Systematic data analysis pipeline for quantitative morphological cell phenotyping.

Quantitative morphological phenotyping (QMP) is an image-based method used to capture morphological features at both the cellular and population level. Its interdisciplinary nature, spanning from data collection to result analysis and interpretation, can lead to uncertainties, particularly among those new to this actively growing field. High analytical specificity for a typical QMP is achieved through sophisticated approaches that can leverage subtle cellular morphological changes. Here, we outline a systematic workflow to refine the QMP methodology. For a practical review, we describe the main steps of a typical QMP; in each step, we discuss the available methods, their applications, advantages, and disadvantages, along with the R functions and packages for easy implementation. This review does not cover theoretical backgrounds, but provides several references for interested researchers. It aims to broaden the horizons for future phenome studies and demonstrate how to exploit years of endeavors to achieve more with less.

Regenerative anemia identification in cats: Red blood cell indices or morphology, what to use?

Background and Aim: Anemia, a clinical condition characterized by reduced erythrocytes, is often observed in cats. Regeneration indicates that the bone marrow can respond appropriately to anemia. The absolute reticulocyte count is the reference for differentiating regenerative and non-regenerative anemia, while red blood cell (RBC) indices and morphology provide supplementary information. This study aimed to identify anemia types and establish the most reliable RBC indices and morphology methods in agreement with the reference method. Materials and Methods: One hundred forty-five cases of cat anemia were prospectively classified using two methods: RBC indices and RBC morphology, and subsequently compared with the absolute reticulocyte count. Results: Based on RBC indices assessment, 27 cases (19%) exhibited regenerative anemia. Based on RBC morphology, 29 (20%) cases were identified as having regenerative anemia. Using the reticulocyte absolute count as a reference method, 34 (23.4%) cases of regenerative anemia were identified. The findings indicated that RBC indices and RBC morphology did not align in evaluating medullary regeneration and that there is a good degree of agreement between RBC morphology assessment and the reticulocyte absolute count in identifying regenerative anemias. Conclusion: Blood smear analysis of RBC morphology was more dependable for classifying regenerative anemia than RBC indices. Further studies should be conducted with a larger number of animals and that allow the identification of the cause of anemia and the monitoring of the animal.

Engineering Microgel Packing to Tailor the Physical and Biological Properties of Gelatin Methacryloyl Granular Hydrogel Scaffolds.

Granular hydrogel scaffolds (GHS) are fabricated via placing hydrogel microparticles (HMP) in close contact (packing), followed by physical and/or chemical interparticle bond formation. Gelatin methacryloyl (GelMA) GHS have recently emerged as a promising platform for biomedical applications; however, little is known about how the packing of building blocks, physically crosslinked soft GelMA HMP, affects the physical (pore microarchitecture and mechanical/rheological properties) and biological (in vitro and in vivo) attributes of GHS. Here, the GHS pore microarchitecture is engineered via the external (centrifugal) force-induced packing and deformation of GelMA HMP to regulate GHS mechanical and rheological properties, as well as biological responses in vitro and in vivo. Increasing the magnitude and duration of centrifugal force increases the HMP deformation/packing, decreases GHS void fraction and median pore diameter, and increases GHS compressive and storage moduli. MDA-MB-231 human triple negative breast adenocarcinoma cells spread and flatten on the GelMA HMP surface in loosely packed GHS, whereas they adopt an elongated morphology in highly packed GHS as a result of spatial confinement. Via culturing untreated or blebbistatin-treated cells in GHS, the effect of non-muscle myosin II-driven contractility on cell morphology is shown. In vivo subcutaneous implantation in mice confirms a significantly higher endothelial, fibroblast, and macrophage cell infiltration within the GHS with a lower packing density, which is in accordance with the in vitro cell migration outcome. These results indicate that the packing state of GelMA GHS may enable the engineering of cell response in vitro and tissue response in vivo. This research is a fundamental step forward in standardizing and engineering GelMA GHS microarchitecture for tissue engineering and regeneration.

A case of t-cell lymphoblastic lymphoma characterized by pleural effusion combined with pericardial effusion.

This case underscores the pivotal role of early cytological examination of bodily fluids in the preliminary detection of lymphoma, a conclusion reinforced by subsequent pathological findings and refined through immunohistochemical characterization. A morphological analysis of pleural effusion cells was conducted in a 25-year-old male presenting initially with concurrent pleural and pericardial effusions. Initial morphological assessment of effusion specimens indicated the likelihood of a lymphoproliferative disorder. Subsequent detailed pathological and immunohistochemical investigations confirmed this suspicion, culminating in a definitive diagnosis of T-cell lymphoblastic lymphoma (T-LBL). The case emphasizes the necessity of employing a comprehensive and synergistic diagnostic approach, facilitating prompt and accurate diagnosis and subtyping of lymphoma.

Quantitative atlas of collagen hydrogels reveals mesenchymal cancer cell traction adaptation to the matrix nanoarchitecture.

Collagen-based hydrogels are commonly used in mechanobiology to mimic the extracellular matrix. A quantitative analysis of the influence of collagen concentration and properties on the structure and mechanics of the hydrogels is essential for tailored design adjustments for specific in vitro conditions. We combined focused ion beam scanning electron microscopy and rheology to provide a detailed quantitative atlas of the mechanical and nanoscale three-dimensional structural alterations that occur when manipulating different hydrogel's physicochemistry. Moreover, we study the effects of such alterations on the phenotype of breast cancer cells and their mechanical interactions with the extracellular matrix. Regardless of the microenvironment's pore size, porosity or mechanical properties, cancer cells are able to reach a stable mesenchymal-like morphology. Additionally, employing 3D traction force microscopy, a positive correlation between cellular tractions and ECM mechanics is observed up to a critical threshold, beyond which tractions plateau. This suggests that cancer cells in a stable mesenchymal state calibrate their mechanical interactions with the ECM to keep their migration and invasiveness capacities unaltered. STATEMENT OF SIGNIFICANCE: The paper presents a thorough study on the mechanical microenvironment in breast cancer cells during their interaction with collagen based hydrogels of different compositions. The hydrogels' microstructure were obtained using state-of-the-art 3D microscopy, namely focused ion beam-scanning electron microscope (FIB-SEM). FIB-SEM was originally applied in this work to reconstruct complex fibered collagen microstructures within the nanometer range, to obtain key microarchitectural parameters. The mechanical microenvironment of cells was recovered using Traction Force Microscopy (TFM). The obtained results suggest that cells calibrate tractions such that they depend on mechanical, microstructural and physicochemical characteristics of the hydrogels, hence revealing a steric hindrance. We hypothesize that cancer cells studied in this paper tune their mechanical state to keep their migration and invasiveness capacities unaltered.

[Chinese expert consensus on the technical and clinical practice specifications of artificial intelligence assisted morphology examination of blood cells (2024)].

Blood cell morphological examination is a crucial method for the diagnosis of blood diseases, but traditional manual microscopy is characterized by low efficiency and susceptibility to subjective biases. The application of artificial intelligence (AI) technology has improved the efficiency and quality of blood cell examinations and facilitated the standardization of test results. Currently, a variety of AI devices are either in clinical use or under research, with diverse technical requirements and configurations. The Experimental Diagnostic Study Group of the Hematology Branch of the Chinese Medical Association has organized a panel of experts to formulate this consensus. The consensus covers term definitions, scope of application, technical requirements, clinical application, data management, and information security. It emphasizes the importance of specimen preparation, image acquisition, image segmentation algorithms, and cell feature extraction and classification, and sets forth basic requirements for the cell recognition spectrum. Moreover, it provides detailed explanations regarding the fine classification of pathological cells, requirements for cell training and testing, quality control standards, and assistance in issuing diagnostic reports by humans. Additionally, the consensus underscores the significance of data management and information security to ensure the safety of patient information and the accuracy of data.

Recent advancements in machine learning for bone marrow cell morphology analysis.

As machine learning progresses, techniques such as neural networks, decision trees, and support vector machines are being increasingly applied in the medical domain, especially for tasks involving large datasets, such as cell detection, recognition, classification, and visualization. Within the domain of bone marrow cell morphology analysis, deep learning offers substantial benefits due to its robustness, ability for automatic feature learning, and strong image characterization capabilities. Deep neural networks are a machine learning paradigm specifically tailored for image processing applications. Artificial intelligence serves as a potent tool in supporting the diagnostic process of clinical bone marrow cell morphology. Despite the potential of artificial intelligence to augment clinical diagnostics in this domain, manual analysis of bone marrow cell morphology remains the gold standard and an indispensable tool for identifying, diagnosing, and assessing the efficacy of hematologic disorders. However, the traditional manual approach is not without limitations and shortcomings, necessitating, the exploration of automated solutions for examining and analyzing bone marrow cytomorphology. This review provides a multidimensional account of six bone marrow cell morphology processes: automated bone marrow cell morphology detection, automated bone marrow cell morphology segmentation, automated bone marrow cell morphology identification, automated bone marrow cell morphology classification, automated bone marrow cell morphology enumeration, and automated bone marrow cell morphology diagnosis. Highlighting the attractiveness and potential of machine learning systems based on bone marrow cell morphology, the review synthesizes current research and recent advances in the application of machine learning in this field. The objective of this review is to offer recommendations to hematologists for selecting the most suitable machine learning algorithms to automate bone marrow cell morphology examinations, enabling swift and precise analysis of bone marrow cytopathic trends for early disease identification and diagnosis. Furthermore, the review endeavors to delineate potential future research avenues for machine learning-based applications in bone marrow cell morphology analysis.

Moderate capsaicin-containing kochujang alleviates memory impairment through the gut-brain axis in rats with scopolamine-induced amnesia.

This study investigated the efficacy and mechanism of traditionally made kochujang(TMK) with different capsaicin levels to alleviate memory impairment in rats with scopolamine-induced amnesia. Sprague-Dawley male rats were administered scopolamine (2 mg/kg bw/day) intraperitoneally to suppress the parasympathetic nervous system(PNS) and induce memory impairment. The rats were divided into four experimental groups, each consuming a diet containing 1 % kochujang in a 43-energy% high-fat diet(HFD) for 8 weeks. The TMK samples used for the study were categorized according to their capsaicin(CPS) content as follows: Low-CPS(0.5 mg%), medium-CPS(1.2 mg%), and high-CPS(1.7 mg%). In addition, factory-made kochujang (FMK; 1.1 mg% capsaicin) was also tested. The effects of kochujang were compared with the Control group(scopolamine), Positive-control(scopolamine+donepezil), and Normal-control(saline) fed HFD. Kochujang consumption reduced body weight and fat mass compared to the Control group. Compared to the Control, memory function measured using passive avoidance, water maze, and novel object recognition tests was enhanced in kochujang-fed rats, especially in the Medium-CPS group, similar to Positive-control. The Medium-CPS and Positive-control groups also exhibited inhibition of hippocampal cell death and increased cholesterol and triglyceride contents and mRNA expression of TNF-α and IL-1ß in the brain tissue compared to the Control group. Additionally, TMK elevated short-chain fatty acid, particularly, butyrate concentration in the portal vein. Scopolamine disturbed large intestine cell morphology and gut microbiota composition, and kochujang improved them. Kochujang in the medium-CPS (1.2 mg%) had a more significant impact on the gut microbiota in the interaction analysis between gut microbiota and memory function. In conclusion, kochujang, especially with medium-CPS (1.2 mg%), is a potential dietary intervention to mitigate memory impairment and promote overall cognitive health through improving eubiosis, potentially linked to the gut-brain axis in PNS-suppressed rats.

Sigma Factor Engineering in Actinoplanes sp. SE50/110: Expression of the Alternative Sigma Factor Gene ACSP50_0507 (σHAs) Enhances Acarbose Yield and Alters Cell Morphology.

Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. Actinoplanes sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50_0507 is a homolog of the developmental and osmotic-stress-regulating Streptomyces coelicolor σHSc. Therefore, the protein encoded by ACSP50_0507 was named σHAs. Here, an Actinoplanes sp. SE50/110 expression strain for the alternative sigma factor gene ACSP50_0507 (sigHAs) achieved a two-fold increased acarbose yield with acarbose production extending into the stationary growth phase. Transcriptome sequencing revealed upregulation of acarbose biosynthesis genes during growth and at the late stationary growth phase. Genes that are transcriptionally activated by σHAs frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its S. coelicolor homolog. The DNA-binding motif of σHAs was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σHAs to the upstream sequence of a strongly upregulated gene. Autoregulation of σHAs was observed, and binding to its own gene promoter region was also confirmed.

Harnessing Enhanced Flame Retardancy in Rigid Polyurethane Composite Foams through Hemp Seed Oil-Derived Natural Fillers.

Over the past few decades, polymer composites have received significant interest and become protagonists due to their enhanced properties and wide range of applications. Herein, we examined the impact of filler and flame retardants in hemp seed oil-based rigid polyurethane foam (RPUF) composites' performance. Firstly, the hemp seed oil (HSO) was converted to a corresponding epoxy analog, followed by a ring-opening reaction to synthesize hemp bio-polyols. The hemp polyol was then reacted with diisocyanate in the presence of commercial polyols and other foaming components to produce RPUF in a single step. In addition, different fillers like microcrystalline cellulose, alkaline lignin, titanium dioxide, and melamine (as a flame retardant) were used in different wt.% ratios to fabricate composite foam. The mechanical characteristics, thermal degradation behavior, cellular morphology, apparent density, flammability, and closed-cell contents of the generated composite foams were examined. An initial screening of different fillers revealed that microcrystalline cellulose significantly improves the mechanical strength up to 318 kPa. The effect of melamine as a flame retardant in composite foam was also examined, which shows the highest compression strength of 447 kPa. Significantly better anti-flaming qualities than those of neat foam based on HSO have been reflected using 22.15 wt.% of melamine, with the lowest burning time of 4.1 s and weight loss of 1.88 wt.%. All the composite foams showed about 90% closed-cell content. The present work illustrates the assembly of a filler-based polyurethane foam composite with anti-flaming properties from bio-based feedstocks with high-performance applications.

A three-dimensional tissue-engineered rostral migratory stream as an in vitro platform for subventricular zone-derived cell migration.

In the brains of most adult mammals, neural precursor cells (NPCs) from the subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to replace olfactory bulb interneurons. Following brain injury, published studies have shown that NPCs can divert from the SVZ-RMS-OB route and migrate toward injured brain regions, but the quantity of arriving cells, the lack of survival and terminal differentiation of neuroblasts into neurons, and their limited capacity to re-connect into circuitry are insufficient to promote functional recovery in the absence of therapeutic intervention. Our lab has fabricated a biomimetic tissue-engineered rostral migratory stream (TE-RMS) that replicates some notable structural and functional components of the endogenous rat RMS. Based on the design attributes for the TE-RMS platform, it may serve as a regenerative medicine strategy to facilitate sustained neuronal replacement into an injured brain region or an in vitro tool to investigate cell-cell communication and neuroblast migration. Previous work has demonstrated that the TE-RMS replicates the basic structure, unique nuclear shape, cytoskeletal arrangement, and surface protein expression of the endogenous rat RMS. Here, we developed an enhanced TE-RMS fabrication method in hydrogel microchannels that allowed more robust and high-throughput TE-RMS assembly. We report unique astrocyte behavior, including astrocyte bundling into the TE-RMS, the presence of multiple TE-RMS bundles, and observations of discontinuities in TE-RMS bundles, when microtissues are fabricated in agarose microchannels containing different critical curved or straight geometric features. We also demonstrate that we can harvest NPCs from the SVZ of adult rat brains and that EGFP+ cells migrate in chain formation from SVZ neurospheres through the TE-RMS in vitro. Overall, the TE-RMS can be utilized as an in vitro platform to investigate the pivotal cell-cell signaling mechanisms underlying the synergy of molecular cues involved in immature neuronal migration and differentiation.

A deep learning approach for automatic recognition of abnormalities in the cytoplasm of neutrophils.

BACKGROUND AND OBJECTIVES: This study aims to develop and evaluate NeuNN, a system based on convolutional neural networks (CNN) and generative adversarial networks (GAN) for the automatic identification of normal neutrophils and those containing several types of inclusions or showing hypogranulation. METHODS: From peripheral blood smears, a set of 5605 digital images was obtained with neutrophils belonging to seven categories: Normal neutrophils (NEU), Hypogranulated (HYP) or containing cryoglobulins (CRY), Döhle bodies (DB), Howell-Jolly body-like inclusions (HJBLI), Green-blue inclusions of death (GBI) and phagocytosed bacteria (BAC). The dataset utilized in this study has been made publicly available. The class of GBI was augmented using synthetic images generated by GAN. The NeuNN classification model is based on an EfficientNet-B7 architecture trained from scratch. RESULTS: NeuNN achieved an overall performance of 94.3% accuracy on the test data set. Performance metrics, including sensitivity, specificity, precision, F1-Score, Jaccard index, and Matthews correlation coefficient indicated overall values of 94%, 99.1%, 94.3%, 94.3%, 89.6%, and 93.6%, respectively. CONCLUSIONS: The proposed approach, combining data augmentation and classification techniques, allows for automated identification of morphological findings in neutrophils, such us inclusions or hypogranulation. The system can be used as a support tool for clinical pathologists to detect these specific abnormalities with clinical relevance.

Cultivation modes affect the morphology, biochemical composition, and antioxidant and anti-inflammatory properties of the green microalga Neochloris oleoabundans.

Microalgae are considered promising sustainable sources of natural bioactive compounds to be used in biotechnological sectors. In recent years, attention is increasingly given to the search of microalgae-derived compounds with antioxidant and anti-inflammatory properties for nutraceutical or pharmacological issues. In this context, attention is usually focused on the composition and bioactivity of algae or their extracts, while less interest is driven to their biological features, for example, those related to morphology and cultivation conditions. In addition, specific studies on the antioxidant and anti-inflammatory properties of microalgae mainly concern Chlorella or Spirulina. The present work was focused on the characterization of the Chlorophyta Neochloris oleoabundans under two combinations of cultivation modes: autotrophy and glucose-induced mixotrophy, each followed by starvation. Biomass for morphological and biochemical characterization, as well as for extract preparation, was harvested at the end of each cultivation phase. Analyses indicated a different content of the most important classes of bioactive compounds with antioxidant/anti-inflammatory properties (lipids, exo-polysaccharides, pigments, total phenolics, and proteins). In particular, the most promising condition able to prompt the production of antioxidant algal biomass with anti-inflammatory properties was the mixotrophic one. Under mixotrophy, beside an elevated algal biomass production, a strong photosynthetic metabolism with high appression of thylakoid membranes and characteristics of high photo-protection from oxidative damage was observed and linked to the overproduction of exo-polysaccharides and lipids rather than pigments. Overall, mixotrophy appears a good choice to produce natural bioactive extracts, potentially well tolerated by human metabolism and environmentally sustainable.

Low-vacuum SEM imaging and viability test of L929 cells exposed to a Euro 6 diesel exhaust gas mixture in a BAT-CELL chamber in comparison with hydrocarbons emission.

Exhaust emissions, which count among the most common causes of premature death worldwide, can cause irreversible changes in cells, leading to their damage or degeneration. In this research, L929 line cells were observed after exposure in the BAT-CELL chamber to exhaust gases emitted from a Euro 6 compression-ignition engine. Real road traffic conditions were simulated, taking into account air resistance while driving at speeds of 50 km/h, 120 km/h and idling engine. Morphological analysis of the cells was performed using an environmental scanning electron microscope. It has been observed that diesel exhaust fumes can cause inflammation, which can induce apoptosis or leads to necrotic cell death. The impact of the vehicle exhaust gases can inhibit cell proliferation by almost three times. Moreover, a correlation has been observed between the speed of the inflammatory reaction in cells and the presence of specific hydrocarbon compounds that determine the toxicity of exhaust gases. Research has shown that the toxicity of the emitted exhaust gases has been the highest at the driving speed of 120 km/h. In order to reduce the harmful effects of exhaust emissions, ecological alternatives and the supplementation of legal provisions regarding the compounds subject to limitation are necessary.

The tumor microenvironment of VETC+ hepatocellular carcinoma is enriched of immunosuppressive TAMs spatially close to endothelial cells.

BACKGROUND AND AIM: VETC (vessel that encapsulate tumor cluster) is a peculiar vascular phenotype observed in hepatocellular carcinoma (HCC), associated with distant metastases and poor outcome. VETC has been linked to the Tie2/Ang2 axis and is characterized by lymphocytes poor (cold) tumor microenvironment (TME). In this setting the role of Tumor Associated Macrophages (TAMs) has never been explored. Aim of the study is to investigate the presence and features of TAMs in VETC+ HCC and the possible interplay between TAMs and endothelial cells (ECs). METHODS: The series under study included 42 HCC. Once separated according to the VETC phenotype (21 VETC+; 21 VETC-) we stained consecutive slides with immunohistochemistry for CD68, CD163 and Tie2. Slides were then scanned and QuPath used to quantify morphological features. RESULTS: VETC+ cases were significantly (p < 0.001) enriched with large, lipid rich CD163+ TAMs (M2 oriented) that were spatially close to ECs; HCC cells significantly (p: 0.002) overexpressed Tie2 with a polarization toward ECs. CONCLUSIONS: The pro-metastatic attitude of VETC is sustained by a strict morphological relationship between immunosuppressive M2-TAMs, ECs and Tie2-expressing HCC cells.

Development of an Azobenzene-Based Cell Culture Photoresponsive Platform for In Situ Modulation of Surface Topography in Wet Environments.

Azopolymers are light-responsive materials that hold promise to transform in vitro cell culture systems. Through precise light illumination, they facilitate substrate pattern formation and erasure, allowing for the dynamic control and creation of active interfaces between cells and materials. However, these materials exhibit a tendency to locally detach from the supporting glass in the presence of aqueous solutions, such as cell culture media, due to the formation of blisters, which are liquid-filled cavities generated at the azopolymer film-glass interface. These blisters impede precise structurization of the surface of the azomaterial, limiting their usage for surface photoactivation in the presence of cells. In this study, we present a cost-effective and easily implementable method to improve the azopolymer-glass interface stability through silane functionalization of the glass substrate. This method proved to be efficient in preventing blister formation, thereby enabling the dynamic modulation of the azopolymer surface in situ for live-cell experiments. Furthermore, we proved that the light-illumination conditions used to induce azopolymer surface variations do not induce phototoxic effects. Consequently, this approach facilitates the development of a photoswitchable azopolymer cell culture platform for studying the impact of multiple in situ inscription and erasure cycles on cell functions while maintaining a physiological wet microenvironment.

cellPLATO - an unsupervised method for identifying cell behaviour in heterogeneous cell trajectory data.

Advances in imaging, segmentation and tracking have led to the routine generation of large and complex microscopy datasets. New tools are required to process this 'phenomics' type data. Here, we present 'Cell PLasticity Analysis Tool' (cellPLATO), a Python-based analysis software designed for measurement and classification of cell behaviours based on clustering features of cell morphology and motility. Used after segmentation and tracking, the tool extracts features from each cell per timepoint, using them to segregate cells into dimensionally reduced behavioural subtypes. Resultant cell tracks describe a 'behavioural ID' at each timepoint, and similarity analysis allows the grouping of behavioural sequences into discrete trajectories with assigned IDs. Here, we use cellPLATO to investigate the role of IL-15 in modulating human natural killer (NK) cell migration on ICAM-1 or VCAM-1. We find eight behavioural subsets of NK cells based on their shape and migration dynamics between single timepoints, and four trajectories based on sequences of these behaviours over time. Therefore, by using cellPLATO, we show that IL-15 increases plasticity between cell migration behaviours and that different integrin ligands induce different forms of NK cell migration.

Single-cell morphology encodes functional subtypes of senescence in aging human dermal fibroblasts.

Cellular senescence is an established driver of aging, exhibiting context-dependent phenotypes across multiple biological length-scales. Despite its mechanistic importance, profiling senescence within cell populations is challenging. This is in part due to the limitations of current biomarkers to robustly identify senescent cells across biological settings, and the heterogeneous, non-binary phenotypes exhibited by senescent cells. Using a panel of primary dermal fibroblasts, we combined live single-cell imaging, machine learning, multiple senescence induction conditions, and multiple protein-based senescence biomarkers to show the emergence of functional subtypes of senescence. Leveraging single-cell morphologies, we defined eleven distinct morphology clusters, with the abundance of cells in each cluster being dependent on the mode of senescence induction, the time post-induction, and the age of the donor. Of these eleven clusters, we identified three bona-fide senescence subtypes (C7, C10, C11), with C10 showing the strongest age-dependence across a cohort of fifty aging individuals. To determine the functional significance of these senescence subtypes, we profiled their responses to senotherapies, specifically focusing on Dasatinib + Quercetin (D+Q). Results indicated subtype-dependent responses, with senescent cells in C7 being most responsive to D+Q. Altogether, we provide a robust single-cell framework to identify and classify functional senescence subtypes with applications for next-generation senotherapy screens, and the potential to explain heterogeneous senescence phenotypes across biological settings based on the presence and abundance of distinct senescence subtypes.

Vascular endothelial cell morphology and alignment regulate VEGF-induced endothelial nitric oxide synthase activation.

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) inhibits platelet and leukocyte adhesion while promoting vasorelaxation in smooth muscle cells. Dysfunctional regulation of eNOS is a hallmark of various vascular pathologies, notably atherosclerosis, often associated with areas of low shear stress on endothelial cells (ECs). While the link between EC morphology and local hemodynamics is acknowledged, the specific impact of EC morphology on eNOS regulation remains unclear. Morphological differences between elongated, aligned ECs and polygonal, randomly oriented ECs correspond to variations in focal adhesion and cytoskeletal organization, suggesting differing levels of cytoskeletal prestress. However, the functional outcomes of cytoskeletal prestress, particularly in the absence of shear stress, are not extensively studied in ECs. Some evidence suggests that elongated ECs exhibit decreased immunogenicity and enhanced NO production. This study aims to elucidate the signaling pathways governing VEGF-stimulated eNOS regulation in the aligned EC phenotype characterized by elongated and aligned cells within a monolayer. Using anisotropic topographic cues, bovine aortic endothelial cells (BAECs) were elongated and aligned, followed by VEGF treatment in the presence or absence of cytoskeletal tension inhibitors. Phosphorylation of eNOS ser1179, AKT ser437 and FAK Tyr397 in response to VEGF challenge were significantly heightened in aligned ECs compared to unaligned ECs. Moreover this response proved to be robustly tied to cytoskeletal tension as evinced by the abrogation of responses in the presence of the myosin II ATPase inhibitor, blebbistatin. Notably, this work demonstrates for the first time the reliance on FAK phosphorylation in VEGF-mediated eNOS activation and the comparatively greater contribution of the cytoskeletal machinery in propagating VEGF-eNOS signaling in aligned and elongated ECs. This research underscores the importance of utilizing appropriate vascular models in drug development and sheds light on potential mechanisms underlying vascular function and pathology that can help inform vascular graft design.

Human plasma-like medium (HPLM) induces Cryptococcus neoformans in vivo cell morphologies.

The fungal pathogen Cryptococcus neoformans (C. neoformans) forms yeast cells of different sizes and morphological characteristics during infection. These features are usually not seen in standard laboratory in vitro conditions. Here, we describe in vivo cell morphologies when C. neoformans is grown in human plasma-like medium at 37°C, 5% CO2. We observed mixed-size populations of cells less than 1 µm up to 16.8 µm in cell diameter, increased capsule size, high chitin, and DNA content in larger cells. Our findings show that serum is not required for human plasma-like medium (HPLM)-induced C. neoformans cellular heterogeneity. Thus, this new method offers an opportunity to investigate factors of C. neoformans that mediate pathogenesis or host-pathogen interactions in a physiologically relevant setting.IMPORTANCEWe provide a description of new in vitro culture condition using the human plasma-like medium that supports the formation of the full range of in vivo cell morphologies of C. neoformans.