Human Foreskin Fibroblasts Research Articles

Preparation and Characterization of Allicin-Loaded Solid Lipid Nanoparticles Surface-Functionalized with Folic Acid-Bonded Chitosan: In Vitro Anticancer and Antioxidant Activities.

This investigation aimed to increase the bioavailability and anticancer effects of allicin (AC) by encapsulating it in solid lipid nanoparticles (SLN) decorated with chitosan (CS)-conjugated folic acid (FA). Nanoparticles (NPs) were synthesized by high-pressure homogenization, and then, Fourier-transform infrared (FTIR), Field-Emission Scanning Electron Microscopy (FESEM), Dynamic Light Scattering (DLS), and zeta potential methods were used to determine their physicochemical characteristics. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess the effect of toxicity and flow cytometry, while fluorescent staining methods were used to investigate the type of cell death. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expression levels of apoptotic genes: Bcl-2, and caspase-8. The presence of AC-SLN-CS-FA with a spherical morphology, an average size of 86.7 ± 9.4 nm, uniform distribution (0.31), a surface charge of +21.3 ± 13.3 mV, an encapsulation percentage of 86.3%, and a folate binding rate of 63% confirmed the success of the preparation method. Suppression of MCF-7 cancer cells and non-toxicity of AC-SLN-CS-FA on Human foreskin fibroblast (HFF) normal cells were confirmed by cytotoxic assay. The results of flow cytometry revealed that the cells were arrested in the sub-G1 phase, and the activation of the intrinsic apoptosis pathway was confirmed by the results of real-time qPCR. In general, AC-SLN-CS-FA has the potential to prevent free radicals and trigger apoptosis in cancer cells by activating the intrinsic apoptosis pathway; thus, making it a promising subject in preclinical research.

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion.

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells' differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures. To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting). In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed forcolony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

Fluorescent poly[N‐(2‐hydroxypropyl) methacrylamide] nanogel by dispersion polymerization as a contrast agent for live‐cell imaging

AbstractHere, we report a novel dispersion polymerization for the preparation of cross‐linked poly[N‐(2‐hydroxypropyl) methacrylamide] (PHPMA)‐based nanogels in water/2‐methoxyethanol mixture (H2O/MetCel), initiated with potassium persulfate (KPS), and stabilized with poly(vinyl alcohol) 25/140 (PVA) and sodium dodecyl sulfate (SDS). Obtained nanogels were characterized using transmission (TEM) and cryogenic transmission electron microscopy (cryo‐TEM), dynamic light scattering (DLS), asymmetric flow field‐flow fractionation (A4F), nuclear magnetic resonance spectroscopy (NMR), and Raman spectroscopy methods in terms of size, particle size distribution, morphology, and structure. N‐(2‐hydroxypropyl) methacrylamide (HPMA) was copolymerized with 20 wt% ethylene dimethacrylate (EDMA) resulting in 138 nm poly[N‐(2‐hydroxypropyl) methacrylamide‐co‐ethylene dimethacrylate] (PHPMA‐EDMA) nanogel dispersion with irregular shape and core‐shell type structure. Next, we copolymerized HPMA with 20 wt% EDMA and 10 wt% propargyl methacrylate (PMA) to incorporate reactive functionality into the final core‐shell type 120 nm poly[N‐(2‐hydroxypropyl) methacrylamide‐co‐ethylene dimethacrylate‐co‐propargyl methacrylate] (PHPMA‐EDMA‐PMA) nanogel dispersion. Then, the biocompatibility of PHPMA‐EDMA‐PMA nanogel was proved using rat mesenchymal stem cells (rMSC), and human foreskin fibroblasts (BJ). PHPMA‐EDMA‐PMA nanogel was fluorescently labeled with sulfo‐cyanine3 azide resulting in 131 nm nanogel. We performed in vitro uptake studies with fluorescently labeled PHPMA‐EDMA‐PMA nanogel using rMSC showing that the fluorescently labeled PHPMA‐EDMA‐PMA nanogel was well‐distributed in the cytosol and taken up into lysosomes.

Toxoplasma gondii inhibits the expression of autophagy-related genes through AKT-dependent inactivation of the transcription factor FOXO3a.

The intracellular parasite Toxoplasma gondii induces host AKT activation to prevent autophagy-mediated clearance; however, the molecular underpinnings are not fully understood. Autophagy can be negatively regulated through AKT-sensitive phosphorylation and nuclear export of the transcription factor Forkhead box O3a (FOXO3a). Using a combination of pharmacological and genetic approaches, herein we investigated whether T. gondii hinders host autophagy through AKT-dependent inactivation of FOXO3a. We found that infection by type I and II strains of T. gondii promotes gradual and sustained AKT-dependent phosphorylation of FOXO3a at residues S253 and T32 in human foreskin fibroblasts (HFF) and murine 3T3 fibroblasts. Mechanistically, AKT-sensitive phosphorylation of FOXO3a by T. gondii required live infection and the activity of PI3K but was independent of the plasma membrane receptor EGFR and the kinase PKCα. Phosphorylation of FOXO3a at AKT-sensitive residues was paralleled by its nuclear exclusion in T. gondii-infected HFF. Importantly, the parasite was unable to drive cytoplasmic localization of FOXO3a upon pharmacological blockade of AKT or overexpression of an AKT-insensitive mutant form of FOXO3a. Transcription of a subset of bona fide autophagy-related targets of FOXO3a was reduced during T. gondii infection in an AKT-dependent fashion. However, parasite-directed repression of autophagy-related genes was AKT-resistant in cells deficient in FOXO3a. Consistent with this, T. gondii failed to inhibit the recruitment of acidic organelles and LC3, an autophagy marker, to the parasitophorous vacuole upon chemically or genetically induced nuclear retention of FOXO3a. In all, we provide evidence that T. gondii suppresses FOXO3a-regulated transcriptional programs to prevent autophagy-mediated killing. IMPORTANCE The parasite Toxoplasma gondii is the etiological agent of toxoplasmosis, an opportunistic infection commonly transmitted by ingestion of contaminated food or water. To date, no effective vaccines in humans have been developed and no promising drugs are available to treat chronic infection or prevent congenital infection. T. gondii targets numerous host cell processes to establish a favorable replicative niche. Of note, T. gondii activates the host AKT signaling pathway to prevent autophagy-mediated killing. Herein, we report that T. gondii inhibits FOXO3a, a transcription factor that regulates the expression of autophagy-related genes, through AKT-dependent phosphorylation. The parasite's ability to block the recruitment of the autophagy machinery to the parasitophorous vacuole is impeded upon pharmacological inhibition of AKT or overexpression of an AKT-insensitive form of FOXO3a. Thus, our study provides greater granularity in the role of FOXO3a during infection and reinforces the potential of targeting autophagy as a therapeutic strategy against T. gondii.

Plant Gel-Mediated Synthesis of Gold-Coated Nanoceria Using Ferula gummosa: Characterization and Estimation of Its Cellular Toxicity toward Breast Cancer Cell Lines.

In this study, a novel method using Ferula gummosa gums as a capping agent was used to synthesize the nanoceria for the first time. The method was economical and performed at room temperature. Furthermore, it was coated with gold (Au/nanoceria) and fully characterized using X-ray powder diffraction (XRD), field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy (FESEM-EDX), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and zeta potential (ζ potential). The crystallite size obtained from the results was 28.09 nm for Au/nanoceria. The energy-dispersive X-ray spectroscopy (EDX) analysis of Au/nanoceria revealed the compositional constituents of the product, which display the purity of the Au/nanoceria. The cell toxicity properties of the non-doped and Au-coated nanoceria were identified by a MTT analysis on a breast cancer cell line (MCF7). Additionally, human foreskin fibroblast cells (HFF) were used as a normal cell line. The cytotoxicity results indicated that the toxicological effect of Au/nanoceria on cancer cells was significant while having little toxic effect on normal cells. The toxicity effect of nanoceria clearly shows the dependence on dose and time, so, with increasing the dose of Au/nanoceria, the death of cancer cells also increases.

Knocking down the expression of the molecular motors, myosin A, C and F genes in Toxoplasma gondii to decrease the parasite virulence

Toxoplasmosis is a serious parasitic infection and novel therapeutic options are highly demanded to effectively eliminate it. In current study, Toxoplasma gondii myosin A, C and F genes were knocked down using small interference RNA (siRNA) method and the parasite survival and virulence was evaluated in vitro and in vivo. The parasites were transfected with specific siRNA, virtually designed for myosin mRNAs, and co-cultured with human foreskin fibroblasts. The transfection rate and the viability of the transfected parasites were measured using flow cytometry and methyl thiazole tetrazolium (MTT) assays, respectively. Finally, the survival of BALB/c mice infected with siRNAs-transfected T. gondii was assessed. It was demonstrated that a transfection rate of 75.4% existed for siRNAs, resulting in 70% (P = 0.032), 80.6% (P = 0.017) and 85.5% (P = 0.013) gene suppression for myosin A, C and F in affected parasites, respectively, which was subsequently confirmed by Western blot analysis. Moreover, lower parasite viability was observed in those with knocked down myosin C with 80% (P = 0.0001), followed by 86.15% (P = 0.004) for myosin F and 92.3% (P = 0.083) for myosin A. Considerably higher mouse survival (about 40 h) was, also, demonstrated in mice challenged with myosin siRNA-transfected T. gondii, in comparison with control group challenged with wild-type parasites. In conclusion, myosin proteins knock down proposes a promising therapeutic strategy to combat toxoplasmosis.

Phytochemical screening and in vitro assessments of antioxidant and cytotoxic potentials of extracts from Aesculus hippocastanum L. green fruit mesocarps

In this study, the in vitro antioxidant and cytotoxic effects of water and methanol extracts obtained from the green fruit mesocarp of Aesculus hippocastanum L. (Hippocastanaceae) were investigated. Phytochemical content of the methanol extract and the water extract were determined by qualitative methods; antioxidant activity was determined by DPPH free radical scavenging test, and total antioxidant capacity was determined by phosphomolybdate test. The effects of the extracts on proliferation and cell viability of BJ normal human foreskin fibroblasts were also evaluated by the WST-8 cell viability test.
 Qualitative phytochemical screening results showed that the methanol extract contains phenols, tannins, flavonoids, and saponins, but no alkaloids and anthraquinones. On the other hand, phenols, flavonoids, anthraquinone, and saponins were found in the water extract, tannins and alkaloids could not be detected.
 In addition, an increase in antioxidant activity was also observed with each increasing concentration of methanol and water extract. When the antioxidant capacity and free radical scavenging activity of methanol and water extracts were compared, it was determined that the methanol extract was more effective than that the water extract. The WST-8 trial results showed that both water and methanol extracts obtained from the green fruit mesocarp of A. hippocastanum did not have cytotoxic effects on BJ cells, on the contrary, treatment concentrations of 10, 20 and, 30 µgmL-1 increased cell proliferation significantly at the 24-hour work.

Biosynthetic of Green Zinc Oxide Nanoparticles with Effect on Cancer Cell Line Hela

The cytotoxic impact of biosynthetic zinc oxide nanoparticles (ZnONPs) was investigated using Vitex agnus-castus, which has been shown to have Effective compounds that suppress cancer cell proliferation. Zinc oxide nanoparticles were biosynthesized in the laboratories of the Biology department /College of Education for the Pure Sciences /University of Diyala. The phenotypic and structural characteristics of biosynthetic nanoparticles were identified using scanning electron microscopy (SEM). The majority of ZnONPs are dense and spherical in shape, with diameters ranging from (20-61) nm, and were discovered on the cervical cancer cell line Hela and compared to the normal line Human Foreskin Fibroblast cells (HFF) using the MTT stain test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium ). Cytotoxicity experiments were conducted at the Iraqi Center for Cancer Research / Al-Mustansiriya University. This study showed inhibitory activity on Hela cervical cancer cells, where the highest inhibition rate reached 93.6% at a concentration of 200 µg/ml. This raises the prospect of finding a viable therapy for cervical cancer (Hela) or any other malignancy using nanoparticle manufacturing technologies. Keywords: Biosynthetic, Green nanoparticles, Cancer cell, Hela, GC-MS.

Antiplasmodial Activity Evaluation of a Bestatin-Related Aminopeptidase Inhibitor, Phebestin.

The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the in vitro multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC50 values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC50 concentration. Using 72-h in vitro exposure of phebestin at concentrations of 1 μM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An in silico study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. In vivo evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.

Targeting Trop2 by Bruceine D suppresses breast cancer metastasis by blocking Trop2/β-catenin positive feedback loop

IntroductionTumor-associated calcium signal transducer 2 (Trop2) has been used as a transport gate for cytotoxic agents into cells in antibody–drug conjugate designs because of its expression in a wide range of solid tumors. However, the specific role of Trop2 itself in breast cancer progression remains unclear and small molecules targeting Trop2 have not yet been reported. ObjectivesTo screen small molecules targeting Trop2, and to reveal its pharmacological effects and the molecular mechanisms of action. MethodsSmall molecule targeting Trop2 was identified by cell membrane chromatography, and validated by cellular thermal shift assay and point mutation analyses. We investigated the pharmacological effects of Trop2 inhibitor using RNA-seq, human foreskin fibroblast (HFF)-derived extracellular matrix (ECM), Matrigel drop invasion assays, colony-forming assay, xenograft tumor model, 4T1 orthotopic metastasis model and 4T1 experimental metastasis model. The molecular mechanism was determined using immunoprecipitation, mass spectrometry, immunofluorescence, immunohistochemistry and Western blotting. ResultsHere we identified Bruceine D (BD) as the inhibitor of Trop2, and demonstrated anti-metastasis effects of BD in breast cancer. Notably, Lys307 and Glu310 residues of Trop2 acted as critical sites for BD binding. Mechanistically, BD suppressed Trop2-induced cancer metastasis by blocking the formation of Trop2/β-catenin positive loop, in which the Trop2/β-catenin complex prevented β-catenin from being degraded via the ubiquitin-proteosome pathway. Destabilized β-catenin caused by BD reduced nucleus translocation, leading to the reduction of transcription of Trop2, the reversal of epithelial-mesenchymal transition (EMT) process, and the inhibition of ECM remodeling, further inhibiting cancer metastasis. Additionally, the inhibitory effects of BD on lung metastatic colonization and the beneficial effects of BD on prolongation of survival were validated in 4T1 experimental metastasis model. ConclusionsThese results support the tumor-promoting role of Trop2 in breast cancer by stabilizing β-catenin in Trop2/β-catenin positive loop, and suggest Bruceine D as a promising candidate for Trop2 inhibition.

Methanolic extract of Kigelia africana and wound healing: an in vitro study.

Kigelia africana (Lam.) Benth. (Bignoniaceae) syn. Kigelia pinnata (Jacq. DC) is a tropical plant that is native to tropical Africa. The aim of this study was to determine if a methanolic extract prepared from Kigelia africana (KAE) can promote wound healing in treated human normal epidermal keratinocyte (HaCaT) cells and human normal foreskin fibroblast cell line (BJ) cells compared with untreated cells. Experimental steps included: the methanolic extraction of the leaf and fruit of the Kigelia africana plant; the preparation of HaCaT and BJ cell lines; cell culture with a stable tetrazolium salt-based proliferation assay; and the evaluation of the wound healing effect of KAE (2μg/ml) in BJ and HaCaT cells. The phytochemical contents of KAE were determined using liquid chromatography quadrupole time-of-flight mass spectrometry. The following molecules were identified as being present in the KAE, among others: cholesterol sulfate; lignoceric acid; embelin; isostearic acid; linoleic acid; dioctyl phthalate; arg-pro-thr; 15-methyl-15(S)-PGE1; sucrose; benzododecinium (Ajatin); and 9-Octadecenamide (oleamide). KAE effected faster wound healing in treated cells compared with untreated cells for both cell lines. HaCaT cells that had been mechanically injured and treated with KAE healed completely in 48 hours compared with 72 hours for untreated HaCaT cells. Treated BJ cells healed completely in 72 hours compared with 96 hours for untreated BJ cells. Concentrations of KAE up to 300μg/ml had a very low cytotoxic effect on treated BJ and HaCaT cells. The experimental data in this study support the potential of KAE-based wound healing treatment to accelerate wound healing.

Enhanced Cosmeceutical Potentials of the Oil from Gryllus bimaculatus de Geer by Nanoemulsions.

This study aimed to extract the oil from Gryllus bimaculatus de Geer, evaluate its potential for cosmeceutical applications, and develop nanoemulsions to promote the cosmeceutical capabilities of the oil. G. bimaculatus oil was produced by the cold pressing method. Its fatty acid compositions were assessed by fatty acid methyl ester/gas chromatographic-mass spectrometry. The antioxidant activities of the oil were investigated in terms of radical scavengers, reducing power, and lipid peroxidation inhibition. The whitening effects were investigated through anti-tyrosinase activities, whilst the anti-aging effects were investigated through inhibition against collagenase, elastase, and hyaluronidase. The irritant effects were investigated by the hen's egg chorio-allantoic membrane test and the cytotoxicity assay in immortalized human epidermal keratinocytes and human foreskin fibroblast cells. The nanoemulsions were developed, characterized, and evaluated for their stability and cosmeceutical properties. G. bimaculatus oil, rich in linoleic acid (31.08 ± 0.00%), oleic acid (30.44 ± 0.01%), palmitic acid (24.80 ± 0.01%), and stearic acid (7.61 ± 0.00%), demonstrated promising cosmeceutical properties in terms of antioxidant, anti-tyrosinase, and anti-skin ageing activities. Besides, the oil was safe since it induced no irritation or cytotoxicity. G. bimaculatus oil was successfully developed into nanoemulsions, and F1, composed of 1% w/w G. bimaculatus oil, 1.12% w/w polysorbate 80, 0.88% w/w sorbitan oleate, and 97% w/w DI water, had the smallest internal droplet size (53.8 ± 0.6 nm), the narrowest polydispersity index (0.129 ± 0.010), and a pronounced zeta potential (-28.23 ± 2.32 mV). All cosmeceutical activities of the oil were significantly enhanced after incorporation in the nanoemulsions (p < 0.001), particularly the whitening effects. G. bimaculatus oil nanoemulsion was an attractive cosmeceutical formulation with potent whitening effects, along with antioxidant and anti-aging properties. Therefore, nanoemulsion technology was found to be an effective strategy for improving the cosmeceutical properties of G. bimaculatus oil.

Autophagy gene Atg7 regulates the development of radiation-induced skin injury and fibrosis of skin.

Radiation-induced skin injury, which may progress to fibrosis, is a severe side effect of radiotherapy in patients with cancer. However, currently, there is a lack of preventive or curative treatments for this injury. Meanwhile, the mechanisms underlying this injury remain poorly understood. Here, we elucidated whether autophagy is essential for the development of radiation-induced skin injury and the potential molecular pathways and mechanisms involved. We used the myofibroblast-specific Atg7 knockout (namely, conditional Atg7 knockout) mice irradiated with a single electron beam irradiation dose of 30Gy. Vaseline-based 0.2% rapamycin ointment was topically applied once daily from the day of irradiation for 30days. On day 30 post irradiation, skin tissues were harvested for further analysis. In vitro, human foreskin fibroblast cells were treated with rapamycin (100nM) for 24h and pretreated with 3-MA (5mM) for 12h. Macroscopic skin manifestations, histological changes, and fibrosis markers at the mRNA and protein expression levels were measured. Post irradiation, the myofibroblast-specific autophagy-deficient (Atg7Flox/Flox Cre+ ) mice had increased fibrosis marker (COL1A1, CTGF, TGF-β1, and α-SMA) levels in the irradiated area and had more severe macroscopic skin manifestations than the control group (Atg7Flox/Flox Cre- ) mice. Treatment with an autophagy agonist rapamycin attenuated macroscopic skin injury scores and skin fibrosis marker levels with decreased epidermal thickness and dermal collagen deposition in Atg7Flox/Flox Cre+ mice compared with the vehicle control. Moreover, in vitro experiment results were consistent with the in vivo results. Together with studies at the molecular level, we found that these changes involved the Akt/mTOR pathway. In addition, this phenomenon might also relate to Nrf2-autophagy signaling pathway under oxidative stress conditions. In conclusion, Atg7 and autophagy-related mechanisms confer radioprotection, and reactivation of the autophagy process can be a novel therapeutic strategy to reduce and prevent the occurrence of radiodermatitis, particularly skin fibrosis, in patients with cancer.

Anti-Toxoplasma gondii activity of Trametes versicolor (Turkey tail) mushroom extract

Toxoplasma gondii (T. gondii) infection continues to rise globally in humans and animals with high socioeconomic and public health challenges. Current medications used against T. gondii infection are limited in efficacy, safety, and affordability. This research was conducted to assess the higher fungi extract effect on T. gondii tachyzoites growth in vitro and possibly decipher its mechanism of action. Furthermore, we evaluated the extract's effect on human foreskin fibroblast viability. The methanol extracts of Turkey tail (TT) mushroom was tested against T. gondii tachyzoites growth using an RH-RFP type I strain that expresses red fluorescent protein throughout culture in a dose-dependent manner using a fluorescent plate reader. Similarly, we tested the effect of the extract on host cell viability. We observed that TT extract inhibited tachyzoites growth with a 50% minimum inhibitory concentration (IC50s), IC50 = 5.98 ± 1.22 µg/mL, and 50% cytotoxic concentration (CC50s), CC50 ≥ 100 µg/mL. It was discovered that TT extract induced strong mitochondria superoxide and reactive oxygen species production and disrupted mitochondria membrane potential in T. gondii tachyzoites. Additionally, scanning electron microscopy depicted that TT extract and pyrimethamine (PY) caused a morphological deformation of tachyzoites in vitro. In conclusion, TT methanol extract made up of phytosterols, bioactive sphingolipids, peptides, phenolic acids, and lactones could be a promising source of new compounds for the future development of anti-Toxoplasma gondii drugs. Extracts were non-cytotoxic, even at higher concentrations.

Facile photosynthesis of novel porphyrin-derived nanocomposites containing Ag, Ag/Au, and Ag/Cu for photobactericidal study

In this research, the one-step synthesis of novel porphyrin-based nanocomposites was performed easily using a photochemical under visible light illumination strategy. As a result, the focus of this research is on synthesizing and using decorated ZnTPP (zinc(II)tetrakis(4-phenyl)porphyrin) nanoparticles with Ag, Ag/AgCl/Cu, and Au/Ag/AgCl nanostructures as antibacterial agents. Initially, ZnTPP NPs were synthesized as a result of the self-assembly of ZnTPP. In the next step, in a visible-light irradiation photochemically process, the self-assembled ZnTPP nanoparticles were used to make ZnTPP/Ag NCs, ZnTPP/Ag/AgCl/Cu NCs, and ZnTPP/Au/Ag/AgCl NCs. A study on the antibacterial activity of nanocomposites was carried out for Escherichia coli, and Staphylococcus aureus as pathogen microorganisms by the plate count method, well diffusion tests, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) values determination. Thereafter, the reactive oxygen species (ROS) were determined by the flow cytometry method. All the antibacterial tests and the flow cytometry ROS measurements were carried out under LED light and in dark. The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to investigate the cytotoxicity of the ZnTPP/Ag/AgCl/Cu NCs, against Human foreskin fibroblast (HFF-1) normal cells. Due to the specific properties such as admissible photosensitizing properties of porphyrin, mild reaction conditions, high antibacterial properties in the presence of LED light, crystal structure, and green synthesis, these nanocomposites were recognized as kinds of antibacterial materials that are activated in visible light, got the potential for use in a broad range of medical applications, photodynamic therapy, and water treatment.

Interleukin‐1α promotes matrix metalloproteinase‐9 expression, cellular motility, and local invasiveness of ameloblastoma cells

AbstractAimAlthough ameloblastoma is a benign tumor, its local invasiveness and recurrence rate are both high. Thus, the regulation of the invasiveness of ameloblastoma cells into the surrounding tissue is required to understand its pathogenesis. Ameloblastoma cells secrete several matrix metalloproteinases (MMPs); however, the factors inducing their secretion remain unclear. We previously suggested that interleukin (IL)‐1α derived from ameloblastoma cells triggers the production of inflammatory cytokines by stromal fibroblasts. In this study, we estimated whether IL‐1α affects the behavior of ameloblastoma cells.MethodsThe gene expression of MMP‐9 was assessed by real‐time reverse transcription–polymerase chain reaction (RT‐PCR). The secretion of MMP‐9 was assessed by enzyme‐linked immunosorbent assay (ELISA). The motility of AM‐3 ameloblastoma and Raw264.7 （macrophage derived cells） cells and the invasiveness of AM‐3 cells were calculated using the Boyden chamber. The invasiveness of AM‐3 cells toward human foreskin fibroblast (HFF)‐2 fibroblasts were assessed using modified double‐layered collagen gel hemisphere (DL‐CGH).ResultsThe mRNA expression and secretion of MMP‐9 by AM‐3 ameloblastoma cells were significantly increased by IL‐1α stimulation. The motilities of AM‐3 and RAW264.7 macrophage derived cells and the invasiveness of AM‐3 cells were significantly enhanced by IL‐1α and suppressed by an IL‐1 receptor antagonist (IL‐1Ra). The invasiveness of AM‐3 cells towards HFF‐2 fibroblasts in a DL‐CGH model was suppressed by a treatment with IL‐1Ra or an anti‐IL‐1α neutralizing antibody.ConclusionIL‐1α itself or the IL‐1α‐dependent production of unidentified chemo attractants by stromal cells may be important for the local invasiveness of ameloblastoma cells, and IL‐1α might be a therapeutic target of the ameloblastoma.

Phosphatase-degradable nanoparticles: A game-changing approach for the delivery of antifungal proteins

HypothesisPolyphosphate nanoparticles as phosphatase-degradable carriers for Penicillium chrysogenum antifungal protein (PAF) can enhance the antifungal activity of the protein against Candida albicans biofilm. ExperimentsPAF-polyphosphate (PP) nanoparticles (PAF-PP NPs) were obtained through ionic gelation. The resulting NPs were characterized in terms of their particle size, size distribution and zeta potential. Cell viability and hemolysis studies were carried out in vitro on human foreskin fibroblasts (Hs 68 cells) and human erythrocytes, respectively. Enzymatic degradation of NPs was investigated by monitoring release of free monophosphates in the presence of isolated as well as C. albicans-derived phosphatases. In parallel, shift in zeta potential of PAF-PP NPs as a response to phosphatase stimuli was determined. Diffusion of PAF and PAF-PP NPs through C. albicans biofilm matrix was analysed by fluorescence correlation spectroscopy (FCS). Antifungal synergy was evaluated on C. albicans biofilm by determining the colony forming units (CFU). FindingsPAF-PP NPs were obtained with a mean size of 300.9 ± 4.6 nm and a zeta potential of −11.2 ± 2.8 mV. In vitro toxicity assessments revealed that PAF-PP NPs were highly tolerable by Hs 68 cells and human erythrocytes similar to PAF. Within 24 h, 21.9 ± 0.4 μM of monophosphate was released upon incubation of PAF-PP NPs having final PAF concentration of 156 μg/ml with isolated phosphatase (2 U/ml) leading to a shift in zeta potential up to −0.7 ± 0.3 mV. This monophosphate release from PAF-PP NPs was also observed in the presence of C. albicans-derived extracellular phosphatases. The diffusivity of PAF-PP NPs within 48 h old C. albicans biofilm matrix was similar to that of PAF. PAF-PP NPs enhanced antifungal activity of PAF against C. albicans biofilm decreasing the survival of the pathogen up to 7-fold in comparison to naked PAF. In conclusion, phosphatase-degradable PAF-PP NPs hold promise as nanocarriers to augment the antifungal activity of PAF and enable its efficient delivery to C. albicans cells for the potential treatment of Candida infections.

ROP16-mediated activation of STAT6 enhances cyst development of type III Toxoplasma gondii in neurons.

Toxoplasma gondii establishes a long-lived latent infection in the central nervous system (CNS) of its hosts. Reactivation in immunocompromised individuals can lead to life threatening disease. Latent infection is driven by the ability of the parasite to convert from the acute-stage tachyzoite to the latent-stage bradyzoite which resides in long-lived intracellular cysts. While much work has focused on the parasitic factors that drive cyst development, the host factors that influence encystment are not well defined. Here we show that a polymorphic secreted parasite kinase (ROP16), that phosphorylates host cell proteins, mediates efficient encystment of T. gondii in a stress-induced model of encystment and primary neuronal cell cultures (PNCs) in a strain-specific manner. Using short-hairpin RNA (shRNA) knockdowns in human foreskin fibroblasts (HFFs) and PNCs from transgenic mice, we determined that ROP16's cyst enhancing abilities are mediated, in part, by phosphorylation-and therefore activation-of the host cell transcription factor STAT6. To test the role of STAT6 in vivo, we infected wild-type (WT) and STAT6KO mice, finding that, compared to WT mice, STAT6KO mice have a decrease in CNS cyst burden but not overall parasite burden or dissemination to the CNS. Finally, we found a similar ROP16-dependent encystment defect in human pluripotent stem cell-derived neurons. Together, these findings identify a host cell factor (STAT6) that T. gondii manipulates in a strain-specific manner to generate a favorable encystment environment.

Inhibition of Cytomegalovirus by Pentacta pygmaea Fucosylated Chondroitin Sulfate Depends on Its Molecular Weight.

Many viruses attach to host cells by first interacting with cell surface proteoglycans containing heparan sulfate (HS) glycosaminoglycan chains and then by engaging with specific receptor, resulting in virus entry. In this project, HS-virus interactions were targeted by a new fucosylated chondroitin sulfate from the sea cucumber Pentacta pygmaea (PpFucCS) in order to block human cytomegalovirus (HCMV) entry into cells. Human foreskin fibroblasts were infected with HCMV in the presence of PpFucCS and its low molecular weight (LMW) fractions and the virus yield at five days post-infection was assessed. The virus attachment and entry into the cells were visualized by labeling the purified virus particles with a self-quenching fluorophore octadecyl rhodamine B (R18). The native PpFucCS exhibited potent inhibitory activity against HCMV specifically blocking virus entry into the cell and the inhibitory activities of the LMW PpFucCS derivatives were proportional to their chain lengths. PpFucCS and the derived oligosaccharides did not exhibit any significant cytotoxicity; moreover, they protected the infected cells from virus-induced lytic cell death. In conclusion, PpFucCS inhibits the entry of HCMV into cells and the high MW of this carbohydrate is a key structural element to achieve the maximal anti-viral effect. This new marine sulfated glycan can be developed into a potential prophylactic and therapeutic antiviral agent against HCMV infection.

In Vitro Evaluation Reveals Effect and Mechanism of Artemether against Toxoplasma gondii.

Due to the limited effectiveness of existing drugs for the treatment of toxoplasmosis, there is a dire need for the discovery of new therapeutic options. Artemether is an important drug for malaria and several studies have indicated that it also exhibits anti-T. gondii activity. However, its specific effect and mechanisms are still not clear. To elucidate its specific role and potential mechanism, we first evaluated its cytotoxicity and anti-Toxoplasma effect on human foreskin fibroblast cells, and then analyzed its inhibitory activity during T. gondii invasion and intracellular proliferation. Finally, we examined its effect on mitochondrial membrane potential and reactive oxygen species (ROS) in T. gondii. The CC50 value of artemether was found to be 866.4 μM, and IC50 was 9.035 μM. It exhibited anti-T. gondii activity and inhibited the growth of T. gondii in a dose-dependent manner. We also found that the inhibition occurred primarily in intracellular proliferation, achieved by reducing the mitochondrial membrane integrity of T. gondii and stimulating ROS production. These findings suggest that the mechanism of artemether against T. gondii is related to a change in the mitochondrial membrane and the increase in ROS production, which may provide a theoretical basis for optimizing artemether derivatives and further improving their anti-Toxoplasma efficacy.