Generation of a NES-mScarlet Red Fluorescent Reporter Human iPSC Line for Live Cell Imaging and Flow Cytometric Analysis and Sorting Using CRISPR-Cas9-Mediated Gene Editing

Advances in the regenerative stem cell field have propelled the generation of tissue-specific cells in the culture dish for subsequent transplantation, drug screening purposes, or the elucidation of disease mechanisms. One major obstacle is the heterogeneity of these cultures, in which the tissue-specific cells of interest usually represent only a fraction of all generated cells. Direct identification of the cells of interest and the ability to specifically isolate these cells in vitro is, thus, highly desirable for these applications. The type VI intermediate filament protein NESTIN is widely used as a marker for neural stem/progenitor cells (NSCs/NPCs) in the developing and adult central and peripheral nervous systems. Applying CRISPR-Cas9 technology, we have introduced a red fluorescent reporter (mScarlet) into the NESTIN (NES) locus of a human induced pluripotent stem cell (hiPSC) line. We describe the generation and characterization of NES-mScarlet reporter hiPSCs and demonstrate that this line is an accurate reporter of NSCs/NPCs during their directed differentiation into human midbrain dopaminergic (mDA) neurons. Furthermore, NES-mScarlet hiPSCs can be used for direct identification during live cell imaging and for flow cytometric analysis and sorting of red fluorescent NSCs/NPCs in this paradigm.

Quantitative Assessment of the Size-dependent Whole-body to Cellular Biodistribution of Fine Particulate Matters Following Inhalation by Fluorescence Imaging

Background: There has been growing concern regarding the impact of air pollution, especially fine dust, on human health. However, it is difficult to estimate the toxicity of fine dust on the human body because of its diverse effects depending on the composition and environmental factors.Results: In this study, we focused on the difference in the biodistribution of fine dust according to the size distribution of particulate matter after inhalation into the body to predict its impact on human health. We synthesized Cy7-doped silica particulate matters (CSPMs) having different particle sizes and employed them as model fine dust, and studied their whole-body in vivo biodistribution in BALB/c nude mice. Image-tracking and quantitative analysis were performed on the ex vivo organs and tissues. Additionally, flow cytometric analysis of single cells isolated from the lungs was performed. Smaller particles with a diameter of less than 100 nm (CSPM0.1) were observed to be removed relatively rapidly from the lungs upon initial inhalation. However, they were confirmed to accumulate continuously over 4 weeks of observation. In particular, smaller particles were found to spread rapidly to other organs during the early stages of inhalation.Conclusions: It is expected that the effect of fine dust on human health can be predicted through the differences in in vivo behavior that arise depending on the particle size. This study might provide with insights on association between CSPM0.1 accumulation in several organs including the lungs and adverse effect to underlying diseases in the organs.

Responses of Bloom-Forming Heterosigma akashiwo to Allelochemical Linoleic Acid: Growth Inhibition, Oxidative Stress and Apoptosis

Algal blooms have been occurring in many regions worldwide, and allelochemicals are important algaecides used to control harmful algal blooms (HABs). The allelopathic effects of linoleic acid (LA) on the harmful raphidophyte Heterosigma akashiwo were studied, and the possible mechanisms were investigated through analyses of population growth dynamics, cellular ultrastructure and the physiological levels of H. akashiwo. The results showed that the inhibitory effect of LA on H. akashiwo cells increased with an increasing LA concentration. The levels of ROS and MDA were significantly elevated, indicating oxidative stress and lipid peroxidation due to LA exposure. At the same time, LA also activated the antioxidant system, including superoxide dismutase (SOD), catalase (CAT), and POD, and non-enzymatic antioxidants such as reduced AsA and glutathione (GSH). Transmission electron microscopy (TEM) revealed that the morphology of the algal cells was impaired in an LA-dependent manner. Annexin V-FITC/PI double staining and flow cytometric analysis revealed that LA exposure decreased the cellular mitochondrial membrane potential (MMP), increased the rate of apoptosis. LA modulated bcl-2/bax homeostasis and increased the expressions of cytochrome c and caspases-3 and -9, proving that LA induced cell death via the mitochondria-mediated apoptotic pathway. It was suggested that LA had allelopathic effects on H. akashiwo, inducing physiological and morphological changes and finally triggering the apoptosis of H. akashiwo. All of these results showed that LA might have the potential as an algaecide to control harmful algae.

Enhanced Anti-Proliferative Effect of Carboplatin in Ovarian Cancer Cells Exploiting Chitosan – Poly (lactic glycolic acid) Nanoparticles.

Objective: The aim of the present article was to enhance the therapeutic efficacy of carboplatin (CP) using the formulation of chitosan – poly (lactic glycolic acid) nanoparticles (CS-PLGA NPs). Methods: Nanoparticles were synthesized by an ionic gelation method and were characterized for their morphology, particle size, and surface potential measurements by TEM and zeta sizer. This study was highlighted for the evaluation of drug entrapment, loading and in vitro drug release capabilities of the prepared nanoparticles by spectrophotometric analysis. The stability study was also conducted after 3 months for their particle size, zeta potential, drug loading and encapsulation efficiencies. Further, ovarian cancer cell line PEO1 were used to evaluate the toxicity and efficacy of nano-formulation by MTT assay. Further, the study was evaluated for apoptosis using flow cytometric analysis. Result: The CS-PLGA-CP NPs were uniform and spherical in shape. The particle size and zeta potential of CS-PLGA-CP NPs were measured 156 ± 6.8 nm and +52 ± 2.4 mV, respectively. High encapsulation (87.4 ± 4.5 %) and controlled retention capacities confirmed the efficiency of the prepared nanoparticles in a time and dose dependant manner. The cytotoxicity assay results also showed that CS-PLGA-CP NPs has high efficiency on PEO1 cells compared to the free drug. The flow cytometric result showed 64.25 % of the PEO1 cells were apoptotic and 8.42 % were necrotic when treated with CS-PLGA-CP NPs. Conclusion: Chitosan-PLGA combinational polymeric nanoparticles were not only steady but also non-toxic. Our experiments revealed that the chitosan- PLGA nanoparticles could be used as a challenging vehicle candidate for drug delivery for the therapeutic treatment of ovarian cancer.

Forsythoside A Alleviates Imiquimod-Induced Psoriasis-like Dermatitis in Mice by Regulating Th17 Cells and IL-17A Expression

Psoriasis is a recurrent inflammatory skin disease characterized by redness and scaly skin lesions with itchy or painful sensations. Forsythoside A, one of the main active compounds isolated from the fruit of Forsythia suspensa, has been widely applied to treat inflammatory diseases in the clinical use of traditional oriental medicine. However, the effect of forsythoside A on psoriasis remains unclear. This study aimed to explore the therapeutic effects and immune regulation of forsythoside A on psoriasis. C57BL/6 mice were divided into six groups and treated with imiquimod cream on their shaved back skin to induce psoriasis-like dermatitis. Different doses of forsythoside A (5 mg/kg, 10 mg/kg, or 20 mg/kg) were administered to the respective treatment groups. Skin redness, scaling, and ear thickness were measured; keratinocyte proliferation and inflammatory cytokine expression were detected by hematoxylin–eosin and immunohistochemical staining. Th17 cells in the inguinal lymph nodes were detected by flow cytometric analysis. IL-17A levels were measured using ELISA. The results showed that forsythoside A relieved psoriatic skin symptoms such as skin redness, thickness, scaling, and reduced epidermal thickening. The expression of IL-6, IL-17, and Ki-67 was downregulated in the forsythoside-A-treated groups. Th17 cell expression in inguinal lymph nodes and IL-17A secretion was suppressed by forsythoside A. In conclusion, forsythoside A was found to alleviate imiquimod-induced psoriasis-like dermatitis in mice by suppressing Th17 development and IL-17A secretion. These findings demonstrate the feasibility of forsythoside A in treating human psoriasis.

B cell overexpression of FCRL5 and PD-1 is associated with low antibody titers in HCV infection

Antibodies targeting the hepatitis C virus (HCV) envelope glycoprotein E2 are associated with delayed disease progression, and these antibodies can also facilitate spontaneous clearance of infection in some individuals. However, many infected people demonstrate low titer and delayed anti-E2 antibody responses. Since a goal of HCV vaccine development is induction of high titers of anti-E2 antibodies, it is important to define the mechanisms underlying these suboptimal antibody responses. By staining lymphocytes with a cocktail of soluble E2 (sE2) glycoproteins, we detected HCV E2-specific (sE2+) B cells directly ex vivo at multiple acute infection timepoints in 29 HCV-infected subjects with a wide range of anti-E2 IgG titers, including 17 persistently infected subjects and 12 subjects with spontaneous clearance of infection. We performed multi-dimensional flow cytometric analysis of sE2+ and E2-nonspecific (sE2-) class-switched B cells (csBC). In sE2+ csBC from both persistence and clearance subjects, frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Across all subjects, plasma anti-E2 IgG levels were positively correlated with frequencies of sE2+ rMBC and sE2+ actMBC, while anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on sE2+ rMBC and PD-1 expression on sE2+ actMBC. Upregulation of FCRL5 on sE2+ rMBC and upregulation of PD-1 on sE2+ actMBC may limit anti-E2 antibody production in vivo. Strategies that limit upregulation of these molecules could potentially generate higher titers of protective antibodies against HCV or other pathogens.

Novel Substituted Purine Isosteres: Synthesis, Structure-Activity Relationships and Cytotoxic Activity Evaluation

A number of pyrrolo[2,3-c]pyridines, pyrrolo[3,2-d]pyrimidines and pyrazolo[4,3-d]pyrimidines were designed and synthesized as antiproliferative agents. The target compounds possessed selected substituents in analogous positions on the central scaffold that allowed the extraction of interesting SARs. The cytotoxic activity of the new derivatives was evaluated against prostatic (PC-3) and colon (HCT116) cell lines, and the most potent analogues showed IC50 values in the nM to low µM range, while they were found to be non-toxic against normal human fibroblasts (WI-38). Flow cytometric analysis of DNA content revealed that the most promising derivative 14b caused a statistically significant accumulation of PC-3 cells at G2/M phase and induced apoptosis in PC-3 cells.

Evaluation of 2-Thioxoimadazolidin-4-One Derivatives as Potent Anti-Cancer Agents through Apoptosis Induction and Antioxidant Activation: In Vitro and In Vivo Approaches

Background: Hepatocellular carcinoma (HCC) is one of the most widespread malignancies and is reported as the fourth most prevalent cause of cancer deaths worldwide. Therefore, we aimed to investigate the probable mechanistic cytotoxic effect of the promising 2-thioxoimidazolidin-4-one derivative on liver cancer cells using in vitro and in vivo approaches. The compounds were tested for the in vitro cytotoxic activity using MTT assay, and the promising compound was tested in colony forming unit assay, flow cytometric analysis, RT-PCR, Western blotting, in vivo using SEC-carcinoma and in silico to highlight the virtual mechanism of action. Both compounds 4 and 2 performed cytotoxic effects against HepG2 cells with IC50 values of 0.017 and 0.18 μM, respectively, compared to Staurosporine and 5-Fu as reference drugs with IC50 values of 5.07 and 5.18 µM, respectively. Compound 4 treatment revealed apoptosis induction by 19.35-fold (11.42% compared to 0.59% in control), arresting the cell cycle at G2/M phase. Moreover, studying gene expression that plays critical roles in cell cycle and apoptosis by RT-PCR demonstrated that compound 4 enhances the expression of the pro-apoptotic genes p53, PUMA, and Caspase 3, 8, and 9, and impedes the anti-apoptotic Bcl-2 gene in the HepG2 cells. It can also inhibit the PI3K/AKT pathway at both gene and protein levels, which was reinforced by the in silico predictions of the molecular docking simulations towards the PI3K/AKT proteins. Finally, in vivo study verified that compound 4 has a promising anti-cancer activity through activating antioxidant levels (CAT, SOD and GSH) and ameliorating hematological, biochemical, and histopathological findings.

β-Estradiol Induces Mitochondrial Apoptosis in Cervical Cancer Through the Suppression of AKT/NF-κB Signaling Pathway

Background: Cervical cancer is the fourth most prevalent gynecological cancer worldwide, which threatens women's health and causes cancer-related mortality. In the search for effective anticervical cancer drugs, we discovered that β-estradiol (E2), a patent drug for estrogen deficiency syndrome treatment, displays the most potent cytotoxicity against HeLa cells. Objective: This study aims to evaluate the growth inhibitory effect of β-estradiol on HeLa cells and explore its underlying mechanisms. Methods: CCK-8 assay was used to evaluate the cytotoxicity of 6 compounds against HeLa cells. Flow cytometric analysis and Hoechst 33258 staining assay were performed to detect cell cycle arrest and apoptosis induction. The collapse of the mitochondrial potential was observed by the JC-1 staining assay. The expression levels of proteins were examined by western blotting. Results: β-Estradiol, at high concentration, displays potent cytotoxicity against HeLa cells with an IC50 value of 18.71 ± 1.57 μM for 72 h treatment. β-Estradiol induces G2/M cell cycle arrest through downregulating Cyclin B1 and p-CDK1. In addition, β-estradiol-induced apoptosis is accompanied by the loss of mitochondrial potential, activation of the Caspase family, and altered Bax/Bcl-2 ratio. β-Estradiol markedly decreased the expression level of p-AKT and p-NF-κB. Conclusion: This study demonstrated that β-estradiol induces mitochondrial apoptosis in cervical cancer through the suppression of the AKT/NF-κB signaling pathway, indicating that β-estradiol may serve as a potential agent for cervical cancer treatment.

Flow cytometric analysis of the inhibition of human basophil activation by histamine high dilutions - a replication study

Background: Inhibition of human basophil activation by highly diluted histamine was reported to be a reliable experimental model to examine biological effects of high dilutions. However, independent replications did not always yield concordant results. Aims: We aimed at performing an independent replication of a former study [1] using rigorously controlled experimental conditions to minimise confounding factors. Materials and Methods: In 20 independent experiments, human basophils were treated with highly diluted histamine (15cH, 16cH, corresponding to 10-30-10-32 M) prior to activation by fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Controls were treated with analogously diluted water (15cH, 16cH). The dilutions were prepared freshly for each experiment in deionised water by successive steps of centesimal dilution and agitation (10 s vortex at high speed). Highly diluted samples were blinded and randomised. All samples were set in triplicates. Activated basophils were determined by flow cytometry using anti-CD203c. 20 independent systematic negative control (SNC) experiments were carried out to investigate possible systematic errors. Results: No difference in basophil activation was observed between the highly diluted histamine samples and the highly diluted water controls. There was no evidence for a blood donor specificity of the results. The SNC experiments demonstrated the stability of the test system. Experimental variability within and between experiments was slightly reduced for the highly diluted histamine samples. Discussion: This study was designed as an independent reproduction of a former study [1]. Though we strictly adopted the experimental procedure described in [1], our results do not confirm the large inhibitory effects observed for histamine 15cH and 16cH. This lack of reproducibility might be due to minor differences in the experimental design, such as blinding and randomising of the samples, which we chose to perform in order to reduce the possibility of artifacts but was omitted in the former study. Conclusions: Laboratory independent replication of homeopathic basic research experiments is still a challenge. Assuming that the results formerly obtained with this model were not due to systematic errors, the quest identifying the crucial factors for successful reproducibility is open for future research. Keywords: Human basophils; histamine; high dilutions; flow cytometry Reference: [1] Sainte-Laudy J, Belon P. Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c. Homeopathy. 2006;95:3-8.