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Cytometry is an advanced technique for simultaneously identifying and quantifying many cell surface and intracellular proteins at a single-cell resolution. Analyzing high-dimensional cytometry data involves identifying and quantifying cell populations based on their marker expressions. This study provided a quantitative review and comparison of various ways to phenotype cellular populations within the cytometry data, including manual gating, unsupervised clustering, and supervised auto-gating. Six datasets from diverse species and sample types were included in the study, and manual gating with two hierarchical layers was used as the truth for evaluation. For manual gating, results from five researchers were compared to illustrate the gating consistency among different raters. For unsupervised clustering, 23 tools were quantitatively compared in terms of accuracy with the truth and computing cost. While no method outperformed all others, several tools, including PAC-MAN, CCAST, FlowSOM, flowClust, and DEPECHE, generally demonstrated strong performance. For supervised auto-gating methods, four algorithms were evaluated, where DeepCyTOF and CyTOF Linear Classifier performed the best. We further provided practical recommendations on prioritizing gating methods based on different application scenarios. This study offers comprehensive insights for biologists to understand diverse gating methods and choose the best-suited ones for their applications.

Microfluidic flow cytometry (MFCM) is considered to be an effective substitute for traditional flow cytometry, because of its advantages in terms of higher integration, smaller device size, lower cost, and higher cell sorting activity. However, MFCM still faces challenges in balancing parameters such as sorting throughput, viability, sorting efficiency, and cost. Here, we demonstrate a cost-effective and high-performance microfluidic cytometry cell sorting system, along with a customized microfluidic chip that integrates hydrodynamic focusing, droplet encapsulation, and sorting for precise cell manipulation. An innovative photon incremental counting-based fluorescence detection method is proposed, which requires only one-fiftieth of the data compared to traditional methods. This significantly simplifies the structure of the system and substantially reduces costs. The system exhibits detection recoveries exceeding 95% across sample solution flow rates ranging from 10 to 80 μL min−1. Moreover, it accurately achieves individual droplet deflections at a droplet generation frequency of 1600 Hz. Ultimately, our cell sorting system offers an impressive sorting efficiency of 90.7% and a high cell viability of 94.3% when operating at a droplet generation frequency of 1316 Hz, highlighting its accuracy and gentleness throughout the entire process. Our work will enhance advances in the life sciences, thereby creating a boom in great applications in single-cell cloning, single-cell analysis, drug screening, etc. [ABSTRACT FROM AUTHOR]

Objective: To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma. Methods: In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously. Results: In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01). Conclusions: Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes. [ABSTRACT FROM AUTHOR]

Auto-immune and inflammatory diseases are heterogenous in their clinical manifestations and prognosis, even among individuals presenting with the same pathology. Understanding the immunological alterations involved in their pathogenesis provides valuable insights in different clinical phenotypes and treatment responses. Immunophenotyping could lead to significant improvements in diagnosis, monitoring, initial treatment decisions and follow-up in autoimmune and inflammatory diseases. Mass cytometry provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, and therefore holds immense potential to evaluate complex cellular systems and for high-dimensional single-cell analysis. The high dimensionality of mass cytometry provides better coverage of immune populations dynamics, with sufficient power to identify rare cell types compared to flow cytometry. In this comprehensive review, we explore how mass cytometry findings contributed in the past decade to a deeper understanding of the cellular actors involved in systemic auto-immune and auto-inflammatory diseases with their respective therapeutic and prognostic impact. We also delve into the bioinformatical approaches applied to mass cytometry to analyze the high volumes of data generated, as well as the impact of the use of complementary single cell RNA sequencing, and their spatial modalities. Our analysis highlights the fact that mass cytometry captures major information on cell populations providing insights on the complex pathogenesis of autoimmune diseases. Future research designs could include mass cytometry findings in association to other -omics to stratify patients in adequate therapeutic arms and provide advancements in personalized therapies in the field of auto-immune and inflammatory diseases. Interest of using mass cytometry for systemic autoimmune and inflammatory diseases immunophenotyping. [ABSTRACT FROM AUTHOR]

The existence of cancer stem cells (CSCs) in various tumors has become increasingly clear in addition to their prominent role in therapy resistance, metastasis, and recurrence. For early diagnosis, disease progression monitoring, and targeting, there is a high demand for clinical-grade methods for quantitative measurement of CSCs from patient samples. Despite years of active research, standard measurement of CSCs has not yet reached clinical settings, especially in the case of solid tumors. This is because detecting this plastic heterogeneous population of cells is not straightforward. This review summarizes various techniques, highlighting their benefits and limitations in detecting CSCs from patient samples. In addition, methods designed to detect CSCs based on secreted and niche-associated signaling factors are reviewed. Spatial and single-cell methods for analyzing patient tumor tissues and noninvasive techniques such as liquid biopsy and in vivo imaging are discussed. Additionally, methods recently established in laboratories, preclinical studies, and clinical assays are covered. Finally, we discuss the characteristics of an ideal method as we look toward the future. [ABSTRACT FROM AUTHOR]

Imaging flow cytometry is a technology that performs microscopy image analysis of cells within flow cytometry and allows high-throughput, high-content cell analysis based on their intracellular molecular distribution and/or cellular morphology. While the technology has been available for a couple of decades, it has recently gained significant attention as technical limitations for higher throughput, sorting capability, and additional imaging dimensions have been overcome with various approaches. These evolutions have enabled imaging flow cytometry to offer a variety of solutions for life science and medicine that are not possible with conventional flow cytometry or microscopy-based screening. It is anticipated that the extent of applications will expand in the upcoming years as the technology becomes more accessible through dissemination. In this review, we will cover the technical advances that have led to this new generation of imaging flow cytometry, focusing on the advantages and limitations of each technique. [ABSTRACT FROM AUTHOR]

In principle, the measurement of mechanical property differences between cancer cells and their benign counterparts enables the detection, diagnosis, and classification of diseases. Despite the existence of various mechanophenotyping methods, the ability to perform high-throughput single-cell deformability measurements on liquid and/or solid tissue biopsies remains an unmet challenge within clinical settings. To address this issue, we present an ultrahigh-throughput viscoelastic microfluidic platform able to measure the mechanical properties of single cells at rates of up to 100,000 cells per second (and up to 10,000 cells per second in real time). To showcase the utility of the presented platform in clinical scenarios, we perform single-cell phenotyping of both liquid and solid tumor biopsies, cytoskeletal drug analysis, and identification of malignant lymphocytes in peripheral blood samples. Our viscoelastic microfluidic methodology offers opportunities for high-throughput, label-free single-cell analysis, with diverse applications in clinical diagnostics and personalized medicine. [ABSTRACT FROM AUTHOR]

Measurable residual disease (MRD) in acute myeloid leukemia (AML) refers to the quantity of residual leukemic cells in a patient after treatment.According to the latest agreements, MRD in AML offering essential prognostic insights. However, there is ongoing debate regarding MRD-based monitoring and treatment strategies. There are multiple platforms for detecting MRD, each varying in sensitivity and suitability for different patients. MRD not only predicts treatment outcomes but also serves as an indicator of treatment effectiveness and a prognostic biomarker. In AML, most retrospective studies indicate that patients who are MRD-positive or show increasing MRD levels at specific time points during remission have significantly higher risks of relapse and mortality compared to MRD-negative patients. Although achieving MRD-negative status can improve patient prognosis, the possibility of relapse remains. Despite the correlation between MRD and clinical outcomes, MRD assessment methods are not yet standardized, leading to discrepancies in results across different techniques. To provide reliable MRD results, it is essential to optimize and standardize MRD detection methods. Methods for assessing MRD include multiparameter flow cytometry (MFC) and molecular assays, chosen based on disease characteristics. This review focuses on currently available MRD detection methods and discusses how the prognostic value of MRD test results informs personalized treatment strategies for AML patients. [ABSTRACT FROM AUTHOR]

Schimke immuno-osseous dysplasia is a rare multisystemic disorder caused by biallelic loss of function of the SMARCAL1 gene that plays a pivotal role in replication fork stabilization and thus DNA repair. Individuals affected from this disease suffer from disproportionate growth failure, steroid resistant nephrotic syndrome leading to renal failure and primary immunodeficiency mediated by T cell lymphopenia. With infectious complications being the leading cause of death in this disease, researching the nature of the immunodeficiency is crucial, particularly as the state is exacerbated by loss of antibodies due to nephrotic syndrome or immunosuppressive treatment. Building on previous findings that identified the loss of IL-7 receptor expression as a possible cause of the immunodeficiency and increased sensitivity to radiation-induced damage, we have employed spectral cytometry and multiplex RNA-sequencing to assess the phenotype and function of T cells ex-vivo and to study changes induced by in-vitro UV irradiation and reaction of cells to the presence of IL-7. Our findings highlight the mature phenotype of T cells with proinflammatory Th1 skew and signs of exhaustion and lack of response to IL-7. UV light irradiation caused a severe increase in the apoptosis of T cells, however the expression of the genes related to immune response and regulation remained surprisingly similar to healthy cells. Due to the disease’s rarity, more studies will be necessary for complete understanding of this unique immunodeficiency. [ABSTRACT FROM AUTHOR]

Metaproteomic analysis of microbiome post-translation modifications (PTMm) is challenging, and little is known about the effects of inflammation on the bacterial PTM landscape in IBD. Here, we adapted and optimised fluorescence in situ hybridisation–flow cytometry (FISH-FC) to study microbiome-wide tyrosine phosphorylation (p-Tyr) in children with and without inflammatory bowel disease (IBD). Microbial p-Tyr signal was significantly higher in children with IBD, compared to those without. Faecalibacterium prausnitzii, Bacteroidota, Gammaproteobacteria and Bifidobacteria tended to be more abundant in IBD than in non-IBD control children but there were only minor differences in p-Tyr among these bacterial communities in those with and without IBD. p-Tyr was significantly lower in non-IBD children older than 9 yrs compared with those less than 9 yrs, and the effect was seen in all four bacterial subgroups studied. The opposite trend was seen in patients with IBD. p-Tyr overall is higher in children with IBD but the effects of inflammation on p-Tyr vary according to the bacterial community. The overall microbiome p-Tyr signal changes with age in healthy children. FISH-FC can be used to study the microbiome-wide PTM landscape. [ABSTRACT FROM AUTHOR]

Objective: Patients with non-small cell lung cancer (NSCLC) have poor prognoses. Sulfatase 1 (SULF1) is an extracellular neutral sulfatase and is involved in multiple physiological processes. Hence, this study investigated the function and possible mechanisms of SULF1 in NSCLC. Material and Methods: Difference in SULF1 expression level between tumors and normal lung tissues was analyzed through bioinformatics and clinical sampling, and the effects of SULF1 expression on prognosis were investigated through Kaplan–Meier analysis. SULF1 level in NSCLC cells was modulated through small interfering ribonucleic acid interference. NSC228155, which is an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling pathway agonist, was for handling NSCLC cells. SULF1 expression level was tested through quantitative reverse transcriptase real-time polymerase chain reaction. Cell proliferation, migration, and invasion were evaluated with cell counting kit-8, 5-ethynyl-2-deoxyuridine, and transwell assays, and the levels of epithelial-to-mesenchymal transition (EMT)- and EGFR/MAPK pathway-related proteins were detected through Western blot. Results: Bioinformatics and clinical samples showed that NSCLC tumor tissues had elevated SULF1 expression levels relative to those of normal tissues (P < 0.05). Patients with NSCLC and high SULF1 expression levels experienced poorer prognosis than those of low SULF1 expression levels (P < 0.05). SULF1 knockdown repressed the malignant biological behavior, including proliferation, migration, and invasion, of the NSCLC cells (P < 0.05). Mechanistically, SULF1 knockdown augmented E-cadherin level and abated N-cadherin and vimentin protein levels (P < 0.05). These results confirmed that EMT was inhibited. In addition, the knockdown of SULF1 reduced the phosphorylation of EGFR, extracellular signal-regulated kinase, p38 MAPK and c-Jun N-terminal kinase, and NSC228155 partially reversed these changes, which were affected by SULF1 knockdown. Meanwhile, NSC228155 partially reversed the inhibition of EMT, migration, and invasion affected by SULF1 knockdown. Conclusion: SULF1 knockdown inhibits the proliferation, migration, invasion, and EMT of NSCLC cells by inactivating EGFR/MAPK pathway. [ABSTRACT FROM AUTHOR]

This work presents an innovative all-electrical platform for selective single-particle manipulation. The platform combines microfluidic impedance cytometry for label-free particle characterization and dielectrophoresis for contactless multi-way particle separation. The microfluidic chip has a straightforward coplanar electrode layout and no particle pre-focusing mechanism is required. An original online algorithm analyzes the impedance signals of each incoming particle and regulates in real time the dielectrophoretic voltages according to a desired control logic. As a proof-of-concept, three operation modes are demonstrated on a mixture of 8, 10, and 12 µm diameter beads: (i) particle position swapping across the channel axis, irrespective of particle size, (ii) size-based particle separation, irrespective of particle position, and (iii) sorting of a selected sequence of particles. As a perspective, the versatility of impedance cytometry and dielectrophoresis, and the possibility of configuring alternative control logics, hold promise for advanced particle and cell manipulation. [ABSTRACT FROM AUTHOR]

Objective: This study aims to explore the regulatory mechanisms of Wnt signaling in the invasion and recurrence of ameloblastoma (AM) to provide a new theoretical basis for its treatment. Methods: Bulk RNA sequencing was employed to analyze samples from AM patients, and identify differentially expressed genes. Subsequently, bioinformatics methods such as Weighted Gene Co-Expression Network Analysis (WGCNA), DESeq2, and KEGG enrichment analysis were utilized to construct gene co-expression networks and identify pathways associated with invasion and recurrence. Furthermore, in vitro experiments, including Cell Counting Kit-8 (CCK-8), Wound healing assays, Western blotting, and qPCR were conducted to validate the effects of Wnt signaling on AM biological functions and the expression of related genes and proteins. Results: Bioinformatics analysis revealed significant activation of the Wnt signaling pathway during AM invasion and recurrence, and differential gene analysis identified specific gene expression patterns associated with the Wnt signaling pathway. In vitro experiments further demonstrated that the standard Wnt/β-catenin pathway activator, Laduviglusib significantly activated Wnt signaling, leading to a marked increase in the mRNA and protein expression levels of TCF7, β-catenin, WNT2B, and LEF1, thereby enhancing the proliferation and migration capabilities of AM cells. Conclusion: This study reveals the critical role of aberrant Wnt signaling activation in AM proliferation and migration, identifying it as a key driver of AM invasion and recurrence. The findings provide new insights into the mechanisms underlying AM invasion and recurrence, laying the foundation for developing novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

The ability of flow cytometry to identify and quantify the presence of cell populations defined by their expression profile of specific markers has made this technique a powerful and routinary tool in clinical diagnostic practice. Specifically in the field of hematological malignancies, flow cytometry allows the identification of the correct type and lineage of each patient's disease and also sensitively quantifies the presence of the disease at precise moments during treatment, that is, levels of measurable residual disease (MRD). The quantification of MRD by flow cytometry has allowed the adaptation of tailored therapies to patients, contributing to the improvement of the results of the different protocols in recent decades. In this context, our objective in the present work was to evaluate the potential impact that spectral flow cytometry can provide compared to conventional cytometry, which is the one usually used in clinics. We present here a comparative study of both technologies, spectral versus conventional flow cytometry, in primary samples corresponding to the diagnosis and follow-up of children and adolescents with acute lymphoblastic leukemia. Our initial experience demonstrates the feasibility of incorporating spectral flow cytometry into the routine workflow of a reference laboratory. [ABSTRACT FROM AUTHOR]

Background: Dental caries, also known as tooth decay or cavity formation, is one of the world's most widespread dental conditions. It is a plaque-related infection caused mainly by Streptococcus mutans. People have relied on several plant species to treat oral infections; Heteropyxis natalensis, for example, has been used to treat toothache and gum infections. Methods: In this study, the antimicrobial and anti-adherence properties of H. natalensis and Camellia sinensis, as well as tea tree and peppermint essential oils were investigated on tooth enamel. Results: The bacterial load of S. mutans was reduced by approximately two orders of a magnitude after 48 h, with a lesser extent on the commensal bacteria, Lactobacillus paracasei. Scanning electron micrographs of enamel blocks showed a reduction in the attachment and chain formation of S. mutans and degraded cell morphology. Lastly, the combination and each component individually, showed low to no cellular toxicity when tested on human macrophages. Conclusions: This is the first report of this polyherbal regarding its selectivity and potential prevention of dental caries. [ABSTRACT FROM AUTHOR]

The correction notice addresses an error in Figure 5 of the original article titled "HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway." The correction does not impact the validity of the conclusions or the overall content of the article. The authors have updated the original article to reflect the accurate information. [Extracted from the article]

High-dimensional cell phenotyping is a powerful tool to study molecular and cellular changes in health and diseases. CyTOF enables high-dimensional cell phenotyping using tens of surface and intra-cellular markers. To utilize the full potential of CyTOF, we need advanced clustering and machine learning methodologies to enable automated gating of the complex data. Here we show that critical modifications to a machine learning based FlowSOM package and precise parameter optimization can enable us to reliably analyze the complex CyTOF data. We show the impact of key parameters on clustering outcomes while addressing bugs within the publicly available package. We modified the FlowSOM pipeline to fix the bugs, enable scalability to handle large datasets and perform parameter optimization. We further validated this modified pipeline on a substantial external immunological dataset demonstrating the need of data-specific tailored parameter optimization to ensure reliable definition and interrogation of immune cell populations associated with immune disorders. Optimized FlowSOM pipeline for reliable clustering of high-dimensional cytometry data. [ABSTRACT FROM AUTHOR]

Kirsten rat sarcoma virus (KRAS)-G12C inhibition causes remodeling of the lung tumor immune microenvironment and synergistic responses to anti-PD-1 treatment, but only in T cell infiltrated tumors. To investigate mechanisms that restrain combination immunotherapy sensitivity in immune-excluded tumors, we used imaging mass cytometry to explore cellular distribution in an immune-evasive KRAS mutant lung cancer model. Cellular spatial pattern characterization revealed a community where CD4+ and CD8+ T cells and dendritic cells were gathered, suggesting localized T cell activation. KRAS-G12C inhibition led to increased PD-1 expression, proliferation, and cytotoxicity of CD8+ T cells, and CXCL9 expression by dendritic cells, indicating an effector response. However, suppressive regulatory T cells (Tregs) were also found in frequent contact with effector T cells within this community. Lung adenocarcinoma clinical samples showed similar communities. Depleting Tregs led to enhanced tumor control in combination with anti-PD-1 and KRAS-G12C inhibitor. Combining Treg depletion with KRAS inhibition shows therapeutic potential for increasing antitumoral immune responses. [ABSTRACT FROM AUTHOR]

Background: Granulocyte concentrates (GCs) are usually prepared by single-donor apheresis after G-CSF pretreatment and have to be transfused within 24 h after cell collection because of the rapid decrease in pH and cell survival due to high lactate production by red blood cell contamination. GCs pooled from buffy coats of whole blood donations could improve the availability of these products. Methods to reduce red blood cell and platelet contamination may improve storability. We developed a manufacturing process for pooled GCs and investigated cell viability and functionality over time. Methods: Six ABO blood group-identical buffy coats were pooled. Subsequently, the red blood cells spontaneously sedimented after the addition of hydroxyethyl starch. The resulting leukocyte-enriched supernatant was washed twice with saline to reduce platelets and was resuspended in ABO-identical donor plasma. The leukocyte concentrate was transferred to a platelet storage bag and stored up to 72 h at 20–24°C w/o agitation. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst activity were monitored. Results: The number of red blood cells and platelets was reduced to 0.4% and 6.1% of the baseline levels. About 50% of the original present leukocytes could be extracted (n = 76). In the course of 72 h of storage, there were no significant changes in white blood cell counts (p = 0.12). The viability exceeded 98% during the entire period. The rate of granulocytes performing phagocytosis and oxidative burst remained above 95% anytime. Conclusion: GCs prepared from pooled buffy coats provide a precious alternative to granulocytes obtained from apheresis. Reduction of red blood cells and platelets by more than 90% extends the maximum shelf life of GCs from 24 h to 72 h. For a therapeutic dose of at least 1 × 1010 granulocytes, 15–20 buffy coats are required. [ABSTRACT FROM AUTHOR]

Flow cytometry is a sophisticated technology used widely in both basic research and as a routine tool in clinical diagnosis. The technology has progressed from single parameter detection in the 1970s and 1980s to high end multicolor analysis, with currently 30 parameters detected simultaneously, allowing the identification and purification of rare subpopulations of cells of interest. Flow cytometry continues to evolve and expand to facilitate the investigation of new diagnostic and therapeutic avenues. The present review gives an overview of basic theory and instrumentation, presents and compares the advantages and disadvantages of conventional, spectral and imaging flow cytometry as well as mass cytometry. Current methodologies and applications in both research, pre- and clinical settings are discussed, as well as potential limitations and future evolution. This finding encourages the reader to promote such relationship between basic science, diagnosis and multidisciplinary approach since the standard methods have limitations (e.g., in differentiating the cells after staining). Moreover, such path inspires future cytometry specialists develop new/alternative frontiers between pre- and clinical diagnosis and be more flexible in designing the study for both human as well as veterinary medicine. [ABSTRACT FROM AUTHOR]

Aim: To use autofluorescence multispectral imaging (AFMI) to develop a non-invasive assay for the in-depth characterisation of human bone marrow derived mesenchymal stromal cells (hBM-MSCs). Methods: hBM-MSCs were imaged by AFMI on gridded dishes, stained for endpoints of interest (STRO-1 positivity, alkaline phosphatase, beta galactosidase, DNA content) then relocated and results correlated. Intensity, texture and morphological features were used to characterise the colour distribution of regions of interest, and canonical discriminant analysis was used to separate groups. Additionally, hBM-MSC lines were cultured to arrest, with AFMI images taken after each passage to investigate whether an assay could be developed for growth potential. Results: STRO-1 positivity could be predicted with a receiver operator characteristic area under the curve (AUC) of 0.67. For spontaneous differentiation this was 0.66, for entry to the cell-cycle it was 0.77 and for senescence it was 0.77. Growth potential (population doublings remaining) was estimated with an RMSPE = 2.296. The Mean Absolute Error of the final prediction model indicated that growth potential could be predicted with an error of ± 1.86 doublings remaining. Conclusions: This non-invasive methodology enabled the in-depth characterisation of hBM-MSCs from a single assay. This approach is advantageous for clinical applications as well as research and stands out for the characterisation of both present status as well as future behaviour. The use of data from five MSC lines with heterogenous AFMI profiles supports potential generalisability. [ABSTRACT FROM AUTHOR]

Objective: To investigate the mechanism of induction of ferroptosis by brazilin in breast cancer cells. Methods: Breast cancer 4T1 cells were divided into 6 groups: control, brazilin 1/2 half maximal inhibitory concentration (IC50), IC50, 2×IC50, erastin (10 µg/mL) and capecitabine (10 µg/mL) groups. The effect of brazilin on the proliferation of 4T1 cells was detected by cell counting kit-8 assay, and the treatment dose of brazilin was screened. The effect of brazilin on the mitochondrial morphology of 4T1 cells, and the mitochondrial damage was evaluated under electron microscopy. The levels of Fe2+, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase 4 (GPX4) were estimated using various detection kits. The invasion and migration abilities of 4T1 cells were detected by scratch assay and transwell assay. The expressions levels of tumor protein p53, solute carrier family 7 member 11 (SLC7A11), GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) proteins were quantified by Western blot assay. Results: Compared to the control group, the 10 (1/2 IC50), 20 (IC50) and 40 (2×IC50) µg/mL brazilin, erastin, and capecitabine groups showed a significant decrease in the cell survival rate, invasion and migration abilities, GSH, SLC7A11 and GPX4 protein expression levels, and mitochondrial volume and ridge (P<0.05), and a significant increase in the mitochondria membrane density, Fe2+, ROS and MDA levels, and p53 and ACSL4 protein expression levels (P<0.05). Conclusions: Brazilin actuated ferroptosis in breast cancer cells, and the underlying mechanism is mainly associated with the p53/SLC7A11/GPX4 signaling pathway. [ABSTRACT FROM AUTHOR]

Objective: To observe the protective effect and mechanism of hydroxyl safflower yellow A (HSYA) from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were treated with oxygen-glucose deprivation reperfusion (OGD/R) to simulate the ischemia reperfusion model, and cell counting kit-8 was used to detect the protective effect of different concentrations (1.25–160 µ mol/L) of HSYA on HUVECs after OGD/R. HSYA 80 µ mol/L was used for follow-up experiments. The contents of inflammatory cytokines interleukin (IL)-18, IL-1 β, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α) and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay. The protein expressions of toll-like receptor, NOD-like receptor containing pyrin domain 3 (NLRP3), gasdermin D (GSDMD) and GSDMD-N-terminal domain (GSDMD-N) before and after administration were detected by Western blot. NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt (CRID3 sodium salt, also known as MCC950) and agonist were added, and the changes of NLRP3, cysteine-aspartic acid protease 1 (Caspase-1), GSDMD and GSDMD-N protein expressions were detected by Western blot. Results: HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18, IL-1 β, MCP-1, TNF-α and IL-6 (P<0.01 or P<0.05). At the same time, by inhibiting NLRP3/Caspase-1/GSDMD pathway, HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3, Caspase-1, GSDMD and GSDMD-N proteins (P<0.01). Conclusions: The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis. [ABSTRACT FROM AUTHOR]

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Background: Dental restorative materials are recognized as artificial niches that facilitate the adherence and accumulation of oral microorganisms. To mitigate oral diseases and extend the lifespan of restorations, it is advantageous to use dental materials that exhibit low susceptibility to bacterial adhesion. Objective: To evaluate and compare bacterial adhesion on two bioactive restorative materials, a glass hybrid restorative, and an alkasite with a nanohybrid resin composite as a positive control. The secondary objectives were to compare the surface roughness (SR) of the materials and determine the correlation between the bacterial adhesion and the SR. Materials and methods: The samples consisted of 33 polished discs of each material: Group A: Tetric® N-Ceram (nanohybrid resin composite), Group B: Equia Forte™ HT Fil (glass hybrid restorative) and Group C: Cention N® (alkasite). Streptococcus mutans cultures were inoculated and after 24-hours of incubation, bacterial adhesion was measured by measuring optical density (OD) and number of colony forming units (CFUs). After 96-hours incubation, the bacterial cell count was determined using scanning electron microscopy (SEM). SR was assessed using surface profilometer. Results: Alkasite had significantly lower OD and CFUs (p < 0.001 and p = 0.015 respectively). According to the SEM analysis, the glass hybrid restorative had lower mean bacterial cell count with no significant difference between the groups. The nanohybrid composite had the smoothest surface that was significantly lower than the alkasite and glass hybrid restorative (p = 0.002). None of the groups demonstrated a correlation between bacterial adhesion and SR. Conclusion: Alkasite impedes bacterial adhesion better than the glass hybrid restorative and nanohybrid composite, while smoother surfaces are achieved with the nanohybrid composite. [ABSTRACT FROM AUTHOR]

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration–response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution. [ABSTRACT FROM AUTHOR]

Thymic involution is a key factor in human immune aging, leading to reduced thymic output and a decline in recent thymic emigrant (RTE) naive T cells in circulation. Currently, the precise definition of human RTEs and their corresponding cell surface markers lacks clarity. Analysis of single-cell RNA-seq/ATAC-seq data distinguished RTEs by the expression of SOX4, IKZF2, and TOX and CD38 protein, whereby surface CD38hi expression universally identified CD8+ and CD4+ RTEs. We further determined the dynamics of RTEs and mature cells in a cohort of 158 individuals, including age-associated transcriptional reprogramming and shifts in cytokine production. Spectral cytometry profiling revealed two axes of aging common to naive CD8+ and CD4+ T cells: (1) a decrease in CD38++ cells (RTEs) and (2) an increase in CXCR3hi cells. Identification of RTEs enables direct assessment of thymic health. Furthermore, resolving the dynamics of naive T cell remodeling yields insight into vaccination and infection responsiveness throughout aging. [Display omitted] • Human RTEs are epigenetically and transcriptionally defined by SOX4, IKZF2, and TOX • Both CD8+ and CD4+ RTEs are characterized by high surface expression of CD38 • CD38 expression is a reliable RTE marker also under chronic and acute inflammation • Naive T cells show an age-related decline in CD38++ RTEs and an increase in CXCR3hi cells Identifying human T cells that have recently emigrated from the thymus remains problematic. Here, Bohacova et al. categorize recent thymic emigrants with high CD38 expression as their universal surface marker. Additionally, they explore the transcriptional and epigenetic landscape of naive CD8+ and CD4+ T cells, revealing shared axes of aging. [ABSTRACT FROM AUTHOR]

Many clinical procedures and biomedical research workflows rely on microscopy, including diagnosis of cancer, genetic disorders, autoimmune diseases, infections, and quantification of cell culture. Despite its widespread use, traditional image acquisition and review by trained microscopists is often lengthy and expensive, limited to large hospitals or laboratories, precluding use in point‐of‐care settings. In contrast, lensless or lensfree holographic microscopy (LHM) is inexpensive and widely deployable because it can achieve performance comparable to expensive and bulky objective‐based benchtop microscopes while relying on components that cost only a few hundred dollars or less. Lab‐on‐a‐chip integration is practical and enables LHM to be combined with single‐cell isolation, sample mixing, and in‐incubator imaging. Additionally, many manual tasks in conventional microscopy are instead computational in LHM, including image focusing, stitching, and classification. Furthermore, LHM offers a field of view hundreds of times greater than that of conventional microscopy without sacrificing resolution. Here, the basic LHM principles are summarized, as well as recent advances in artificial intelligence integration and enhanced resolution. How LHM is applied to the above clinical and biomedical applications is discussed in detail. Finally, emerging clinical applications, high‐impact areas for future research, and some current challenges facing widespread adoption are identified. [ABSTRACT FROM AUTHOR]

Emphysema in patients with chronic obstructive pulmonary disease (COPD) is characterized by progressive inflammation. Preclinical studies suggest that lung volume reduction surgery (LVRS) and mesenchymal stromal cell (MSC) treatment dampen inflammation. We investigated the effects of bone marrow-derived MSC (BM-MSC) and LVRS on circulating and pulmonary immune cell profiles in emphysema patients using mass cytometry. Blood and resected lung tissue were collected at the first LVRS (L1). Following 6–10 weeks of recovery, patients received a placebo or intravenous administration of 2 × 106 cells/kg bodyweight BM-MSC (n = 5 and n = 9, resp.) in week 3 and 4 before the second LVRS (L2), where blood and lung tissue were collected. Irrespective of BM-MSC or placebo treatment, proportions of circulating lymphocytes including central memory CD4 regulatory, effector memory CD8 and γδ T cells were higher, whereas myeloid cell percentages were lower in L2 compared to L1. In resected lung tissue, proportions of Treg (p = 0.0067) and anti-inflammatory CD163− macrophages (p = 0.0001) were increased in L2 compared to L1, while proportions of pro-inflammatory CD163+ macrophages were decreased (p = 0.0004). There were no effects of BM-MSC treatment on immune profiles in emphysema patients. However, we observed alterations in the circulating and pulmonary immune cells upon LVRS, suggesting the induction of anti-inflammatory responses potentially needed for repair processes. [ABSTRACT FROM AUTHOR]

Recent therapeutic approaches in animal diseases involve stem cell-based therapies which are showing promising results, particularly with the use of mesenchymal stem cells (MSCs). Our study aimed at isolating and characterizing adipose tissue-derived stem cells (ADSCs) in goats and sheep, evaluating their characteristics mesenchymal nature, and adipogenic, chondrogenic, and osteogenic differentiation potentials. For this purpose, subcutaneous adipose tissues were collected from the inguinal region under sterile conditions from five healthy adult sheep and five goats each slaughtered at a slaughterhouse. MSCs were isolated, cultured, and differentiated into adipogenic, chondrogenic, and osteogenic lineages, followed by respective histochemical staining to confirm differentiation. In passage 3 (P3), the surface markers CD44, CD90, CD105, and CD45 were analyzed using flow cytometry to characterize mesenchymal properties. The cells expressed CD44, CD90, and CD105 but did not express hematopoietic marker CD45, confirming their mesenchymal nature. This study successfully identified ADSCs from sheep and goats as mesenchymal stem cells and characterized their strong trilineage differentiation potential, highlighting their strong therapeutic capabilities and revealing interspecies differences in MSCs properties. These findings provide valuable insights for future MSCs-based therapeutic applications in veterinary regenerative medicine, particularly for economically and clinically important species. [ABSTRACT FROM AUTHOR]

Introduction: Immune checkpoint inhibitors (ICI) have changed the prognostic outlook for several malignancies. Despite the unprecedented durable responses and improvement in survival outcomes with ICIs, exclusion of oncology patients living with human immunodeficiency virus (HIV) from most ICI-related trials has limited utility of these agents. Clinical outcomes related to concomitant use of antiretroviral therapy and ICI remain unclear. We present a case series based on our institution's experience to address this unmet need of clinical outcomes with ICI in oncology patients living with HIV. Methods: Electronic medical records were queried to identify patients living with HIV who were also diagnosed with cancer and treated with ICI from May 2019 to September 2022. Results: A total of five patients were on concurrent antiretroviral therapy and immunotherapy. From an efficacy perspective, three patients were observed to have a response (one complete response, one partial response, and one stable disease). There were three patients with known cluster of differentiation (CD4 +) levels who had an increase in CD4 + cell count with ICI treatment. The HIV viral load remained undetected in most of the patients on ICI treatment. No confirmed immune-related adverse effects were documented for any patients in this review. Conclusion: Immune checkpoint inhibitors may be efficacious and tolerable for treatment of cancer in patients living with HIV. Upward trends in CD4 + cell counts observed in this case series suggest that immune checkpoint inhibitors may enhance HIV disease control. Further research is needed for this patient population to supply more robust evidence for clinical practice. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are mediators of intercellular communication, recently recognised for their clinical applications. Accurate characterisation and quantification of EVs are critical for understanding of their function and clinical relevance. Many platforms utilise fluorescence for EV characterisation, frequently labelling surface proteins to identify EVs. The heterogeneity of EVs and the lack of a universal protein marker encourages the use of generic EV labelling methods, including membrane labelling. Using nano‐flow cytometry, we evaluated six membrane dyes, including MemGlow and CellMask. Evaluation criteria included EV labelling efficacy, non‐specific labelling of very low‐density lipoproteins (VLDLs), brightness and dye aggregation. Significant variation was observed in dye performance, with certain dyes showing poor EV labelling efficacy or high affinity to VLDLs. Importantly, several promising candidates were identified for further investigation. Overall, this study highlights the importance of selecting appropriate membrane dyes for EV staining tailored to the aims of the study and the EV origin. MemGlow and CellMask proved favourable, allowing bright, sensitive staining of EV membranes with minimal aggregation. However, MemGlow showed an affinity to VLDLs, and CellMask requires additional sample handling for optimal labelling. These results contribute to deepening our understanding of EV membrane dyes, allowing for better dye selection and EV identification in future studies. [ABSTRACT FROM AUTHOR]

Motivation Single cell profiling by cytometry has emerged as a key technology in biology, immunology and clinical-translational medicine. The correct annotation, which refers to the identification of clusters as specific cell populations based on their marker expression, of clustered high-dimensional cytometry data, is a critical step of the analysis. Its accuracy determines the correct interpretation of the biological data. Despite the progress in various clustering algorithms, the annotation of clustered data still remains a manual, time consuming and error-prone task. We developed a user-friendly cluster annotation and differential abundance detection tool that can be applied on data generated with Self Organizing Map clustering algorithms, thus simplifying the annotation process of datasets that consist of hundreds or thousands of clusters. Results We present Cytometry Cluster Annotation and Differential Abundance Suite (CyCadas), a semi-automated software tool that facilitates cluster annotation in cytometry data by offering both visual and computational guidance. CyCadas addresses the critical need for efficient and accurate annotation of high-resolution clustered cytometry data, significantly reducing the time needed to perform the analysis compared to both manual gating approaches and manual annotation of clustered data. The tool features a user-friendly interface, visual tools enabling data exploration and automated threshold estimation to separate negative and positive marker expression. It facilitates the definition and annotation of cell phenotypes among multiple clusters in a tree-based data structure. Finally, it calculates the abundance of various cell populations across the conditions with statistical interpretation. It is an ideal resource for researchers aiming to streamline their cytometry workflow. Availability and implementation CyCadas is available as open source at: https://github.com/DII-LIH-Luxembourg/cycadas. [ABSTRACT FROM AUTHOR]

Malignant peritoneal mesothelioma (MPeM) is a rare primary malignant tumor originating from peritoneal mesothelial cells. Insufficient specificity of the symptoms and their frequent reappearance following surgery make it challenging to diagnose, creating a need for more efficient treatment options. Natural killer cells (NK cells) are part of the innate immune system and are classified as lymphoid cells. Under the regulation of activating and inhibiting receptors, NK cells secrete various cytokines to exert cytotoxic effects and participate in antiforeign body, antiviral, and antitumor activities. This review provides a comprehensive summary of the specific alterations observed in NK cells following MPeM treatment, including changes in cell number, subpopulation distribution, active receptors, and cytotoxicity. In addition, we summarize the impact of various therapeutic interventions, such as chemotherapy, immunotherapy, and targeted therapy, on NK cell function post-MPeM treatment. [ABSTRACT FROM AUTHOR]

Background: In this study, Schisandrin B (SCHB), the main active component of Schisandra chinensis extract (SCE), was taken as the research object. From gene, microRNA (miR-124), and the level of protein expression system to study the influences of microglia phenotype to play the role of nerve inflammation. Methods: In this study, we investigated the role of miR-124 in regulating microglial polarization alteration and NF-κB/TLR4 signaling and MAPK signaling in the LPS-induced BV2 by PCR, western blot, ELISA, immunofluorescence, and cytometry. Results: SCE and SCHB significantly reduced the NO-releasing, decreased the levels of TNF-α, iNOS, IBA-1, and ratio of CD86+/CD206+, and increased the levels of IL-10, Arg-1. In addition, SCE and SCHB inhibited the nucleus translocation of NF-κB, decreased the expressions of IKK-α, and increased the expressions of IκB-α. Besides, the expressions of TLR4 and MyD88, and the ratios of p-p38/p38, p-ERK/ERK, and p-JNK/JNK were reduced by SCE and SCHB treatments. Furthermore, SCHB upregulated the mRNA levels of miR-124. However, the effects of SCHB were reversed by the miR-124 inhibitor. Conclusions: These findings suggested SCHB downregulated NF-κB/TLR4/MyD88 signaling pathway and MAPK signaling pathway via miR-124 to restore M1/M2 balance and alleviate depressive symptoms. [ABSTRACT FROM AUTHOR]

Digitization in pathology and cytology labs is now widespread, a significant shift from a decade ago when few doctors used image processing tools. Despite unchanged scanning times due to excitation in fluorescent imaging, advancements in computing power and software have enabled more complex algorithms, yielding better-quality results. This study evaluates three nucleus segmentation algorithms for ploidy analysis using propidium iodide-stained digital WSI slides. Our goal was to improve segmentation accuracy to more closely match DNA histograms obtained via flow cytometry, with the ultimate aim of enhancing the calibration method we proposed in a previous study, which seeks to align image cytometry results with those from flow cytometry. We assessed these algorithms based on raw segmentation performance and DNA histogram similarity, using confusion-matrix-based metrics. Results indicate that modern algorithms perform better, with F1 scores exceeding 0.845, compared to our earlier solution's 0.807, and produce DNA histograms that more closely resemble those from the reference FCM method. [ABSTRACT FROM AUTHOR]

Background/Objectives: Flow cytometry is a convenient tool in immunophenotyping for monitoring the status of immunological conditions and diseases. The aim of this study was to investigate the effect of isolation and cryopreservation by flow cytometric analysis on subpopulations of CD4+ T helper (Th), T regulatory (Treg), CD8+ T cytotoxic (Tc), CD56+ NK, CD19+ B and monocytes. Freshly isolated and cryopreserved peripheral blood mononuclear cells (PBMCs) were compared to fresh whole blood. Methods: Peripheral blood was collected from healthy donors and prepared for flow cytometric analysis using the same panels of antibodies throughout the study. Results: Comparisons between fresh (F)- and cryopreserved (C)-PBMCs showed no major differences in percentages of CD4+, Th1, Th2 and CD4+CD25+CD127low Treg cells. No differences in percentage of CD8+ or subpopulations of naive/stem, central or effector memory cells were observed between F- and C-PBMCs. The percentage of CD56+ NK cells, CD19+ B cells or classical and nonclassical monocytes did not differ between F-and C-PBMCs either. On the contrary, whole blood had lower percentages of Th and NK cells but higher percentages of Th1, Th17, Th1Th17, Tregs, Tc and B cells compared to C-PBMCs, while it had a higher proportion of Tc compared to F-PBMCs. Conclusions: Flow cytometric immunophenotyping minimally differs between freshly isolated and cryopreserved PBMCs. This implies the possibility of cryostorage of cohorts for later analysis. Importantly, care must be taken when comparing results from whole blood with isolated and cryopreserved PBMCs. Collectively, these results can contribute to the standardization of flow cytometric protocols in both clinical and research settings. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to investigate how L1 cell adhesion molecule (L1CAM) interacting with protein tyrosine kinase 2 (PTK2) affects endometrial cancer (EC) progression and determine its association with the focal adhesion kinase (FAK)–growth factor receptor-bound protein 2 (GRB2)–son of sevenless (SOS)–rat sarcoma (RAS) pathway. EC is a female cancer of major concern in the world, and its incidence has increased rapidly in recent years. L1CAM is considered a reliable marker of poor prognosis in patients with EC. Material and Methods: A single-center and prospective study was conducted using data from the Cancer Genome Atlas and samples from normal and EC tissues to explore the differential expression of L1CAM. Additional experimental models included human immortalized endometrial epithelium cells (hEECs) and EC cell lines such as KLE, RL95-2, and Ishikawa. L1CAM expression was regulated using lentiviruses designed for either overexpression or interference, and PTK2/focal adhesion kinase (FAK) signaling was inhibited with PF431396. Transfected KLE cells were injected into mice, and tumor growth was monitored over 14 days. Cellular proliferation and survival were assessed using cell counting kit, colony formation, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling assays. Metastatic behavior was evaluated through Transwell assays for cell migration and invasion. The expression levels of matrix metallopeptidase (MMP) 2 and MMP9 were determined by Western blot. In addition, the activation of the FAK–GRB2–SOS–RAS pathway was examined by assessing the protein levels of FAK, GRB2, SOS, and RAS. Results: There was a significant difference in L1CAM expression between EC tumor tissues and normal tissues, and L1CAM messenger RNA (1.85-fold) and L1CAM protein (2.59-fold) were significantly more expressed in EC tissues (P < 0.01) than in normal tissues. The tumor growth of L1CAM overexpressing EC cells was faster than that of negative control EC cells (6.43 fold; P < 0.001). L1CAM promoted the expression of FAK (1.43-2.72-fold; P < 0.001); enhanced EC cell proliferation (P < 0.01), survival and motility (P < 0.001), migration (P < 0.001), and invasion (P < 0.001); and activated the FAK–GRB2–SOS–RAS pathway, all of which were reversed when FAK expression was not upregulated (P < 0.001). Conclusion: By upregulating PTK2 and its encoded protein FAK, L1CAM was found to promote tumor progression and increase the activation of the FAK–GRB2–SOS–RAS pathway. These findings establish L1CAM and PTK2 as reference genes for poor prognostic prediction in EC and as targets for EC therapy, providing a valuable basis for distinguishing between benign and malignant endometrial conditions and justifying the necessity of targeted therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background: The aim of the present study was to investigate the function of novel circular RNA hsa_circ_0036683 (circ‐36683) in non‐small cell lung cancer (NSCLC). Methods: RNA sequencing was used to screen out differentially expressed miRNAs. Expression levels of miR‐4664‐3p and circ‐36683 were evaluated in lung carcinoma cells and tissues by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The effects of miR‐4664‐3p and circ‐36683 on proliferation and migration were assessed using cell counting kit‐8 (CCK‐8), wound healing and transwell migration assays and xenograft experiments. The targeting relationship of circ‐36683/miR‐4664‐3p/CDK2AP2 was assessed by luciferase reporter assays, western blot, qRT‐PCR and argonaute2‐RNA immunoprecipitation (AGO2 RIP). Co‐immunoprecipitation (Co‐IP), 5‐ethynyl‐2′‐deoxyuridine (EdU) staining and CCK‐8 were used to validate the indispensable role of CDK2AP2 in suppressing cell proliferation as a result of CDK2AP1 overexpression. Results: By RNA sequencing, miR‐4664‐3p was screened out as an abnormally elevated miRNA in NSCLC tissues. Transfection of miR‐4664‐3p could promote cell proliferation, migration and xenograft tumor growth. As a target of miR‐4664‐3p, CDK2AP2 expression was downregulated by miR‐4664‐3p transfection and CDK2AP2 overexpression could abolish the proliferation promotion resulting from miR‐4664‐3p elevation. Circ‐36683, derived from back splicing of ABHD2 pre‐mRNA, was attenuated in NSCLC tissue and identified as a sponge of miR‐4664‐3p. The functional study revealed that circ‐36683 overexpression suppressed cell proliferation, migration and resulted in G0/G1 phase arrest. More importantly, the antioncogenic function of circ‐36683 was largely dependent on the miR‐4664‐3p/CDK2AP2 axis, through which circ‐36683 could upregulate the expression of p53/p21/p27 and downregulate the expression of CDK2/cyclin E1. Conclusion: The present study revealed the antioncogenic role of circ‐36683 in suppressing cell proliferation and migration and highlighted that targeting the circ‐36683/miR‐4664‐3p/CDK2AP2 axis is a promising strategy for the intervention of NSCLC. [ABSTRACT FROM AUTHOR]

PUPAID is a workflow written in R + ImageJ languages which is dedicated to the semi-automated processing and analysis of multi-channel immunofluorescence data. The workflow is designed to extract fluorescence signals within automatically-segmented cells, defined here as Areas of Interest (AOI), on whole multi-layer slides (or eventually cropped sections of them), defined here as Regions of Interest (ROI), in a simple and understandable yet thorough manner. The included (but facultative) R Shiny-based interactive application makes PUPAID also suitable for scientists who are not fluent with R programming. Furthermore, we show that PUPAID identifies significantly more cells, especially in high-density regions, as compared to already published state-of-the-art methods such as StarDist or Cellpose. For extended possibilities and downstream compatibility, single cell information is exported as FCS files (the standardized file format for single cell-based cytometry data) in order to be openable using any third-party cytometry analysis software or any analysis workflow which takes FCS files as input. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is one of the major threats to human life worldwide. MiR‐190 has been found to perform essential roles in multiple cancer progression; however, there have been no studies focused on its function and underlying regulatory mechanism in lung cancer. Method: The miR‐190 expression was detected by real‐time quantitative polymerase chain reaction (RT‐qPCR). The cell functional experiments, including cell counting kit‐8 (CCK‐8), colony formation and transwell assay were conducted in vitro, as well as animal experiments performed in vivo. The regulation and potential binding sites of CBX4 on miR‐190 were predicted by TCGA data set and JASPAR website and verified by ChIP assay and dual‐luciferase reporter assay. The prospects binding site of miR‐190‐3p on CBX4 3′UTR region was predicted by StarBase and verified by dual‐luciferase reporter assay. Results: MiR‐190 was decreased in lung cancer cells. The overexpression of miR‐190 had no effects on cell proliferation, but significantly inhibited cancer metastasis both in vitro and in vivo. Moreover, miR‐190 expression could be transcriptionally inhibited by CBX4, and CBX4 was the direct target of miR‐190‐3p. Conclusion: MiR‐190 served as a cancer metastasis inhibitor in lung cancer and formed a regulatory loop with CBX4. These findings provided emerging insights into therapeutic targets and strategies for metastatic lung cancer. [ABSTRACT FROM AUTHOR]

CD163, a scavenger receptor with anti-inflammatory function expressed exclusively on monocytes/macrophages, is dysregulated in cases of diabetes complications. This study aimed to characterize circulating CD163+ monocytes in the presence (D+Comps) or absence (D−Comps) of diabetes-related complications. RNA-sequencing and mass cytometry were conducted on CD163+ monocytes in adults with long-duration diabetes and D+Comps or D−Comps. Out of 10,868 differentially expressed genes identified between D+Comps and D−Comps, 885 were up-regulated and 190 were down-regulated with a ≥ 1.5-fold change. In D+Comps, 'regulation of centrosome cycle' genes were enriched 6.7-fold compared to the reference genome. MIR27A, MIR3648-1, and MIR23A, the most up-regulated and CD200R1, the most down-regulated gene, were detected in D+Comps from the list of 75 'genes of interest'. CD163+ monocytes in D+Comps had a low proportion of recruitment markers CCR5, CD11b, CD11c, CD31, and immune regulation markers CD39 and CD86. A gene–protein network identified down-regulated TLR4 and CD11b as 'hub-nodes'. In conclusion, this study reports novel insights into CD163+ monocyte dysregulation in diabetes-related complications. Enriched centrosome cycle genes and up-regulated miRNAs linked to apoptosis, coupled with down-regulated monocyte activation, recruitment, and immune regulation, suggest functionally distinct CD163+ monocytes in cases of diabetes complications. Further investigation is needed to confirm their role in diabetes-related tissue damage. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103− counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function. [ABSTRACT FROM AUTHOR]

Compared to animal cells, phenotypic characterization of single plant cells on microfluidic platforms is still rare. In this work, we collated population statistics on the morphological, biochemical, physical and electrical properties of Arabidopsis protoplasts under different external and internal conditions, using progressively improved microfluidic platforms. First, we analyzed the different effects of three phytohormones (auxin, cytokinin and gibberellin) on the primary cell wall (PCW) regeneration process using a microfluidic flow cytometry platform equipped with a single-channel fluorescence sensor. Second, we correlated the intracellular reactive oxygen species (ROS) level induced by heavy metal stress with the concurrent PCW regeneration process by using a dual-channel fluorescence sensor. Third, by integrating contraction channels, we were able to effectively discriminate variations in cell size while monitoring the intensity of intracellular ROS signaling. Fourth, by combining an electrical impedance electrode with the contraction channel, we analyzed the differences in electrical and mechanical properties of wild-type and mutant plant cells before and after primary cell wall regeneration. Overall, our work demonstrates the feasibility and sensitivity of microfluidic flow cytometry in high-throughput phenotyping of plant cells and provides a reference for assessing metabolic and physiological indicators of individual plant cells in multiple dimensions. [ABSTRACT FROM AUTHOR]

Mass cytometry is a cutting-edge high-dimensional technology for profiling marker expression at the single-cell level, advancing clinical research in immune monitoring. Nevertheless, the vast data generated by cytometry by time-of-flight (CyTOF) poses a significant analytical challenge. To address this, we describe ImmCellTyper (https://github.com/JingAnyaSun/ImmCellTyper), a novel toolkit for CyTOF data analysis. This framework incorporates BinaryClust, an in-house developed semi-supervised clustering tool that automatically identifies main cell types. BinaryClust outperforms existing clustering tools in accuracy and speed, as shown in benchmarks with two datasets of approximately 4 million cells, matching the precision of manual gating by human experts. Furthermore, ImmCellTyper offers various visualisation and analytical tools, spanning from quality control to differential analysis, tailored to users' specific needs for a comprehensive CyTOF data analysis solution. The workflow includes five key steps: (1) batch effect evaluation and correction, (2) data quality control and pre-processing, (3) main cell lineage characterisation and quantification, (4) in-depth investigation of specific cell types; and (5) differential analysis of cell abundance and functional marker expression across study groups. Overall, ImmCellTyper combines expert biological knowledge in a semi-supervised approach to accurately deconvolute well-defined main cell lineages, while maintaining the potential of unsupervised methods to discover novel cell subsets, thus facilitating high-dimensional immune profiling. [ABSTRACT FROM AUTHOR]

Microfluidic cytometry is an emerging technology for single-cell analysis and offers rich biochemical information about cells. Three-dimensional focusing of cells is a key function for microfluidic cytometry. However, the existing microfluidic chips have fixed geometries and are designed for specific cells, limiting the applicability of microfluidic cytometry. In this work, we develop the ultrastretchable microchannel for size-tunable three-dimensional elasto-inertial focusing of particles and cells. This channel can be modulated by stretching the chip, enabling the focusing of particles and cells with a wide range in sizes. The focusing performance of this ultrastretchable channel is characterized experimentally at different particle sizes, flow rates, polyethylene oxide concentrations, and stretch ratios, showing the great capability in three-dimensional focusing of particles. Finally, the applicability of our ultrastretchable microchannel to biological cells is verified by three-dimensional focusing of yeast cells and fibroblast cells (3T3 cells). The ultrastretchable microchannel is easy for mass production and can be integrated with optical sensing modules for downstream single-cell imaging and analysis. [ABSTRACT FROM AUTHOR]

Characterization of inflammation in chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) is an ongoing research process. To overcome limitations of current cytologic techniques, we investigated whether immunofluorescence multiplex image cytometry could quantify intact neutrophils, eosinophils, and other immune cells in solid upper airway mucosa. We used a four-channel immunofluorescence-microscopy technique for the simultaneous detection of the leukocyte marker CD45, the neutrophil marker myeloperoxidase, two eosinophil markers, i.e., major basic protein and eosinophil peroxidase, and DAPI (4′,6-diamidin-2-phenylindole), in formalin-fixed paraffin-embedded upper airway tissue samples of patients with CRSwNP and CRSsNP, as well as of patients free of CRS with inferior turbinate hypertrophy (controls). Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively. Positive and negative immunostaining were differentiated with a specific fluorescence signal/background signal ratio. Isotype controls were used as negative controls. In six controls, nine patients with CRSsNP, and 11 patients with CRSwNP, the median area scanned and median cell count per patient were 14.2 mm2 and 34,356, respectively. In CRSwNP, the number of eosinophils was three times higher (23%) than that of neutrophils (7%). Three times more immune cells were encountered in CRSwNP (33%) compared to CRSsNP (11%). In controls, inflammation was balanced between the epithelial layer and lamina propria, in contrast to CRS (three times more pronounced inflammation in the lamina propria). The quantification of intact neutrophils, eosinophils, and other immune cells in solid tissue with undisrupted architecture seems feasible with immunofluorescence multiplex image cytometry. [ABSTRACT FROM AUTHOR]