Search Results (122)

Large-scale bioprocesses often suffer from spatial heterogeneities, which impact microbial performance and often lead to phenotypic population heterogeneity. To better understand these effects at the single-cell level, this study applied, for the first time, automated real-time flow cytometry (ART-FCM) to monitor L-phenylalanine production with an Escherichia coli triple reporter strain in a fed-batch process with glycerol as the carbon source. The strain was cultivated in both a well-mixed stirred-tank bioreactor (STR) and a scale-down two-compartment bioreactor (TCB), consisting of an STR and a coiled flow inverter (CFI) in bypass, to simulate spatial heterogeneities. ART-FCM enabled autonomous, high-frequency sampling every 20 min, allowing for real-time tracking of fluorescence signals linked to growth (rrnB-mEmerald), oxygen availability (narGHIJ-CyOFP1), and product formation (aroFBL-mCardinal2). The STR exhibited uniform reporter expression and higher biomass accumulation, while the TCB showed delayed product formation and pronounced phenotypic diversification depending on the set mean residence time in the CFI. Single-cell fluorescence distributions revealed that the shorter mean residence time in the CFI resulted in pronounced subpopulation formation, whereas longer exposure attenuated heterogeneity, indicating transcriptional adaptation. This finding highlights a critical aspect of scale-down studies: increased exposure duration to perturbations can enhance population robustness. Overall, this study demonstrates the relevance of ART-FCM, in combination with a multi-reporter strain, as a pioneering tool for capturing dynamic cellular behavior and correlating it to process performance, providing deeper insights into microbial heterogeneity under fluctuating bioprocess conditions. Full article

Flow cytometry is widely applied to infer the ploidy and genome size (GS) of plant nuclei. The conventional approach of sample preparation, reliant on fresh plant material to release intact nuclei, often results in poor yields of nuclei in conditions when a plant material cannot be kept fresh due to logistical constraints. Previous attempts to use frozen plant material were mainly limited to ploidy analysis and relied on chopping methods, which restrict the material input and often result in poor nuclei yield, especially in frozen samples, due to incomplete disruption. Here, we present a modified protocol for GS estimation using frozen plant material that facilitates larger volumes of tissue to be processed while improving debris removal. Nuclei isolated from this protocol can also be used for DNA or RNA extraction. Genome size estimates from frozen material are similar to those from fresh material, with a reduction in error range, although not always significant (p > 0.05). In certain species, frozen samples can yield substantially more nuclei than fresh material. With the addition of specific debris compensation algorithms, coefficient of variation (CV%) can be maintained below 5%. This method has special value in estimating the GS of samples collected from remote locations and frozen for use in plant genome sequencing. Freezing preserves high-quality DNA and RNA, enabling the same sample to be used for both flow cytometry and genome sequencing. Full article

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that survives and replicates within host cells. Macrophages, key immune cells in infection defense, play a vital role in pathogen clearance through polarization (M1/M2) and NLRP3 inflammasome activation. While TRIM67 regulates macrophage recruitment in the liver, its role in S. Typhimurium infection remains unclear. In this study, a S. Typhimurium infection model was established by orally infecting streptomycin-pretreated TRIM67 WT and KO mice with 1 × 10⁹ CFU of S. Typhimurium. TRIM67 expression in the ileum, colon, mesenteric lymph nodes (MLNs), and peritoneal macrophages (PMs) was assessed via qRT-PCR and Western blotting. Histopathological changes were analyzed using HE and PAS staining. IHC staining, flow cytometry (FCM), qRT-PCR, and Western blotting were used to evaluate TRIM67 knockout effects on macrophage recruitment, polarization, and NLRP3 inflammasome activation. In vitro, PMs were infected with S. Typhimurium (MOI 1:20), and TRIM67’s role in macrophage polarization and NLRP3 activation was validated. S. Typhimurium infection significantly upregulated TRIM67 in the ileum, colon, and MLN. TRIM67 knockout reduced intestinal inflammatory cell infiltration but worsened goblet cell loss and impaired digestion. Bacterial load assays revealed weakened pathogen clearance, leading to weight loss and increased mortality. TRIM67 knockout inhibited intestinal macrophage recruitment, M1 polarization in MLN, and NLRP3 activation. In vitro, TRIM67 knockout increased PMs’ intracellular bacterial load and suppressed NLRP3, caspase-1, and IL-1β expression. TRIM67 knockout impairs the host’s ability to clear S. Typhimurium by inhibiting M1 macrophage polarization and NLRP3 inflammasome activation. Full article

A rapid and accurate evaluation of a chemical disinfectant’s bactericidal efficacy is crucial for ensuring effective infection control, preventing the spread of pathogens, and supporting the development of new disinfectant formulations. In this study, we report a rapid, label-free flow cytometry (FCM) protocol for evaluating the bactericidal efficacy of disinfectants. Five commercial disinfectants (alcohols, oxidizing agents, and alkylating agents) were evaluated against type strains recommended by EN 13727+A2 and ten clinical strains. The label-free FCM method allowed the determination of disinfectant efficacy through assessment of scatter light profiles (FSC-H/SSC-H) and count differences. The label-free FCM provided the results in approximately 4 h and showed strong correlation with standard tests (91.4%, sensitivity 0.94 and specificity 0.98) that can take up to 48 h. Our results represent a proof-of-principle that label-free FCM can reliably assess the efficacy of chemical disinfectants, the same day, and substantially faster than the current growth-based methods. Additionally, the study highlights the potential of the FCM method for detecting the occurrence of viable but non-culturable bacteria following treatment with chlorine-based disinfectants. With its speed, accuracy, and capability to identify bacterial injuries at a single-cell level, the FCM method is a powerful tool for assessing the efficacy of new disinfectant formulations. Full article

In the field of fish genetic breeding, accurately determining the DNA content and ploidy of fish is of great significance. This article introduces the use of flow cytometry (FCM) to measure the DNA content and conduct ploidy analysis by sampling different tissues of freshwater fish species. It describes the FCM detection methods and their effectiveness for different individual tissues. These tissues include embryos and fry, as well as the blood, caudal fins, and sperm of adult live fish, and also specific tissues such as testes, ovaries, gills, spleens, and livers under anatomical conditions. Moreover, the application of ploidy detection to different tissues or individuals in different stages in the practice of fish genetic breeding is analyzed. This research covers samples from different growth stages and a variety of tissue types. The results show that this method exhibits high stability and reliability in the detection of different tissue samples, providing solid data support for subsequent research. It holds significant application value in fish genetic breeding. Full article

Background/Objectives: Minimal residual disease (MRD) refers to the resistant clonal population of leukemia cells that survive induction chemotherapy, serving as a critical indicator of treatment response in pediatric Acute Lymphoid Leukemia (ALL). While flow cytometry (FCM) and molecular methods are standard for MRD detection, novel leukemia-associated immunophenotype (LAIP) markers are needed when conventional markers are insufficient. Methods: MRD was assessed in 218 pediatric B-ALL patients using a combinatory approach of Different-from-Normal (DfN) and LAIP strategies. An eight-color flow cytometry panel included routine MRD markers (e.g., CD10, CD19, and CD20) and less commonly used markers (e.g., CD123, CD73, CD86). Cytogenetic and molecular profiling were integrated to evaluate the association between genetic abnormalities and MRD positivity. Results: The combined DfN and LAIP approach enhanced MRD detection sensitivity compared to individual methods. CD7 showed a significant association with MRD positivity (p = 0.003), whereas CD73 (p = 0.000) and CD86 (p = 0.002) correlated with MRD-negative status. CD123 exhibited the highest aberrancy among MRD-positive cases, while CD81 had the lowest. These findings highlight the prognostic potential of CD73 and CD86 for MRD-negative status, complementing the established utility of CD123. Conclusions: Incorporating novel markers (CD123, CD73, CD86, and CD81) into MRD panels enhances detection sensitivity and clinical applicability. These markers are compatible with standard flow cytometry, supporting their integration into routine practice for comprehensive MRD evaluation, ultimately improving therapeutic outcomes in pediatric B-ALL. Full article

Monitoring measurable residual disease (MRD) is critical for the management of B-cell acute lymphoblastic leukemia (B-ALL). While a quantitative assessment of BCR::ABL1 transcripts is standard for Philadelphia chromosome-positive cases (Ph+ ALL), a multiparameter flow cytometry (FCM) is commonly used for MRD detection in other genetic subtypes. A total of 106 B-ALL patients underwent genetic and phenotypic analyses. Among them, 27 patients (20 adults and 7 children) harbored the t(9;22)(q34.1;q11.2) translocation and/or the BCR::ABL1 rearrangement. A high correlation between the BCR::ABL1 transcript levels (PCR-MRD) and a standardized FCM-based method for MRD detection (FCM-MRD) was observed (r = 0.7801, p < 0.001), with a concordance rate of 88% (κ = 0.761). The FCM detected MRD in 82.9% of the samples with transcript levels of > 0.01%. The CD34+CD38−/dim blast pattern was significantly more frequent in Ph+ ALL (77.7%), compared to other B-ALL cases (20.2%, p < 0.0001). Additionally, Ph+ ALL exhibited a higher expression of CD66c+/CD73+ (94.0% vs. 56.9%), CD66c+/CD304+ (58.8% vs. 6.9%), and CD73+/CD304+ (75.5% vs. 15.5%) than the other B-ALL subtypes (p < 0.001). In conclusion, this high-sensitivity FCM-MRD demonstrated comparable performance to the PCR-MRD, serving as a complementary tool for MRD assessment in Ph+ ALL. Moreover, a distinct leukemia-associated immunophenotype was identified, highlighting potential biomarkers for MRD monitoring. Full article

Effective antimicrobial and biofilm control strategies require an understanding of the differential effects of antimicrobial agents on the viability and culturability of microbial cells. A viable but non-culturable (VBNC) state, a survival strategy of non-spore-forming bacteria in response to adverse conditions, poses a significant challenge for public health and food safety. In the present study, we investigated the antimicrobial and antibiofilm effects of nisin and gallium (III) nitrate hydrate against the Gram-positive strain Lactiplantibacillus plantarum subsp. plantarum DSM 20174 and the Gram-negative strain Pseudomonas fluorescens ATCC 13525, respectively. Both strains were chosen as model systems for their relevance to food and clinical settings. Culture-based methods and flow cytometry (FCM) were used to evaluate the culturability and viability of both planktonic and sessile cells, providing insights into their physiological response to antimicrobial treatment-induced stress at different concentrations (100, 250, 350, and 500 ppm). The findings highlight the strain-specific action of nisin on L. plantarum and the promising antibiofilm effects of Ga (III) against P. fluorescens. This study underscores the promising potential of FCM as a powerful tool for high-throughput analyses of antimicrobial efficacy, providing valuable insights into developing targeted biofilm control strategies for food safety and clinical applications. Full article

Rubella infection (RuV) during early pregnancy is a known cause of congenital rubella syndrome (CRS). However, the mechanisms by which the virus crosses the placenta and infects the fetus are not fully understood. It has been known that various kinds of cell stresses can occur during the placenta formation. Previously, we demonstrated that low-glucose-induced endoplasmic reticulum stress could drastically enhance RuV infection in immortalized human first-trimester trophoblast cells. In this study, we investigated the roles of oxidative stress in RuV infection in these cells. Oxidative stress was induced in Swan.71 cells by culturing them in medium containing hydrogen peroxide (H₂O₂) in various concentrations and durations (50 µM or 100 µM for 24 h, or 150 µM for 1 h). RuV infection with a clinical strain was performed 24 h post-treatment, and capsid proteins were visualized at 24 and 48 h post-infection (hpi) using flow cytometry (FCM) and fluorescence microscopy (IF), respectively. The findings demonstrated that oxidative stress significantly enhanced RuV infection, as evidenced by FCM analysis, showing a twofold increase in infection rate, and confirmed by IF assay. Additionally, significantly increased intracellular viral replication was observed at 3 dpi. These findings suggest that oxidative stress during early pregnancy may promote the maternal-to-fetal transmission of rubella, contributing to the development of CRS. Full article

Encyclia Hook. is a tropical orchid known for its delightful fragrance and distinctive flower shapes, which have been widely used as superior parents for inter and intrageneric breeding. In this study, karyotype analysis shows that the number of chromosomes in diploid plants of Encyclia is 40, with all species exhibiting mesial and central filamentous chromosomes. The karyotype of diploid plants is primarily 2B. Among the three detected intergeneric hybrids, both Enanthleya Circus Lady ‘Coastal Star’ and Guaricyclia Kyoguchi ‘Fumi’ are triploid, while Robertsara ‘Green Wonder’ is tetraploid. By evaluating various lysates and tissues, a flow cytometry system (FCM) is developed with Galbraith’s buffer (GLB) and tender leaves. By using Dendrobium officinale as an external reference, the genome size of the Encyclia species and its intra and intergeneric hybrids are assessed, which ranges from 1.07 Gb to 3.23 Gb. These results will establish a foundation for high-throughput identification of germplasms, advance crossbreeding efforts, and enhance the understanding of the genome in Encyclia. Full article

Polyploidy is a powerful mechanism driving genetic, physiological, and phenotypic changes among cytotypes of the same species across both large and small geographic scales. These changes can significantly shape population structure and increase the evolutionary and adaptation potential of cytotypes. Alyssum moellendorfianum, an edaphic steno-endemic species with a narrow distribution in the Balkan Peninsula, serves as an intriguing case study. We conducted a comprehensive analysis of genetic diversity and population structure across the species’ range, employing an array of genetic techniques (nuclear microsatellites, amplified fragment length polymorphisms, and plastid DNA sequences), flow cytometry (FCM), morphometry, and pollen analysis. The study reveals two genetic lineages: spatially distributed diploid and tetraploid cytotypes. Clear divergence between diploids and tetraploids was shown by AFLP, while plastid DNA sequences confirmed private haplotypes in each of the studied populations. Higher genetic diversity and allelic richness following the north-south pattern were documented in tetraploids compared to diploids, as indicated by nuclear microsatellites. Morphometric analysis via principal component analysis (PCA) and canonical discriminant analysis (CDA) did not reveal any divergence between diploid and tetraploid cytotypes. Nonetheless, a distinction in pollen size was clearly observed. The results suggest an autopolyploid origin of tetraploids from diploid ancestors. Despite the population fragmentation in a very small geographic range, these populations harbour high genetic diversity, which would allow them to remain stable if natural processes remain undisturbed. Full article

Aerides rosea (Orchidaceae) boasts high ornamental value due to its pleasant aroma, foxtail spike, and elegant floral morphology. Inducing A. rosea to become tetraploid enhances horticultural traits and facilitates fertile intergeneric hybrids through crosses with other market-available tetraploid species. The experimental design involved the application of colchicine at varying concentrations—0.05%, 0.1%, and 0.2%—to a solid medium. Exposure durations were 5, 10, and 15 days, with treatments conducted under sterile conditions on 6-week-old protocorms post-germination. Results indicated that the protocorms were sensitive to colchicine concentrations exceeding 0.05%, with high concentrations leading to a mortality rate exceeding 50%. Flow cytometry (FCM) with 4′,6-diamidino-2-phenylindole (DAPI) staining confirmed a doubling of chromosome numbers in tetraploid plants (2n = 4x = 76) compared to diploid controls (2n = 2x = 38). Induction efficiency was significantly influenced by colchicine concentration and treatment duration. A 10-day treatment with 0.2% colchicine yielded a 70.00% tetraploid induction rate; however, considering protocorm survival, a 5-day treatment with 0.05% colchicine was preferable, achieving a 63.55% survival rate and a 56.67% tetraploid induction rate. Tetraploid plants exhibited distinct morphological traits, such as a more compact growth habit, thicker leaves, and increased stem and root thickness. Leaf morphology changes included larger stomata with reduced density, denser spongy mesophyll, and more pronounced venation. Tetraploids also demonstrated a 1.94-fold increase in genome size compared to diploids. The tetraploid genotypes developed in this study hold significant potential for future Aerides breeding programs. Full article

Idesia polycarpa from Sichuan is a valuable germplasm with high economic potential, but it faces variety scarcity. To address this, this study collected 16 varieties (lines), identifying IpHT1 as a promising parent due to its high oil content (38.5%) and red fruits. Polyploid induction via adding 0.50% colchicine to Murashige and Skoog (MS) medium yielded 520 IpHT1 mutagenized seedlings. Subsequently, flow cytometry (FCM) was performed on 401 morphologically variant seedlings which had been initially screened, resulting in the identification of 15 suspected triploids, 35 suspected tetraploids, and 3 chimeras. Furthermore, fluorescence in situ hybridization (FISH) analysis found that the probe (AG₃T₃)₃ had terminal signals at both ends of each chromosome, allowing for the counting of 42 chromosomes in diploids and 84 in tetraploids. The probe 5S rDNA showed 2, 3, and 4 hybridization signals in the interphase nuclei of diploid, triploid, and tetraploid cells, respectively, but the probe (GAA)₆ failed to produce any signal on I. polycarpa chromosomes. Ultimately, 18 polyploids were selected, including 7 triploids and 11 tetraploids. Triploids and tetraploids showed significant leaf morphological and physiological differences from diploids. Consequently, this study successfully established a polyploid breeding system for I. polycarpa, thereby enhancing its genetic diversity and breeding potential. Full article

Objective: The aim of this study was to evaluate the photothermal effect of a 970 nm diode laser on Enterococcus faecalis biofilms. Methods: 72 extracted human single-rooted teeth were prepared, sterilized, and inoculated with Enterococcus faecalis to establish a two-week-old biofilm. The specimens were divided into six groups (n = 12): Group 1 (G1)—negative control (PBS—no laser), Group 2 (G2)—positive control (1% NaOCl rinse—no laser), Group 3 (G3)—a 970 nm laser in 1.5 W pulse mode, Group 4 (G4)—a 970 nm laser in 2 W pulse mode, Group 5 (G5)—a 970 nm laser in 1.5 W continuous mode, Group 6 (G6)—a 970 nm laser in 2 W continuous mode. Bacterial viability was evaluated using the LIVE/DEAD BacLight kit and analyzed by flow cytometry (FCM). Temperature changes on the root surface during irradiation were analyzed using a K-type thermocouple. Data were statistically analyzed using one-way ANOVA and Tukey’s multiple comparison test (α = 0.05). Results: Bacterial viability was significantly reduced after laser irradiation in continuous mode using 1.5 W (21% of live bacteria) and 2 W (14% of live bacteria). When the pulsed mode was applied, the reduction in bacterial viability was less, with a mean survival of 53% (1.5 PF, whereas 29% of bacteria survived after 2 W irradiation). Conclusions: The 970 nm diode laser at 2 W continuous mode effectively reduced the viability of E. faecalis biofilms in root canals without causing unacceptable temperature rises at the root surface. Full article

Nano- and micro-sized vesicular and colloidal structures mediate cell–cell communication. They are important players in the physiology of plants, animals, and humans, and are a subject of increasing interest. We investigated the effect of three surfactants, N-cetylpyridinium chloride (CPC), sodium dodecyl sulfate (SDS), and Triton X-100 (TX100), and two anionic polyelectrolytes, sodium polystyrene sulfonate (NaPSS) and sodium polymethacrylate (NaPMA), on nanoliposomes. In addition, the effect of SDS and TX100 on selected biological membranes (erythrocytes and microalgae) was investigated. The liposomes were produced by extrusion and evaluated by microcalorimetry and light scattering, based on the total intensity of the scattered light (I_tot), hydrodynamic radius (R_h), radius of gyration (R_g), shape parameter p (=R_h/R_g,0), and polydispersity index. The EPs shed from erythrocytes and microalgae Dunaliella tertiolecta and Phaeodactylum tricornutum were visualized by scanning electron microscopy (SEM) and analyzed by flow cytometry (FCM). The R_h and I_tot values in POPC liposome suspensions with added CPC, SDS, and TX100 were roughly constant up to the respective critical micelle concentrations (CMCs) of the surfactants. At higher compound concentrations, I_tot dropped towards zero, whereas R_h increased to values higher than in pure POPC suspensions (R_h ≈ 60–70 nm), indicating the disintegration of liposomes and formation of larger particles, i.e., various POPC–S aggregates. Nanoliposomes were stable upon the addition of NaPSS and NaPMA, as indicated by the constant R_h and I_tot values. The interaction of CPC, SDS, or TX100 with liposomes was exothermic, while there were no measurable heat effects with NaPSS or NaPMA. The SDS and TX100 increased the number density of EPs several-fold in erythrocyte suspensions and up to 30-fold in the conditioned media of Dunaliella tertiolecta at the expense of the number density of cells, which decreased to less than 5% in erythrocytes and several-fold in Dunaliella tertiolecta. The SDS and TX100 did not affect the number density of the microalgae Phaeodactylum tricornutum, while the number density of EPs was lower in the conditioned media than in the control, but increased several-fold in a concentration-dependent manner. Our results indicate that amphiphilic molecules need to be organized in nanosized particles to match the local curvature of the membrane for facilitated uptake. To pursue this hypothesis, other surfactants and biological membranes should be studied in the future for more general conclusions. Full article