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Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

Flow cytometry, single-cell RNA sequencing, and other analyses enable us to capture immune profiles of the tumor microenvironment. Here, we present a protocol to characterize the immune profile of tumor-bearing mice. We describe steps for establishing mouse models and preparing single-cell suspensions from tumor tissue and other immune-related organs, which can be further analyzed by flow cytometry and other omics assays. We then detail procedures for staining, flow cytometry analysis, and phenotyping of the immune cell populations. For complete details on the use and execution of this protocol, please refer to Miyauchi et al.1.

Dye-encoded bead-based assays are widely used for diagnostics. Multiple bead populations are required for multiplexing and can be produced using different dye colors, labeling levels, or combinations of dye ratios. Ready-to-use multiplex bead populations restrict users to specific targets, are costly, or require specialized instrumentation. In-house methods produce few bead plexes or require many fine-tuning steps. To expand bead encoding strategies, we present a simple, safe, and cost-effective bench-top system for generating bead populations using photobleaching. By photobleaching commercially available dye-encoded magnetic beads for different durations, we produce three times as many differentiable bead populations on flow cytometry from a single dye color. Our photobleaching system uses a high-power LED module connected to a light concentrator and a heat sink. The beads are photobleached in solution homogeneously by constant mixing. We demonstrate this photobleaching method can be utilized for cross-testing antibodies, which is the first step in developing immunoassays. The assay uses multiple photobleached encoded beads conjugated with capture antibodies to test many binding pairs simultaneously. To further expand the number of antibodies that can be tested at once, several antibodies were conjugated to the same bead, forming a pooled assay. Our assay predicts the performance of antibody pairs used in ultrasensitive Simoa assays, narrowing the number of cross-tested pairs that need to be tested by at least two-thirds and, therefore, providing a rapid alternative for an initial antibody pair screening. The photobleaching system can be utilized for other applications, such as multiplexing, and for photobleaching other particles in solution. [ABSTRACT FROM AUTHOR]

Inertial focusing excels at the precise spatial ordering and separation of microparticles by size within fluid flows. However, this advantage, resulting from its inherent size-dependent dispersion, could turn into a drawback that challenges applications requiring consistent and uniform positioning of polydisperse particles, such as microfiltration and flow cytometry. To overcome this fundamental challenge, we introduce Dispersion-Free Inertial Focusing (DIF). This new method minimizes particle size-dependent dispersion while maintaining the high throughput and precision of standard inertial focusing, even in a highly polydisperse scenario. We demonstrate a rule-of-thumb principle to reinvent an inertial focusing system and achieve an efficient focusing of particles ranging from 6 to 30 μm in diameter onto a single plane with less than 3 μm variance and over 95% focusing efficiency at highly scalable throughput (2.4–30 mL h−1) – a stark contrast to existing technologies that struggle with polydispersity. We demonstrated that DIF could be applied in a broad range of applications, particularly enabling high-yield continuous microparticle filtration and large-scale high-resolution single-cell morphological analysis of heterogeneous cell populations. This new technique is also readily compatible with the existing inertial microfluidic design and thus could unleash more diverse systems and applications. [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]

Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics technology has provided significant progress in our understanding of oomycete biology, however, transformation studies of Phytophthora for gene functionalisation are still in their infancy. Only a limited number of Phytophthora species have been successfully transformed and gene edited to elucidate the role of particular genes. There is a need to escalate our efforts to understand molecular processes, gene regulation and infection mechanisms of the pathogen to enable us to develop new disease management strategies. The primary obstacle hindering the advancement of transformation studies in Phytophthora is their challenging and unique nature, coupled with our limited comprehension of why they remain such an intractable system to work with. In this study, we have identified some of the key factors associated with the recalcitrant nature of P. cinnamomi. We have incorporated fluorescence microscopy and flow cytometry along with the organelle-specific dyes, fluorescein diacetate, Hoechst 33342 and MitoTracker™ Red CMXRos, to assess P. cinnamomi-derived protoplast populations. This approach has also provided valuable insights into the broader cell biology of Phytophthora. Furthermore, we have optimized the crucial steps that allow transformation of P. cinnamomi and have generated transformed isolates that express a cyan fluorescent protein, with a transformation efficiency of 19.5%. We therefore provide a platform for these methodologies to be applied for the transformation of other Phytophthora species and pave the way for future gene functionalisation studies. [ABSTRACT FROM AUTHOR]

Purpose: To develop diagnostic recommendations for diffuse large B-cell vitreoretinal lymphoma (VRL) in Chinese patients. Methods: Retrospective observational case series. Seventy-three eyes of 40 VRL patients and 8 control patients were analyzed. Eighteen patients from Beijing Tongren Hospital and 46 patients from literature were involved as validations. Results: Diagnostic methods included (1) typical clinical manifestations; (2) vitreous cytology; (3) immunohistochemical examination of vitreous or choroid/retina; (4) aqueous humor or vitreous cytokine; (5) vitreous cell gene rearrangement; (6) vitreous flow cytometry. If patients meet (1)+(2)+(3), or if they meet (1), and two of (4), (5), (6) are positive, they can be diagnosed as VRL. The sensitivity and specificity values for accurate diagnosis were 0.975 and 1.00. One hundred percent eyes from Beijing Tongren Hospital and 92.7% eyes from literature can be diagnosed. Conclusion: We developed diagnostic recommendations for diffuse large B-cell VRL through vitreous cytology combined with multiple auxiliary examinations. [ABSTRACT FROM AUTHOR]

Flavonoids play a significant role in mitigating the production of reactive oxygen species (ROS) and subsequent oxidative stress. In the present study we aimed to evaluate the cytoprotective and antioxidant defense mechanism of flavonoid fraction from Carica papaya L. (FFCP) leaf in human PBMC. For this investigation, the human PBMCs were lotted into three groups viz., negative control, positive control (treated with H 2 O 2), and experiment group (pretreated with FFCP and challenged with H 2 O 2). The cytoprotective mechanism was evaluated by assessing the intracellular ROS and Ca2+ mobilization, membrane and DNA damages, antioxidant enzymes, and Nrf2 activation. In-silico analysis was employed for assessing the interactions of flavonoids in FFCP with the Nrf2 binding pocket of Keap 1 protein. The results of the experiment unveiled that FFCP protected the human PBMCs from H 2 O 2 induced oxidative damages such as lipid peroxidation, LDH leakage, DNA breakage, reduced the intracellular ROS and Ca2+ mobilization as compared to positive control. FFCP pretreatment effectively improved the activity of key antioxidant enzyme markers, such as SOD (1.66 ± 0.032-fold increase), CAT (2.24 ± 0.09-fold increase), GPx (1.94 ± 0.05-fold increase), and GR (1.81 ± 0.14-fold increase). Furthermore, the FFCP modulated the Nrf2 gene expression (a 2.38 ± 0.25 -fold) and its translocation into the nucleus as compared to the positive control. In-silico analysis enunciated that all flavonoids exhibited strong affinity with the Nrf2 binding pocket of Keap1 protein via stable hydrogen bonding and hydrophobic interactions. These observations suggest that FFCP is a natural source of bioactive compounds with nutraceutical benefits for combating oxidative stress-associated diseases by protecting the cells from oxidative stress induced damages as well as by instigating antioxidant defense mechanisms. [Display omitted] • Flavonoid fraction of Carica papaya L. (FFCP) protected the human PBMCs from oxidative damages- Lipid peroxidation, LDH leakage and DNA damage. • FFCP pretreatment effectively reduced the intracellular calcium mobilization and ROS production induced by oxidative stress. • FFCP pretreatment enhanced the activity of key antioxidant enzymes under oxidative stress. • FFCP upregulated the expression and translocation of Nrf2 transcription factor into nucleus. • Flavonoids in FFCP exhibited good binding affinity with the Nrf2 binding pocket of Keap 1 • All flavonoids exhibited good ADME properties. [ABSTRACT FROM AUTHOR]

Adequate re-establishment of thymopoiesis is critical for long-term immune reconstitution after hematopoietic cell transplantation (HCT), potentially impacting patient survival rates. This study aimed to evaluate immune reconstitution in pediatric HCT recipients by quantifying recent thymic emigrants (RTEs), specifically CD3+CD31+CD45RA+ cells. We conducted a retrospective analysis of 186 pediatric patients transplanted between 2013 and 2020, undergoing their first allogeneic HCT, who were alive in the first 100 days after transplantation with immune recovery evaluation at three time points: day 100, day 180 and day 360 after HCT. We analyzed the distribution of peripheral blood subsets of T, B and natural killer lymphocytes and assessed the impact of underlying disease, HCT type, stem cell source, recipient age, conditioning regimen, graft-versus-host disease (GVHD) occurrence and cytomegalovirus (CMV) reactivation on immune recovery. At day 100, patients under 10 years exhibited higher RTE CD4+ and CD8+CD31+CD45RA+ counts compared with older patients (5.3 versus 2.2 cells/µL, P = 0.022 and 48 versus 72.8 cells/µL, P = 0.049, respectively). Patients with haploidentical HCT had lower RTE CD4+ counts compared with those with unrelated or related donors (2.4 versus 4.4 versus 7.9 cells/µL, P = 0.024). Administration of rabbit anti-thymocyte globulin negatively impacted RTE CD4+ production (median, 6.5 versus 2.4 cells/µL, P = 0.007). At day 180, the presence of GVHD had a negative influence on RTE production (11.7 versus 56.8 cells/µL, P < 0.001), particularly higher-grade acute GVHD (without, 56.8 cells/µL, grade 1–2, 28.1 cells/µL, grade 3–4, 6.0 cells/µL, P < 0.001). Patients with CMV reactivation had higher CD8+CD31+CD45RA+ compared with those without reactivation (median, 204.6 versus 100.2 cells/µL, P = 0.022). At day 360, no variables significantly affected RTE recovery. Overall survival at 5-year follow-up was 87.7%, with a median of 1170 days (range, 122–3316). Multivariate analysis showed that age >10 years (P = 0.038), negative CMV donor serology (P = 0.0029) and acute GVHD (P = 0.0026) had a negative impact on survival. This study highlights variations in RTE production based on patient age, donor type and immunosuppression regimen employed. [ABSTRACT FROM AUTHOR]

HIV-1 chronically infects host CD4+ T lymphocytes and further affects a variety of immune cells, including CD8+ T cells. In our previous study, by analyzing unbiased high-dimensional single-cell RNA-seq data (scRNA-seq), we found that the frequency of GZMK+CD8+ T cells expressing granzyme K (GZMK) was increased in people living with HIV-1 (PLWHs). However, the phenotypic and functional characteristics of these cells in chronic HIV-1 infection and their correlation with disease are not well understood. In this study, we conducted a comprehensive analysis of scRNA-seq and matched T-cell receptor repertoire (TCR) sequencing data to delve into the characterizations of GZMK+CD8+ T cells, which was further validated by flow cytometry. We observed heterogeneity within the GZMK+CD8+ T cells, which could be further subdivided into a GZMK+GZMB- subset and a GZMK+GZMB+ subset, with the latter being significantly enriched in PLWHs. The GZMK+GZMB+ cells are a unique subset within CD8+ T cells, characterized by high proliferation, activation, inflammatory response, clone transition, etc., and are one of the differentiation endpoints by pseudotemporal analysis of CD8+αβ T cells. Despite being predominantly composed of effector memory T cells (Tem), similar to the GZMK+GZMB- subset, the GZMK+GZMB+ subset exhibits differentiation at a later stage than the GZMK+GZMB- subset. We also observed that the frequency/count of GZMK+GZMB+CD8+ T cells was negatively correlated with CD4/CD8 ratio, and positively correlated with HIV DNA, IP-10, and MIG levels in PLWHs. In vitro experiments demonstrate that GZMK can potentiate the stimulatory effects of lipopolysaccharide (LPS) on THP-1 macrophages via the TLR-4 pathway, significantly enhancing the secretion of IP-10, MIG, and MCP-1, as well as increasing the proportion of TNF-α+ cells. In conclusion, in PLWHs, GZMK+GZMB+CD8+ T cells are a highly reactive and inflammatory-inducing subset that may be associated with systemic inflammation. • We characterized GZMK+CD8+ T cells and identified a new subset in HIV-1 patients. • We used scRNA-seq, TCR-seq, flow cytometry, and functional experiments. • The GZMK+GZMB+ subset is a subset of GZMK+ T cells that is enriched in HIV-1 patients. • The GZMK+GZMB+ subset is characterized by proliferation, activation, inflammation. • The GZMK+GZMB+ subset is highly reactive and associated with systemic inflammation. [ABSTRACT FROM AUTHOR]

Objective: This study assessed the cellular composition and effects of leukocyte-platelet-rich fibrin (L-PRF) exudate on whole blood platelets from healthy volunteers. Key objectives included evaluating leukocyte subpopulations, platelet activation markers, platelet-leukocyte interactions and quantifying inflammatory cytokines within the L-PRF exudate. Materials and methods: L-PRF was obtained from 20 healthy donors. Flow cytometry methodologies were used to assess intracellular calcium kinetics and activated GPIIbIIIa, and P-selectin expression. Leukocyte subpopulations and platelet-leukocyte interactions were characterized using monoclonal antibodies. Inflammatory cytokines (IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70) within L-PRF exudate were quantified using a cytometric bead array. Results: The expression of activated GPIIbIIIa, and P-selectin exhibited a significant increase (p < 0.001) when L-PRF exudate was added to platelets of whole blood. Regarding intracellular Ca2+ mobilization, the L-PRF exudate elicited significant responses (p < 0.001). L-PRF exudate contained different leukocytes populations, being TCD4 + the most representative of T cells. It was possible to stablish a profile of cytokines produced by the L-PRF exudate, with human IL-8 cytokine exhibiting the highest average (16.90 pg/mL). Conclusions: Despite the study limitations, the research yielded important insights: 1- L-PRF exudate can stimulate platelet activation, essential in healing, tissue inflammation and remodeling. 2-The presence of leukocyte subpopulations within L-PRF exudate reflexes its complexity and potential to enhance immune responses. 3-The analysis of inflammatory cytokines within L-PRF exudate revealed its immunomodulatory potential. These findings are valuable evidences for understanding the potential role of L-PRF exudate in regenerative dentistry and medicine, offering innovative therapeutic strategies. Clinical relevance: This research highlights crucial aspects that could significantly influence the clinical use of L-PRF exudate in the oral cavity. The findings support the application of L-PRF exudate in both surgical and regenerative dentistry, facilitating the development of innovative therapeutic strategies to enhance patient outcomes. [ABSTRACT FROM AUTHOR]

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer's software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay's ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established. [ABSTRACT FROM AUTHOR]

SET domain‐containing 2 (SETD2) is a histone methyltransferase. It regulates the activity of H3K36me3 to enhance gene transcription. Macrophages (Mϕs) are one of the cell types involved in immune response. The purpose of this study is to clarify the role of SETD2 in regulating the immune property of Mϕ. The Mφs were isolated from the bronchoalveolar lavage fluid (BALF) and analysed through flow cytometry and RNA sequencing. A mouse strain carrying Mφs deficient in SETD2 was used. A mouse model of airway allergy was established with the ovalbumin/alum protocol. Less expression of SETD2 was observed in airway Mϕs in patients with allergic asthma. SETD2 of M2 cells was associated with the asthmatic clinical response. Sensitization reduced the expression of SETD2 in mouse respiratory tract M2 cells, which is associated with the allergic reaction. Depletion of SETD2 in Mφs resulted in Th2 pattern inflammation in the lungs. SETD2 maintained the immune regulatory ability in airway M2 cells. SETD2 plays an important role in the maintenance of immune regulatory property of airway Mφs. [ABSTRACT FROM AUTHOR]

Diabetic kidney disease (DKD) is a complication of diabetic mellitus. New treatments need to be developed. This study aimed to investigate the effects of quercetin‐4′‐O‐β‐D‐glucopyranoside (QODG) on podocyte injury. Podocytes were cultured in high glucose (HG) medium, treated with QODG, and overexpressing or knocking down SIRT5. Oxidative stress indicators were assessed using corresponding kits. Pyroptosis was detected by flow cytometry and western blot analysis. Succinylation modification was detected using immunoprecipitation (IP) and western blot analysis. The interaction between NEK7 and NLRP3 was determined by co‐IP. The results indicated that QODG inhibited oxidative stress and pyroptosis of podocytes induced by HG. Besides, QODG suppressed succinylation levels in HG‐induced podocytes, with the upregulation of SIRT5. Knockdown of SIRT5 reversed the effects of QODG on oxidative stress and pyroptosis. Moreover, SIRT5 inhibited the succinylation of NEK7 and the interaction between NLRP3 and NEK7. In conclusion, QODG upregulates SIRT5 to inhibit the succinylation modification of NEK7, impedes the interaction between NEK7 and NLRP3, and then inhibits the pyroptosis and oxidative stress injury of podocytes under HG conditions. The findings suggested that QODG has the potential to treat DKD and explore a novel underlying mechanism of QODG function. [ABSTRACT FROM AUTHOR]

Background: Type-2 diabetes mellitus is a metabolic disorder characterised by hyperglycemia and insulin resistance. This study aims to explore the role and mechanism of DIRAS family GTPase 3 (DIRAS3) in mediating pancreatic β-cell death and insulin secretion. Materials and Methods: A bioinformatic analysis of the GSE118230 and GSE150281 datasets was performed to screen differentially expressed genes. The pancreatic β-cell lines, INS-1 and MIN6, were treated with palmitic acid (PA) to mimic the cell models of type-2 diabetes mellitus. CCK-8 assay, 5-ethynyl-2'-deoxyuridine staining, flow cytometry, enzyme-linked immunoassay, immunofluorescence, qRT-PCR, and western immunoblotting were conducted to illustrate the role of DIRAS3 in the cell models. Results: Unlike in normal controls, DIRAS3 was highly expressed in PA-treated pancreatic β-cells in a dose- and time-dependent manner. Moreover, the silencing of DIRAS3 in the INS-1 cells attenuated PA-induced cell loss by improving cell proliferation and inhibiting apoptosis and prevented the PA-induced impairment of insulin secretion. Consistently, the overexpression of DIRAS3 in the MIN6 cells accelerated PA-induced cell loss and impaired insulin secretion. A Gene Set Enrichment Analysis predicted that phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) was a downstream signalling pathway of DIRAS3, as the inhibitory effects of DIRAS3 on the activation of the PI3K/AKT signalling pathway were confirmed in the INS-1 and MIN6 cells. Moreover, the PI3K inhibitor, LY294002, effectively reversed the protective effects of DIRAS3 silencing on the INS-1 cells. Conclusion: DIRAS3 was highly expressed in the cell models of type-2 diabetes mellitus, contributing to PA-induced cell death and impaired insulin secretion in pancreatic β-cells through the inhibition of the PI3K/AKT signalling pathway. [ABSTRACT FROM AUTHOR]

Summary: The role of maternal tissue in embryogenesis remains enigmatic in many complex organisms. Here, we investigate the contribution of maternal tissue to apical–basal patterning in the kelp embryo.Focussing on Undaria pinnatifida, we studied the effects of detachment from the maternal tissue using microsurgery, staining of cell wall modifications, morphometric measurements, flow cytometry, genotyping and a modified kelp fertilisation protocol synchronising kelp embryogenesis.Detached embryos are rounder and often show aberrant morphologies. When a part of the oogonial cell wall remains attached to the zygote, the apical–basal patterning is rescued. Furthermore, the absence of contact with maternal tissue increases parthenogenesis, highlighting the critical role of maternal signals in the initial stages of development.These results show a key role for the connection to the maternal oogonial cell wall in apical–basal patterning in kelps. This observation is reminiscent of another brown alga, Fucus, where the cell wall directs the cell fate. Our findings suggest a conserved mechanism across phylogenetically distant oogamous lineages, where localised secretion of sulphated F2 fucans mediates the establishment of the apical–basal polarity. In this model, the maternal oogonial cell wall mediates basal cell fate determination by providing an extrinsic patterning cue to the future kelp embryo. [ABSTRACT FROM AUTHOR]

Considering the established pharmacokinetics and toxicity profiles, drug repurposing has emerged as an alternative therapeutic approach for treating cancer. Mefloquine has previously demonstrated inhibitory effects on multiple cancer types. This study aims to explore the impact of mefloquine on nasopharyngeal carcinoma (NPC). We found that mefloquine, at pharmacologically achievable concentrations, displayed anti-NPC activity while sparing normal counterparts. Mefloquine inhibits proliferation and induces death by reducing the levels of Cyclin A2, Bcl-2, and Bcl-xL. Intriguingly, we observed an increase in the levels of the anti-apoptotic protein Mcl-1. Mefloquine exerts its effects on NPC cells by inducing lysosomal-mediated ROS production, and the heightened expression of Mcl-1 is a consequence of ROS generation in mefloquine-treated NPC cells. The combination of an Mcl-1 inhibitor with mefloquine synergistically inhibits NPC growth in mice without causing substantial toxicity. These findings demonstrate the effectiveness and limited toxicity of mefloquine as a monotherapy and in combination with an Mcl-1 inhibitor. Our research underscores the promise of the mefloquine and Mcl-1 inhibitor combination as a potential treatment for NPC. Additionally, the elevation of Mcl-1 is a compensatory response in cells exposed to oxidative stress, offering a potential target to overcome resistance induced by pro-oxidant therapies. [ABSTRACT FROM AUTHOR]

Flow cytometry (FC) was developed in the late 1950s and is nowadays the gold standard for automated quantitative cellular analysis. In the early 1990s, a new generation of cytometry became commercially available—laser scanning cytometry (LSC). While the hardware of LSC was related to FC, LSC provided images of forward scatter and all fluorochromes. This new technique allowed the analysis of solid tissues and reanalysis of samples and provided data similar to those of FC. A number of peer-reviewed, frequently cited studies were published using the features of LSC. Only a minority, however, must have believed that LSC would replace FC by that time. Currently, the LSC is still sold but withheld from any further development. It seems an epoque has come to an end. How did it come? The presented literature review tries to answer some questions. [ABSTRACT FROM AUTHOR]

In-vivo flow cytometry is a noninvasive real-time diagnostic technique that facilitates continuous monitoring of cells without perturbing their natural biological environment, which renders it a valuable tool for both scientific research and clinical applications. However, the conventional approach for improving classification accuracy often involves labeling cells with fluorescence, which can lead to potential phototoxicity. This study proposes a label-free in-vivo flow cytometry technique, called dynamic YOLOv4 (D-YOLOv4), which improves classification accuracy by integrating absorption intensity fluctuation modulation (AIFM) into YOLOv4 to demodulate the temporal features of moving red blood cells (RBCs) and platelets. Using zebrafish as an experimental model, the D-YOLOv4 method achieved average precisions (APs) of 0.90 for RBCs and 0.64 for thrombocytes (similar to platelets in mammals), resulting in an overall AP of 0.77. These scores notably surpass those attained by alternative network models, thereby demonstrating that the combination of physical models with neural networks provides an innovative approach toward developing label-free in-vivo flow cytometry, which holds promise for diverse in-vivo cell classification applications. [ABSTRACT FROM AUTHOR]

Introduction: Oral squamous cell carcinoma (OSCC), while common and with a favorable prognosis in early stages, presents a marked reduction in survival rate upon metastasis to lymph nodes. Early detection of lymph node metastasis via biomarkers could enhance the therapeutic strategy for OSCC. Here, we explored dendritic cells (DCs) and cytotoxic T-cells in tumour-draining lymph nodes (TDLNs) as potential biomarkers. Method: Dendritic cells and cytotoxic T-cells in 33 lymph nodes were analyzed with multi-parameter flow cytometry in TDLNs, regional non-TDLNs surgically excised from 12 OSCC patients, and compared to 9 lymph nodes from patients with benign conditions. Results: Our results displayed a higher proportion of conventional cDC1s with immunosuppressive features in TDLN. Further, high PD-L1 expression on cDC1 in TDLNs was associated with metastasis and/or recurrent disease risk. Also, elevated levels of memory CD8+ T-cells and terminally exhausted PD-1+TCF-1−CD8+ T-cells were observed in TDLNs and non-TDLNs compared to healthy lymph nodes. Conclusions: We conclude that TDLNs contain cells that could trigger an anti-tumor adaptive response, as evidenced by activated cDC1s and progenitor-like TCF-1+ T-cells. The detection of high PDL1 expression on cDC1s was indicative of TDLN metastasis and an adverse prognosis, proposing that PD-L1 on dendritic cells in TDLN could serve as a predictive biomarker of OSCC patients with a worse prognosis. [ABSTRACT FROM AUTHOR]

Objective: To present a comprehensive flow cytometry panel for idiopathic subglottic stenosis (iSGS). Study Design: Controlled ex vivo cohort study. Setting: Tertiary care academic hospital in a metropolitan area. Methods: Flow cytometry and single‐cell RNA sequencing were performed on 9 paired normal and scar tissue samples from iSGS patients. Flow cytometry was used to assess the presence of myeloid (CD11b, CD14, CD15, Siglec8), lymphoid (CD3, CD4, CD8, gamma delta [γδ], FOXP3), endothelial (CD31), fibroblast (CD90, SMA), and epithelial (CD326, CK5) markers. Results: On flow cytometry, iSGS scar is characterized by an increased presence of myeloid, lymphoid, endothelial, and fibroblast cell types, but a decreased presence of epithelial cells. In the myeloid lineage, iSGS scar samples demonstrated increased CD11b+ monocytes (P <.001), Siglec8+ eosinophils (P =.03), and CD14+ monocytes (P =.02). In the lymphoid lineage, iSGS scar demonstrated increased CD3+ T‐cells (P <.001), CD4+ helper T‐cells (P <.001), γδ+ T‐cells (P <.001), and FOXP3+ regulatory T‐cells (P =.002). iSGS scar exhibited specific increases in CD90+ (P =.04) and SMA+ (P <.001) fibroblasts but decreased CD326+ (E‐cadherin) epithelial cells (P =.01) relative to normal samples. Conclusion: We present a comprehensive flow cytometry panel for iSGS. This flow panel may serve as a common platform among airway scientists to elucidate the cellular mechanisms underpinning iSGS and other upper airway pathologies. Scar iSGS samples demonstrate a distinct cellular profile relative to normal iSGS specimens, exhibiting increased fibroblast, endothelial, and inflammatory cell types but decreased epithelium. [ABSTRACT FROM AUTHOR]

Objective: To explore the potential mechanism of lysionotin in treating glioma. Methods: First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively. Results: Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling. Conclusion: Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway. [ABSTRACT FROM AUTHOR]

Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia–ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation. [ABSTRACT FROM AUTHOR]

Trypanosomosis due to Trypanosoma evansi (surra) is one of the most important diseases with a significant impact on camel health and production. Trypanosoma-induced immunosuppression mechanisms, which are key factors of disease pathogenesis, have been characterized in several animal species. The present study investigated, therefore, the impact of trypanosomosis on the immunophenotype of blood leukocytes in camels. For this, the relative and absolute values of blood leukocyte populations, their expression pattern of cell surface molecules, and the numbers of the main lymphocyte subsets were compared between healthy camels and camels with clinical symptoms of chronic surra and serological evidence of exposure to Trypanosoma infection. Leukocytes were separated from the blood of healthy and diseased camels, labeled with fluorochrome-conjugated antibodies, and analyzed by flow cytometry. Compared to healthy camels, the leukogram of diseased camels was characterized by a slightly increased leukocyte count with moderate neutrophilia and monocytosis indicating a chronic inflammatory pattern that may reflect tissue injury due to the long-lasting inflammation. In addition, the analysis of lymphocyte subsets revealed a lower number and percentage of B cells in diseased than healthy camels. In vitro incubation of camel mononuclear cells with fluorochrome-labeled T. evansi revealed a higher capacity of camel B cells than T cells to bind the parasite in vitro. Furthermore, cell viability analysis of camel PBMC incubated in vitro with T. evansi whole parasites but not the purified antigens resulted in Trypanosoma-induced apoptosis and necrosis of camel B cells. Here we demonstrate an association between trypanosomosis in camels and reduced numbers of blood B cells. In vitro analysis supports a high potential of T. evansi to bind to camel B cells and induce their elimination by apoptosis and necrosis. [ABSTRACT FROM AUTHOR]

The main objective of this study was to investigate the effect of insulin on drone semen quality after cryopreservation. Semen samples were obtained from sexually mature drones. The semen samples were pooled and diluted with varying concentrations of insulin lispro, specifically 5 IU (I-5), 10 IU (I-10), 15 IU (I-15), and 20 IU (I-20), as well as a control. The assessment of motility, plasma membrane integrity and DNA integrity was conducted using microscopy. Flow cytometry was used to assess acrosome integrity, mitochondrial membrane potential, and nitric oxide level. The preservation of motility, plasma membrane functional integrity, and acrosome integrity was significantly superior in the I-5 group compared to the control group. However, increasing levels of insulin had a detrimental impact on the integrity of the acrosome in both the I-15 and I-20 groups. The mitochondrial membrane potential was significantly elevated in the I-5 and I-10 groups compared to the control group. However, a statistically significant decrease was observed in both the I-15 and I-20 groups. We observed a significant decrease in nitric oxide levels in both the I-5 and I-10 groups as compared to the control group. The preservation of DNA integrity was significantly higher in the insulin containing groups compared to the control group. The study findings revealed that the inclusion of insulin lispro in drone semen extenders yielded beneficial outcomes for drone semen. [ABSTRACT FROM AUTHOR]

Background: miR-155 is involved in the generation and maintenance of inflammation and pain, endothelial function and immune system homeostasis, all functions that are relevant for migraine. The present study aims to assess the levels of miR-155 in migraine subtypes (episodic and chronic) in comparison to age- and sex-matched healthy controls. Methods: This is a cross-sectional, controlled, study involving three study groups: I) episodic migraine (n = 52, EM), II) chronic migraine with medication overuse (n = 44, CM-MO), and III) healthy controls (n = 32, HCs). We assessed the interictal gene expression levels of miR-155, IL-1β, TNF-α, and IL-10 in peripheral blood monocytes using rtPCR. The monocytic differentiation toward the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes was assessed in circulating monocytes with flow cytometry analysis and cell sorting. Results: miR-155 gene expression was higher in CM-MO group (2.68 ± 2.47 Relative Quantification - RQ) when compared to EM group (1.46 ± 0.85 RQ, p = 0.006) and HCs (0.44 ± 0.18 RQ, p = 0.001). In addition, miR-155 gene expression was higher in EM group when compared to HCs (p = 0.001). A multivariate analysis confirmed the difference between EM and CM-MO groups after correction for age, sex, smoking habit, preventive treatment, aura, presence of psychiatric or other pain conditions. We found higher gene expression of IL-1β, TNF-α, and lower gene expression of IL-10 in migraine participants when compared to HCs (p = 0.001 for all comparisons). TNF-α and IL-10 genes alterations were more prominent in CM-MO when compared to EM participants (p = 0.001). miR-155 positively correlated with IL-1β (p = 0.001) and TNF-α (p = 0.001) expression levels. Finally, in people with CM-MO, we described an up-regulated percentage of events in both M1 and M2 monocytic profiles. Conclusions: Our study shows for the first time a specific profile of activation of miR-155 gene expression levels in monocytes of selected migraine subpopulations, more pronounced in subjects with CM-MO. Interestingly, mir-155 expression correlated with markers of activation of the inflammatory and immune systems. The CM-MO subpopulation showed a peculiar increase of both pro-inflammatory and anti-inflammatory monocytes which worths further investigation. Trial registration: www.clinicaltrials.gov. (NCT05891808). [ABSTRACT FROM AUTHOR]

Background: Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. Results: We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. Conclusions: The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: Understanding immune cell dynamics in kidney transplantation may provide insight into the mechanisms of rejection and improve patient management. B cells have gained interest with a special relevance of the "regulatory" subsets and their graft outcome prognostic value. In this study, we aimed to prove that the direct immunophenotyping and target gene expression analysis of kidney transplant patients' fresh whole blood will help to identify graft rejection risk and assist in the monitoring of kidney transplanted patients. Methods: We employed flow cytometry and qPCR techniques to characterize B and T cell subsets within fresh whole blood samples, with particular emphasis on transitional B cells (TrB) identified as CD19+CD24hiCD38hi. TrB are a relevant population in the context of kidney transplantation and are closely associated with regulatory B cells (Bregs) in humans. Patients were monitored, tracking pertinent clinical parameters and kidney-related events, including alterations in graft function and episodes of biopsy proven rejection. Results: Higher percentages of TrB cells at 3 months after transplantation were positively associated with better graft outcomes and lower biopsy-proven acute rejection risk. Furthermore, a novel panel of B cell regulatory associated genes was validated at 3 months post-transplantation by qPCR analysis of peripheral blood mononuclear cell (PBMC) mRNA, showing high predictive power of graft events and prognostic value. Discussion: These findings suggest that monitoring TrB may provide interesting patient management information, improve transplant outcomes, and allow for personalized drug regimens to minimize clinical complications. [ABSTRACT FROM AUTHOR]

Methionine, an indispensable amino acid crucial for dietary balance, intricately governs metabolic pathways. Disruption in its equilibrium has the potential to heighten homocysteine levels in both plasma and tissues, posing a conceivable risk of inducing inflammation and detriment to the integrity of vascular endothelial cells. The intricate interplay between methionine metabolism, with a specific focus on S-adenosyl-L-methionine (SAM), and the onset of thoracic aortic dissection (TAD) remains enigmatic despite acknowledging the pivotal role of inflammation in this vascular condition. In an established murine model induced by β-aminopropionitrile monofumarate (BAPN), we delved into the repercussions of supplementing with S-adenosyl-L-methionine (SAM) on the progression of TAD. Our observations uncovered a noteworthy improvement in aortic dissection and rupture rates, accompanied by a marked reduction in mortality upon SAM supplementation. Notably, SAM supplementation exhibited a considerable protective effect against BAPN-induced degradation of elastin and the extracellular matrix. Furthermore, SAM supplementation demonstrated a robust inhibitory influence on the infiltration of immune cells, particularly neutrophils and macrophages. It also manifested a notable reduction in the inflammatory polarization of macrophages, evident through diminished accumulation of MHC-IIhigh macrophages and reduced expression of inflammatory cytokines such as IL1β and TNFα in macrophages. Simultaneously, SAM supplementation exerted a suppressive effect on the activation of CD4 + and CD8 + T cells within the aorta. This was evidenced by an elevated proportion of CD44- CD62L + naïve T cells and a concurrent decrease in CD44 + CD62L- effector T cells. In summary, our findings strongly suggest that the supplementation of SAM exhibits remarkable efficacy in alleviating BAPN-induced aortic inflammation, consequently impeding the progression of thoracic aortic dissection. [ABSTRACT FROM AUTHOR]

Mutations detected in the AVPR2 gene (arginine vasopressin type 2 receptor) are known to cause nephrogenic diabetes insipidus (NDI). Several pharmacological chaperones (PCs) target misfolded AVPR2 proteins and rescue them from the quality control system of the cell. In this study, we investigated the effect of YM087 and VPA985, which are PCs, on T273M-AVPR2 mutant that are known to cause NDI.The total and cell surface expressions of T273M in COS-1 cells were measured by sandwich ELISA and flow cytometry after the cells were treated with YM087 and VPA985 separately. In addition, the cAMP accumulation assay was performed.It was observed that VPA985 treatment significantly increased the cell surface expression and slightly increased the maximum cAMP response of T273M. Both YM087 and VPA985 decreased the ligand concentrations which were responsible for making half of the maximum response of the T273M mutant receptor.PCs have different potential effects on different AVPR2 mutants. Therefore, studying the effectiveness of PCs in rescuing AVPR2 mutants and making them functional again may contribute to the development of new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Microplastics, widely present in the environment, are implicated in disease pathogenesis through oxidative stress and immune modulation. Prevailing research, primarily based on animal and cell studies, falls short in elucidating microplastics' impact on human cardiovascular health. This cross-sectional study detected blood microplastic concentrations in patients presenting with chest pain using pyrolysis–gas chromatography/mass spectrometry and evaluating inflammatory and immune markers through flow cytometry, to explore the potential effects of microplastic on acute coronary syndrome. Results: The study included 101 participants, comprising 19 controls and 82 acute coronary syndrome cases. Notably, acute coronary syndrome patients exhibited elevated microplastic concentrations, with those suffering from acute myocardial infarction presenting higher loads compared to those with unstable angina. Furthermore, patients at intermediate to high risk of coronary artery disease displayed significantly higher microplastic accumulations than their low-risk counterparts. A significant relationship was observed between increased microplastic levels and enhanced IL-6 and IL-12p70 contents, alongside elevated B lymphocyte and natural killer cell counts. Conclusion: These results suggest an association between microplastics and both vascular pathology complexity and immunoinflammatory response in acute coronary syndrome, underscoring the critical need for targeted research to delineate the mechanisms of this association. Highlights: Blood microplastic levels escalate from angiographic patency, to angina patients, peaking in myocardial infarction patients. Microplastics in acute coronary syndrome patients are predominantly PE, followed by PVC, PS, and PP. Microplastics may induce immune cell-associated inflammatory responses in acute coronary syndrome patients. [ABSTRACT FROM AUTHOR]

Background: To investigate the effects of Isoorientin on the apoptosis, proliferation, invasion, and migration of human gastric cancer cells (HGC27 cells). Methods: We used network pharmacology to predict the targets of drugs and diseases. The CCK-8 assay was used to determine the effects of Isoorientin on the proliferation of HGC27 cells. Flow cytometry was employed to analyze the effects of Isoorientin on cell apoptosis and cell cycle distribution of HGC27 cells. Scratch test and transwell chamber test were conducted to assess the effects of Isoorientin on invasion and migration, respectively. Additionally, qPCR and western blot were performed to examine the impact of Isoorientin on apoptosis-related genes and protein expression, respectively. Results: The Isoorientin significantly inhibited the proliferation, migration, and invasion of HGC27 cells compared to the control group. Furthermore, Isoorientin induced apoptosis in HGC27 cells by upregulating the relative expression of Bax and caspase-3 while downregulating the relative expression of p-PI3K, p-AKT, and Bcl-2 proteins. Conclusion: The Isoorientin exhibits inhibitory effects on the proliferation, invasion, and migration of HGC27 cells, and induces apoptosis in gastric cancer cells. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most aggressive and common type of malignant brain tumor diagnosed in adults. Preclinical immunocompetent mouse tumor models generated using mouse tumor cells play a pivotal role in testing the therapeutic efficacy of emerging immune-based therapies for GBMs. However, the clinical translatability of such studies is limited as mouse tumor lines do not fully recapitulate GBMs seen in inpatient settings. In this study, we generated three distinct, imageable human-GBM(hGBM)models in humanized mice using patientderived GBM cells that cover phenotypic and genetic GBM heterogeneity in primary (invasive and nodular) and recurrent tumors. We developed a pipeline to first enrich the tumor-initiating stem-like cells and then successfully established robust patient-derived GBMtumor engraftment and growth in bonemarrow-liverthymus (BLT) humanized mice. Multiplex immunofluorescence of GBM tumor sections revealed distinct phenotypic features of the patient GBM tumors, with myeloid cells dominating the immune landscape. Utilizing flow cytometry and correlative immunofluorescence, we profiled the immune microenvironment within the established human GBM tumors in the BLT mouse models and showed tumor infiltration of variable human immune cells, creating a unique immune landscape compared with lymphoid organs. These findings contribute substantially to our understanding of GBMbiologywithin the context of the human immune system in humanized mice and lay the groundwork for further translational studies aimed at advancing therapeutic strategies for GBM. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence shows the pivotal significance of miRNAs in the pathogenesis of osteoporosis. miR-381-3p has been identified as an inhibitor of osteogenesis. This study explored the role and mechanism of miR-381-3p in postmenopausal osteoporosis (PMOP), the most common type of osteoporosis. Methods: Bilateral ovariectomy (OVX) rat model was established and miR-381-3p antagomir was administrated through the tail vein in vivo. The pathological changes in rats were assessed through the evaluation of serum bone turnover markers (BALP, PINP, and CTX-1), hematoxylin and eosin (H&E) staining, as well as the expression of osteoblast differentiation biomarkers. Moreover, isolated bone marrow mesenchymal stem cells from OVX-induced rats (OVX-BMMSCs) were utilized to explore the impact of miR-381-3p on osteoblast differentiation. In addition, the target gene and downstream pathway of miR-381-3p were further investigated both in vivo and in vitro. Results: miR-381-3p expression was elevated, whereas KLF5 was suppressed in OVX rats. miR-381-3p antagomir decreased serum levels of bone turnover markers, improved trabecular separation, promoted osteoblast differentiation biomarker expression in OVX rats. ALP activity and mineralization were suppressed, and levels of osteoblast differentiation biomarkers were impeded after miR-381-3p overexpression during osteoblast differentiation of OVX-BMMSCs. While contrasting results were found after inhibition of miR-381-3p. miR-381-3p targets KLF5, negatively affecting its expression as well as its downstream Wnt/β-catenin pathway, both in vivo and in vitro. Silencing of KLF5 restored Wnt/β-catenin activation induced by miR-381-3p antagomir. Conclusion: miR-381-3p aggravates PMOP by inhibiting osteogenic differentiation through targeting KLF5/Wnt/β-catenin pathway. miR-381-3p appears to be a promising candidate for therapeutic intervention in PMOP. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OS) is a malignant bone tumor that commonly occurs in children and adolescents under the age of 20. Dysregulation of microRNAs (miRNAs) is an important factor in the occurrence and progression of OS. MicroRNA miR-744-5p is aberrantly expressed in various tumors. However, its roles and molecular targets in OS remain unclear. Methods: Differentially expressed miRNAs in OS were analyzed using the Gene Expression Omnibus dataset GSE65071, and the potential hub miRNA was identified through weighted gene co-expression network analysis. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-744-5p in OS cell lines. In vitro experiments, including CCK-8 assays, colony formation assays, flow cytometry apoptosis assays, and tube formation assays, were performed to explore the effects of miR-744-5p on OS cell biological behaviors. The downstream target genes of miR-744-5p were predicted through bioinformatics, and the binding sites were validated by a dual-luciferase reporter assay. Results: The lowly expressed miRNA, miR-744-5p, was identified as a hub miRNA involved in OS progression through bioinformatic analysis. Nuclear factor I X (NFIX) was confirmed as a direct target for miR-744-5p in OS. In vitro studies revealed that overexpression of miR-744-5p could restrain the growth of OS cells, whereas miR-744-5p inhibition showed the opposite effect. It was also observed that treatment with the conditioned medium from miR-744-5p-overexpressed OS cells led to poorer proliferation and angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, NFIX overexpression restored the suppression effects of miR-744-5p overexpression on OS cell growth and HUVECs angiogenesis. Conclusion: Our results indicated that miR-744-5p is a potential tumor-suppressive miRNA in OS progression by targeting NFIX to restrain the growth of OS cells and angiogenesis in HUVECs. [ABSTRACT FROM AUTHOR]

AbstractReactive pleocytosis in the CSF has been observed with blinatumomab but has not been well-described. We performed a retrospective study of 88 patients who received intrathecal chemotherapy (IT) while on blinatumomab with CSF analyzed to determine if pleocytosis had an impact efficacy and safety. Blinatumomab was used for relapsed/refractory 62.5%, MRD-positive 31.8%, and consolidation in MRD-negative 5.7%. The incidence of pleocytosis in CSF was 51% and was more frequent after day 15 (55.8% vs. 18.2%, p = 0.025). Pleocytosis did not impact CR, clearance of MRD positivity, PFS and OS rates. Lower incidence of non-CNS extramedullary relapse was seen (3.7% vs. 30.8%, p = 0.011) with pleocytosis in CSF. Analysis of CSF by flow cytometry showed median CD4:CD8 ratio of 1.34. In conclusion, CSF pleocytosis is prevalent with blinatumomab but only demonstrated lower rates of non-CNS extramedullary relapse but no impact on CNS relapse or neurotoxicity. [ABSTRACT FROM AUTHOR]

Tumor vaccine therapy offers significant advantages over conventional treatments, including reduced toxic side effects. However, it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods. Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors, thereby limiting their clinical efficacy. In this study, we developed a nanoparticle-based tumor vaccine, OVA@HA-PEI, utilizing ovalbumin (OVA) as the presenting antigen and hyaluronic acid (HA) and polyethyleneimine (PEI) as adjuvants and carriers. This formulation significantly enhanced the proliferation of immune cells and cytokines, such as CD3, CD8, interferon-γ, and tumor necrosis factor-α, in vivo, effectively activating an immune response against B16–F10 tumors. In vivo fluorescence flow cytometry (IVFC) has already become an effective method for monitoring circulating tumor cells (CTCs) due to its direct, noninvasive, and long-term detection capabilities. Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1 (PD-1) antibody. The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone. Additionally, this combination therapy substantially reduced the number of CTCs in vivo, increased the clearance rate of CTCs by the immune system, and slowed tumor progression. These findings greatly enhance the clinical application prospects of IVFC and tumor vaccines. [ABSTRACT FROM AUTHOR]

ABSTRACT Objective Materials and Methods Results Conclusion To investigate the association between interleukin‐8 (IL‐8) levels in gingival crevicular fluid (GCF) and total oral fluid (TOF) and the responsiveness to steps 1 and 2 of periodontal therapy.One‐hundred and fifty‐nine patients affected by periodontitis received steps 1 and 2 of periodontal therapy. At baseline, TOF and GCF samples were collected and analysed for IL‐8 (Il‐8TOF/IL‐8GCF) using flow cytometry. Therapy outcomes were relative proportions of residual periodontal pockets (PPD%), pocket closure (PC) rates and pocket probing depth (PPD) reductions; these were associated with IL‐8TOF/IL‐8GCF.High IL‐8TOF was significantly associated with higher residual PPD% (p = 0.044) and lower PPD reduction compared to low IL‐8TOF (high 0.79 ± 1.20 mm vs. low 1.20 ± 1.20 mm, p < 0.001) in non‐smokers, while in smokers high IL‐8GCF was related to lower PPD reduction (high 0.62 ± 1.22 mm vs. low 0.84 ± 1.12 mm, p = 0.009). Furthermore, high baseline IL‐8TOF was significantly associated with poorer PC rates compared to medium and low concentrations in both non‐smokers (high 41% vs. medium 55% vs. low 58%, p < 0.001) and smokers (high 34% vs. medium 44% vs. low 46%, p < 0.001).High IL‐8 concentrations at baseline had a significant impact on residual PPD%, PC rates and PPD reduction. The findings suggest that, especially in non‐smokers, baseline IL‐8 levels collected from the TOF could serve as a component in the estimation of responsiveness to steps 1 and 2 of periodontal therapy. [ABSTRACT FROM AUTHOR]

Transportation of medical samples between laboratories or hospital sites is typically performed by motorized ground transport. Due to the increased traffic congestions in urban environments, drone transportation has become an attractive alternative for fast shipping of samples. In accordance with the CLSI guidelines and the ISO 15189 standard, the impact of this transportation type on sample integrity and performance of laboratory tests must be thoroughly validated.Blood samples from 36 healthy volunteers and bacterial spiked urine samples were subjected to a 20–40 min drone flight before they were analyzed and compared with their counterparts that stayed on the ground. Effects on stability of 30 routine biochemical and hematological parameters, immunohematology tests and flow cytometry and molecular tests were evaluated.No clinically relevant effects on blood group typing, flow cytometry lymphocyte subset testing and on the stability of the multicopy opacity-associated proteins (Opa) genes in bacterial DNA nor on the number of Abelson murine leukemia viral oncogene homolog 1 (abl) housekeeping genes in human peripheral blood cells were seen. For three of the 30 biochemistry and hematology parameters a statistically significant difference was found: gamma-glutamyl transferase (gamma-GT), mean corpuscular hemoglobin (MCH) and thrombocyte count. A clinically relevant effect however was only seen for potassium and lactate dehydrogenase (LDH).Multi-rotor drone transportation can be used for medical sample transportation with no effect on the majority of the tested parameters, including flow cytometry and molecular analyses, with the exception of a limited clinical impact on potassium and LDH. [ABSTRACT FROM AUTHOR]

Background: Several studies have been reported previously on the bioactivities of different extracts of marine molluscs. Therefore, we decided to evaluate the cytotoxic and antimicrobial activities of S. pharaonis ink as a highly populated species in the Red Sea. We extracted the flavonoids from the ink and analyzed their composition. Then we evaluated systematically the cytotoxic and antimicrobial properties of this extract. A pharmacokinetic study was also conducted using SwissADME to assess the potential of the identified flavonoids and phenolic compounds from the ink extract to be orally active drug candidates. Results: Cytotoxic activity was evaluated against 5 cell lines (MCF7, Hep G2, A549, and Caco2) at different concentrations (0.4 µg/mL, 1.6 µg/mL, 6.3 µg/mL, 25 µg/mL, 100 µg/mL). The viability of examined cells was reduced by the extract in a concentration-dependent manner. The highest cytotoxic effect of the extract was recorded against A549 and Hep G2 cancer cell lines cells with IC50 = 2.873 and 7.1 µg/mL respectively. The mechanistic analysis by flow cytometry of this extract on cell cycle progression and apoptosis induction indicated that the extract arrests the cell cycle at the S phase in Hep G2 and MCF7, while in A549 cell arrest was recorded at G1 phase. However, it causes G1 and S phase arrest in Caco2 cancer cell line. Our data showed that the extract has significant antimicrobial activity against all tested human microbial pathogens. However, the best inhibitory effect was observed against Candida albicans ATCC 10,221 with a minimum inhibitory concentration (MIC) of 1.95 µg/mL. Pharmacokinetic analysis using SwissADME showed that most flavonoids and phenolics compounds have high drug similarity as they satisfy Lipinski's criteria and have WLOGP values below 5.88 and TPSA below 131.6 Å2. Conclusion: S. pharaonis ink ethanolic extract showed a promising cytotoxic potency against various cell lines and a remarkable antimicrobial action against different pathogenic microbial strains. S. pharaonis ink is a novel source of important flavonoids that could be used in the future in different applications as a naturally safe and feasible alternative of synthetic drugs. [ABSTRACT FROM AUTHOR]

Background: Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches. Method: We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1. Results: We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity. Conclusion: rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8α hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8α hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8α TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs. [ABSTRACT FROM AUTHOR]

Objective Methods Results Conclusion Hydrolyzed conchiolin protein (HCP) derived from pearl and nacre extracts exerts skin‐lightening effects; however, the underlying molecular mechanisms are not fully understood. Herein, we investigated the effect of HCP on melanogenesis and the signalling pathways involved.B16F10 cells and PIG cells were treated with HCP to verify its ability to inhibit melanin. Western Blot, immunofluorescence, and flow cytometry methods were performed to investigate the effect of HCP on melanogenesis signalling pathway proteins. The inhibitors were used to further validate the effect of HCP on PKA/CREB and MEK/ERK signalling pathways. To further evaluate the whitening ability of HCP, changes in melanin were detected using 3D melanin skin model and zebrafish model.HCP was found to significantly inhibit melanin synthesis and decrease the expression of melanogenesis‐related proteins, such as microphthalmia‐associated transcription factor (MITF), tyrosinase, and tyrosinase‐related protein‐2, in a dose‐dependent manner. Additionally, we revealed that HCP suppresses melanogenesis via the regulation of the PKA/cAMP response element‐binding (CREB) and MEK/extracellular signalling‐regulated kinase (ERK) signalling pathways. Using 3D melanin skin models, we demonstrated that HCP can achieve skin‐lightening effects by improving apparent chroma, increasing apparent brightness, and inhibiting melanin synthesis. Furthermore, HCP exhibits skin‐whitening effects in a zebrafish model.These results suggest that HCP suppresses the melanogenesis signalling cascade by inhibiting the PKA/CREB, MEK/ERK signalling pathway and downregulating MITF and its downstream signalling pathways, resulting in decreased melanin synthesis. In summary, HCP is a potential anti‐pigmentation agent with promising applications in cosmetics and pharmaceutical products. [ABSTRACT FROM AUTHOR]

Flow cytometry plays a pivotal role in biotechnology by providing quantitative measurements for a wide range of applications. Nonetheless, achieving precise particle quantification, particularly without relying on counting beads, remains a challenge. In this study, we introduce a novel exhaustive counting method featuring a sample loop–based injection system that delivers a defined sample volume to a detection system to enhance quantification in flow cytometry. We systematically assess the performance characteristics of this system with micron-sized polystyrene beads, addressing issues related to sample introduction, adsorption, and volume measurement. Results underscore the excellent analytical performance of the proposed method, characterized by high linearity and repeatability. We compare our approach to counting bead–based measurements, and while an approximate bias value was observed, the measured values were found to be similar between the methods, demonstrating its comparability and reliability. This method holds great promise for improving the accuracy and precision of particle quantification in flow cytometry, with implications for various fields including healthcare and environmental monitoring. [ABSTRACT FROM AUTHOR]

AbstractPurposeMethodsResultsConclusionHead and neck squamous cell carcinoma (HNSCC) is globally prevalent with high recurrence, low survival rate, and poor quality of life for patients. Derived from PAC-1, SM-1 can activate procaspase-3 and induce apoptosis in cancer cells to exert anti-tumor effects. However, the inhibitory effect of SM-1 on HNSCC after combination with radiation are unclear. This study aims to investigate the radiosensitizing effect of SM-1 on HNSCC in vitro and in vivo.MTT method was used to detect the effect of SM-1 on the viability of HNSCC cell lines (HONE1, HSC-2, and CAL27). The effects of SM-1 combined with radiation on the survival index of HONE1, HSC-2, and CAL27 cell lines were determined by colony formation assay. Flow cytometry was used to investigate the effects of SM-1 and radiation combination on cell apoptosis and cell cycle, and western blot experiments were performed to detect the expression of apoptosis and cell cycle-related proteins. Finally, a xenograft tumor model of CAL27 was established to evaluate the anti-tumor effect of SM-1 combined with radiation in vivo.In vitro, SM-1 effectively inhibited the activity of HNSCC cell lines HONE1, HSC-2, and CAL27 cells, and synergistically showed anti-proliferation activity during combined irradiation. Meanwhile, anti-tumor effect of SM-1 on HNSCC was higher than that of Debio1143, and the radiosensitivity of cells was greatly increased. Flow cytometry and western blot analysis showed that SM-1 induced G2/M phase arrest of head and neck squamous cell carcinoma cells via inhibiting the expression of CyclinB1 and CDC2. Moreover, SM-1 activated caspase-3 activity and up-regulated the cleaved form of PARP1 to induce cell apoptosis. In vivo, SM-1 combined irradiation showed a good anti-tumor effect.SM-1 enhances HNSCC cell radiation sensitivity in vitro and in vivo, supporting its potential as a radiosensitizer for clinical trials in combination with radiotherapy. [ABSTRACT FROM AUTHOR]

Background: Proper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation. Methods: We utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance. Results: In this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p. Conclusions: miR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs. [ABSTRACT FROM AUTHOR]

Premise Methods Results Conclusions Intraspecific variation in drought resistance traits, such as drought escape, appear to be frequent within wild, ruderal forb species. Understanding how these traits are arrayed across the landscape, particularly in association with climate, is critical to developing forbs for wildland restoration programs. Use of forbs is requisite for maintaining biological diversity and ecological services.Using 6074 greenhouse‐grown Chaenactis douglasii seedlings from 95 wild, seed‐sourced populations across the western United States, we recorded bolting phenology and estimated genome size using flow cytometry. Mixed‐effects regression models were used to assess whether climate of seed origin was predictive for bolting phenology and genome size.Variation in bolting, reflecting an annual vs. perennial lifespan in this species, was observed in 8.7% of the plants, with bolting plants disproportionately occurring in locations with warm, arid climates. Populations with increasing heat and aridity were positively correlated with observed bolting (r = 0.61, p < 0.0001). About one‐third (22%) of the total (61%) lifespan variation was attributed to seed source climate and annual heat moisture index, a measure of aridity. Genome size had no significant effect on bolting. Projected climate modeling for mid‐century (2041–2070) supports an increasing occurrence of annual lifespan.Our analyses support a drought escape, bet‐hedging strategy in C. douglasii. Populations exposed to greater aridity exhibited a higher proportion of individuals with an annual lifespan. Drought escape leading to an annual lifespan can affect how seeds are propagated and deployed for climate‐informed restoration. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusions Eosinophils are key therapeutic targets in severe asthma that are suppressed by IL5 (mepolizumab) and IL5 receptor (benralizumab) blockade. The effect of IL5 pathway biologics on recently described homeostatic (hEOs) and inflammatory (iEOs) eosinophil subsets is unknown. We aimed to determine the relative impact of mepolizumab and benralizumab treatment on eosinophil subset and phenotype, and explore clinical associations of eosinophil subsets with severe asthma characteristics and treatment response.We performed a cross‐sectional observational study of severe asthma (eosinophilic n = 32, non‐eosinophilic n = 23, mepolizumab‐treated n = 25), with longitudinal follow‐up of 30 eosinophilic participants at two timepoints (4–24 weeks, >24 weeks) post‐commencement of mepolizumab (n = 20) or benralizumab (n = 10). Blood hEOs and iEOs were measured by flow cytometry assessment of surface CD62L protein.iEO proportion was significantly lower in mepolizumab‐treated participants in both the cross‐sectional and longitudinal study. Mepolizumab and benralizumab depleted iEOs to a similar extent, however a significantly greater number of hEOs remained in mepolizumab participants at follow‐up. Greater iEO proportion correlated with poorer asthma control in eosinophilic but not non‐eosinophilic asthma. Higher residual iEO proportion correlated with poorer asthma control in mepolizumab‐treated individuals. Reduced blood eosinophil viability was observed in around half of mepolizumab‐treated participants, which was associated with significantly better asthma control and spirometry.Mepolizumab depletes iEOs and reduces circulating eosinophil viability in severe asthma but preserves a residual population of circulatory hEOs. In contrast benralizumab depleted both iEOs and hEOs. Higher iEO abundance and eosinophil viability are associated with poorer clinical outcomes following mepolizumab‐treatment. Monitoring circulating eosinophil phenotype and viability may be useful to predict biologic treatment response in severe asthma. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusion Alopecia areata (AA) is a chronic, nonscarring hair‐loss disorder associated with significant quality‐of‐life impairment and limited treatment options. AA has been recently linked to atopy and shown to exhibit both Th1‐ and Th2‐driven inflammation. However, a comprehensive molecular and cellular characterization across blood and scalp compartments in both atopic and nonatopic patients is lacking.Lesional and nonlesional scalp biopsies obtained from AA patients with (n = 16) or without (n = 20) atopic history, and 17 demographically matched healthy controls were analyzed with RNA‐seq, RT‐PCR, and immunohistochemistry. Flow cytometry was also performed on peripheral blood mononuclear cells (PBMCs) from a subset of patients. Differential expression was defined using |fold‐change| > 1.5 and false‐discovery rate <0.05.AA scalp exhibited robust upregulation of Th1‐ (IFNG, CXCL9, CXCL10, CXCL11) and Th2‐related products (CCL26, CCR4, IL10, IL13, TSLP, TNFRSF4/OX40) and shared downregulation of hair keratins, regardless of atopic background, with variable Th17/Th22 modulation. AA patients with atopy exhibited greater inflammatory tone and Th2‐skewing (IL10, IL13, IL33, CCR4, CCL26). Disease severity correlated significantly with immune and hair keratin biomarkers and with perifollicular cellular infiltrates. Cutaneous OX40/OX40L upregulation was paralleled by increases in circulating OX40+ and OX40L+ leukocytes, regardless of atopic background.Our results suggest some atopy‐associated immune differences in AA and highlight the OX40 axis as a potential novel therapeutic target that may broadly benefit AA patients. [ABSTRACT FROM AUTHOR]

Introduction: Programmed Death-Ligand 1 is a well-known immune checkpoint molecule. Recent studies evaluated its expression in different canine cancer types through different laboratory techniques. The present study aims to evaluate the surface membrane protein expression (mPD-L1) by means of flow cytometry (FC) in different canine lymphoma immunophenotypes. Furthermore, in a subset of cases, mRNA and plasmatic soluble protein (sPD-L1) have been assessed in the same patient, and correlations among results from the three analyses investigated. Methods: Samples obtained for diagnostic purpose from untreated dogs with a confirmed lymphoma immunophenotype were included: surface protein was assessed via FC and quantified with median fluorescence index ratio (MFI ratio), gene expression was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and plasmatic concentration of soluble protein (sPD-L1) measured with ELISA. Statistical analyses were performed to investigate any difference among FC immunophenotypes, updated Kiel cytological classes, and in the presence of blood infiltration. Results: Considering FC, most B-cell lymphomas (BCL) were positive, with higher MFI ratios than other subtypes (81%, median MFI ratio among positive samples = 1.50, IQR 1.21-2.03, range 1.01-3.47). Aggressive T-cell lymphomas had a lower percentage of positive samples (56%) and showed low expression (median MFI ratio in positive samples = 1.14, IQR 1.07-1.32, range 1.02-2.19), while T-zone lymphomas (TZL) were frequently positive (80%) but with low expression (median MFI ratio in positive samples = 1.19, IQR 1.03-1.46, range 1.02-6.03). Cellular transcript and sPD-L1 were detected in all samples, without differences among immunophenotypes. No correlation between results from different techniques was detected, but sPD-L1 resulted significantly increased in FC-negative lymphomas (p = 0.023). Discussion: PD-L1 molecule is involved in canine lymphoma pathogenesis, with differences among immunophenotypes detected by FC. Specifically, BCL have the highest expression and aggressive T-cell lymphomas the lowest, whereas TZL need further investigations. [ABSTRACT FROM AUTHOR]