Morphological Characteristics Of Cells Research Articles

Optimizing the Cell Painting assay for image-based profiling.

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years' experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105.

Effects of culture conditions on isolated microspore culture of melon (Cucumis melo L.)

Isolated microspore culture is a useful tool to produce pure, fully homozygous parental lines in a short time. This study evaluated factors including the microspore developmental stage, cell culture density, and heat treatment influencing callus formation from melon microspores (Cucumis melo L.). The results showed that the obtained number of calli induced was highest (11.00±1.02) when cultivating flower buds with sizes 7.0-7.9 mm that contained microspores at middle-to-late uninucleate stages. The optimal microspore density for culture is4×104 cells. The cultured medium was NLN, containing 130 g/l of sucrose at pH 5.8. Heat treatment at 40ºC for 48 hours was best suited for callus induction of all flower bud sizes. The survival rate of microspores after 7 days of culture was lower than before inoculation and was only 75.6%. The development of the microspores and the arising of calli and embryos have been observed and evaluated for morphological cell characteristics. However, in this study, no mature embryo formation or seedling regeneration was observed.

Characterisation of cell lines derived from prostate cancer patients with localised disease.

Prostate cancer is a broad-spectrum disease, spanning from indolent to a highly aggressive lethal malignancy. Prostate cancer cell lines are essential tools to understanding the basic features of this malignancy, as well as in identifying novel therapeutic strategies. However, most cell lines routinely used in prostate cancer research are derived from metastatic disease and may not fully elucidate the molecular events underlying the early stages of cancer development and progression. Thus, there is a need for new cell lines derived from localised disease to better span the disease spectrum. Prostatic tissue from the primary site, and adjacent non-cancerous tissue was obtained from four patients with localised disease undergoing radical prostatectomy. Epithelial cell outgrowths were immortalised with human papillomavirus type 16 (HPV16) E6 and E7 to establish monoclonal cell lines. Chromosomal ploidy was imaged and STR profiles were determined. Cell morphology, colony formation and cell proliferation characteristics were assessed. Androgen receptor (AR) expression and AR-responsiveness to androgen treatment were analysed by immunofluorescence and RT-qPCR, respectively. RNA-seq analysis was performed to identify prostate lineage markers and expression of prostate cancer tumorigenesis-related genes. Two benign cell lines derived from non-cancer cells (AQ0420 and AQ0396) and two tumour tissue derived cancer cell lines (AQ0411 and AQ0415) were immortalised from four patients with localised prostatic adenocarcinoma. The cell lines presented an epithelial morphology and a slow to moderate proliferative rate. None of the cell lines formed anchorage independent colonies or displayed AR-responsiveness. Comparative RNA-seq expression analysis confirmed the prostatic lineage of the four cell lines, with a distinct gene expression profile from that of the metastatic prostate cancer cell lines, PC-3 and LNCaP. Comprehensive characterization of these cell lines may provide new in vitro tools that could bridge the current knowledge gap between benign, early-stage and metastatic disease.

Alterations of bovine nucleus pulposus cells with aging.

Aging is one of the major etiological factors driving intervertebral disc (IVD) degeneration, the main cause of low back pain. The nucleus pulposus (NP) includes a heterogeneous cell population, which is still poorly characterized. Here, we aimed to uncover main alterations in NP cells with aging. For that, bovine coccygeal discs from young (12 months) and old (10-16 years old) animals were dissected and primary NP cells were isolated. Gene expression and proteomics of fresh NP cells were performed. NP cells were labelled with propidium iodide and analysed by flow cytometry for the expression of CD29, CD44, CD45, CD146, GD2, Tie2, CD34 and Stro-1. Morphological cell features were also dissected by imaging flow cytometry. Elder NP cells (up-regulated bIL-6 and bMMP1 gene expression) presented lower percentages of CD29+, CD44+, CD45+ and Tie2+ cells compared with young NP cells (upregulated bIL-8, bCOL2A1 and bACAN gene expression), while GD2, CD146, Stro-1 and CD34 expression were maintained with age. NP cellulome showed an upregulation of proteins related to endoplasmic reticulum (ER) and melanosome independently of age, whereas proteins upregulated in elder NP cells were also associated with glycosylation and disulfide bonds. Flow cytometry analysis of NP cells disclosed the existence of 4 subpopulations with distinct auto-fluorescence and size with different dynamics along aging. Regarding cell morphology, aging increases NP cell area, diameter and vesicles. These results contribute to a better understanding of NP cells aging and highlighting potential anti-aging targets that can help to mitigate age-related disc disease.

Enhanced protein-glutaminase production from Chryseobacterium proteolyticum by the addition of leucine

Protein-glutaminase (EC 3.5.1.44, PG) derived from Chryseobacterium proteolyticum can significantly improve the solubility, emulsification and foaming properties of food proteins and is a promising deaminase. PG has broad application prospects in the food industry. However, the low yield of the enzyme is not sufficient for industrial applications. Mutagenesis and molecular breeding can improve PG yield, which consumes considerable time and has high costs. In contrast, it is a feasible way to achieve high PG yields to optimize the fermentation conditions of the strains. In this work, amino acids were screened to improve PG production. The addition of 0.80 g/L leucine (Leu) could increase PG production by 27.80%. Then, through the response surface method, it was found that under the combination of 1.60 g/L Leu, 5.65 g/L MgSO4.7 H2O and 5.13 g/L KH2PO4, PG production reached 2.93 U/mL, which was 1.87 times that of the original medium. The growth and metabolism curves of Chryseobacterium proteolyticum were measured under this formula. The effect of Leu on PG production was further investigated by measuring the transcription of the PG gene, the conductivity of fermentation fluid and the morphological characteristics of cells. This work lays a foundation for the industrial production of PG.

Microendoscopy in vivo for the pathological diagnosis of cervical precancerous lesions and early cervical cancer

BackgroundCervical cancer is an important public health problem. Conventional colposcopy is inefficient in the diagnosis of cervical lesions and massive biopsies result in trauma. There is an urgent need for a new clinical strategy to triage women with abnormal cervical screening results immediately and effectively. In this study, the high-resolution microendoscopy combined with methylene blue cell staining technology was used to perform real-time in vivo imaging of the cervix for the first time.MethodsA total of 41 patients were enrolled in the study. All patients underwent routine colposcopy and cervical biopsy, and high-resolution images of methylene blue-stained cervical lesions were obtained in vivo using microendoscopy. The cell morphological features of benign and neoplastic cervical lesions stained with methylene blue under microendoscopy were analyzed and summarized. The microendoscopy and histopathology findings of the high-grade squamous intraepithelial lesion (HSIL) and more severe lesions were compared.ResultsThe overall consistency of microendoscopy diagnosis with pathological diagnosis was 95.12% (39/41). Diagnostic cell morphological features of cervicitis, low-grade squamous intraepithelial lesion (LSIL), HSIL, adenocarcinoma in situ, and invasive cancer were clearly demonstrated in methylene blue stained microendoscopic images. In HSIL and more severe lesions, microendoscopic methylene blue cell staining technology can show the microscopic diagnostic features consistent with histopathology.ConclusionsThis study was an initial exercise in the application of the microendoscopy imaging system combined with methylene blue cell staining technology to cervical precancerous lesions and cervical cancer screening. The results provided the basis for a novel clinical strategy for triage of women with abnormal cervical screening results using in vivo non-invasive optical diagnosis technology.

Multisite assessment of reproducibility in high-content cell migration imaging data.

High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction.

Automatic granular and spinous epidermal cell identification and analysis on in vivo reflectance confocal microscopy images using cell morphological features.

Reflectance confocal microscopy (RCM) allows for real-time in vivo visualization of the skin at the cellular level. The study of RCM images provides information on the structural properties of the epidermis. These may change in each layer of the epidermis, depending on the subject's age and the presence of certain dermatological conditions. Studying RCM images requires manual identification of cells to derive these properties, which is time consuming and subject to human error, highlighting the need for an automated cell identification method. We aim to design an automated pipeline for the analysis of the structure of the epidermis from RCM images of the Stratum granulosum and Stratum spinosum. We identified the region of interest containing the epidermal cells and the individual cells in the segmented tissue area using tubeness filters to highlight membranes. We used prior biological knowledge on cell size to process the resulting detected cells, removing cells that were too small and reapplying the used filters locally on detected regions that were too big to be considered a single cell. The proposed full image analysis pipeline (FIAP) was compared with machine learning-based approaches (cell cutter, different U-Net configurations, and loss functions). All methods were evaluated both on simulated data (four images) and on manually annotated RCM data (seven images). Accuracy was measured using recall and precision metrics. Both accuracy metrics were higher in the proposed FIAP for both real ( , ) and synthetic images ( , ). The tested machine learning methods failed to identify and segment keratinocytes on RCM images with a satisfactory accuracy. We showed that automatic cell segmentation can be achieved using a pipeline based on membrane detection, with an accuracy that matches expert manual cell identification. To our knowledge, this is the first method based on membrane detection to study healthy skin using RCM images evaluated against manually identified cell positions.

Stenomitos nagquensis sp. nov. (Leptolyngbyaceae, Cyanobacteria) from a Meadow Wetland in the Tibet Plateau: A Novel Species Studied Based on a Polyphasic Approach

A novel cyanobacterial strain (CSML-F035) here described as Stenomitos nagquensis sp. nov. was isolated from a plateau meadow wetland habitat in the Tibet Autonomous Region. The novel strain was studied based on a polyphasic approach by combining characteristics of cell morphology and evidence from molecular data and ecology. Phylogenetic analyses based on the 16S rRNA and rbcL genes indicated that the novel strain is a member of the genus Stenomitos (Leptolyngbyaceae) but is positioned in a unique clade, with strongly supported values from BI, ML, and NJ methods. The percentage similarity of the 16S rRNA gene sequence of CSML-F035 compared with known strains of Stenomitos was >97%. Morphologically, S. nagquensis sp. nov. possesses overlapping morphological characteristics with other Stenomitos species, with thin trichomes enclosed in a firm, colorless, hyaline, persistent mucilage. However, the strain appeared to have slightly smaller cells compared to most known species of Stenomitos. Further analyses of the 16S–23S ITS region showed dissimilarity at 11.2–22.8% compared to known species of Stenomitos. The secondary helices—including D1–D1, V2, Box–B, and V3—that were predicted based on the 16S–23S ITS region for Stenomitos nagquensis sp. nov. were also distinct, thereby providing strong additional support for the separation of this species. This study highlights the importance of unexplored habitats in the Tibet region in which cyanobacterial diversity can be deemed high, with many potential new species that remain poorly explored.

Abstract 1633: ADT-030, a novel dual-acting RAS and β-catenin inhibitor with robust antitumor activity

Abstract BACKGROUND: Previous publications have reported that the antineoplastic activity of sulindac occurs by a cyclooxygenase independent mechanism, possibly involving direct inhibition of RAS. Suppression of β-catenin/TCF transcription has also been reported by inhibiting cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) and the activation of cGMP-dependent protein kinase (PKG). Here we characterize a potent and selective non-COX inhibitory sulindac derivative, ADT-030, that inhibits cancer cell growth by blocking RAS and β-catenin signaling. METHODS AND RESULTS: ADT-030 inhibited the proliferation of lung cancer cells expressing the PDE10A isozyme, while normal airway cells lacking PDE10A displayed reduced sensitivity. ADT-030 inhibited recombinant PDE10 activity at concentrations that inhibit lung cancer cells proliferation. ADT-030 induced cell cycle arrest, apoptosis, and morphological characteristics of autophagy blockage. A cellular thermal stability assay showed that ADT-030 binds both RAS and PDE10 in lysates from cancer cells with nanomolar affinity. Consistent with its PDE10 inhibitory activity, ADT-030 activated PKG and phosphorylated β-catenin on amino acid residues known to induce proteasomal degradation, and reduced levels of the unphosphorylated (oncogenic stabilized) form of β-catenin. Consistent with its ability to directly bind RAS, ADT-030 also inhibited EGF-stimulated MAPK/AKT signaling. Oral administration of ADT-030 inhibited tumor growth in an orthotopic xenograft mouse model of lung cancer, providing a durable survival response with 25% tumor-free mice at dosages having no discernable toxicity. ADT-030 was also highly efficacious in two different chemical-induced mouse models of lung tumorigenesis suppressing the carcinogen-activation of RAS, as well as in multiple mouse subcutaneous tumor models, including highly aggressive models of metastasis. CONCLUSIONS: These results support further development of ADT-030, a novel inhibitor of RAS and β-catenin signaling for the treatment of lung cancer and other RAS driven cancers, including those with co-occurring mutations in the Wnt/β-catenin pathway. Supported by NIH grants R01 CA238514, R01 CA197147, R01 CA254197. Citation Format: Veronica Ramirez-Alcantara, Xi Chen, Michele A. Schuler, Dennis Otali, William Grizzle, Kristy Berry, Antonio Ward, Ivan Babic, Elmar Nurmemmedov, Gang Zhou, Chengguo Xing, Greg Gorman, Lori Coward, Yulia Maxuitenko, Adam B. Keeton, Gary A. Piazza. ADT-030, a novel dual-acting RAS and β-catenin inhibitor with robust antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1633.

Silk fibroin-hydroxyapatite scaffolds promote the proliferation of adipose-derived mesenchymal stem cells by activating the ERK signal.

Adipose-derived mesenchymal stem cell (Ad-MSC) with capacities of releasing trophic factors and chondrogenic differentiation was a promising candidate for tracheal reconstruction. Silk fibroin (SF)- hydroxyapatite (HA) scaffolds were fabricated by the freeze-drying method. And Ad-MSCs were co-cultured on the scaffolds for 14days in vitro. The role of the SF-HA scaffold in regulating the adhesion, growth, and proliferation of Ad-MSCs, and its potential mechanisms were investigated. The identity of Ad-MSCs was confirmed by cell morphology, surface markers, and differentiation characteristics. Cell proliferation, viability, and morphology were observed via CCK-8, live/dead assay, and scanning electron microscopy (SEM). Gene mRNA and protein levels were examined using quantitative real-time polymerase chain reaction and western blotting, respectively. SF-HA scaffolds showed excellent properties of promoting Ad-MSCs adhesion, growth, and proliferation for at least 14days. In the CCK-8 assay, the relative OD value of Ad-MSCs cultured on SF-HA scaffolds increased (p < 0.001). Furthermore, live/dead staining showed that the fluorescent coverage increased with time (p < 0.05). SEM also showed that 3days after inoculation, the coverage of Ad-MSCs on the SF-HA scaffolds was 78.15%, increased to 92.91% on day 7, and reached a peak of 94.38% on day 14. Extracellular signal-regulated kinase (ERK) mRNA and phosphorylated ERK (pERK) protein expression increased at day 3 (p < 0.05), followed by a significant decline at day 7 (p < 0.05). And ERK mRNA expression was positively correlated with Ad-MSCs proliferation (p < 0.05). In summary, the SF-HA scaffold co-cultured with Ad-MSCs is a promising biomaterial for tracheal repair by activating the ERK signal pathway.

Deletion of pbpC Enhances Bacterial Pathogenicity on Tomato by Affecting Biofilm Formation, Exopolysaccharides Production, and Exoenzyme Activities in Clavibacter michiganensis.

Penicillin-binding proteins (PBPs) are considered essential for bacterial peptidoglycan biosynthesis and cell wall assembly. Clavibacter michiganensis is a representative Gram-positive bacterial species that causes bacterial canker in tomato. pbpC plays a significant role in maintaining cell morphological characteristics and stress responses in C. michiganensis. The current study demonstrated that the deletion of pbpC commonly enhances bacterial pathogenicity in C. michiganensis and revealed the mechanisms through which this occurs. The expression of interrelated virulence genes, including celA, xysA, xysB, and pelA, were significantly upregulated in △pbpC mutants. Compared with those in wild-type strains, exoenzyme activities, the formation of biofilm, and the production of exopolysaccharides (EPS) were significantly increased in △pbpC mutants. It is noteworthy that EPS were responsible for the enhancement in bacterial pathogenicity, with the degree of necrotic tomato stem cankers intensifying with the injection of a gradient of EPS from C. michiganensis. These findings highlight new insights into the role of pbpC affecting bacterial pathogenicity, with an emphasis on EPS, advancing the current understanding of phytopathogenic infection strategies for Gram-positive bacteria.

Deep learning-based morphological feature analysis and the prognostic association study in colon adenocarcinoma histopathological images.

Colorectal cancer (CRC) is now the third most common malignancy to cause mortality worldwide, and its prognosis is of great importance. Recent CRC prognostic prediction studies mainly focused on biomarkers, radiometric images, and end-to-end deep learning methods, while only a few works paid attention to exploring the relationship between the quantitative morphological features of patients' tissue slides and their prognosis. However, existing few works in this area suffered from the drawback of choosing the cells randomly from the whole slides, which contain the non-tumor region that lakes information about prognosis. In addition, the existing works, which tried to demonstrate their biological interpretability using patients' transcriptome data, failed to show the biological meaning closely related to cancer. In this study, we proposed and evaluated a prognostic model using morphological features of cells in the tumor region. The features were first extracted by the software CellProfiler from the tumor region selected by Eff-Unet deep learning model. Features from different regions were then averaged for each patient as their representative, and the Lasso-Cox model was used to select the prognosis-related features. The prognostic prediction model was at last constructed using the selected prognosis-related features and was evaluated through KM estimate and cross-validation. In terms of biological meaning, Gene Ontology (GO) enrichment analysis of the expressed genes that correlated with the prognostically significant features was performed to show the biological interpretability of our model.With the help of tumor segmentation, our model achieved better statistical significance and better biological interpretability compared to the results without tumor segmentation. Statistically, the Kaplan Meier (KM) estimate of our model showed that the model using features in the tumor region has a higher C-index, a lower p-value, and a better performance on cross-validation than the model without tumor segmentation. In addition, revealing the pathway of the immune escape and the spread of the tumor, the model with tumor segmentation demonstrated a biological meaning much more related to cancer immunobiology than the model without tumor segmentation. Our prognostic prediction model using quantitive morphological features from tumor regions was almost as good as the TNM tumor staging system as they had a close C-index, and our model can be combined with the TNM tumor stage system to make a better prognostic prediction. And to the best of our knowledge, the biological mechanisms in our study were the most relevant to the immune mechanism of cancer compared to the previous studies.

Collagen Nanoyarns: Hierarchical Three-Dimensional Biomaterial Constructs.

Hierarchical fibrous scaffolds (HFS) consist of nanoscale fibers arranged in larger macroscale structures, much in the same pattern as in native tissue such as tendon and bone. Creation of continuous macroscale nanofiber yarns has been made possible using modified electrospinning set-ups that combine electrospinning with techniques such as twisting, drawing, and winding. In this paper, a modified electrospinning setup was used to create continuous yarns of twisted type I collagen nanofibers, also known as collagen nanoyarns (CNY), from collagen solution prepared in acetic acid. Fabricated CNYs were cross-linked and characterized using SEM imaging and mechanical testing, while denaturation of collagen and dissolution of the scaffolds were assessed using circular dichroism (CD) and UV-vis spectroscopy, respectively. HeLa cells were then cultured on the nanoyarns for 24 h to assess cell adhesion on the scaffolds. Scanning electron micrographs revealed a twisted nanofiber morphology with an average nanofiber diameter of 213 ± 60 nm and a yarn diameter of 372 ± 23 μm that shrank by 35% after covalent cross-linking. Structural denaturation assessment of native collagen using circular dichroism (CD) spectroscopy showed that 60% of the triple-helical collagen content in CNYs was retained. Cross-linking of CNYs significantly improved their mechanical properties as well as stability in buffered saline with no sign of degradation for 14 days. In addition, CNY strength and stiffness increased significantly with cross-linking although in the wet state, significant loss in these properties, with a corresponding increase in elasticity, was observed. HeLa cells cultured on cross-linked CNYs for 24 h adhered to the yarn surface and oriented along the nanofiber alignment axis, displaying the characteristic spindle-like morphology of cells grown on surfaces with aligned topography. Collectively, the results demonstrate the promising potential of collagen nanoyarns as a new class of shapable biomaterial scaffold and building block for generating macroscale fiber-based tissues.

Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture.

Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.

Colec12 and Trail signaling confine cranial neural crest cell trajectories and promote collective cell migration.

Collective and discrete neural crest cell (NCC) migratory streams are crucial to vertebrate head patterning. However, the factors that confine NCC trajectories and promote collective cell migration remain unclear. Computational simulations predicted that confinement is required only along the initial one-third of the cranial NCC migratory pathway. This guided our study of Colec12 (Collectin-12, a transmembrane scavenger receptor C-type lectin) and Trail (tumor necrosis factor-related apoptosis-inducing ligand, CD253) which we show expressed in chick cranial NCC-free zones. NCC trajectories are confined by Colec12 or Trail protein stripes in vitro and show significant and distinct changes in cell morphology and dynamic migratory characteristics when cocultured with either protein. Gain- or loss-of-function of either factor or in combination enhanced NCC confinement or diverted cell trajectories as observed in vivo with three-dimensional confocal microscopy, respectively, resulting in disrupted collective migration. These data provide evidence for Colec12 and Trail as novel NCC microenvironmental factors playing a role to confine cranial NCC trajectories and promote collective cell migration.

Disrupting bacterial cytoskeletal protein interaction partners modulates geometric localization and alters cell shape.

The shape a bacterial cell assumes is an essential component in its ability to interact with its physical environment. For many bacterial species, shape is genetically encoded in the action of a small number of cell shape determining proteins. These proteins guide the building of a rigid peptidoglycan cell wall of a given geometry. We use a computational imaging framework to reconstruct the 3D shape of individual cells from fluorescence microscopy images and determine where a secondary fluorescent probe is enriched. In the straight-rod bacterium Escherichia coli, the bacterial actin MreB is enriched in areas with low Gaussian curvature, avoiding the poles. Disrupting MreB's interactions with the transmembrane protein RodZ alters MreB's geometric localization and cells lose their characteristic rod-like morphology. In the helical-rod bacterium Helicobacter pylori, the bactofilin CcmA is preferentially enriched at regions of small positive Gaussian curvature along the outer or major helical axis. Disrupting CcmA interaction partners results in less CcmA on the membrane, altered geometric localization of CcmA, and loss of the characteristic helical morphology of cells. These results are consistent with a model of cell shape determination that includes proper geometric localization of bacterial cytoskeletal proteins.

Morphodynamic and motility feature-based deep learning classification for subtypes of cancer-associated fibroblasts.

The cancer-associated fibroblasts (CAFs) have risen to prominence as key players in cancer progression. It is now widely appreciated that CAFs are composed of heterogeneous populations, either pro-tumorigenic or anti-tumorigenic. In addition, a growing body of evidence supports the multifaceted nature of CAFs. Therefore, researchers in the field have been trying to identify subtypes of CAFs with molecular markers. However, those markers cannot accurately classify the subpopulations and can be co-expressed in different subtypes. In order to utilize CAFs as a target for cancer treatment, issues with subtypes of CAFs must be resolved such that specific pro-tumorigenic subtypes can be suppressed or reprogrammed to anti-tumorigenic ones. The morphology and the motile characteristics of CAFs result from gene expression combinations. Thus, those characteristics can be holistic readouts of CAFs. Unlike biomolecular analysis, a fixed cell-based end-point assay, morphology or motility features of cells can be traced with live-cell imaging. Those features can provide information on the dynamic changes of CAFs. Here, in order to comprehensively identify subtypes CAFs, we adopt a deep learning-based cell classification strategy. Using multiple unsupervised and supervised machine learning algorithms, we extract the morphodynamic and motility features of cells from label-free live-cell imaging data of CAFs. To this end, we established in vitro breast CAFs, which were fibroblasts cocultured with two different breast cancer cell lines with different aggressiveness. As a result, we show that the morphodynamic and motility features can successfully classify heterogeneous subpopulations of cells.

Morphological and cytochemical characteristics of Varanus niloticus (Squamata, Varanidae) blood cells.

Varanus niloticus is a lizard residing within the Varanidae family. To date no studies detailing its blood morphology and characteristics have been conducted. This study used histologically stained blood and bone marrow samples to visualize the cells and their characteristics. The erythrocytes were nucleated, these nuclei were located in the middle of the elliptical cells. Hemoglobin filled the erythrocyte cytoplasm. Eosinophils were large cells with lobed nuclei and spherical acidophilic granules. Large granulocytes called heterophils were present and characterized by their fusiform/pleomorphic cytoplasmic granules. Small spherical granulocytes, known as basophils, presented with round, deeply stained metachromatic granules that gave the cytoplasm a dusty or cobblestoned appearance which was able to cover the nucleus, which in turn had an unusual shape. Thrombocytes ranged in shape from ellipsoidal to fusiform. They featured an elliptical, centrally located nucleus and a pale cytoplasm, with small vacuoles, and fine acidophilic granulation. The smallest variety of non-granular leukocytes was the lymphocytes. Their cytoplasm was sparse, finely granular, light blue, had tiny cytoplasmic projections, featuring a high nucleus: cytoplasm ratio. Larger and smaller sized populations of lymphocytes were distinguished, with the larger cells similar in size to azurophils. In general, the pleomorphic monocytes were the biggest mononuclear leucocytes, displaying cytoplasmic projections. Their nuclei were ovoid, kidney- or bean-shaped, with vacuolated and granular cytoplasms. Round cells were common among the monocytic azurophils, and they had a granular cytoplasm, and their nuclei were typically eccentric. The present research identifies the cell types and morphologies within the Varanus niloticus. HIGHLIGHTS: H&E, PAS, toluidine blue, methylene blue, and Safranin O stains provided morphological and morphometric descriptions of Varanus niloticus blood cells from blood smears and bone marrow. The Varanus niloticus had nucleated erythrocytes and white blood cells, mostly granulocytes (heterophils, eosinophils, and basophils) and mononuclear cells (azurophils, lymphocytes, and monocytes). Aquatic vertebrate Varanus niloticus had larger erythrocytes than terrestrial counterparts. Blood cell morphological and cytochemical features were similar to other reptilian species, with some species-specific differences, which likely accommodate differing environmental conditions. These results may help clinical researchers track the pathological conditions and support conservation of these wild animals.

Role of Prebiotic, Probiotic and Symbiotic Diets on Bacterial proliferation in Feed and Intestine of African (Clarias gariepinus) Catfish

The influence of prebiotic, probiotic and symbiotic diet on microbial proliferation was studied using an in vitro method. In the present trial, formulated diets were supplemented with prebiotic (Sargassum muticum), probiotic (Parkia biglobosa) and combination of Parkia biglobosa and Sargassum muticum (symbiotic diet). Bacteria proliferation in supplemented feeds, small and large intestine of the African catfish, Clarias gariepinus fed the formulated diets were evaluated. The feeding trial that lasted 12 weeks was conducted in plastic aquaria, with each treatment replicated three times. A control diet containing only the feed ingredients was also formulated and fed for the same period. The result showed bacteria proliferation was lowest in the control diet and highest in feed supplemented with prebiotic. Low bacteria proliferation was observed in the small intestine of fish fed symbiotic diet while highest proliferation was recorded in the fish fed prebiotic diet. The result also revealed the lowest bacteria proliferation in the large intestine of fish fed symbiotic diet and highest in fish fed probiotic diet. Using the cell morphology and biochemical characteristics of bacteria isolates in supplemented feed, Clarias gariepinus small and large intestine, the result indicated the presence of some Lactic Acid Bacteria (LAB) known to produce a variety of antimicrobial substances which are able to stop the development of foodborne diseases by inhibiting the growth of food spoilage and pathogenic organisms.