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This study aims to investigate the characteristics of Epstein-Barr virus associated-hemophagocytic lymphohistiocytosis (EBV-HLH) and HLH caused by a severe form of infectious mononucleosis (IM-HLH) compared to IM by EBV, and thus also to assist in early diagnosis and providing appropriate treatment. Data for this analysis were collected from patients at the Department of General Medicine, Nara Medical University, between April 1, 2012, and August 1, 2020. EBV infection was diagnosed using clinical presentation and laboratory tests. HLH diagnosis followed the HLH-2004 protocol, supplemented by plasma EBV DNA detection. A range of clinical and laboratory parameters were collected, including age, sex, clinical outcomes, blood cell counts, hemoglobin, platelets, and various serum values. Plasma EBV DNA levels and flow cytometric analysis (FCM) of bone marrow were performed for HLH cases. Among 1850 hospitalized patients, 14 cases were identified, including 2 HLH cases and 12 IM cases. Comparative analysis revealed distinctive features of HLH, including lower lymphocyte and platelet counts and higher levels of ferritin, soluble interleukin 2 receptor (sIL-2R), and D dimer compared to IM. Notably, one HLH case responded well to corticosteroid monotherapy, while the other case did not, resulting in a fatal outcome. Detection of a cluster of CD5-CD7 lymphocytes in bone marrow is a hallmark of EBV-HLH and useful to distinguish from IM-HLH. This study underscores the importance of early differentiation among EBV-HLH, IM-HLH, and IM in adults to guide appropriate treatment strategies. While specific laboratory markers help distinguish HLH from IM, a more detailed analysis of FCM is crucial for precise diagnosis of HLH cases and tailored therapeutic interventions.

The inflammatory cytokine interleukin-17 (IL17) plays an important role in innate immunity by binding to its receptors (IL17Rs) to activate immune defense signals. To date, information on members of the IL17 family is still very limited in molluscan species. Here, a novel member of the IL17 family was identified and characterized from thick shell mussel Mytilus coruscus, and this gene was designated as McIL17-1 by predicting structural domains and phylogenetic analysis. McIL17-1 transcripts existed in all examined tissues with high expression levels in gills, hemocytes and digestive glands. After the stimuli of different pathogen associated molecular patterns (PAMPs) for 72 h, transcriptional expression of McIL17-1 was significantly upregulated, except for poly I:C stimulation. Cytoplasm localization of McIL17-1 was shown in HEK293T cells by fluorescence microscopy. Further, in vivo and in vitro assays were performed to evaluate the potential function of McIL17-1 played in immune response. McIL17-1 was either knocked down or overexpressed in vivo through RNA inference (RNAi) and recombinant protein injection, respectively. With the infection of living Vibrio alginolyticus, a high mortality rate was exhibited in the McIL17-1 overexpressed group compared to the control group, while a lower mortality rate was observed in the McIL17-1 knocked down group than control group. In vitro, the flow cytometric analysis showed that the apoptosis rate of McIL17-1 inhibited hemocytes was significantly lower than that of the control group after lipopolysaccharide stimulation. These results collectively suggested that the newly identified IL17 isoform is involved in the inflammatory response to bacterial infection in M. coruscus.

In the present study, we explored the potential of coumarin-based compounds, known for their potent anticancer properties, by designing and synthesizing a novel category of 8-methoxycoumarin-3-carboxamides. Our aim was to investigate their antiproliferative activity against liver cancer cells. Toward this, we developed a versatile synthetic approach to produce a series of 8-methoxycoumarin-3-carboxamide analogues with meticulous structural features. Assessment of their antiproliferative activity demonstrated their significant inhibitory effects on the growth of HepG2 cells, a widely studied liver cancer cell line. Among screened compounds, compound 5 exhibited the most potent antiproliferative activity among the screened compounds (IC50 = 0.9 µM), outperforming the anticancer drug staurosporine (IC50 = 8.4 µM), while showing minimal impact on normal cells. The flow cytometric analysis revealed that compound 5 induces cell cycle arrest during the G1/S phase and triggers apoptosis in HepG2 cells by increasing the percentage of cells arrested in the G2/M and pre-G1 phases. Annexin V-FITC/PI screening further supported the induction of apoptosis without significant necrosis. Further, compound 5 exhibited the ability to activate caspase3/7 protein and substantially inhibited β-tubulin polymerization activity in HepG2 cells. Finally, molecular modelling analysis further affirmed the high binding affinity of compound 5 toward the active cavity of β-tubulin protein, suggesting its mechanistic involvement. Collectively, our findings highlight the therapeutic potential of the presented class of coumarin analogues, especially compound 5, as promising candidates for the development of effective anti-hepatocellular carcinoma agents.

Background Cancer is a major global health challenge, driving the need for new therapeutic agents. This study explored the anticancer potential of microorganisms isolated from healthy bee workers collected from 35 sites in three Egyptian governorates to identify novel bioactive metabolites. Results A total of 113 microbial isolates (35 fungal and 78 bacterial) were obtained and screened. Thirty-two selected isolates were fermented, and their cytotoxicity against MCF-7 breast cancer cells was assessed using the MTT assay. Eight isolates showed strong anticancer activity, reducing cell viability below 30%. Among them, isolate Tm2 demonstrated the highest selectivity and potency across multiple cancer cell lines (Caco-2, MCF-7, HepG-2, A549), with IC50 values between 30.63 and 113.39 µg/ml, and minimal toxicity toward normal cells (IC50 > 976 µg/ml; selectivity index = 18.21). Morphological and molecular analyses identified Tm2 as Aspergillus oryzae . Optimization of fermentation parameters via Plackett-Burman and Box-Behnken designs maximized extract yield to 1.86 g/100 ml under specific conditions (yeast extract 1.5 g/L, pH 7.6, 25 °C). Chemical profiling using GC-MS and UPLC-MS/MS detected 65 bioactive compounds, mainly fatty acids such as oleic, hexadecanoic, and linoleic acids. Live-dead staining showed dose-dependent cytotoxic effects, while flow cytometry revealed induction of apoptosis through multiple pathways in cancer cells treated with the extract. Conclusions Aspergillus oryzae isolated from bee microbiota exhibits potent and selective anticancer activity mediated by a complex mixture of bioactive fatty acids. These results highlight its potential as a novel source for developing effective anticancer therapies with low toxicity to normal cells.

Equine breeding would benefit greatly from reliable biomarkers of stallion or ejaculate fertility. The aim of the study was to investigate how several in vitro sperm characteristics correlate with fertility after artificial insemination, to explore the potential to build a fertility prediction model for stallions. Cooled insemination doses (3–5 per stallion) were obtained from various studs. Sperm membrane integrity, acrosome integrity, chromatin integrity, mitochondrial membrane potential, and reactive oxygen species production were evaluated by flow cytometry 24–30 h after semen collection, and sperm motility was assessed by computer aided sperm analysis. Calcein violet was used to differentiate viable spermatozoa. Per season pregnancy rates for these stallions were available the following year. Positive correlations were found between pregnancy rate and straightness (r = 0.43, p ≤ 0.001), as well as pregnancy rate and the proportion of living hydrogen peroxide positive spermatozoa (r = 0.32, p ≤ 0.05). There were negative correlations between pregnancy rate and amplitude of lateral head displacement (r = -0.26, p ≤ 0.05), and between pregnancy rate and the mean fluorescence of dead superoxide positive spermatozoa (r = -0.46, p < 0.001). Principal component analysis indicated that motility, membrane integrity, DNA fragmentation, and reactive oxygen species production were associated with pregnancy rate. Therefore, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates. However, data from more individuals would be required to construct a model for fertility prediction.

Venous thromboembolism is a common complication following trauma. We investigated the dynamics of plasma microparticles (MPs) levels and explored their potential as biomarkers of deep vein thromboembolism (DVT) after trauma. A total of 775 patients with traumatic fractures were recruited in this nested study. About 106 trauma patients (53 DVT subjects and 53 age-, sex-, and fracture site-matched non-DVT subjects) and 53 healthy volunteers met the enrollment criteria. MPs were characterized by transmission electron microscope, nanoparticle tracking analysis, and western blotting. Circulating levels of MPs were measured using a flow cytometer. Meanwhile, routine laboratory parameters were examined in all patients. Compared to non-DVT patients, DVT patients had higher circulating phosphatidylserine (PS) + MPs, hepatocyte-derived MPs (HMPs), PS + HMPs, and platelet-derived MPs (PMPs). Notably, PS + HMPs had the best predictive value for DVT diagnosis in trauma patients (area under the curve [AUC] 0.8939, 95% CI 0.8326 to 0.9552), which was superior to d-dimer (AUC 0.5881). The Hepatic Procoagulant Index combined plasma levels of PS + HMPs and albumin, increasing the AUC to 0.8978 (95% CI 0.8396 to 0.9561). This is the first study that addressed circulating PS + HMPs are promising biomarkers with high performance in diagnosing DVT. The Hepatic Procoagulant Index is a potential predictor of DVT in trauma patients.

Tetraploid poplar plants were induced by colchicine from tissue-cultured shoots; the induction efficiency varied with colchicine concentration. Morphologically, induced tetraploid plants were significantly distinguishable from diploid plants; their leaves were larger, thicker, and rounder, with larger stomata and more chloroplast numbers but smaller stomata index in the abaxial leaf epidermis. Data from validation with chromosome counting and flow cytometry corresponded well with changes in stomata size and chloroplast number. This ready use protocol would facilitate poplar breeding.Key messageTetraploid induction by colchicine in Populus euroamericana from tissue cultured shoots.

Reliable and sensitive characterization assays are important determinants of the successful clinical translation of immunotherapies. For the assessment of cytolytic potential, the chromium 51 ( 51 Cr) release assay has long been considered the gold standard for testing effector cells. However, attaining the approvals to access and use radioactive isotopes is becoming increasingly complex, while technical aspects [i.e. sensitivity, short (4-6 hours) assay duration] may lead to suboptimal performance. This has been the case with our ex vivo expanded, polyclonal (CD4+ and CD8+) multivirus-specific T cell (multiVST) lines, which recognize 5 difficult-to-treat viruses [Adenovirus (AdV), BK virus (BKV), cytomegalovirus (CMV), Epstein Barr virus (EBV), and human herpes virus 6 (HHV6)] and when administered to allogeneic hematopoietic stem cell (HCT) or solid organ transplant (SOT) recipients have been associated with clinical benefit. However, despite mediating potent antiviral effects in vivo , capturing in vitro cytotoxic potential has proven difficult in a traditional 51 Cr release assay. Now, in addition to cytotoxicity surrogates, including CD107a and Granzyme B, we report on an alternative, vital dye -based, flow cytometric platform in which superior sensitivity and prolonged effector:target co-culture duration enabled the reliable detection of both CD4- and CD8-mediated in vitro cytolytic activity against viral targets without non-specific effects.

The authors describe a flow cytometric immunoassay for aflatoxin B1 (AFB1). It has three distinct features: (a) Magnetic microspheres encoded with upconverting nanocrystals (UCNMMs) are used as fluorescent labels. These have the advantage of non-overlapping spectra and lacking crosstalk between the encoding signal and reporter signal via the low-energy near-infrared (NIR) light excitation; (b) phycoerythrin-labeled secondary antibodies are used to amplify the reporter signal; (c) The use of magnetic nanoparticles facilitates the rapid separation and specific purification of the analyte (AFB1). This assay has a detection limit of 9 pg·mL −1 and a broad working range for AFB1, requires a 50 μL sample only, and can be completed within 2 h with good accuracy and high reproducibility. It is perceived that such multifluorescent UCNMMs, whose color depends on the kind of dopants (Yb, Er, Tm, Mn) in the NaYF 4 host lattice, represent a promising tool for the analysis of mycotoxins and other analytes. Graphical abstract Schematic of the UCNMM-based indirective competitive immunoassay for AFB1 using the flow cytometric analysis (FCA) technology. The UCNMMs are prepared by doping the upconversion nanocrystals and magnetic nanoparticles inside the mesoporous polystyrene microspheres as the self-healing encapsulation strategy.