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Inappropriate platelet activation is the key point of thrombogenesis. The aim of the present study was to investigate the effects of resveratrol (RESV), a compound extracted from the Chinese medicinal herb Polygonum cuspidatum sieb et Zucc, on the platelet activation induced by adenosine diphosphate (ADP) and its possible mechanism. The percentage of platelet aggregation and surface P-selectin-positive platelets, and the activity of protein kinase C (PKC) of platelet were observed with platelet aggregometer, flow cytometry and phosphorimaging system, respectively. RESV at 25, 50 and 100 μM showed anti-platelet aggregation and inhibition of surface P-selectin-positive platelets in a concentration-dependent manner. RESV (50 μM) inhibited the activity of PKC in the membrane fraction of platelets and decreased the percentage of membrane associated PKC activity in total PKC activity. Moreover, DL-erythro-1,3-Dihydroxy-2-aminooctadecane, an elective protein kinase C inhibitor (PKCI), and RESV had additive effects of inhibiting the percentage of platelet aggregation and surface P-selectin-positive platelets. It is suggested that RESV may inhibit platelet aggregation, the percentage of surface P-selectin-positive platelets and subsequent thrombus formation. The mechanisms may be partly relative to the decrease of the activity of PKC of platelets.

The aim of the present work was to explore the anti-hepatoma effects of icariin both in vitro and in vivo and to elucidate its potential mechanism of action. The MTT assay was applied to test the anti-proliferative effects of icariin in vitro. HepG2 bearing NMRI nu/nu mice were used to test the anticancer effects of icariin in vivo. Immunohistochemical assay and flow cytometry assay (FACS) were applied to detect the possible mechanisms of action of icariin. MTT assay illustrated that icariin inhibited the proliferation of HepG2 cells in a concentration dependent manner; meanwhile, icariin inhibited the tumor growth in HepG2 bearing NMRI nu/nu mice. The tumor weight was inhibited by 55.6% and tumor volume was inhibited by 47.2%. Icariin did not influence the spleen and body weights or blood parameters. Immunohistochemical analysis indicated that the expressions of both CD31 and Ki67 in the icariin treated group were significantly lower than those in the control group (p < 0.01). FACS assay showed that icariin dramatically decreased the percentage of CD4+ and CD8+ cells in bone marrow and CD19+ cells in blood on day 8. On day 17, the percentage of CD8+ cells in blood was lower than those in the control group. CD4/CD8 ratio in icariin group was significantly elevated in bone marrow on day 17. Icariin showed anticancer efficacy both in vitro and in vivo. The possible mechanism of action could be related to its anti-angiogenesis and anti-proliferative effects in tumors.

Ajuga bracteosa (AB) has been widely used in folk medicine in Asian countries against gout, hepatitis, pneumonia, rheumatism, and various neuro inflammatory disorders. The aim of this study was to investigate the possible immunoregulatory effects of the ethanolic extract of Ajuga bracteosa (ABEE) on systemic Th1/Th2 immunity in SRBC immunized Balb/C mice. Animals were orally administered with graded doses of ABEE from 6.25 mg/kg to 100 mg/kg. Post sub-cutaneous immunization with SRBCs and circulating antibody titers, DTH responses and splenocyte proliferation was monitored as markers of Th2 and Th1 responses. Cyclophosphamide and levamisole were used as controls. Lymphocyte immunophenotying (CD4/CD8 cell counts) and intracellular Th1/Th2 cytokine concentrations were determined using flow cytometry. Treatment with ABEE demonstrated significant biphasic immunostimulation of effector T-helper immunity. ABEE at 50 mg/kg dose resulted in maximal increase in antibody titers, DTH responses and CD4+/CD8+ T-cell percentages indicating maximal activation and proliferation of T and B lymphocytes at this dose. ABEE, at the same dose, also showed maximal up regulation of LPS and CON A stimulated splenocyte proliferation and also maximal up-regulation of both Th1 (IL-2, IFN-γ) and Th2 (IL-4) cytokines which suggest its mixed Th1/Th2 immunostimulatory activity. Comparatively at higher doses (100 mg/kg), significant down regulation of all these effector T-helper (Th) immune responses was observed. The study therefore suggests mixed biphasic immunostimulatory Th1/Th2 activity of ABEE that could support its immunoadjuvant potential.

A major component of flow cytometry (FCM) data analysis involves gating, which is the process of identifying homogeneous groups of cells. With the rapid development of the portable flow cytometer, manual gating techniques have been unable to meet the demand for accurate and rapid analysis of samples. To provide a practical application for portable devices, we propose a flexible, statistical model-based clustering approach for identifying cell populations in FCM data. This approach, which mimics the manual gating process, employs a finite mixture model with a density function of skew t distribution and estimates parameters via an expectation maximization algorithm. Data analysis from an experiment on a patient’s peripheral blood samples have proven that the proposed methodology yields better results in terms of robustness against outliers than current state-of-the-art automated gating methods, has more flexibility in clustering symmetric data and leads to lower misclassification rates (misclassification rates of skew t method is 0.06442) when handling highly asymmetric data. The method we proposed will improve data analysis of portable flow cytometers, especially when the users have no professional training.

Recently, using gelatin nanoparticles as a biocompatible carrier in drug delivery systems is growing up. Drug delivery is one of the most common applications of nanoparticles in cancer treatment in order to optimize the drug efficiency. In this study, gelatin nanoparticles were firstly synthesized and loaded with tamoxifen that subsequently characterized by SEM, TGA and FT-IR analyses. The approximate drug loading efficiency was calculated about 17.43% for tamoxifen-loaded gelatin (TG). Then, the effect of TG on apoptosis induction and cytotoxicity of MCF-7 cell line was evaluated and compared with flow cytometry and MTT assay. The MTT results showed that tamoxifen and TG nanoparticles could inhibit the proliferation of MCF-7 cells in a dose-responsive manner, with an IC${}_{50}$ of IC${}_{50}$ of 200 $\mu$g/mL and 50 $\mu$g/mL after 24h and 48h, respectively. Moreover, from flow cytometric results, it can be suggested that TG nanoparticles are more potent in inducing apoptosis and cell death through programmed cell death. Actually, TG nanoparticles primarily increased the early apoptotic cells during the 24-h incubation period Our results revealed that tamoxifen-loaded gelatin nanoparticles are more potent than tamoxifen alone. These findings support the use of tamoxifen-loaded gelatin nanoparticles in target-specific therapy for cancer treatment.

Tetraphenylporphyrins (TPPs) have been proposed for the treatment of retinoblastoma by photodynamic therapy. Glycoconjugated compounds were synthesized for improving TPP solubility and amphipathy, and to specifically target mannose receptors overexpressed at the surface of cells. The efficiency of four TPP derivatives with different chemical structures was compared by phototoxicity tests and flow cytometry experiments. Interestingly, the absence/presence and distribution of saccharide moieties in the various compounds affected differently their mechanism of interaction with cancer cells and their phototoxic efficiency. For glycodendrimeric TPP-1 and TPP-2 incubated with retinoblastoma cells, a fast two-step uptake-equilibrium process was observed, whereas for a dendrimeric TPP without saccharide moieties (TPP-1c) and a glycoconjugated compound with no dendrimeric structure (TPP(DegMan)${}_3)$ uptake was very slow. The difference in uptake profiles and kinetics between TPP-1c on the one hand and TPP-1 and TPP-2 on the other hand would account for the interaction of the two glycodendrimeric compounds with a mannose receptor. These TPPs encapsulated in endosomes would induce less damage to cells upon illumination. TPP(DegMan)${}_3$ showed the highest phototoxicity, but its efficiency was unaffected by pretreatment of cells by mannan. The penetration of this glycoconjugated compound in cells and its phototoxic effect appeared independent of its interaction with a mannose receptor. Thus, if glycoconjugation influenced TPPs behavior in solution and interaction with serum proteins, phototoxicity was not necessarily related to upfront molecular recognition.

Selenium nanoparticles (SeNPs), due to their unique properties, have attracted researchers’ attention. Though SeNPs have been used for wide applications, the chemically synthesized one lacks stability due to aggregation, and it releases toxic byproducts. These drawbacks can be overcome by producing SeNPs using natural sources as reducing and capping agents. Luffa cylindrica is an immense source of phytochemical compounds reported for its potential therapeutical value towards cancer, asthma, and sinusitis. In the current study, we have synthesized SeNPs using leaf extract of L. cylindrica and evaluated its biocompatibility and haemocompatibility using peripheral blood mononuclear cells and erythrocytes respectively. The formation of SeNPs was confirmed by a color change from greenish yellow to ruby red during 6 h incubation at 40${}^{\circ }$C and further confirmed by the maximum absorbance at 266nm and 380nm in the UV–Vis spectrum. The fingerprint regions of the Fourier-transform infrared spectroscopy spectrum between 1500cm${}^{-1}$ and 500cm${}^{-1}$ revealed the presence of phytoconstituents of L. cylindrica. The particle size analysis showed a size range of 100nm and zeta potential of −13.6 mV. Scanning electron micrograph showed flower-shaped surface morphology with a size range of 100nm. The erythrocytes treated with higher concentrations of LC-SeNPs showed less than 5% lysis compared to the positive control. Similarly, in the apoptosis assay, 80.45% of cells remained viable after being treated with LC-SeNPs, which is comparable with that of untreated control. Since the synthesized SeNPs possess biocompatibility and are less cytotoxic, they could be used in cardiac tissue engineering applications. However, further in vitro and in vivo studies are required to confirm its role in cardiac tissue engineering.

Time-resolved flow cytometry (TRFC) was used to measure metabolic differences in estrogen receptor-positive breast cancer cells. This specialty cytometry technique measures fluorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening. Differences in fluorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen. Differences in fluorescence lifetime are attributed to the binding states of the autofluorescent metabolite NAD(P)H. The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate. NAD(P)H fluorescence lifetimes depend on the bound or unbound state of the metabolite, which also relates to metabolic transitions between oxidative phosphorylation and glycolysis. To determine if fundamental metabolic profiles differ for cells that are sensitive to tamoxifen compared to those that are resistant, large populations of MCF-7 breast cancer cells were screened and fluorescence lifetimes were quantified. Additionally, metabolic differences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer (Agilent Technologies Inc. Santa Clara, CA) and confocal imaging. Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells. This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be differentiated for drug sensitivity in a label-free fashion.

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.

Tetraphenylporphyrins (TPPs) have been proposed for the treatment of retinoblastoma by photodynamic therapy. Glycoconjugated compounds were synthesized for improving TPP solubility and amphipathy, and to specifically target mannose receptors overexpressed at the surface of cells. The efficiency of four TPP derivatives with different chemical structures was compared by phototoxicity tests and flow cytometry experiments. Interestingly, the absence/presence and distribution of saccharide moieties in the various compounds affected differently their mechanism of interaction with cancer cells and their phototoxic efficiency. For glycodendrimeric TPP-1 and TPP-2 incubated with retinoblastoma cells, a fast two-step uptake-equilibrium process was observed, whereas for a dendrimeric TPP without saccharide moieties (TPP-1c) and a glycoconjugated compound with no dendrimeric structure (TPP(DegMan)₃) uptake was very slow. The difference in uptake profiles and kinetics between TPP-1c on the one hand and TPP-1 and TPP-2 on the other hand would account for the interaction of the two glycodendrimeric compounds with a mannose receptor. These TPPs encapsulated in endosomes would induce less damage to cells upon illumination. TPP(DegMan)₃ showed the highest phototoxicity, but its efficiency was unaffected by pretreatment of cells by mannan. The penetration of this glycoconjugated compound in cells and its phototoxic effect appeared independent of its interaction with a mannose receptor. Thus, if glycoconjugation influenced TPPs behavior in solution and interaction with serum proteins, phototoxicity was not necessarily related to upfront molecular recognition.

The cell cycle of slowly growing bacteria consists of three phases that resemble the eukaryotic cell cycle: the pre-initiation, replication and post-replication periods, termed B, C and D, respectively. However, bacteria are also capable of very rapid growth, whereas the replication period remains long and hence the B period disappears and a round of replication is triggered before the previous one is finished. Several frequently used methods yield an incomplete analysis of these rapidly growing bacterial cultures. However, DNA distributions obtained by flow cytometry can be used to fully unravel the bacterial cell cycle independently of the duration of each period. In the present work, we have compared the accuracy of the cell cycle parameters estimation in different cultures. Whereas, for a given DNA per cell content, the accuracy of the C period determination by flow cytometry is essentially independent of the cell cycle, the precision of the D period estimation is inversely correlated with the number of replication forks per chromosome. We have established a data analysis routine to estimate the accuracy of the cell cycle parameters determination.

Plasmid copy number (pcn) is a key parameter in studies on antibiotic resistance, recombinant protein production or plasmid biology. We have analysed a set of plasmids derivatives of F, P1ΔincA, pSC101, R1, p15A and pBR322 replicons in Escherichia coli.All of them harbour the GFPmut2 gene under the control of pBAD. We have made use of constant GFP expression in the presence of arabinose to quantify pcn by spectrofluorometry (pcn/mass) and flow cytometry (pcn/cell). Exponentially growing cultures at 30°C, 37°C and 41°C were analysed. Plasmid copy number showed a dependence on temperature incubation in a growth rate-dependent manner. Except for F and pBR322 derivatives, the faster the growth rate, the higher the pcn. GFP fluorescence quantification from cell cultures was a straightforward approach to pcn determination. Comparing to Southern method, pcn determinations by spectrofluorometry gave more precise determination than flow cytometry.