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Breast cancer ranks as the second leading reason of cancer mortality among females globally, emphasizing the critical need for novel anticancer treatments. In current work, berberine-zinc oxide conjugated chitosan nanoparticles were synthesized and characterized using various characterization techniques. The cytotoxic effects of CS-ZnO-Ber NPs on MCF-7 cells were assessed using the MTT assay. Also, annexin V-FITC/PI double staining, Hoechst 33,342 staining, caspases-8 and 9 activity assays, and cell cycle analysis were performed. Furthermore, the mRNA levels of Bax and Bcl-2 genes were quantified using qPCR. Additionally, cell migration was evaluated using a scratch assay. The IC50 value of NPs against MCF-7 cells measured 7.41 µg/mL. Apoptosis induction in NP-treated cells was confirmed by Annexin V/PI staining, accompanied by the observation of condensed chromatin and fragmented DNA. Moreover, the pro-apoptotic potential of NPs was evidenced by significant increases in caspases-8 and 9 activity and a decreased Bcl-2/Bax ratio. Furthermore, cell cycle arrest at the sub-G1 was noticed in the treated cells. Additionally, the NPs markedly inhibited the migration rate of MCF-7 cells. These findings suggest that CS-ZnO-Ber NPs induce cell-cycle arrest and activate the apoptotic pathways in MCF-7 cells, highlighting their potential as a hopeful therapeutic agent for breast cancer. [ABSTRACT FROM AUTHOR]

A protocol for micropropagation of potato (Solanum tuberosum L.) cv. Cooch Behar local retaining the fidelity of the in vitro regenerants was established for the first time. Initially, tuber sprouts were inoculated in Murashige and Skoog (MS) basal medium supplemented with 0.5–1.5 mg/l 6-benzyladenine (BA), kinetin (Kin), meta-Topolin (mT), and zeatin (Zea). A maximum number of shoots was induced in 0.5 mg/l Zea followed by 0.5 mg/l mT. For subsequent rooting, the shoots were inoculated in MS medium supplemented with 1.0–3.0 mg/l indole-3-acetic acid and indole-3-butyric acid (IBA), wherein 1.0 mg/l IBA-fortified medium recorded the maximum number of roots. The mono-phasic micropropagation, i.e., simultaneous multiple shoots and roots formation was achieved in MS medium with combinations of Zea (0.25–1.0 mg/l) with 1.0 mg/l IBA, wherein 0.5 mg/l Zea + 1.0 mg/l IBA exhibited the best results. The micropropagated plantlets were then acclimatized in cocopeat with 100% survival before field evaluation. To ensure the true-to-type nature of the micropropagated plants, the cytology, flow cytometry, inter simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism primers based fidelity analyses were carried out. Cytology and flow cytometry confirmed that the micropropagated plants had the same ploidy levels. Likewise, the molecular marker-based genetic fidelity study via ISSR and SCoT primers showed monomorphic banding patterns. The present protocol has the potential for large-scale propagation, conservation, and commercialization of indigenous potatoes and can also be used to study the response of other potato cultivars to in vitro regeneration. [ABSTRACT FROM AUTHOR]

Detecting alterations in plasmid structures is often performed using conventional molecular biology. However, these methods are laborious and time-consuming for studying the conditions inducing these mutations, which prevent real-time access to cell heterogeneity during bioproduction. In this work, we propose combining both flow cytometry and fluorescence-activated cell sorting, integrated with mechanistic modelling to study conditions that lead to plasmid recombination using a limonene-producing microbial system as a case study. A gene encoding GFP was introduced downstream of the key enzymes involved in limonene biosynthesis to enable real-time kinetics monitoring and the identification of cell heterogeneity according to microscopic and flow cytometric analyses. Three different plasmid configurations (one correct and two incorrect) were identified through cell sorting based on subpopulations expressing different levels of GFP at 10 and 50 µM IPTG. Higher limonene production (530 mg/L) and lower subpopulation proportion carrying the incorrect plasmid (12%) were observed for 10 µM IPTG compared to 50 µM IPTG (96 mg/L limonene and more than 70% of cell population carrying the incorrect plasmid, respectively) in 100 mL production culture. We also managed to derive exploratory hypotheses regarding the plasmid recombination region using the model and successfully validated them experimentally. Additionally, the results also showed that limonene production was proportional to GFP fluorescence intensity. This correlation could serve as an alternative to using biosensors for a high-throughput screening process. The developed method enables rapid identification of plasmid recombination at single-cell level and correlates the heterogeneity with bioproduction performance. Key points: • Strategy to study plasmid recombination during bioproduction. • Different plasmid structures can be identified and monitored by flow cytometry. • Mathematical modelling suggests specific alterations in plasmid structures. [ABSTRACT FROM AUTHOR]

In this work, an electrochemical system combined with low-cost UV-LEDs was implemented for the inactivation of Escherichia coli and Pseudomonas aeruginosa. The individual elimination of these bacteria was followed by plate counting and flow cytometry, as complementary techniques to establish the cell inactivation and non-viability, respectively. The contribution of the different parts of the disinfection system (anode, cathode, and light) was determined. In addition, the efficiency of the UV-LEDs/GDE/DSA system in the disinfection of an irrigation water sample was studied. It was found that the combination of the electrochemical system with UV-LEDs was highly synergistic (φ > 7), having low electric energy consumptions per order of magnitude (EEO: 1.13 × 10–2 and 1.55 × 10–2 kWh/m3 order). Moreover, some differences in the inactivation kinetics and synergy between E. coli and P. aeruginosa were observed and linked to the structural/morphological characteristics of the two bacteria. Remarkably, the electrochemical system combined with low-cost UV-LEDs inactivated both target microorganisms after only 2 min of treatment. The flow cytometry analyses evidenced the damage to the cell membrane of the bacteria by the simultaneous and synergistic action of the electrogenerated H2O2 and active chlorine species (ACS), plus the attacks of photo-generated reactive oxygen species. This synergistic combination in the UV-LEDs/GDE/DSA system demonstrated remarkable efficiency in the disinfection of an irrigation water sample, achieving the elimination of culturable bacteria in 45 min of treatment. The results of this research demonstrated the capacity and great potential of an easy combination of electrochemistry with UV-LEDs as an alternative system for the elimination of gram-negative bacteria in water. [ABSTRACT FROM AUTHOR]

The most common method for detection of apoptosis is flow cytometry. In previously published studies there are some uncertainties and problems about the preparation of adherent cell lines for analysis. Thus, the aim of this study is to determine and describe how preparing the sample of SW-480 cells in two different ways affects the reliability of the results. In Protocol 1 the total cell number, cells in flow media and cells which adhere, were used, while in Protocol 2 the medium was removed after cell incubation and only the adherent cells were used. Results show statistically significant changes in percentages of different cell types (viable, apoptosis, and necrosis) between two different protocols. Protocol 2, where the first medium with dead cells were removed and only the cells that were attached to the bottom were used for analysis, give better cell viability in the control sample. Removing the medium is especially recommended for long-term treatments, where the cells consume nutrients and, due to lack, initiate apoptosis. After 72 h, spontaneous apoptosis is detectable in control cells and indicates low viability of the control sample, while in treated cells by proapoptotic substances, together with induced apoptosis leads to cumulative or synergistic effects. [ABSTRACT FROM AUTHOR]

Biosynthesized nanoparticles are becoming more prevalent because of their distinctive biological applications and the availability of naturally occurring bioactive secondary metabolites from plants that facilitate green synthesis. In the current study, silver nitrate solution was reduced in order to create silver nanoparticles using Trachyspermum ammi (T. ammi) seed extract. The production of bionanoparticles was confirmed by using UV–visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and X-ray diffraction analysis (XRD). The green synthetic AgNPs are spherical in form and range in size from 50 to 90 nm. The antibacterial properties of the produced silver nanoparticles were tested by the minimum inhibitory concentration (MIC) method and disc diffusion assay. The antibacterial efficacy expressed was maximum against Escherichia coli and Enterococcus faecalis with ZOI of 15.33 ± 0.4 and 15.0 ± 0.1 mm, respectively. The MIC values for E. faecalis and E. coli of the biosynthesized AgNPs were observed to be 12.5 μg/ml. To ascertain the mechanism of action of the created biosynthesized nanoparticles, estimates of the production of reactive oxygen species (ROS), lipid peroxidation (LPO), and reduced glutathione (GSH) levels were measured. The effect of biosynthesized AgNPs on cell proliferation, ROS induction, and cell cycle arrest was investigated through MTT, DCFDA staining, and flow cytometry assay. The cytotoxic efficiency of synthesized AgNPs against the human colorectal carcinoma (HCT) cell line was evaluated through MTT assays and showed 50% inhibitory concentration (IC50) values at 62.5 μg/ml. The biosynthesized AgNPs presented high ROS production against MCF-7 cancer cells compared to control cells and revealed growth arrest in the G2/M phase through flow cytometry analysis. Hence, the present study suggested that the biosynthesized AgNPs using T. ammi could be used as a viable source for antibacterial and anticancer medicines with additional in vivo research. [ABSTRACT FROM AUTHOR]

Background: The lack of genomic and genetic research on wild sweetpotato species has hindered the advancement of sweetpotato variety development through modern crop improvement techniques. Methods and results: To facilitate the use of genomic and genetic approaches in sweetpotato variety development, we conducted a comprehensive assessment of the genome size and ploidy of six closely related wild sweetpotato species using flow cytometry and chromosome counting. Additionally, we acquired insights into their genomic characteristics through high-throughput sequencing. Based on the 17-mer frequency distribution, the genome sizes of these species ranged from 518.47 Mb to 1,505.04 Mb. Notably, most diploid species exhibited genome sizes of approximately 500 Mb, with the diploid wild species I. purpurea standing out as having a significantly larger genome size compared to other diploid species. A substantial proportion of repeats (ranging from 57.47 to 81.07%) was identified across the genomes of the six species. Heterozygosity levels varied from 0.24 to 2.21%. SSR analysis revealed that the distribution of microsatellite patterns was largely consistent among the genomes of I I. lacunosa, I. tenuissima, and I. tiliacea, with mono-, di-, and trinucleotide motifs dominated by A/T, AT/AT, and AAT/ATT, respectively, indicating a strong A/T base preference. SNPs in this study were unevenly distributed across chromosomes, and non-synonymous SNVs in exonic accounted for 3.199% of the total number of SNPs, which may lead to genetic functional variation between species. In addition, the cross-regional annotation of SNPs highlights the diversity of gene regulatory regions and may provide insights into gene regulation, the underlying genetics of complex traits, and genetic differences between species. Conclusion: The current data reinforce the established positive correlation between genome size and ploidy in the genus Ipomoea. In particular, the diploid I. purpurea had a larger genome compared to other diploid species. The genome survey indicated that I. lacunosa(2x), I. tiliacea(2x), and I. tenuissima(2x) possess simple genomes with low heterozygosity (0.36%, 0.37%, and 0.24%, respectively). In contrast, I. purpurea(2x) has a simple genome but exhibits high heterozygosity (1.95%), while I. tabascana(4x) and I. trifida(6x) have complex genomes with high heterozygosity (2.21% and 1.54%, respectively). These results provide a reasonable basis for the selection of whole genome sequencing strategies for these species and would provide references for research into the genetic diversity of wild relatives of sweetpotato. [ABSTRACT FROM AUTHOR]

Objective: To investigate the inhibitory effect of Tanreqing Injection (TRQ) on the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in macrophages infected with influenza A virus and the underlying mechanism based on mitophagy pathway. Methods: The inflammatory model of murine macrophage J774A.1 induced by influenza A virus [strain A/Puerto Rico/8/1934 (H1N1), PR8] was constructed and treated by TRQ, while the mitochondria-targeted antioxidant Mito-TEMPO and autophagy specific inhibitor 3-methyladenine (3-MA) were used as controls to intensively study the anti-inflammatory mechanism of TRQ based on mitophagy-mitochondrial reactive oxygen species (mtROS)-NLRP3 inflammasome pathway. The levels of NLRP3, Caspase-1 p20, microtubule-associated protein 1 light chain 3 II (LC3II) and P62 proteins were measured by Western blot. The release of interleukin-1β (IL-1β) was tested by enzyme linked immunosorbent assay, the mtROS level was detected by flow cytometry, and the immunofluorescence and co-localization of LC3 and mitochondria were observed under confocal laser scanning microscopy. Results: Similar to the effect of Mito-TEMPO and contrary to the results of 3-MA treatment, TRQ could significantly reduce the expressions of NLRP3, Caspase-1 p20, and autophagy adaptor P62, promote the expression of autophagy marker LC3II, enhance the mitochondrial fluorescence intensity, and inhibit the release of mtROS and IL-1β (all P<0.01). Moreover, LC3 was co-localized with mitochondria, confirming the type of mitophagy. Conclusion: TRQ could reduce the level of mtROS by promoting mitophagy in macrophages infected with influenza A virus, thus inhibiting the activation of NLRP3 inflammasome and the release of IL-1β, and attenuating the inflammatory response. [ABSTRACT FROM AUTHOR]

Cryopreservation enhances the availability of "off-the-shelf" cell therapies. However, the choice between tissue culture polystyrene (TCP) and hollow fiber system (HFB) system for adipose-derived stem cell (ASC) production remains a critical decision, with implications for scalability, reproducibility, and the clinical efficacy. Therefore, the characteristics of ASCs expanded in TCP and HFB and cryopreserved were compared. TCP and HFB cultures were established, and cells were cryopreserved. Surface markers were analyzed to identify immunophenotypic changes and subpopulations. Clonogenicity, differentiation capability, and proliferation potentials were determined along with surrogate tests on wound healing. The expressions of the most markers were consistent before and after thawing for both systems. However, CD105 expression of TCP cells was significantly decreased by the freeze-thawing procedure. Also, CD274 was significantly less expressed on HFB-expanded cells before freezing, however, post-thawing, the proportion of CD274 positive cells was comparable to TCP cells. Besides, two expansions supported different subpopulations, influencing the heterogeneity within ASC cultures. Despite this heterogeneity, no statistical differences were observed as for ASC functional characteristics and the effects on fibroblasts. This study highlighted freeze-thaw does not interfere with the production of fully functional ASCs in either system, although it drives some differential changes in the subpopulations between systems. [ABSTRACT FROM AUTHOR]

Objective(s): Some forms of breast cancer such as triple-negative phenotype, are serious challenge because of high metastatic cases, high mortality and resistance to conventional therapy motivated the search for alternative treatment approaches. Nanomaterials are promising candidates and suitable alternatives for improving tumor and cancer cell treatments. Materials and methods: Biosynthesis of ZnO NPs by help of Berberis integerrima fruit extract, has been done. Analysis of Zinc Oxide NPs using DLS, FTIR, SEM, and EDS techniques have been performed. Moreover, biological activities of ZnO NPs evaluated through MTT method, Flow cytometry, and real time PCR methods. Biocatalytic and apoptotic activity of ZnO NPs on healthy HFF (human fibroblast cell line), MDA-MB 231, and MDA-MB 468 (triple negative breast cancer cell lines, (TNBC)) evaluated. Furthermore, Bax, Bcl-2 and caspase-3 apoptotic genes expression changes in cancer cells assessed in compare to GAPDH as a house keeping gene. Results: Physico-chemical investigation demonstrated ZnO NPs were confirmed by Berberis integerrima fruit extract for the first time. The MTT assay and Flow cytometry results indicated biocompatibility of the ZnO NPs in normal cell line and high anticancer potential against TNBC MDA-MB-231 and MDA-MB-468 cell lines. The IC50 of ZnO NPs were 104.4 and 44.86, 20.96 after 24 hours for HFF, MDA-MB-231 and MDA-MB-468 cells, respectively. Conclusion: The current research showed a fast, cost effective and ecofriendly method for ZnO NPs nanoparticle synthesis. Furthermore, In vitro data analysis demonstrated biocompatibility and highly anticancer effects of biosynthesized ZnO NPs against TNBC cancerous cells. [ABSTRACT FROM AUTHOR]

The aim of our research was to explore the character of autophagy related 12 (ATG12) in the development of hepatocellular carcinoma (HCC). A total of 145 HCC tissues as well as paired adjacent normal tissues were collected, then immunohistochemistry was conducted to access the expression of ATG12. HCC cells were transfected with pcDNA ATG12 or si-ATG12 to overexpress ATG12 or downregulate ATG12. The vitality of HCC cells was accessed using CCK-8 assay, and the ability of invasion of them was tested through Transwell assay. The apoptotic rate of HCC cells was calculated by flow cytometry. The expression of ATG12 was lower in HCC tissues than that in normal tissues, and HCC patients with high ATG12 level survived longer. Overexpressed of ATG12 restrained vitality and invasion of HCC cells, while elevated apoptotic rate of HCC cells. Silence of ATG12 expression yielded opposite results to overexpression of ATG12 in HCC cells. In conclusion, ATG12 is low expressed in HCC, which attenuated the growth and invasion of HCC, while induced the apoptosis of HCC cells. Current research suggested that ATG12 might be a potential target for the diagnosis and treatment of HCC. [ABSTRACT FROM AUTHOR]

Background: The genus Allium is known for its high chromosomal variability, but most chromosome counts are based on a few individuals and genome size (GS) reports are limited in certain taxonomic groups. This is evident in the Allium sect. Codonoprasum, a species-rich (> 150 species) and taxonomically complex section with weak morphological differences between taxa, the presence of polyploidy and frequent misidentification of taxa. Consequently, a significant proportion of older karyological reports may be unreliable and GS data are lacking for the majority of species within the section. This study, using chromosome counting and flow cytometry (FCM), provides the first comprehensive and detailed insight into variation in chromosome number, polyploid frequency and distribution, and GS in section members, marking a step towards understanding the unresolved diversification and evolution of this group. Results: We analysed 1578 individuals from 316 populations of 25 taxa and reported DNA ploidy levels and their GS, with calibration from chromosome counts in 22 taxa. Five taxa had multiple ploidy levels. First estimates of GS were obtained for 16 taxa. A comprehensive review of chromosome number and DNA-ploidy levels in 129 taxa of the section revealed that all taxa have x = 8, except A. rupestre with two polyploid series (x = 8, descending dysploidy x = 7), unique for this section. Diploid taxa dominated (72.1%), while di- & polyploid (12.4%) and exclusively polyploid (15.5%) taxa were less common. Ploidy diversity showed that diploid taxa dominated in the eastern Mediterranean and decreased towards the west and north, whereas only polyploid cytotypes of di- & polyploid taxa or exclusively polyploid taxa dominated in northern and northwestern Europe. A 4.1-fold variation in GS was observed across 33 taxa analysed so far (2C = 22.3–92.1 pg), mainly due to polyploidy, with GS downsizing observed in taxa with multiple ploidy levels. Intra-sectional GS variation suggests evolutionary relationships, and intraspecific GS variation within some taxa may indicate taxonomic heterogeneity and/or historical migration patterns. Conclusions: Our study showed advantages of FCM as an effective tool for detecting ploidy levels and determining GS within the section. GS could be an additional character in understanding evolution and phylogenetic relationships within the section. [ABSTRACT FROM AUTHOR]

Objective: Juvenile Idiopathic Arthritis (JIA) comprises diverse chronic inflammatory conditions driven by malfunction of the immune system. The intestinal microbiota is considered a crucial environmental factor correlating with chronic inflammatory diseases, and for JIA certain alterations in the microbiota have already been described. Methods: Here, we have characterized intestinal microbiota samples from 54 JIA patients and 38 pediatric healthy controls by conventional 16S rRNA sequencing and by single-cell analysis for phenotypic features by multi-parameter microbiota flow cytometry (mMFC), which complements the population-based taxonomic profiling with the characterization of individual bacterial cells. Results: We found age to be a crucial confounder in microbiota analyses of JIA patients. Age stratification revealed specific microbiota alterations neglected by the general comparison of JIA patients and pediatric controls. Conclusion: Age groups presented distinct taxonomic profiles and microbiota phenotypic signatures which transitioned with age, highlighting changes in the microbiota-immune system interaction with age. [ABSTRACT FROM AUTHOR]

With ca. 60 species, Mollinedia is the second most diverse genus of Monimiaceae and the richest genus of the family in the Neotropics. In Brazil, the Atlantic Forest is the richest biome for Mollinedia, with 34 species, most of which are endemic to this biome. Here we describe a new species of Mollinedia endemic to Rio de Janeiro state, based partly on molecular evidence. [ABSTRACT FROM AUTHOR]

The objective of this work was to study the expression of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes responsible for the regulation of the differentiation of various T-helper subpopulations in individuals chronically exposed to radiation. The object of the study was peripheral blood cells obtained from 120 persons chronically exposed to radiation in a wide range of doses on the Techa River. The mean cumulative absorbed dose to red bone marrow in the examined exposed individuals was 742.7 ± 78.6 mGy (dose range, 73.5–3516.1 mGy); in the comparison group, 17.4 ± 2.2 mGy (dose range, 0.0–55.5 mGy). The subpopulation composition of T-helpers (Th1, Th2, and Th17) was analyzed by flow cytofluorometry. The relative mRNA content of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes was estimated by real-time PCR. The study made it possible to note a decrease in the relative number of T-helpers 2 in the populations of T-helpers of the central memory in the group of chronically exposed persons compared to the comparison group. In the population of T-helpers of the central memory, a statistically significant increase in the relative number of T-helpers 1 was shown, depending on the accumulated absorbed dose to red bone marrow. No changes in mRNA expression of the studied genes were observed. The analysis of the correlation between the expression of GATA3, MAPK8, STAT3, RORC, and TBX21 mRNA and the relative number of cells in subpopulations of T-helper types 1, 2, and 17 in the examined people did not reveal statistically significant patterns. [ABSTRACT FROM AUTHOR]

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), a member of the Cruciferous family, can achieve efficient germplasm resource innovation through the regeneration of microspores. In this study, 18 genotypes, including 'I105' and 'I112,' were cultured to investigate the impact of genotype on the regeneration of microspores in non-heading Chinese cabbage. Simultaneously, optimization of conditions, such as the low-temperature time of microspore's donor plants and the addition of suberoylanilide hydroxamic acid (SAHA), was conducted to obtain a substantial number of regenerated plants. By using flow cytometry for ploidy identification and conducting phenotype verification, a highly efficient system for regenerating microspores in non-heading Chinese cabbage was established. [ABSTRACT FROM AUTHOR]

A protocol for in vitro induction of autoallododecaploid Echinochloa crus-galli with colchicine treatment was established. Colchicine concentration and exposure time had significant effects on the callus survival rate, shoot differentiation rate, and chromosome doubling rate. With the increase of colchicine concentration and the extension of treatment time, the callus survival rate and shoot differentiation rate both decreased. Treatment with 0.05% colchicine for 48 hr was the most effective condition for polyploidization, yielding 42.99% dodecaploids. The authenticity of the induced dodecaploid was confirmed by nuclear DNA content, chromosome number, and stomatal and morphological characteristics. Dodecaploid plants showed the obvious "giant" effects of polyploid organs, as well as significantly reduced seed set rate. The concentrations of calcium, iron, most free amino acids, and total free amino acids in dodecaploid plants were significantly higher than those in hexaploid plants. The results showed that the chromosome of allohexaploid E. crus-galli could be redoubled. The dodecaploid E. crus-galli could still grow normally and showed advantages associated with polyploidization, such as enlarged organs and increased nutrient concentrations. The study enriched the germplasm resources of barnyard grass and laid a foundation for the utilization and theoretical research of E. crus-galli in the future. [ABSTRACT FROM AUTHOR]

Cardiopulmonary fitness (CRF) is on the decline among youth. This study aimed to investigate the effects of a low-intensity serial blood flow restriction training exercise protocol on CRF and body composition indices in youth. It also aimed to reveal changes in immune status following low-intensity continuous training exercise in adolescents, shedding light on the regulation of human immunity by exercise. We recruited 50 non-smoking, healthy, and non-exercising young volunteers and randomly divided them into a low-intensity continuous training group (LICT-BFR, n = 25) and a no-exercise Control group (n = 25), with the experimental intervention lasting for ten weeks. Physical function was assessed before and after the intervention. Peripheral blood leukocytes were analyzed for microRNA (miRNA) using Agilent human microRNA 2.0 and genetic software. MiRNA quantification was conducted through reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Flow cytometry was used to measure the percentage of various lymphocytes. The results showed that maximum oxygen uptake (VO2 max, p < 0.01) and the percentage of body fat (p < 0.05) were significantly higher in the exercise group compared to the Control group. The LICT-BFR group exhibited a significant decrease in fat mass and visceral fat area (p < 0.05) and an increase in muscle mass (p < 0.05) compared to the Control group. Additionally, six miRNAs (hsa-miR150-5p, 31-5p, 3659, 4419a, 650, and 8485) exhibited reduced expression, with miR-150 showing a significant decrease identified by RT-qPCR (p = 0.021). The percentage of CD3 + T cells was lower in the LICT-BFR group than in the non-exercising subjects (p < 0.001). However, the CD4 + to CD8 + ratio and the percentage of CD4 + T cells showed no significant differences between these two groups. In summary, low-intensity blood flow-restricted continuous training improved CRF in youth and resulted in a statistically significant reduction in T cell numbers and hsa-miR150-5p expression during prolonged exercise training. LICT-BFR was found to modulate the immune system by suppressing T cell and hsa-miR150-5p levels. This study supports the use of low-intensity blood flow restriction continuous training as an ideal exercise protocol. [ABSTRACT FROM AUTHOR]

Inborn errors of immunity (IEI) are defined as genetic disorders affecting the immune system and resulting in diverse clinical signs and symptoms. Despite the lack of diagnosis and unavailability of IEI estimation in the Pakistani population, consanguinity is exacerbating its prevalence. The current study focuses on severe combined immunodeficiency (SCID) and leukocyte adhesion deficiency type 1 (LAD1). SCID is associated with the life-threatening symptoms developing at post-birth. LAD1 is clinically characterized by recurrent bacterial infections related to the skin, mouth, and respiratory tract owing to impaired leukocytes. Herein, in six consanguineous families, flow cytometry was used to evaluate the patient's immune status. Whole-exome sequencing (WES) was then conducted to search for the causative variations in immunodeficiency genes. Sanger sequencing was used to assess the segregation of the variants with the disorder within the families. Sequence analysis revealed five homozygous variants in four different causative genes. This included four novel nonsense variants in CD70 p.(Thr126Profs*33), CD3e p.(Trp151*), IL7R p.(Val138Ilefs*10), and ITGB2 p.(Ser627Valfs*61), and one previously reported in ITGB2 p.(Cys62*). In one of the families, two variants in two different genes, including DNAH6 p.(Tyr2653His) and NIPAL4 p.(Gly121Ser), were detected in an unclassified patient. All the identified variants were found in a homozygous state in the patient but in a heterozygous state in the available parents. The study will facilitate the diagnosis and management of IEI patients. [ABSTRACT FROM AUTHOR]

Phosphatidylserine- (PS-) positive platelets play an important role in thrombosis and hemostasis. They have high procoagulant activity, the ability to vesiculate, and can aggregate with activated PS-negative platelets. They are found in growing thrombus in vitro, but numerous mysteries remain regarding them. In particular, intracellular signaling and cytoskeletal reorganization in these platelets is poorly studied, since they have the ability to be destroyed by permeabilization, which is necessary for the penetration of antibodies to intracellular markers into the cell. In this work, we propose an approach that allows the analysis of intracellular signaling in calcium ionophore A23187-induced PS-positive platelets using flow cytometry or confocal microscopy. We used the mildest permeabilization of fixed PS-positive platelets using saponin and showed that such permeabilization allows us to significantly preserve PS-positive platelets. As an example, we analyzed the state of the polymerized form of actin in PS-positive platelets and showed that, despite the significant rearrangement of the cytoskeleton that occurs upon activation in such platelets, actin in them is partially presented in a polymerized form. [ABSTRACT FROM AUTHOR]

The Trigonella species possess medicinal, nutraceutical and pharmaceutical properties due to the presence of many bioactive compounds. Its therapeutic effects are mostly valuable in medicine, cosmetics and the functional food industry. Correct genetic characterisation of plant material is needed to increase the potential of Trigonella species by breeding and conservation programs. The aim of this study was to develop a reliable marker system to support the morphological and phytochemical analysis in Trigonella taxonomic research, species identification and characterization as well as determination of the interspecific variation within this genus along with relationships between species. For this purpose, flow cytometry and SCoT molecular markers were combined. Flow cytometric analyses revealed that Trigonella species possess very small and small genomes. The range of genome sizes was from 1.10 to 5.76 pg/2C, with most species possessing very small genomes (< 2.8 pg/2C). In seeds of 14 species endopolyploid nuclei were detected. Flow cytometric analysis of genome size enabled quick identification of four out of 20 species, while combined with endopolyploidy detection in seeds, facilitated distinction of the next seven species. ScoT molecular markers helped to identify closely related species with similar genome size and cell cycle activity. Therefore, flow cytometry was proposed as the first-choice method for quick accession screening, while the more detailed genetic classification was obtained using SCoT molecular markers. [ABSTRACT FROM AUTHOR]

Purpose: Trauma triggers a systemic inflammatory cellular response due to tissue damage, potentially leading to a secondary immune deficiency. Trauma severity is quantified by the Injury Severity Score (ISS). Severe Traumatic Brain Injury (TBI) is associated with high ISSs due to high lethality, despite limited tissue damage. Therefore, ISS might overestimate the post-traumatic inflammatory cellular response. This study investigated the effect of TBI on the occurrence of different systemic neutrophil phenotypes as alternative read-out for systemic inflammation. Methods: A single-center retrospective cohort study was conducted at a level-1 trauma center. Patients aged ≥ 18 years, admitted between 01-03-2021–01-11-2022 and providing a diagnostic blood sample were included. Four groups were created: isolated TBI, isolated non-TBI, multitrauma TBI and multitrauma non-TBI. Primary outcome was occurrence of different neutrophil phenotypes determined by automated flow cytometry. Secondary outcome was infectious complications. Results: In total, 404 patients were included. TBI and non-TBI patients demonstrated similar occurrences of different neutrophil phenotypes. However, isolated TBI patients had higher ISSs than their isolated non-TBI controls who suffered similar post-traumatic inflammatory cellular responses. Regardless of the type of injury, patients exhibiting higher systemic inflammation had a high infection risk. Conclusion: When TBI is involved, ISS tends to be higher compared to similar patients in the absence of TBI. However, TBI patients did not demonstrate an increased inflammatory cellular response compared to non-TBI patients. Therefore, TBI does not add much to the inflammatory cellular response in trauma patients. The degree of the inflammatory response was related to the incidence of infectious complications. [ABSTRACT FROM AUTHOR]

Summary: The immune checkpoint inhibitor therapy represented by blocking programmed cell death protein 1/ programmed cell death-ligand 1 (PD-1/PD-L1) has made significant progress in melanoma treatment. However, the response rate and therapeutic effect of immunotherapy alone are still not ideal for melanoma. In this study, we aimed to evaluate the defects of treating anti-PD-L1 alone and the therapeutic effect and molecular mechanism of combined therapy with anti-PD-L1 and MnCl2. We detected the changes of immune cell populations after anti-PD-L1 treatment in melanoma xenograft mouse model. Further, we evaluated the regulatory effect of MnCl2 on dendritic cells (DCs) maturation in vitro. Next, we tested the therapeutic effect and regulatory effect on the tumor microenvironment with anti-PD-L1 and MnCl2 via combining treatment with anti-PD-L1 and MnCl2. Anti-PD-L1 therapy has a certain tumor suppressive function, but the effect is not ideal. The results of flow cytometry showed that the number of CD4+ T cells and CD8+ T cells significantly increased after anti-PD-L1 treatment. However, the number of DCs remained basically unchanged after anti-PD-L1 treatment. In vitro, we confirmed that MnCl2 significantly promoted DCs maturation vis activating cGAS-STING signaling pathway. The combination of anti-PD-L1 and MnCl2 displayed the best tumor suppression effect in melanoma xenograft mouse model. In tumor microenvironment, the infiltration of T cells and the maturation of DCs were significantly promoted, demonstrating a strong anti-tumor immune response. In summary, we conclude that combining anti-PD-L1 with MnCl2 is a promising therapeutic strategy for melanoma. [ABSTRACT FROM AUTHOR]

Merkel cell carcinoma is a very aggressive primary skin tumour with a high risk of local recurrences and lymphatic and distant metastases. The frequent association between this carcinoma and other skin tumour and lymphoid malignancies, its similar cellular morphology with leukocytes, and limited infiltration in bone marrow constituted a challenging diagnosis. We report an unusual case of an 82-year-old male who simultaneously presented Merkel cell carcinoma and acute myeloid lymphoma. The diagnosis was established through flow cytometry, immunohistochemical studies and next generation sequencing (NGS) analysis. Flow cytometry allowed for the differentiation of the two cell populations in bone marrow aspirate, which was crucial to the diagnosis of Merkel cell carcinoma and acute myeloid leukaemia (AML), after confirmed by immunohistochemistry. AML could be classified based on NGS results. Following diagnosis, the patient received palliative care and died 50 days later. immunophenotypic analysis by flow cytometry and Immunohistochemical study was crucial to establish the diagnosis of simultaneous affection of Merkel cell carcinoma and hematologic disorder. [ABSTRACT FROM AUTHOR]

Background: Pomalidomide, a third-generation oral immunomodulatory drug, exhibits efficacy in patients with relapsed multiple myeloma or those refractory to bortezomib and lenalidomide (RRMM). Methods: In this clinical context, we employed flow cytometry and CDR3 spectratyping to monitor the dynamics of the T-cell repertoire during Pomalidomide treatment, aiming to investigate its potential to reverse the immunological abnormalities characteristic of RRMM. Results: By flow cytometry at baseline we found a significant decrease in CD4 + frequency in MM patients, while CD8 + frequency were significantly higher in patients when compared to controls. Most T cell populations remained stable across all time points, except for CD4 + frequency, which notably decreased from t1 to subsequent assessments. Our investigation revealed as most relevant finding the notable increase in CD4 + expansions and the growing prevalence of patients manifesting these expansions. This pattern is even more evident in patients receiving their treatment until t3 and therefore still responding to treatment with Pomalidomide. We also conducted a comparison of spectratyping data before and after treatment, substantially demonstrating a relatively stable pattern throughout the course of Pomalidomide treatment. Conclusions: These observations imply that Pomalidomide treatment influences the T-cell repertoire, particularly in the CD4 + subpopulation during the later stages of treatment, raising speculation about the potential involvement of these lymphocyte expansions in mechanisms related to antitumor immunity. [ABSTRACT FROM AUTHOR]

Aspirin is one of the most frequently detected non-steroidal anti-inflammatory drugs in aquatic environments. Despite its prevalence, toxicity possessed by aspirin to non-target organisms like fish is poorly explored. In the present study, cell death induced by different concentrations of aspirin (1, 10, and 100 µg/L) has been investigated in the liver of fish, Labeo rohita exposed for 28 days. A significant increase (p < 0.05) in the density of caspase-3 positive cells in a dose and duration-dependent manner assessed through immunofluorescent staining indicates caspase-dependent pathway of cell death which may be either through intrinsic or extrinsic pathway. The flow cytometric analysis, in addition, revealed a significant (p < 0.05) decline in the live cells and an increase in apoptotic cells in the liver of fish exposed to aspirin. Cell death due to apoptosis is further indicated by a significant increase (p < 0.05) in the Kupffer cells and tumor necrosis factor-α. The decrease in the activity of cyclooxygenase (COX) enzyme significantly (p < 0.05) in all three exposure concentrations of aspirin suggests COX-dependent pathway of cell death. The present study provides in-depth insights into aspirin-induced cell death in the liver of fish at environmentally realistic concentrations. [ABSTRACT FROM AUTHOR]

Objective: To investigate the therapeutic effect of Sanhuang Xiexin Decoction (SXD) on triple-negative breast cancer (TNBC) in mice and its underlying mechanism. Methods: The high-performance liquid chromatography (HPLC) was used to quantitate and qualify SXD. A total of 15 female BALB/c mice were inoculated subcutaneously on the right hypogastrium with 3×105 of 4T1-Luc cells to establish TNBC mouse model. All mice were divided randomly into 3 groups, including phosphate buffered solution (PBS), SXD and doxorubicin (DOX) groups (positive drug). Additionally, tumor growth, pathological changes, serum lipid profiles, expression of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) signaling pathway and its key targets including inflammatory factors, cell cycle and epithelial-mesenchymal transition (EMT) markers were investigated. Besides, the biosafety of SXD was also evaluated in mice. Results: Rhein, coptisine, berberine hydrochloride and baicalin were all found in SXD, and the concentrations of these 4 components were 0.57, 2.61, 2.93, and 46.04 mg/g, respectively. The mouse experiment showed that SXD could notably suppress the development of tumors and reduce the density of tumor cells (P<0.01). The serum lipid analysis and Oil-Red-O staining both showed the differences, SXD group exhibited higher serum adiponectin and HDL-C levels with lower TC and LDL-C levels compared to the PBS and DOX groups (P<0.05 or P<0.01), respectively. SXD also decreased the levels of phospho-JAK2 (p-JAK2), phospho-STAT3 (p-STAT3) expressions and its downstream factors, including mostly inflammatory cytokine, EMT markers, S phase of tumor cells and vascular endothelial growth factor (VEGF) expression (P<0.05 or P<0.01), respectively. The biosafety assessment of SXD revealed low levels of toxicity in mice. Conclusion: SXD could inhibit TNBC by suppressing JAK2-STAT3 phosphorylation which may be associated with modulation of lipid metabolism. [ABSTRACT FROM AUTHOR]

In May and June of 2021, marine microbial samples were collected for DNA sequencing in East Sound, WA, USA every 4 hours for 22 days. This high temporal resolution sampling effort captured the last 3 days of a Rhizosolenia sp. bloom, the initiation and complete bloom cycle of Chaetoceros socialis (8 days), and the following bacterial bloom (2 days). Metagenomes were completed on the time series, and the dataset includes 128 size-fractionated microbial samples (0.22–1.2 µm), providing gene abundances for the dominant members of bacteria, archaea, and viruses. This dataset also has time-matched nutrient analyses, flow cytometry data, and physical parameters of the environment at a single point of sampling within a coastal ecosystem that experiences regular bloom events, facilitating a range of modeling efforts that can be leveraged to understand microbial community structure and their influences on the growth, maintenance, and senescence of phytoplankton blooms. [ABSTRACT FROM AUTHOR]

Background: Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment. Methods: Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase–activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking. Results: CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls. Conclusions: These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization. [ABSTRACT FROM AUTHOR]

Evidence supports that hyaluronic acid (HA) can promote tissue regeneration and reduce inflammation. This study aimed to assess the effects of a bilayered cellulose-coated HA scaffold on oral wound healing. A film-type 3% HA scaffold with bilayer cellulose coating was prepared and compared with an HA scaffold without coating. To evaluate cytocompatibility, human gingival fibroblasts were exposed to both scaffolds, and cell viability, flow cytometry, and scratch wound assays were performed. In addition, in vivo and ex vivo wound-healing assays were performed. Cytocompatibility tests showed no cytotoxicity for either HA scaffold. The scratch wound assay revealed a significant reduction in the open wound area in both HA scaffolds compared with that in the control (p < 0.05); however, no differences were observed between the scaffolds with and without cellulose coating. In vivo wound healing analysis showed significantly higher healing rates on day 3 in the HA scaffolds than in the control (p < 0.05), with no significant differences between the scaffolds. HA scaffolds with coating showed lower CD68 and higher vimentin expression than the control (p < 0.05), whereas HA scaffolds without coating did not. Ex vivo wound healing analysis revealed significantly higher re-epithelialization rates in both scaffolds than in the control (p < 0.05). Within the limits of this study, the HA scaffold with coating showed enhanced wound healing efficacy, indicating its potential for oral wound healing applications. [ABSTRACT FROM AUTHOR]

Research on central nervous system tumors (CNSTs) has a significant impact on the diagnosis and prognosis of patients. Currently, CNSTs are classified according to the schema proposed by the World Health Organization (WHO), which considers clinical, histopathological, and molecular characteristics, highlighting the importance of tumor biology for accurate diagnosis and optimal treatment approaches. Despite these advances, assessing DNA ploidy—a marker of tumor aggressiveness—remains complex in CNSTs. This review investigates the utility of DNA index (DNAi) and DNA ploidy analysis by flow cytometry in diagnosing CNSTs and prognosing their outcomes. We systematically reviewed studies in the PubMed database from 1990 to the present using the keywords "DNA Index", "Brain", "Flow cytometry", and "Ploidy". We identified 151 studies, 36 of which met our inclusion criteria. We found considerable variation in sample sizes and methodological variation across the studies. Discrepancies between the reported DNAi and ploidy values were observed. Aneuploidy is generally associated with more aggressive tumors, although exceptions exist. Higher DNAi levels correlate with increased malignancy, notably in glioblastomas, astrocytomas, and meningiomas, whereas diploid astrocytomas and oligodendrogliomas are associated with shorter survival rates. DNA ploidy assessment via flow cytometry could predict CNST behavior, yet methodological issues with tissue selection, adequate control samples, and technique variability remain. DNAi and ploidy assessments show promise as prognostic markers in CNSTs. However, the standardization of flow cytometry protocols and alignment with the current WHO classification schema are essential steps to integrate ploidy analysis in routine CNST assessment. [ABSTRACT FROM AUTHOR]

Self-assembling DNA nanoparticles have the potential to significantly advance the targeted delivery of molecular cargo owing to their chemical and architectural flexibility. Recently, it has been demonstrated that the genetic code embedded in DNA nanoparticles produced by the method of DNA origami or related techniques can be recognized and copied by RNA polymerase in vitro. Further, sculpted DNA nanoparticles can serve as a substrate for Cas9-mediated gene modification and gene expression in cell culture. In the present study, we further investigate the ability of DNA origami nanoparticles to be expressed in a human cell line with emphasis on the impact of single-stranded DNA (ssDNA) domains and the contributions of the architectural disposition of genetic control elements, namely promoter and enhancer sequences. Our findings suggest that while cells possess the remarkable capability to express genes within highly folded architectures, the presence and relative density and location of ssDNA domains appears to influence overall levels of gene expression. These results suggest that it may be possible to nuance folded DNA nanoparticle architecture to regulate the rate and/or level of gene expression. Considering the highly malleable architecture and chemistry of self-assembling DNA nanoparticles, these findings motivate further exploration of their potential as an economic nanotechnology platform for targeted gene editing, nucleic acid-based vaccines, and related biotherapeutic applications. [ABSTRACT FROM AUTHOR]

Eosinophils were previously reported to play a role in intestinal inflammation and fibrosis. Whether this is as a bystander or as an active participant is still up for debate. Moreover, data describing a causal relationship between eosinophils and intestinal fibrosis are scarce. We here aimed to elucidate the role of eosinophils in the pathogenesis of intestinal inflammation and fibrosis. Therefore, we stimulated fibroblasts with (active) eosinophils or with Eosinophil Cationic Protein (ECP), and assessed fibroblast activation via flow cytometry and immunocytochemistry. We observed decreased fibroblast activation when fibroblasts were co-cultured with active eosinophils or after stimulation with ECP in comparison to monoculture conditions, but not in case of co-culturing with inactivated eosinophils. Furthermore, eosinophil depletion in a RAG−/− chronic DSS colitis model resulted in decreased inflammation, but increased development of fibrosis. In this model, we could show increased expression of the anti-inflammatory protein IL-10 and the pro-fibrotic factors IL-1β, FGF-21 and TGF-β3 in the eosinophil-depleted mice compared to the control mice. In conclusion, our in vitro data revealed an anti-fibrotic role for eosinophils. In line, in a chronic murine colitis model, we observed a pro-inflammatory, but an anti-fibrotic, role for eosinophils. Furthermore, we identified an increased presence of anti-inflammatory and pro-fibrotic cytokines in the eosinophil depleted group. [ABSTRACT FROM AUTHOR]

MBL is a precursor condition to chronic lymphocytic leukemia (CLL), characterized by monoclonal B-cells in blood. Mosaic chromosomal alterations (mCAs) are a form of clonal hematopoiesis that include gains, losses, and copy-neutral loss-of-heterozygosity of large DNA segments. Both MBL and mCAs have been found to increase the risk of CLL and lymphoid malignancies, and the aim of our study was to investigate how mCAs relate to MBL, which is currently unknown. We analyzed genetic, flow cytometric, and hematologic data from 4632 individuals from the Mayo Clinic Biobank and CLL Database. MBL was detected using flow cytometry and classified as high-count (HC) or low-count (LC) MBL based on clone size. mCAs were detected primarily from whole blood DNA using sensitive SNP-array-based analyses. mCAs commonly altered in CLL (deletion of 6q, 11q, 13q, 17p, and trisomy 12) were specific (>99%) to individuals with MBL and CLL. HC-MBL and LC-MBL individuals were 881-fold and 8-fold, respectively, more likely to harbor CLL-associated mCAs than those without MBL. The cell fraction bearing these mCAs typically exceeded the B-cell fraction, suggesting their origin prior to the B-cell lineage. Integrating genetic and blood count data enabled detecting HC-MBL with high specificity in a biobank sample. These results quantify the contribution of mCAs to MBL and could enable large studies of HC-MBL without the need for flow cytometric screening. [ABSTRACT FROM AUTHOR]

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. Key points: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins [ABSTRACT FROM AUTHOR]

The incidence of primary hepatocellular carcinoma (HCC) has recently ranked fifth in the world, and the incidence rate is increasing year by year worldwide. Therefore, early diagnosis is the highest priority in the treatment of HCC. In this paper, four anti-HCC aptamers were obtained using magnetic bead SELEX technology. Among them, Apt-1 had the smallest Kd value(5.9 nM) and the highest affinity. Flow cytometry results showed that the FITC-aptamers only specifically recognized HCC serum. Circular dichronism (CD) spectral characterization showed a positive peak near 275 nm and a negative peak near 250 nm for all aptamers, elucidating that the secondary structure formed by the candidate aptamers was a stem-loop B-DNA structure. In addition, molecular docking simulations showed that the binding of the HCC target to the candidate aptamer sequences was mainly dominated by hydrogen bonding. The results of the aptamer sensing performance analysis showed that under the optimized assay conditions, a linear relationship (ranging from 1 nM to 1 µM) was achieved, with a limit of detection (LOD) down to 0.75 nM and a LOQ of 2.32 nM. This was further validated in clinical samples, with a positive detection rate of more than 90%. Furthermore, aptamer-mediated in vivo delivery of luciferase mRNA showed that Apt-1-luciferase mRNA could be targeted to the liver and hepatic luciferase expression was significantly increased. These results demonstrate that the aptamer paves the way for clinical application, evidencing significant potential to offer reference information for early diagnosis. [ABSTRACT FROM AUTHOR]

Background: Dysfunctional immune regulation plays a crucial role in the pathogenesis of airway allergies. Macrophages are one of the components of the immune regulation cells. The aim of this study is to elucidate the role of lysine demethylase 5 A (KDM5A) in maintaining macrophages' immune regulatory ability. Methods: DNA was extracted from Lactobacillus rhamnosus GG to be designated as LgDNA. LgDNA was administered to the mice through nasal instillations. M2 macrophages (M2 cells) were isolated from the airway tissues using flow cytometry. Results: We found that airway M2 cells of mice with airway Th2 polarization had reduced amounts of IL-10 and KDM5A. Mice with Kdm5a deficiency in M2 cells showed the airway Th2 polarization. The expression of Kdm5a in airway M2 cells was enhanced by nasal instillations containing LgDNA. KDM5A mediated the effects of LgDNA on inducing the Il10 expression in airway M2 cells. Administration of LgDNA mitigated experimental airway allergy. Conclusions: M2 macrophages in the airway tissues of mice with airway allergy show low levels of KDM5A. By upregulating KDM5A expression, LgDNA can increase Il10 expression and reconcile airway Th2 polarization. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) play an important role in the progression of atherosclerosis (AS). This study aimed to explore the exact role and mechanism of circ_0002984 in oxidized low-density lipoprotein (ox-LDL)-mediated human vascular smooth muscle cells (HVSMCs). The model of smooth muscle cell phenotype switching was constructed by treating HVSMCs with ox-LDL. The levels of circ_0002984, let-7a-5p, and kruppel-like factor 5 (KLF5) were measured by quantitative real-time PCR or western blot assay. Cell proliferation, migration, and apoptosis were detected by Cell Counting Kit-8 (CCK-8), EdU staining, wound healing assay, transwell assay, and flow cytometry. The expression of cleaved-caspase-3 and KLF5 was examined by western blot. The relationship between let-7a-5p and circ_0002984 or KLF5 was verified by dual-luciferase reporter assay or RIP assay. The results showed that circ_0002984 and KLF5 were up-regulated, while let-7a-5p was down-regulated in AS patients and ox-LDL-disposed HVSMCs. Silence of circ_0002984 suppressed proliferation and migration, and promoted apoptosis in ox-LDL-stimulated HVSMCs. Moreover, circ_0002984 sponged let-7a-5p to regulate the proliferation, migration, and apoptosis in ox-LDL-resulted HVSMCs. In addition, KLF5 was a target of let-7a-5p and its overexpression reversed the effect of let-7a-5p on the proliferation, migration, and apoptosis in ox-LDL-treated HVSMCs. Also, circ_0002984 positively regulated KLF5 expression by absorbing let-7a-5p. The promotion effect of circ_0002984 on the proliferation and migration of ox-LDL-treated HVSMCs was reversed by KLF5 silencing. Taken together, depletion of circ_0002984 inhibited the proliferation and migration of ox-LDL-stimulated HVSMCs, which might be achieved by modulating the let-7a-5p/KLF5 axis. [ABSTRACT FROM AUTHOR]

Accumulation studies confirmed that oxidative stress caused by ischemia after myocardial infarction (MI) is an important cause of ventricular remodeling. Exosome secretion through hypoxic pretreatment adipose-derived mesenchymal stem cells (ADSCs) ameliorates myocardial damaging post-MI. However, if ADSCs exosome can improve the microenvironment and ameliorate cardiac damage post-MI still unknown. Next-generation sequencing (NGS) was used to study abnormally expressed circRNAs in hypoxic pretreatment ADSC exosomes (HExos) and untreated ADSC exosomes (Exos). Bioinformatics and luciferase reporting were used to elucidate interaction correlation related to circRNA, mRNA, and miRNA. HL-1 cells were used to analyze the reactive oxygen species (ROS) and apoptosis under hypoxic conditions using immunofluorescence and flow cytometry. An MI mouse model was constructed and the therapeutic effect of Exos was determined using immunohistochemistry, immunofluorescence, and ELISA. The results showed that HExos had a more pronounced treatment effect than ADSC Exos on cardiac damage amelioration after MI. NGS showed that circ-Stt3b plays a role in HExo-mediated cardiac damage repair after MI. Overexpression of circ-Stt3b decreased apoptosis, ROS level, and inflammatory factor expression in HL-1 cells under hypoxic conditions. Bioinformatics and luciferase reporting data validated miR-15a-5p and GPX4 as downstream circ-Stt3b targets. GPX4 downregulation or miR-15a-5p overexpression reversed protective effect regarding circ-Stt3b upon HL-1 cells after exposure to a hypoxic microenvironment. Overexpression of circ-Stt3b increased the treatment effect of ASDSC Exos on cardiac damage amelioration after MI. Taken together, the study results demonstrated that Exos from hypoxic pretreatment ADSCs ameliorate cardiac damage post-MI through circ-Stt3b/miR-15a-5p/GPX4 signaling activation and decreased ferroptosis. [ABSTRACT FROM AUTHOR]

Objective: To investigate the inhibitory effect of Celastrus orbiculatus extracts (COE) on the proliferation of lymphoma cells and the immune regulation ability on inflammation and thrombophilia in vivo. Methods: The 38B9 lymphoma cells were treated with COE (160 µ g/mL) and CTX (25 µ mol/L). The apoptosis rate and cell cycle of each group were detected by flow cytometry. The secretion of inflammatory factors, including interleukin (IL)-6, IL-10, and tumor necrosis factor α (TNF-α), in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). In vivo, BALB/c mice were subcutaneously injected with 38B9 lymphoma cells to establish lymphoma model. COE (3 mg·kg−1·d−1) and CTX (40 mg·kg−1·d−1) were administered to the model mice, respectively. The expression of plasma inflammatory factors (IL-6, IL-10 and TNF-α) and thrombus indexes, including D-dimer (D-D), von Willebrand factor (vWF) and tissue factor (TF), were detected by ELISA before tumor bearing (1 d), after tumor formation (14 d) and after intervention (21 d). PicoGreen dsDNA was used to detect the level of serum neutrophil extracellular traps (NETs). Flow cytometry was used to detect the expression of platelet activation marker calcium-dependent lectin-like receptor 2 (CLEC-2). The tumor growth and survival of mice were recorded. Results: The 38B9 lymphoma cells were apoptotic after the intervention of COE and CTX. The ratio of G2-M phase cells decreased in COE intervented cells compared with the control cells (P<0.05), and S phase cells decreased in CTX intervented cells (P<0.05). Also, the secretion level of IL-6 was significantly reduced after COE or CTX intervention (P<0.05), and IL-10 was significantly increased (P<0.05). Furthermore, the tumor mass was reduced, and the median survival time was longer in COE and CTX intervented tumor-bearing mice than in non-intervented mice. The significantly lower levels of TNF-α, IL-6, NETs, TF, DD and CLEC-2, as well as higher IL-10 were observed in COE and CTX treatment mice in comparision with the control mice (P<0.05). Conclusion: COE has a mild and stable anti-tumor effect, which can reduce the secretion of inflammatory factors by lymphoma cells and regulate thrombophilic state caused by tumor inflammatory microenvironment. [ABSTRACT FROM AUTHOR]

Poor aqueous solubility and stability hinder the clinical translation of pyrazolone-based derivatives despite their various biological activities. This study aimed to address these issues by developing water-soluble nanoformulations of two specific pyrazolone derivatives, Compounds I and II, selected based on their promising structural features and previous biological activity data. PLGA/poloxamer-based nanoformulations were prepared and optimized for size, PDI, zeta potential (ZP), and entrapment efficiency (EE). The optimized formulations demonstrated sizes of 166.6 ± 7.12 nm and 192.5 ± 1.08 nm, PDI of 0.129 ± 0.042 and 0.132 ± 0.025, ZP of − 14.14 ± 2.90 mV and − 10.77 ± 1.515 mV, and %EE of 84.20 ± 0.930 and 81.5 ± 2.051, respectively. A sustained drug release was observed over 48 h, with cumulative releases of approximately 37% and 53%, for both formulations, and characterized by a complex drug release behavior. The formulations exhibited significant antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus (SA), with greater than 90% cell death for SA and greater than 80% for MRSA, observed using the flow cytometer. Also, enhanced antioxidant activity was observed using DPPH, FRAP, and NO methods, showing better radical scavenging than standard gallic acid and bare compounds. The hemolysis assay confirmed the biocompatibility of the developed formulation, with a hemolysis percentage of less than 5%. This study highlights the successful development of water-soluble nanoformulations with significant antibacterial and antioxidant activities, emphasizing the impact of solubility enhancement on biological efficacy and suggesting potential pharmaceutical applications for these agents. [ABSTRACT FROM AUTHOR]

The synthesis of silver nanoparticles from biological sources has fascinated the research fraternities due to their distinctive non-toxic features and benign synthesis methodologies. In this pipeline, we have synthesized silver nanoparticles (AgNPs) from the carthamidin (CT) pigment. The yellow-colored water-soluble pigment reduces the silver nitrate into metallic silver in a simple one-pot method. The CTAgNPs were exclusively characterized by UV–Vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive analysis spectroscopy (EDAX). The characterization techniques indicate that CTAgNPs are spherical with an average size of 59 nm and face-centered cubic with a face-centered cubic crystalline structure. The CTAgNPs were evaluated as a cytotoxic agent in the MCF 7 cells, and in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, CTAgNPs inhibited the growth of MCF 7 cells with an IC50 value of 112 µg/mL. The MTT assay signifies that CTAgNPs possess cytotoxic attributes which can be further developed as an anticancer agent. Further, we molecularly analyzed the apoptosis induction by CTAgNPs by acridine orange (AO)/ethidium bromide (EB) staining, a reactive oxygen species (ROS) assay, quantification of lactate dehydrogenase (LDH), and cell cycle arrest using flow cytometry. Finally, CTAgNPs exhibit remarkable cytotoxicity in MCF-7 cells and induce apoptosis. Thus, CTAgNPs may be proposed as a drug molecule for breast cancer treatment. [ABSTRACT FROM AUTHOR]

Grasses are one of the largest angiosperm families, widespread and economically important. Variation in genome size has functional consequences and is an essential parameter for understanding evolutionary patterns. In this study, we report the nuclear genome sizes (2C values) of 32 species and subspecies from 27 genera of Poaceae, including most of its subfamilies, examined by flow cytometry. Obtained genome sizes were analyzed together with the chromosome numbers to give information on the size of monoploid chromosome sets with the chromosome base number x and the mean chromosome size and then supplemented with the previously published data to obtain a deeper insight into the genome size evolution in grasses. Monoploid genomes of < 0.6 pg/1Cx and chromosomes of < 0.1 pg are presumably characteristic of the subfamilies Arundinoideae, Chloridoideae, Micrairoideae and the Oryzoideae. The larger 1Cx values (1.2–1.8 pg) of the evolutionarily ‘early diverging’ subfamilies Anomochlooideae and Pharoideae are discussed in context with the origin of grasses and the pan-grass whole-genome duplication. The data indicate that the ancestral grass had a monoploid genome of this size, which is less than half the size previously assumed. Genome size data and available chromosome numbers support the concept of the ancestral grass karyotype (AGK) with x = 12. The AGK seems to have been conserved in some grass subfamilies (Bambusoideae, Oryzoideae, Pharoideae, parts of the Pooideae), while the major genome rearrangements are lineage-specific and occurred after the separation of the BOP and the PACMAD clades, i.e. when the diversification of their subfamilies had begun. [ABSTRACT FROM AUTHOR]

Schizachyrium scoparium is a kind of ornamental grass that has been planted in China and abroad. In this study, the calli of tetraploid S. scoparium was used as the material and colchicine was used as the inducer of polyploid. The effect of different treatment concentration and time on polyploid induction was studied. The induction system of S. scoparium octaploid was established. The results provide a novel avenue for germplasm innovation and breeding of new cultivars of S. scoparium. The difference between the two plants was compared by stomatal parameter analysis, flow cytometry analysis, chromosome counting, growth parameter and phenological analysis. The optimal colchicine treatment involved exposure to 0.2% colchicine for 72 h. The highest percentage induction of octaploid plants was 26.7%, resulting in the regeneration of 48 independent octaploid individuals from calli. Notably, octaploid plants exhibited morphological changes compared with tetraploids, including a reduction in stomatal density and an increase in stomatal size. Plant height was reduced by 50% and leaf width was significantly increased. The average number of spikes per plant was reduced by 8.7 and flowering was delayed by 21 d. These findings suggest that the induced octaploid plants have potential as a valuable resource for germplasm innovation for new cultivars of S. scoparium.Key message: Treated with 0.2% colchicine for 72 h, 48 Schizachyrium scoparium octaploid were obtained, which provided a new way to solve its lodging issues. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the dose-dependent adverse effects of long-term dietary lithium administration on specific aspects of the defense system in rats. Additionally, the study aimed to explore the inflammatory activities of lithium beyond its recognized anti-inflammatory properties. Forty Wistar Albino rats were involved, which were randomly allocated into the control and four treatment groups. The control group received standard rat feed, and the experimental groups' diet was added 1 g/kg, 1.4 g/kg, 1.8 g/kg, and 2.2 g/kg lithium bicarbonate, respectively. CD4+, CD8+, and CD161 + cells were assessed by flow cytometry. TNF-α, IFN-γ, IL-1β, and IL-2 and IL-4, IL-6, and IL-10 levels were measured. The proportion of CD4 + cells and the CD4+/CD8 + ratio (P = 0.005 and P = 0.038, respectively) were reduced with the highest dose of lithium compared to the control group. The data regarding pro-inflammatory cytokines showed a dose-dependent increase in serum TNF-α and IFN-γ levels (P = 0.023 and P = 0.001, respectively). On the other hand, serum IL-1β and IL-2 levels were decreased in a dose-dependent manner (P = 0. 001 and P = 0. 001, respectively). As for anti-inflammatory cytokines, a dose-dependent decrease was determined in serum IL-4 level (P = 0.002), while no significant changes were noted in IL-6 and IL-10 levels (P = 0.507 and P = 0.732, respectively). In conclusion, lithium adversely impacted the cellular defense system. Furthermore, apart from its anti-inflammatory properties, lithium exhibited cytokine-mediated inflammatory activities. Therefore, lithium's potential adverse effects on the immune system should be considered in immunodeficient patients and those with an inflammatory status treated with high doses of lithium. [ABSTRACT FROM AUTHOR]

This study aims to characterize the majority of immune cell subsets in peripheral blood mononuclear cells in children with Mycoplasma pneumoniae pneumonia (MPP) by a 21-color flow cytometry panel. Patients who met the predetermined eligibility criteria for pneumonia diagnosis were recruited for the research study. Multi-color flow cytometry was conducted on the peripheral blood mononuclear cells of each patient group, which were then subjected to dimensionality reduction and cluster analysis. In our study, the proportion of activated CD4 + T cell and naïve CD8 + T in children with MPP was higher than that of children with non-MPP, and the proportion of CD8 + T cell and central memory CD8 + T cell in MPP children was lower. Central memory CD4 + T cell and activated CD4 + T cell in the severe MPP were higher than those in the mild MPP. The highest proportions of CD8 + T cell, CD8 + Tn cell, activated CD8 + T cell, and total activated T cell were observed in the pulmonary consolidation-mucous group when compared to the pulmonary consolidation-necrosis and bronchiolitis groups. In the pulmonary consolidation-necrosis group, the proportions of central memory CD4 + T cell and T helper 17 cell were higher than those in pulmonary consolidation-mucous and bronchiolitis groups. In the bronchiolitis group, the percentages of CD4 + T cell, naïve CD4 + T cell, and T helper 2 cell were higher than those in pulmonary consolidation-mucous and the pulmonary consolidation-necrosis groups. The T lymphocyte subsets were different among various groups, offering new insights into the immune system of pediatric patients with Mycoplasma pneumoniae pneumonia. [ABSTRACT FROM AUTHOR]

Cisplatin-based chemotherapy serves as the standard of care for individuals with advanced stages of gastric cancer. Nevertheless, the emergence of chemoresistance in GC has detrimental impacts on prognosis, yet the underlying mechanisms governing this phenomenon remain elusive. Level of mitophagy and ferroptosis of GC cells were detected by fluorescence, flow cytometry, GSH, MDA, Fe2+ assays, and to explore the specific molecular mechanisms between NPR1 and cisplatin resistance by performing western blot and coimmunoprecipitation (co-IP) assays. These results indicates that NPR1 positively correlated with cisplatin-resistance and played a crucial part in conferring resistance to cisplatin in gastric cancer cells. Mechanistically, NPR1 affected levels of mitophagy and ferroptosis in human cisplatin-resistance GC cells with cisplatin treatment. Specifically, NPR1 inhibited mitophagy-dependent ferroptosis by reducing the ubiquitination-mediated degradation of PARL; moreover, NPR1 promoted PARL stabilization by disrupting the PARL–MARCH8 complex, which ultimately led to the development of chemoresistance in GC cells. Considering our findings, NPR1 appears to play an important role in chemotherapy for GC. NPR1 could potentially be used to overcome chemotherapy resistance. 1. NPR1 is highly expressed in cisplatin-resistant GC cells and promotes the cisplatin resistance. 2. According to IP-MS, NPR1 inhibits mitophagy-dependent ferroptosis through its downstream protein PARL. 3. NPR1 promoted PARL stabilization by disrupting the PARL–MARCH8 complex to inhibit the ubiquitination-mediated degradation of PARL. [ABSTRACT FROM AUTHOR]

Objective: This study aims to analyze the clinical features, Radiological findings and diagnostic characteristics (tissue biopsy, flow cytometry and cerebrospinal fluid cytology) of primary central nervous system lymphoma (PCNSL). To improve the diagnosis of the disease. Methods: A total of 19 patients with primary central nervous system lymphoma admitted to the Second Hospital of Hebei Medical University from 2014 to 2023 were selected. The clinical data of the patients were retrospectively analyzed. Combined with the relevant literature, the clinical characteristics of PCNSL patients were analyzed to assist the diagnosis. Result: Most of the patients were middle-aged and elderly patients (65 years old), and the main clinical manifestations showed no obvious specificity, including headache, dizziness and limb weakness. Radiological findings showed that common site of currence were located in the brain parenchyma. The PCNSL subtype of 19 patients was all diffuse large B cell subtype by cytology, flow cytometry or histopathology. Six cases were diagnosed by biopsy pathology and 11 cases were diagnosed by flow cytometry of cerebrospinal fluid. In all patients diagnosed with PCNSL by clinical cytology (CSF cytology and flow cytometry), abnormal cells were found on CSF examination. Conclusion: The pathological type of primary central nervous system lymphoma was diffuse large B-cell subtype, with diverse clinical manifestations and multiple. Both tissue directed biopsy and flow cytometry could effectively assist clinical diagnosis. Early and timely diagnosis and intervention are of great significance for delaying the disease process and reducing the economic burden of patients and families. [ABSTRACT FROM AUTHOR]

Our research focuses on semen collection from 42 African penguin males, grouped by age, habituation levels, and reproductive season timing. We assess behavioral and physiological responses to dorso-abdominal massage, evaluating species-specific seminal traits using conventional and advanced methods. Positive behavioral responses corresponded with successful semen collection. Young and human-reared males exhibited more favorable behaviors, and samples containing spermatozoa were more likely collected during the reproductive season. Age did not influence sample collectability or spermatozoa presence, though mature males produced higher semen volumes. Young males exhibited more sperm morphological abnormalities, especially outside the breeding season. Sperm concentration and number per ejaculate showed no significant differences across age or seasonal groups. Young males had higher motile and progressive sperm percentages, while mature males had more static sperm. Additionally, percentages of live sperm and intact acrosomes were higher in mature males. Young males and samples from non-reproductive season presented more dead sperm with damaged acrosomes. Plasma membrane integrity positively correlated with age for live sperm and negatively for dying sperm. This research demonstrates the feasibility of semen collection from African penguins regardless of habituation level, fills the knowledge gap by describing sperm morphological abnormalities, and provides insights into using flow cytometry in Sphenisciformes. [ABSTRACT FROM AUTHOR]

As Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are easily accessible, easy to isolate, and ethically acceptable, they represent a promising source of MSCs for use in regenerative medicine. Considering decisions on WJ-MSCs collection requires extensive knowledge of the factors that impact their yield. This study's aim was to evaluate the influence of parameters related to mothers and newborns on the WJ-MSCs yield. The WJ-MSCs were isolated and expanded after being isolated from 79 umbilical cord (UC) samples. Population doubling time and cell proliferation were assessed. By flow cytometry analysis, WJ-MSCs were identified by positivity of CD105, CD90, and CD73 and negativity of CD45 and CD34. There was a statistically significant negative correlation between UC width and P1 doubling time. Maternal age and WJ-MSC yield were shown to be negatively correlated. Birth weight and gestational age showed a significant positive correlation between WJ-MSCs yield and neonatal variables. No significant correlations were detected between the WJ-MSCs and the mother parity, nor the neonatal sex, fetal presentation, or head circumference. The WJ-MSCs yield increases with younger maternal age, higher gestational age, and increased neonatal birth weight. Hence, consideration should be given to these factors when selecting the ideal donors. [ABSTRACT FROM AUTHOR]