Search Results (221)

Apelin-13, a neuropeptide, has been recognized for its neuroprotective properties. Our previous study found apelin-13 improves cognitive function in Alzheimer’s disease (AD) rats by inhibiting neuroinflammation through upregulation of BDNF/TrkB signaling pathway. However, the precise mechanism by which apelin-13 modulates BDNF remains unclear. Thus, this study aimed to unravel the specific regulatory mechanism by which apelin-13 regulates BDNF. Bilaterally intracerebroventricular injection with Aβ25–35 was used to establish an in vivo model of AD. For the generation of METTL3 KO rats, the Crispr/Cas9 method was applied. PC12 cells were treated with Aβ25–35 to establish an in vitro model of AD. The cognitive function of the rats was evaluated with the Morris water maze and the novel object recognition test. Hippocampal damage and neuron loss were detected through H&E and immunofluorescent staining. METTL3, BDNF, TrkB, and p-TrkB were examined by Western blotting. Inflammation-related cytokines, IBA1, GFAP, IL-1β, and TNF-α were detected by Western blotting, immunofluorescent staining, ELISA, and qRT-PCR. m6A modification level was evaluated through MeRIP. A flow cytometer was applied to evaluate cell apoptosis. Cell proliferation was examined using MTT. m6A methylation inhibitor DAA reverses the improvement effect of apelin-13 on cognitive function, hippocampal nerve damage, neuron loss, and neuroinflammation in Aβ25–35-treated rats. Further results showed that apelin-13 upregulated METTL3, BDNF-AS m6A methylation, inhibited BDNF-AS expression, and subsequently upregulated BDNF/TrkB signaling pathway and reduced neuroinflammation in in vivo and in vitro AD models in a dose-dependent manner. Knockdown of METTL3 abolished apelin-13’s improvement effect in AD rats. Apelin-13-mediated upregulation of METTL3 enhances neuroinflammation inhibition and BDNF/TrkB signaling pathway via m6A-dependent downregulation of lncRNA BDNF-AS, thus ameliorating AD. Our study offers novel insights into the pathogenesis of AD and identifies potential drug targets for its treatment. Full article

The prostate gland, a male accessory reproductive organ, is regulated by hormonal inputs and autonomic innervation from the major pelvic ganglion. This study examined the effects of major pelvic ganglion denervation on prostate histology, immune cell infiltration, and systemic levels of prolactin, testosterone, and cytokines in rats. Male Wistar rats (300–350 g) were divided into groups receiving bilateral axotomy of the hypogastric nerve, the pelvic nerve, or both, alongside with a sham-operated control. After 15 days, the animals were killed, and prostate tissue was dissociated in DMEM medium containing DNase I and collagenase. The dissociated cells were stained with fluorochrome-conjugated antibodies, and cell characterization was performed using a flow cytometer. Hematoxylin and eosin (H&E) staining was used to analyze histological characteristics, while testosterone, prolactin, and interleukin levels were measured via ELISA. Histological analysis revealed inflammatory atypical hypertrophy e hiperplasia. Immunological assessments demonstrated increased leukocytes, T lymphocytes (CD4⁺ and CD8⁺), B lymphocytes, and macrophages following double nerve axotomy. Serum analyses showed elevated pro-inflammatory cytokines IL-1β, IL-6, and IFN-γ, as well as anti-inflammatory IL-10, in denervated animals. Hormonal assessments revealed significant increases in serum prolactin and testosterone levels after double axotomy. Loss of neural control may promote pathological prostate changes via inflammation and hormonal dysregulation, offering insights into neuroimmune and neuroendocrine mechanisms underlying prostate pathologies. Full article

Diploid Vaccinium fuscatum is a wild blueberry species with a low chilling requirement, an evergreen growth habit, and soil adaptability to southeast US growing regions. Regardless of its potential to improve the abiotic and biotic resilience of cultivated blueberries, this species has rarely been used for blueberry breeding. One hurdle is the ploidy barrier between diploid V. fuscatum and tetraploid cultivated highbush blueberries. To overcome the ploidy barrier, vegetative shoots micro-propagated from one genotype of V. fuscatum, selected because it grew vigorously in vitro and two southern highbush cultivars, ‘Emerald’ and ‘Rebel,’ were treated with colchicine. While shoot regeneration was severely repressed in ‘Emerald’ and ‘Rebel,’ shoot production from the V. fuscatum clone was not compromised at either 500 µM or 5000 µM colchicine concentrations. Due to the high number of shoots produced in vitro via the V. fuscatum clone shoots of this clone that had an enlarged stem diameter in vitro were subjected to flow cytometer analysis to screen for induced polyploidy. Sixteen synthetic tetraploid V. fuscatum, one synthetic octoploid ‘Emerald,’ and three synthetic octoploid ‘Rebel’ were identified. Growth rates of the polyploid-induced mutants were reduced compared to their respective wildtype controls. The leaf width and length of synthetic tetraploid V. fuscatum and synthetic octoploid ‘Emerald’ was increased compared to the wildtypes, whereas the leaf width and length of synthetic octoploid ‘Rebel’ were reduced compared to the wildtype controls. Significant increases in stem thickness and stomata guard cell length were found in the polyploidy-induced mutant lines compared to the wildtypes. In the meantime, stomata density was reduced in the mutant lines. These morphological changes may improve drought tolerance and photosynthesis in these mutant lines. Synthetic tetraploid V. fuscatum can be used for interspecific hybridization with highbush blueberries to expand the genetic base of cultivated blueberries. Full article

Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS’ identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24^TIP population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications. Full article

To clarify T-cell responses in myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD), we cultured the peripheral blood mononuclear cells of 24 patients with MOGAD and 20 with aquaporin-4 (AQP4) antibody-positive neuromyelitis optica spectrum disorders (NMOSD), and those of 17 healthy controls (HCs), in the presence of fourteen MOG peptides covering the full-length MOG, five AQP4 peptides, two myelin basic protein peptides, or two proteolipid protein peptides. Then, we measured T-cell activation markers, such as cell surface CD69 and the intracellular production of granulocyte–macrophage colony-stimulating factor (GM-CSF) and interferon-γ in CD4-positive T-cells, with a flow cytometer. The expression of CD69 in response to MOG p16–40 and MOG p181–205 was significantly higher (Stimulation Index > 2) in MOGAD than in HCs. Also, CD69 for AQP4 p21–40, AQP4 p211–230, and MOG p166–190 were significantly increased in NMOSD than in HCs. Intracellular GM-CSF production responding to MOG p16–40 was significantly higher in MOGAD than in HCs (p < 0.05), although intracellular interferon-γ was not elevated. None of the responses to the other peptides were different between the groups. The present study showed subtle CD4-positive T-cell activation elicited by some MOG peptides alone in patients with MOGAD. Further studies of cytokines or other stimulation and alternative assay markers and metrics are needed to delineate the immunopathological roles of T-cells in MOGAD. Full article

Erythrocyte osmotic fragility is an excellent parameter for evaluating the red blood cell (RBC) membrane, which may be abnormal in several pathological conditions. The flow cytometer is a powerful tool that analyzes a single cell in a solution and can detect alterations in RBCs, providing key differential diagnostic information. Both the osmotic fragility test (OFT) and flow cytometry are valuable diagnostic tools in veterinary medicine, but their diagnostic usefulness in anemic cats has not yet been determined. This study aimed to evaluate RBC membrane strength using an OFT in non-anemic and anemic cats and to compare RBC size and density using a flow cytometer in non-anemic and anemic cats. A total of 18 cats in the non-anemic group and 18 cats in the anemic group, divided into adults and seniors, were included in this study. Blood samples were collected for a complete blood count (CBC) and blood chemistry. The remaining blood was used for OFT to evaluate 50% hemolysis from the hemolysis curve and for the flow cytometer to measure forward scatter characteristics (FSC) and side scatter characteristics (SSC). The result of OFT showed no significant difference in OF between normal and anemic cats in the adult and senior groups. In terms of flow cytometry analysis, normal and anemic cats in the adult group showed no significant difference in the FSC (p = 0.769). On the other hand, the FSCs of normal senior cats were significantly higher than those of anemic cats (p = 0.0486). The SSCs of normal cats were significantly higher than those of anemic cats in the adult group (p = 0.048). However, the SSCs of the senior group showed no significant difference (p = 0.074). Based on these results, we concluded that, in the senior group, normal cats had higher FSCs than anemic cats, and in the adult group, normal cats had higher SSCs than anemic cats. However, both normal and anemic cats exhibited similar osmotic fragility. Further studies on various diseases are suggested. Full article

Plant pathogen zoospores play a vital role in the transmission of several significant plant diseases, with their early detection being important for effective pathogen management. Current methods for pathogen detection involve labour-intensive specimen collection and laboratory testing, lacking real-time feedback capabilities. Methods that can be deployed in the field and remotely addressed are required. In this study, we have developed an innovative zoospore-sensing device by combining a microfluidic sampling system with a microfluidic cytometer and incorporating a chemotactic response as a means to selectively detect motile spores. Spores of Phytophthora cactorum were guided to swim up a detection channel following a gradient of attractant. They were then detected by a transient change in impedance when they passed between a pair of electrodes. Single-zoospore detection was demonstrated with signal-to-noise ratios of ~17 when a carrying flow was used and ~5.9 when the zoospores were induced to swim into the channel following the gradient of the attractants. This work provides an innovative solution for the selective, sensitive and real-time detection of motile zoospores. It has great potential to be further developed into a portable, remotely addressable, low-cost sensing system, offering an important tool for field pathogen real-time detection applications. Full article

Urinary tract infections caused by Gram-negative bacteria (GNB) are among the most common infections and a significant cause of sepsis. The increasing prevalence of multidrug-resistant (MDR) bacteria poses challenges to empirical treatment. Colistin may be used a last-resort antibiotic for treating MDR infections, but this requires the rapid determination of susceptibility to colistin. Traditional susceptibility testing methods can take up to 48 h, and there are specific challenges in determining colistin susceptibility. This study evaluates a novel, rapid method for determining colistin susceptibility directly from positive urine samples using the FASTcolistin MIC kit from FASTinov^®. A total of 100 urine samples positive for Gram-negative bacilli when screened by the UF-1000i system were included in this study. After a simple sample prep, the same bacterial suspension was used for identification on MALDI-TOF and inoculated in the FASTcolistin MIC panel for our AST; after incubation at 37 °C for 1 h, it was analyzed via flow cytometry using a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA). The categorical susceptibility to colistin according to EUCAST or CLSI standards as well as the MIC values were given by bioFAST software (bioFAST 2.0). The essential agreement (EA) and bias were calculated. Different species of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter spp. were correctly identified by MALDI-TOF directly from the FASTcolistin MIC sample prep. The essential agreement between the two methods was 99%, with a bias of −17%. Both identification and susceptibility were obtained in less than 2 h. This study presents a rapid and accurate method for determining colistin MIC directly from urine samples. The shortness of time required to produce a result, 2 h versus 48 h with the conventional methods, will significantly impact treatment decisions, especially in urinary tract infections difficult to treat. Full article

Background/Objectives: Gundelia tournefortii (Kenger) is a traditional medicinal plant and exhibits potential anticancer properties. This study investigates the cytotoxic and apoptotic effects of its water extract on human lung carcinoma A549 cells. Methods: A lung cancer cell line was treated with Gundelia tournefortii extract. The metabolic content of the extract that plays key roles in anticancer was detected by high-performance liquid chromatography. Anticancer properties were further detected by a flow cytometer apoptosis assay, and signaling pathways were determined by a PCR array through hub gene expression alteration. Gene enrichment analysis and network pharmacology correlated metabolites and pathways that were involved in anticancer effects. Results: The metabolite content of G. tournefortii was analyzed, and gallic acid, clorogenic acid, hydroxybenzoic acid, caffeic acid, epicatechin, p-coumaric acid, salicylic acid, apigenin 7 glucoside, and cinnamic acid were detected as key compounds. Lung cancer cell line A549 was treated with the extract at increasing concentrations for 24, 48, and 72 h, and its effects on cell viability were determined by MTT analysis. A statistically significant difference was observed for IC₅₀ concentrations depending on incubation times. It was also observed that the G. tournefortii water extract significantly increased apoptosis in A549 cells in comparison with the control group. G. tournefortii extract’s effect on lung cancer cell line was measured using the signal pathway PCR array gene set. Gene enrichment analysis of the array expression data confirmed activation of apoptosis-related pathways, particularly the upregulation of BAX and downregulation of HSP90. Conclusions: These findings suggest that G. tournefortii metabolites provide promising selective anticancer drug candidates and potential drug templates to prevent side effects and resistance of current clinical drug treatments. Full article

Background/Objectives: Liquid biopsy methods have gained prominence as minimally invasive tools to improve cancer treatment outcomes. Circulating tumor cells (CTCs) offer valuable insights into both primary and metastatic lesions. However, validating the CTC test results requires confirmation that the detected cells originate from cancer tissue. While studies have identified CTCs in colorectal cancer (CRC) patients using molecular markers, simultaneous validation of their cancer tissue origin remains unexplored. Methods: This study introduces a simple approach to detect adenomatous polyposis coli (APC) gene abnormalities alongside established CTC markers using a molecular imaging flow cytometer (MI-FCM). Given that APC gene abnormalities occur in 60–70% of CRC patients, their detection serves as strong evidence of cancer origin. Results: Our method achieved 92% concordance with DNA sequence analysis of tumor-derived cells. In a proof-of-concept study using 5 mL of whole blood from CRC patients, we observed a high frequency of cells exhibiting APC abnormalities, cytokeratin (CK), and vimentin (Vim) expression. Extending the study to 80 CRC patients across pathological stages I–IV confirmed CK and Vim as valid CTC markers. Three distinct cell populations were identified in blood: CK+/Vim−, CK+/Vim+, and CK−/Vim+. CTC number and frequency increased progressively with cancer stage. Conclusions: This is the first report demonstrating CK and Vim as effective markers for direct CTC detection in CRC patients. Our findings provide evidence-based validation of CTC markers, offering new insights and advancing approaches for patient care. Full article

CD4 T lymphocytes play a key role in initiating the adaptive immune response, releasing cytokines that mediate numerous signal transduction pathways across the immune system. Therefore, CD4 T cell counts are widely used as an indicator of overall immunological health. HIV, one of the leading causes of death in the developing world, specifically targets and gradually depletes CD4 cells, making CD4 counts a critical metric for monitoring disease progression. As a result, accurately counting CD4 cells represents a pressing challenge in global healthcare. Flow cytometry remains the gold standard for enumerating CD4 T cells; however, flow cytometers are expensive, difficult to transport, and require skilled medical staff to prepare samples, operate the equipment, and interpret results. This highlights the critical need for novel, rapid, cost-effective, and portable methods of CD4 enumeration that are suitable for deployment in resource-limited countries. This review will survey and analyze emerging research in CD4 counting, with a focus on microfluidic systems, which represent a promising area of investigation. Full article

A novel approach to protein quantification utilizing a microfluidic platform activated by a magnetic assembly of functionalized magnetic beads around soft magnetic capture centers is presented. Functionalized magnetic beads, known for their high surface area and facile manipulation under external magnetic fields, are injected inside microfluidic channels and immobilized magnetically on the surface of glass-coated soft magnetic microwires placed along the symmetry axis of these channels. A fluorescent (Cy5) immunomagnetic sandwich ELISA is then performed by sequentially flowing the sample and all necessary reagents in the microfluidic channels. Direct protein quantification is performed by magnetically releasing the beads from the microwire and evaluating their fluorescence intensity with the help of a miniature (microfluidic-based) flow cytometer. Measurements of ICAM-1 protein concentration in human blood plasma samples confirm the feasibility of the approach through extensive performance benchmarking. The automation and multiplexing capabilities of the proposed platform further demonstrate its potential for protein quantification in point-of-care settings using microfluidics and miniature flow cytometry instruments. Full article

Background: Monitoring of measurable residual disease (MRD) requires highly sensitive flow cytometry protocols to provide an accurate prediction of shorter progression-free survival. High assay sensitivity generally requires rapid processing to avoid cell loss from small bone marrow sample volumes, but this requirement conflicts with the need in most clinical cytometry laboratories for long processing and acquisition times, especially when multiple MRD studies coincide on the same day. Methods: The proposed protocol was applied to 226 human bone marrow and 45 peripheral blood samples submitted for the study of MRD or the detection of rare cells. Samples were processed within 24 h of extraction and acquired with an eight-color flow cytometer. Results: The FACSLyse-Bulk protocol allows for the labelling of millions of cells in under 90 min in small sample volumes without affecting the FSC/SSC pattern or antigen expression, and it also allows antigens to be fixed to the membrane, thus avoiding the capping phenomenon. Conclusions: The proposed protocol would allow clinical flow cytometry laboratories to perform MRD studies in house and easily achieve a limit of detection and limit of quantification <0.001%, thus avoiding the need to outsource analysis to specialized cytometry laboratories. Full article

Cistus monspeliensis L. (C. monspeliensis) is used in Italian folk medicine. This study was performed to determine genotoxic and antigenotoxic effects of C. monspeliensis leaf extract against mitomycin C (MMC) using an in vitro cytokinesis-block micronucleus assay (CBMN) in the Chinese Hamster Ovarian K1 (CHO-K1) cell line. The phytochemical composition of C. monspeliensis extract was evaluated using an untargeted metabolomic approach by employing UPLC-PDA-ESI/MS. The automated in vitro CBMN assay was carried out using image analysis systems with a widefield fluorescence microscope and the ImageStreamX imaging flow cytometer. The phytochemical profile of C. monspeliensis extract showed, as the most abundant metabolites, punicalagin, myricetin, gallocathechin, and a labdane-type diterpene. C. monspeliensis, at the tested concentrations of 50, 100, and 200 μg/mL, did not induce significant micronuclei frequency, thus indicating the absence of a genotoxic potential. When testing the C. monspeliensis extract for antigenotoxicity in the presence of MMC, we observed a hormetic concentration-dependent effect, where low concentrations resulted in a significant protective effect against MMC-induced micronuclei frequency, and higher concentrations resulted in no effect. In conclusion, our findings demonstrate that C. monspeliensis extract is not genotoxic and, at low concentration, exhibits an antigenotoxic effect. In relation to this final point, C. monspeliensis may act as a potential chemo-preventive against genotoxic agents. Full article

Sucralose, a commonly nonnutritive sweetener used in daily products of habitual diet, is related to impairing the gut microbiome by disrupting inflammatory response, promoting weight gain by increasing adipose tissue and promoting chronic inflammatory processes. Considering the impact of sucralose in the development of metabolic diseases, in this work, we focused on the impact of sucralose on the adipocyte differentiation process to determine if sucralose can promote adipogenesis and increase adipose tissue depots in PCS 210 010 human preadipocytes cell line. Sucralose at 25 (S25) and 100 ng/µL (S100) concentrations were tested against control with no edulcorant (NS) during the adipocyte differentiation process at 48 h and 96 h. The genetic expression of adipogenesis markers such as CEBP-α, PPARγ, EBF-2, UCP-1, and lipogenesis regulator ACC was determined by qPCR. A panel of human cytokines related to inflammatory response was measured by a flow cytometer using the kit Legend Plex Human Cytokine panel of BIOLUMINEX. Our results indicate that sucralose increased the expression of white adipocyte differentiation marker CEBP-α and lipogenesis regulator ACC at 96 h before complete differentiation. Also, sucralose triggers an inflammatory response by synthesizing adiponectin, resistin, IL-6, IL-8, and Il-1B. To summarize, sucralose stimulates the expression of genes related to adipogenesis and negatively affects the secretion of inflammatory cytokines and adipokines during preadipocyte differentiation. Full article