlation between MTs, actin networks, and vimentin in response to the underlying densities of cRGD.

The Chinese Herbal Formula Weichang'an Reduces the Proliferation of Human Gastric Cancer Cells via Mitochondrial Apoptosis

Abstract Objective The aim of the study was to investigate the effects of Chinese herbal formula Weichang'an (WCA) on the proliferation and mitochondria-mediated apoptosis of human gastric cancer cells. Methods Cell Counting Kit-8 (CCK8) was used to evaluate the antiproliferative activity of WCA on MKN45 cells; Giemsa staining was used to investigate cell colony formation; flow cytometry was used to analyze cell cycle, apoptosis rate, and caspases activation; and Hoechst staining was used to analyze the morphology of cell nuclei. The mitochondrial membrane potential was analyzed by both flow cytometric measurements and fluorescence microscopy. Western blot was used to analyze the protein levels of pro-caspase-3, Bcl-2, Bax, and Bcl-X. MKN45 cells were subcutaneously injected into the right forelimb of 16 nude mice to establish the tumor xenograft model, and a total of 12 nude mouse tumor xenograft models were successfully created. The nude mice were divided into the control group and the WCA-treated group. The two groups received normal saline and WCA treatment at 35.49 g/kg by a single daily oral gavage for 21 days. The body weight and tumor size of the nude mice were measured twice a week. The ultrastructural changes of subcutaneous tumors were observed by a transmission electron microscope. Results WCA can suppress cell proliferation and colony formation. It can also induce changes in the mitochondrial membrane potential and increase the activities of caspases-3, caspases-8, and caspases-9 in MKN45 cells. WCA induced S-phase arrest and apoptosis in MKN45 cells, and decreased the expression levels of antiapoptotic proteins Bcl-2 and Bcl-X in MKN45 cells, while increasing the expression levels of the proapoptotic proteins Bax and pro-caspase-3 compared with the control group. WCA inhibited the growth of a xenografted MKN45 tumor in nude mice, and the protein levels of Bax and pro-caspase-3 were significantly increased, while Bcl-X and Bcl-2 were reduced compared with the control group. The differences are statistically significant (p < 0.05). Conclusion WCA could suppress the proliferation of gastric cancer cells via mitochondrial apoptosis.

DB-FCN: An end-to-end dual-branch fully convolutional nucleus detection model

Detecting cells or nuclei in histopathological image analysis is a crucial prerequisite for subsequent cancer diagnosis. By precisely locating cell nuclei, pathologists can quantitatively analyze the morphology of each cell nucleus. This enables accurate cancer grading, allowing for the implementation of tailored treatment plans based on distinct cancer stages. Manual nucleus detection methods are labor-intensive, making the development of automatic cell nucleus detection algorithms highly necessary. However, due to the small size of the nucleus and increased adhesions between cells, tissue staining may be uneven, leading to a higher occurrence of false positives in the results of the current automatic nucleus detection algorithm. This paper introduces an end-to-end dual-branch-based fully convolutional neural network (DB-FCN) that effectively addresses the aforementioned challenges, thereby enhancing the accuracy of automatic nucleus detection. This algorithm introduces for the first time the use of two detection branches, namely the coarse detection branch and the fine detection branch, to accomplish the detection task. The role of the coarse detection branch is to identify all cell regions in the pathological image as comprehensively as possible and then transmit the detection results as prior information to the fine detection branch. The fine detection branch is necessary solely to conduct more precise detection based on the coarse detection results. Given that the coarse detection branch has already eliminated interference from many background regions, the fine detection branch can focus on detecting the nucleus region, thereby significantly enhancing the efficiency and accuracy of model detection. The detection model proposed in this paper was evaluated on three classic datasets and compared with many existing detection algorithms. Compared with other algorithms, the detection algorithm proposed in this paper has made significant progress in detecting cell nuclei in histopathological images.

Comprehensive Biosafety Profile of Carbomer-Based Hydrogel Formulations Incorporating Phosphorus Derivatives.

Determining the safety of a newly developed experimental product is a crucial condition for its medical use, especially for clinical trials. In this regard, four hydrogel-type formulations were manufactured, all of which were based on carbomer (Blank-CP940) and encapsulated with caffeine (CAF-CP940), phosphorus derivatives (phenyl phosphinic (CAF-S1-CP940) and 2-carboxyethyl phenyl phosphinic acids (CAF-S2-CP940)). The main aim of this research was to provide a comprehensive outline of the biosafety profile of the above-mentioned hydrogels. The complex in vitro screening (cell viability, cytotoxicity, morphological changes in response to exposure, and changes in nuclei morphology) on two types of healthy skin cell lines (HaCaT-human keratinocytes and JB6 Cl 41-5a-murine epidermal cells) exhibited a good biosafety profile when both cell lines were treated for 24 h with 150 μg/mL of each hydrogel. A comprehensive analysis of the hydrogel's impact on the genetic profile of HaCaT cells sustains the in vitro experiments. The biosafety profile was completed with the in vivo and in ovo assays. The outcome revealed that the developed hydrogels exerted good biocompatibility after topical application on BALB/c nude mice's skin. It also revealed a lack of toxicity after exposure to the hen's chicken embryo. Further investigations are needed, regarding the in vitro and in vivo therapeutic efficacy and safety for long-term use and potential clinical translatability.

Cholinergic nucleus degeneration and its association with gait impairment in Parkinson’s disease

BackgroundThe contribution of cholinergic degeneration to gait disturbance in Parkinson’s disease (PD) is increasingly recognized, yet its relationship with dopaminergic-resistant gait parameters has been poorly investigated. We investigated the association between comprehensive gait parameters and cholinergic nucleus degeneration in PD.MethodsThis cross-sectional study enrolled 84 PD patients and 69 controls. All subjects underwent brain structural magnetic resonance imaging to assess the gray matter density (GMD) and volume (GMV) of the cholinergic nuclei (Ch123/Ch4). Gait parameters under single-task (ST) and dual-task (DT) walking tests were acquired using sensor wearables in PD group. We compared cholinergic nucleus morphology and gait performance between groups and examined their association.ResultsPD patients exhibited significantly decreased GMD and GMV of the left Ch4 compared to controls after reaching HY stage > 2. Significant correlations were observed between multiple gait parameters and bilateral Ch123/Ch4. After multiple testing correction, the Ch123/Ch4 degeneration was significantly associated with shorter stride length, lower gait velocity, longer stance phase, smaller ankle toe-off and heel-strike angles under both ST and DT condition. For PD patients with HY stage 1–2, there were no significant degeneration of Ch123/4, and only right side Ch123/Ch4 were corrected with the gait parameters. However, as the disease progressed to HY stage > 2, bilateral Ch123/Ch4 nuclei showed correlations with gait performance, with more extensive significant correlations were observed in the right side.ConclusionsOur study demonstrated the progressive association between cholinergic nuclei degeneration and gait impairment across different stages of PD, and highlighting the potential lateralization of the cholinergic nuclei’s impact on gait impairment. These findings offer insights for the design and implementation of future clinical trials investigating cholinergic treatments as a promising approach to address gait impairments in PD.

Multi-headed U-Net: an automated nuclei segmentation technique using Tikhonov filter-based unsharp masking

ABSTRACT An automated nuclei segmentation is the key technique for understanding and analyzing cellular properties, which are helpful for disease diagnosis and support computer-aided digital pathology. However, this task is challenging because of the variability in nuclei size and morphology, which results in blurry boundaries and overlapping nuclei. To address such issues, a multi-headed U-Net convolutional neural network (CNN) architecture has been proposed. This architecture has multiple heads to extract multi-resolution features of the source image by using different kernel sizes of the filters. The source images are pre-processed using an unsharp masking approach based on the Tikhonov filter. The Tikhonov filter decomposes the input image into low-frequency and high-frequency band images. The unsharp masking method improves the high-frequency information of the input image by primarily enhancing features such as boundaries, contours, and fine details. We have incorporated intersection over union (IOU) and F1Score as measures along with accuracy for our proposed objective functions. The proposed objective functions are tried to be maximized by the optimization algorithm, and the higher value of the metrics indicates better segmentation performance in the spatial domain during the testing phase. The proposed method attained IOU(JI), Accuracy, Precision, and F1Score values as 0.8299, 0.9642, 0.8918, and 0.9070, respectively. The quantitative and qualitative experimental outcomes indicate that our proposed technique outperforms the state-of-the-art techniques.

A novel BSA-coated nano selenium-impregnated scaffold showed improved strength, cellular attachment and proliferation in C2C12 cell

This study aims to develop scaffolds (CCS, CCS-SeNPs, CCS-SA-SeNPs, and CCS-PET-SeNPs) with enhanced mechanical strength, thermal stability, and antioxidant properties to improve cell attachment and proliferation of the C2C12 cell line for cardiac health applications. Collagen and chitosan are popular biopolymers in tissue engineering for their biocompatibility, biodegradability, and support for cell growth. Selenium nanoparticles (SeNPs) are incorporated into scaffolds for their antioxidant, anti-inflammatory, and antimicrobial properties, which enhance tissue regeneration. Sodium alginate and pectin, natural polysaccharides, further modify these scaffolds to improve structural and functional properties. Developing these composite scaffolds aims to optimize biomaterial performance in regenerative medicine. In the present study, various types of scaffolds (CCS, CCS-SeNPs, CCS-SA-SeNPs, and CCS-PET-SeNPs scaffolds) were prepared and characterized using X-ray diffraction (XRD), Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDX), and Thermo-Gravimetric Analysis (TGA). The XRD data revealed that all the scaffolds (CCS, CCS-SeNPs, CCS-SA-SeNPs, and CCS-PET-SeNPs) displayed an amorphous structure. SEM analysis demonstrated that the CCS and CCS-SA-SeNPs scaffolds exhibit solid-walled metrics and a smooth surface. EDX confirmed the homogeneous Se distribution within the scaffolds. The tensile strength of CCS, CCS-SeNPs, CCS-SA-SeNPs, and CCS-PET-SeNPs scaffolds was found to be 5.15 N, 4.77 N, 4.05 N, and 4.01 N, respectively. Thermal stability assessments showed that CCS-SeNPs, CCS-SA-SeNPs and CCS-PET-SeNPs scaffolds displayed good thermal stability, with maximum decomposition occurring at 250 °C. Furthermore, the CCS-SeNPs, CCS-SA-SeNPs, and CCS-PET-SeNPs scaffolds showed notable antioxidant activity of ∼ 57 %, compared to the CCS scaffold’s 42 %. Morphological studies of C2C12 cells on these scaffolds showed better proliferation on SeNPs-enhanced scaffolds. Nucleus morphology remained unchanged, indicating no adverse effects from SeNPs. The results indicate that the scaffolds (CCS, CCS-SeNPs, CCS-SA-SeNPs, and CCS-PET-SeNPs) demonstrate notable enhancements in mechanical strength, thermal stability, and antioxidant efficacy. These advancements are anticipated to promote enhanced cell adhesion and proliferation of the C2C12 cell line, thereby emphasizing their potential utility in cardiac health applications.

Activation of stress reactions in the dinophyte microalga Prorocentrum cordatum as a consequence of the toxic effect of ZnO nanoparticles and zinc sulfate

According to the results of the experimental study, the main regularities of changes in morphological, structural-functional and fluorescent indices of P. cordatum were established when zinc oxide nanoparticles ZnO NPs (0.3–6.4 mg L−1) and Zn in form of salt (0.09–0.4 mg L−1) were added to the medium. The studied pollutants have cytotoxic (growth inhibition, development of oxidative stress, destruction of cytoplasmic organelles, disorganization of mitochondria) and genotoxic (changes in the morphology of nuclei, chromatin condensation) effects on microalgae, affecting almost all aspects of cell functioning. Despite the similar mechanism of action of zinc sulfate and ZnO NPs on P. cordatum cells, the negative effect of ZnO NPs is also due to the inhibition of photosynthetic activity of cells (significant decrease in the maximum quantum yield of photosynthesis and electron transport rate), reduction of chlorophyll concentration from 3.5 to 1.8 pg cell−1, as well as mechanical effect on cells: deformation and damage of cell membranes, aggregation of NPs on the cell surface. Apoptosis-like signs of cell death upon exposure to zinc sulfate and ZnO NPs were identified by flow cytometry and laser scanning confocal microscopy methods: changes in cell morphology, cytoplasm retraction, development of oxidative stress, deformation of nuclei, and disorganization of mitochondria. It was shown that the first signs of cell apoptosis appear at 0.02 mg L−1 Zn and 0.6 mg L−1 ZnO NPs after 72 h of exposure. At higher concentrations of pollutants, a dose-dependent decrease in algal enzymatic activity (up to 5 times relative to control) and mitochondrial membrane potential (up to 4 times relative to control), and an increase in the production of reactive oxygen species (up to 4–5 times relative to control) were observed. The results of the presented study contribute to the disclosure of fundamental mechanisms of toxic effects of pollutants and prediction of ways of phototrophic microorganisms reaction to this impact.

From Epimedium to Neuroprotection: Exploring the Potential of Wushanicaritin.

Epimedium has been used for functional foods with many beneficial functions to human health. Wushanicaritin is one of the most important chemicals int Epimedium. This study investigated the neuroprotective effects of wushanicaritin and potential underlying mechanisms. The results demonstrated that wushanicaritin possessed superior intercellular antioxidant activity compared to icaritin. Wushanicaritin, with an EC50 value of 3.87 μM, showed better neuroprotective effect than quercetin, a promising neuroprotection agent. Wushanicaritin significantly reversed lactate dehydrogenase release, reactive oxygen species generation, cell apoptosis, and mRNA expression related to cell apoptosis and oxidative defense, in glutamate-induced PC-12 cells. Wushanicaritin could also maintain the enzymatic antioxidant defense system and mitochondrial function. The suppression of caspase-3 activation and amelioration of mitochondrial membrane potential loss and nucleus morphology changes were involved in the antiapoptotic effect of wushanicaritin. These findings suggested that wushanicaritin possesses excellent intercellular antioxidant and neuroprotective activities, showing potential promise in functional foods.

Mitochondria-localizing triphenylphosphine-8-hydroxyquinoline Ru complexes induce ferroptosis and their antitumor evaluation

Ruthenium complexes are one of the most promising anticancer drugs and ferroptosis is a novel form of regulated cell death, the study on the effect of Ru complexes on ferroptosis is helpful to find more effective antitumor drugs. Here, the synthesis and characterization of two Ru complexes containing 8-hydroxylquinoline and triphenylphosphine as ligands, [Ru(L1) (PPh3)2Cl2] (Ru-1), [Ru(L2) (PPh3)2Cl2] (Ru-2), were reported. Complexes Ru-1 ∼ Ru-2 showed good anticancer activity in Hep-G2 cells. Researches indicated that complexes Ru-1 ∼ Ru-2 could be enriched and appear as red fluorescence in the mitochondria, arouse dysfunction of mitochondria, induce the accumulation of reactive oxygen species (ROS) and lipid peroxidation (LPO), while the morphology of nuclei and cell apoptosis had no significant change. Further experiments proved that GPX4 and Ferritin were down-regulated, which eventually triggered ferroptosis in Hep-G2 cells. Remarkably, Ru-1 showed high inhibitory activity against xenograft tumor growth in vivo (TGIR = 49%). This study shows that the complex Ru-1 could act as a novel drug candidate by triggering cell ferroptosis.

Exposure of the stingless bee Melipona scutellaris to imidacloprid, pyraclostrobin, and glyphosate, alone and in combination, impair its walking activity and fat body morphology and physiology

The stingless bee Melipona scutellaris performs buzz pollination, effectively pollinating several wild plants and crops with economic relevance. However, most research has focused on honeybees, leaving a significant gap in studies concerning native species, particularly regarding the impacts of pesticide combinations on these pollinators. Thus, this study aimed to evaluate the sublethal effects of imidacloprid (IMD), pyraclostrobin (PYR), and glyphosate (GLY) on the behavior and fat body cell morphology and physiology of M. scutellaris. Foragers were orally exposed to the different pesticides alone and in combination for 48 h. Bees fed with contaminated solution walked less, moved slower, presented morphological changes in the fat body, including vacuolization, altered cell shape and nuclei morphology, and exhibited a higher count of altered oenocytes and trophocytes. In all exposed groups, alone and in combination, the number of cells expressing caspase-3 increased, but the TLR4 number of cells expressing decreased compared to the control groups. The intensity of HSP70 immunolabeling increased compared to the control groups. However, the intensity of the immunolabeling of HSP90 decreased in the IMD, GLY, and I + G (IMD + GLY) groups but increased in I + P-exposed bees (IMD + PYR). Alternatively, exposure to PYR and P + G (PYR + GLY) did not affect the immunolabeling intensity. Our findings demonstrate the hazardous effects and environmental consequences of isolated and combined pesticides on a vital neotropical pollinator. Understanding how pesticides impact the fat body can provide crucial insights into the overall health and survival of native bee populations, which can help develop more environmentally friendly approaches to agricultural practices.

N-Rich Bilayer Solid Electrolyte Interphase toward Highly Reversible Lithium Metal Batteries.

Continuous lithium (Li)/electrolyte interfacial reactions and uncontrollable Li dendrites severely hamper the application of paradigmatic Li metal batteries (LMBs). Aiming to address the above-mentioned crucial issues, N-rich polymer-inorganic bilayers at the Li/electrolyte interface are designed via nitrate-rich electrolytes, achieving high-energy-density and long-lifespan LMBs. The inner layer of Li3N favors rapid and uniform Li+ deposition, while the outer layer of N-containing flexible polymers facilitates uniform Li+ distribution at the interlayer and accommodates volume changes during cycling. The synergistic effect of N-rich polymer-inorganic bilayers promotes the formation of dense uniform spherical nuclei morphology instead of dendrites, thus significantly improving the plating-stripping reversibility of LMBs. Attributed to the unique interphase, the Li|Li cell can stably run for over 1000 h at 1.0 mA cm-2 with an even deposition morphology, which is monitored and proven by in situ optical microscopy. Moreover, the assembled Li|S cell displays a high capacity of 697.6 mA h g-1 for over 150 cycles and a 99% Coulombic efficiency. This work paves the way for designing high-energy and long-lifespan LMBs.

Application of confocal microscopy and flow cytometry to identify physiological responses of Prorocentrum micans to the herbicide glyphosate

The physiological response of the dinoflagellate P. micans to the effect of the herbicide glyphosate at a concentration of 25–200 μg L−1 was evaluated. It has been shown that P. micans is able to grow due to the consumption of dissolved organic phosphorus formed as a result of the mineralization of glyphosate by bacteria. The addition of glyphosate to the medium inhibits the photosynthetic activity of cells; there is a pronounced inhibition of the relative electron transfer rate along the electron transport chain and the maximum quantum efficiency of the use of light energy. Morphological and ultrastructural changes in P. micans cells were evaluated at sublethal (150 μg L−1) and lethal (200 μg L−1) glyphosate concentrations. It has been shown that at a herbicide concentration of 150 μg L−1, the first signs of apoptosis appear in most P. micans cells: a decrease in lateral light scattering, cytoplasmic retraction, partial destruction of cytoplasmic organelles, a change in the morphology of nuclei, mitochondria, a change in the potential of mitochondrial membranes, and a decrease in the autofluorescence of chlorophyll in cells. At a glyphosate concentration of 200 μg L−1, P. micans showed signs of a late stage of apoptosis: violation of the integrity of intracellular organelles and chromatin organization, fragmentation of nuclei, condensation of cytoplasm, disorganization of chloroplasts in the cells, and the release of cell contents beyond the cell membrane. The effectiveness of using flow cytometry and laser scanning confocal microscopy methods for identifying signs and stages of cell apoptosis when exposed to glyphosate is discussed.

Morphological Changes of 3T3 Cells under Simulated Microgravity.

Cells are sensitive to changes in gravity, especially the cytoskeletal structures that determine cell morphology. The aim of this study was to assess the effects of simulated microgravity (SMG) on 3T3 cell morphology, as demonstrated by a characterization of the morphology of cells and nuclei, alterations of microfilaments and microtubules, and changes in cycle progression. 3T3 cells underwent induced SMG for 72 h with Gravite®, while the control group was under 1G. Fluorescent staining was applied to estimate the morphology of cells and nuclei and the cytoskeleton distribution of 3T3 cells. Cell cycle progression was assessed by using the cell cycle app of the Cytell microscope, and Western blot was conducted to determine the expression of the major structural proteins and main cell cycle regulators. The results show that SMG led to decreased nuclear intensity, nuclear area, and nuclear shape and increased cell diameter in 3T3 cells. The 3T3 cells in the SMG group appeared to have a flat form and diminished microvillus formation, while cells in the control group displayed an apical shape and abundant microvilli. The 3T3 cells under SMG exhibited microtubule distribution surrounding the nucleus, compared to the perinuclear accumulation in control cells. Irregular forms of the contractile ring and polar spindle were observed in 3T3 cells under SMG. The changes in cytoskeleton structure were caused by alterations in the expression of major cytoskeletal proteins, including β-actin and α-tubulin 3. Moreover, SMG induced 3T3 cells into the arrest phase by reducing main cell cycle related genes, which also affected the formation of cytoskeleton structures such as microfilaments and microtubules. These results reveal that SMG generated morphological changes in 3T3 cells by remodeling the cytoskeleton structure and downregulating major structural proteins and cell cycle regulators.

Abstract A032: Cellos: High-throughput deconvolution of 3D organoid dynamics at cellular resolution for cancer pharmacology

Abstract Three-dimensional (3D) culture models, such as organoids, are flexible systems to interrogate cellular evolution, cellular growth and morphology, multicellular spatial architecture, and cell interactions in response to drug treatment. However, new computational methods to segment and analyze 3D models at cellular resolution with sufficiently high throughput are needed to realize these possibilities. Here we report Cellos (Cell and Organoid Segmentation), an accurate, high throughput image analysis pipeline for 3D organoid and nuclear segmentation analysis. Cellos segments organoids in 3D using classical algorithms and segments nuclei using a Stardist-3D convolutional neural network which we trained on manually annotated dataset of 3,862 cells. To evaluate the capabilities of Cellos we then analyzed 74,450 organoids with 1.65 million cells, from multiple experiments on triple negative breast cancer organoids containing clonal mixtures with complex cisplatin sensitivies. Cellos was able to accurately distinguish ratios of distinct fluorescently labeled cell populations in organoids, with < 3% deviation from seeding ratios in each well and was effective for both fluorescently labelled nuclei and independent Hoechst stained datasets. Cellos was able to recapitulate traditional luminescence-based drug response quantification by analyzing 3D images, including parallel analysis of multiple cancer clones in the same well. Moreover, Cellos was able to identify organoid and nuclei morphology features associated with treatment and unique to each of the clones. Finally, Cellos enables 3D analysis of cell spatial relationships, which we used to detect ecological affinity between cancer clones beyond what arises from local cell division and organoid composition. Cellos provides powerful tools to perform high throughput analysis for pharmacological testing and biological investigation of organoids based on 3D imaging. Citation Format: Patience Mukashyaka, Pooja Kumar, David J. Mellert, Shadae Nicholas, Javad Noorbakhsh, Mattia Brugiolo, Olga Anczukow, Edison T. Liu, Jeffrey H. Chuang. Cellos: High-throughput deconvolution of 3D organoid dynamics at cellular resolution for cancer pharmacology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A032.

COVID-19 plasma induces subcellular remodelling within the pulmonary microvascular endothelium

BackgroundCOVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can affect multiple organ systems, including the pulmonary vasculature. Endothelial cells (ECs) are thought to play a key role in the propagation of COVID-19, however, our understanding of the exact scale of dysregulation sustained by the pulmonary microvasculature (pMV) remains incomplete. Here we aim to identify transcriptional, phenotypic, and functional changes within the pMV induced by COVID-19. Methods and resultsHuman pulmonary microvascular endothelial cells (HPMVEC) treated with plasma acquired from patients hospitalised with severe COVID-19 were compared to HPMVEC treated with plasma from patients hospitalised without COVID-19 but with other severe illnesses. Exposure to COVID-19 plasma caused a significant functional decline in HPMVECs as seen by a decrease in both cell viability via the WST-1 cell-proliferation assay and cell-to-cell barrier function as measured by electric cell-substrate impedance sensing. High-content imaging using a Cell Painting image-based assay further quantified morphological variations within sub-cellular organelles to show phenotypic changes in the whole endothelial cell, nucleus, mitochondria, plasma membrane and nucleolus morphology. RNA-sequencing of HPMVECs treated with COVID-19 plasma suggests the observed phenotype may, in part, be regulated by genes such as SMAD7, BCOR, SFMBT1, IFIT5 and ZNF566 which are involved in transcriptional regulation, protein monoubiquitination and TGF-β signalling. Conclusion and impactDuring COVID-19, the pMV undergoes significant remodelling, which is evident based on the functional, phenotypic, and transcriptional changes seen following exposure to COVID-19 plasma. The observed morphological variation may be responsible for downstream complications, such as a decline in overall cellular function and cell-to-cell barrier integrity. Moreover, genes identified through bulk RNA sequencing may contribute to our understanding of the observed phenotype and assist in developing strategies that can inform the rescue of the dysregulated endothelium.

Single-cell manipulation by two-dimensional micropatterning

Cells are highly sensitive to their geometrical and mechanical microenvironment that directly regulate cell shape, cytoskeleton and organelle, as well as the nucleus morphology and genetic expression. The emerging two-dimensional micropatterning techniques offer powerful tools to construct controllable and well-organized microenvironment for single-cell level investigations with qualitative analysis, cellular standardization, and in vivo environment mimicking. Here, we provide an overview of the basic principle and characteristics of the two most widely-used micropatterning techniques, including photolithographic micropatterning and soft lithography micropatterning. Moreover, we summarize the application of micropatterning technique in controlling cytoskeleton, cell migration, nucleus and gene expression, as well as intercellular communication.