Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba.

BACKGROUND: Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba. METHODOLOGY/PRINCIPAL FINDINGS: Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF. CONCLUSIONS: The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.

Discovering SNP-disease relationships in genome-wide SNP data using an improved harmony search based on SNP locus and genetic inheritance patterns.

Advances in high-throughput sequencing technologies have made it possible to access millions of measurements from thousands of people. Single nucleotide polymorphisms (SNPs), the most common type of mutation in the human genome, have been shown to play a significant role in the development of complex and multifactorial diseases. However, studying the synergistic interactions between different SNPs in explaining multifactorial diseases is challenging due to the high dimensionality of the data and methodological complexities. Existing solutions often use a multi-objective approach based on metaheuristic optimization algorithms such as harmony search. However, previous studies have shown that using a multi-objective approach is not sufficient to address complex disease models with no or low marginal effect. In this research, we introduce a locus-driven harmony search (LDHS), an improved harmony search algorithm that focuses on using SNP locus information and genetic inheritance patterns to initialize harmony memories. The proposed method integrates biological knowledge to improve harmony memory initialization by adding SNP combinations that are likely candidates for interaction and disease causation. Using a SNP grouping process, LDHS generates harmonies that include SNPs with a higher potential for interaction, resulting in greater power in detecting disease-causing SNP combinations. The performance of the proposed algorithm was evaluated on 200 synthesized datasets for disease models with and without marginal effect. The results show significant improvement in the power of the algorithm to find disease-related SNP sets while decreasing computational cost compared to state-of-the-art algorithms. The proposed algorithm also demonstrated notable performance on real breast cancer data, showing that integrating prior knowledge can significantly improve the process of detecting disease-related SNPs in both real and synthesized data.

Purified Native Protein Extracted from the Venom of Agelena orientalis Attenuates Memory Defects in the Rat Model of Glutamate-Induced Excitotoxicity.

Spider venom contains various peptides and proteins, which can be used for pharmacological applications. Finding novel therapeutic strategies against neurodegenerative diseases with the use of purified peptides and proteins, extracted from spiders can be greatly precious. Neurodegenerative diseases are rapidly developing and expanding all over the world. Excitotoxicity is a frequent condition amongst neuro-degenerative disorders. This harmful process is usually induced through hyper-activation of N-Methyl-D-Aspartate (NMDA) receptor, and P/Q-type voltage-gated calcium channels (VGCCs). The omega-agatoxin-Aa4b is a selective and strong VGCCblocker. This study aimed to investigate the effects of this blocker on the NMDA-induced memory and learning defect in rats. For this purpose, nineteen spiders of the funnel-weaver Agelena orientalis species were collected. The extracted venom was lyophilized andpurified through gel-filtration chromatography, and capillary electrophoresis techniques. Subsequently, mass spectrometry (HPLC-ESI-MS) was used for identification of this bio-active small protein. Afterward, the effect of the omega-agatoxin-Aa4b (2 µg, intra-cornu ammonis-3 of the hippocampus) on the NMDA-induced learning and memory deficits in rats was evaluated. Learning and memory performances were evaluated by the use of passive avoidance test. For synaptic quantification and memory function the amount of calcium/calmodulin-dependent protein kinase ІІ (CaCdPKІІ) gene expression was measured using the Real-time PCR technique. To compare the experimental groups, hematoxylin and eosin (H&E) staining of hippocampus tissues was performed. Our results rendered that the omega-Agatoxin-Aa4b treatment can ameliorate and reverse the learning and memory impairment caused by NMDA-induced excitotoxicity in rat hippocampus.

Induction of Tyrosine Hydroxylase Gene Expression in Embryonal Carcinoma Stem Cells Using a Natural Tissue-Specific Inducer.

A large number of studies have focused on the generation of dopaminergic neurons from pluripotent cells. Differentiation of stem cells into distinct cell types is influenced by tissue-specific microenvironment. Since, central nervous system undergoes further development during postnatal life, in the present study neonatal rat brain tissue extract (NRBE) was applied to direct the differentiation of embryonal carcinoma stem cell line, P19 into dopaminergic (DA) phenotypes. Additionally, a neuroprotective drug, deprenyl was used alone or in combination with the extract. Results from morphological, immunofluorescence, and qPCR analyses showed that during a period of one to three weeks, a large percentage of stem cells were differentiated into neural cells. The results also indicated the greater effect of NRBE on the differentiation of the cells into tyrosine hydroxylase-expressing cells. MS analysis of NRBE showed the enrichment of gene ontology terms related to cell differentiation and neurogenesis. Network analysis of the studied genes and some DA markers resulted in the suggestion of potential regulatory candidates such as AVP, ACHE, LHFPL5, and DLK1 genes. In conclusion, NRBE as a natural native inducer was apparently able to simulate the brain microenvironment and support neural differentiation of P19 cells.

Curcumin nanoemulsion as a novel chemical for the treatment of acute and chronic toxoplasmosis in mice.

BACKGROUND: The aim of this study was to prepare curcumin nanoemulsion (CR-NE) to solve the problems associated with poor water solubility and low bioavailability of CR and to test its efficiency in the treatment of acute and chronic toxoplasmosis in mouse models. MATERIALS AND METHODS: CR-NE 1% was prepared using spontaneous emulsification by soybean as oil phase; a mixture of Tween 80 and Tween 85 as surfactant; ethanol as cosurfactant and distilled water. Particle size and zeta potential of NE were assessed using Nano-ZS90 dynamic light scattering. Stability testing of NE was assessed after storage for 2 months at room temperature. In vivo experiments were carried out using 50 BALB/c mice inoculated with virulent RH strain (type I) and 50 BALB/c mice inoculated with avirulent Tehran strain (type II) of Toxoplasma gondii and treated with CR-NE (1% w/v), CR suspension (CR-S, 1% w/v), and NE without CR (NE-no CR). RESULTS: The mean particle size and zeta potential of CR-NE included 215.66±16.8 nm and -29.46±2.65 mV, respectively, and were stable in particle size after a three freeze-thaw cycle. In acute phase experiment, the survival time of mice infected with RH strain of T. gondii and treated with CR-NE extended from 8 to 10 days postinoculation. The differences were statistically significant between the survival time of mice in CR-NE-treated group compared with negative control group (P<0.001). Furthermore, CR-NE significantly decreased the mean counts of peritoneum tachyzoites from 5,962.5±666 in negative control group to 627.5±73 in CR-NE-treated mice (P<0.001). Growth inhibition rates of tachyzoites in peritoneum of mice receiving CR-NE, CR-S, and NE-no CR included 90%, 21%, and 11%, respectively, compared with negative control group. In chronic phase experiment, the average number and size of tissue cysts significantly decreased to 17.2±15.6 and 31.5±6.26 µm, respectively, in mice inoculated with bradyzoites of T. gondii Tehran strain and treated with CR-NE compared with that in negative control group (P<0.001). Decrease of cyst numbers was verified by downregulation of BAG1 in treatment groups compared with negative control group with a minimum relative expression in CR-NE (1.12±0.28), CR-S (11.76±0.87), and NE-no CR (14.67±0.77), respectively, (P<0.001). CONCLUSION: Results from the current study showed the potential of CR-S and CR-NE in treatment of acute and chronic toxoplasmosis in mouse models for the first time. However, CR-NE was more efficient than CR-S, and it seems that CR-NE has a potential formula for the treatment of acute and chronic toxoplasmosis, especially in those with latent bradyzoites in brain.

The combined effects of three-dimensional cell culture and natural tissue extract on neural differentiation of P19 embryonal carcinoma stem cells.

Tissue engineering, as a novel transplantation therapy, aims to create biomaterial scaffolds resembling the extracellular matrix in order to regenerate the damaged tissues. Adding bioactive factors to the scaffold would improve cell-tissue interactions. In this study, the effect of chitosan polyvinyl alcohol nanofibres containing carbon nanotube scaffold with or without active bioglass (BG+ /BG- ), in combination with neonatal rat brain extract on cell viability, proliferation, and neural differentiation of P19 embryonic carcinoma stem cells was investigated. To induce differentiation, the cells were cultured in α-MEM supplemented with neonatal rat brain extract on the scaffolds. The expression of undifferentiated stem cell markers as well as neuroepithelial and neural-specific markers was evaluated and confirmed by real-time Reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence procedures. Finally, the three-dimensional (3D) cultured cells were implanted into the damaged neural tubes of chick embryos, and their fates were followed in ovo. Based on the histological and immunofluorescence observations, the transplanted cells were able to survive, migrate, and penetrate into the host embryonic tissues. Gene network analysis suggested the possible involvement of neurotransmitters as a downstream target of synaptophysin and tyrosine hydroxylase. Overall, the results of this study indicated that combining the effects of 3D cell culture and natural brain tissue extract can accelerate the differentiation of P19 embryonic carcinoma cells into neuronal phenotype cells.

Larvicidal Activity of Essential Oil of Syzygium aromaticum (Clove) in Comparison with Its Major Constituent, Eugenol, against Anopheles stephensi.

BACKGROUND: In this study, larvicidal activity of clove essential oil (EO), as a green and relatively potent larvicide, was compared with its main constituent, Eugenol, against Anopheles stephensi. METHODS: High-performance liquid chromatography (HPLC) was used to determine the amount of eugenol, major constituent of clove EO. In addition, larvicidal activity of clove EO and eugenol was evaluated against An. stephensi. RESULTS: The amount of eugenol in clove EO was determined as 67% using HPLC analysis. LC50 and LC90 of clove EO (57.49 and 93.14ppm, respectively) were significantly lower than those of eugenol (86.96 and 128.18 ppm, respectively). CONCLUSION: EO showed more effective than its major component. Considering the lower cost of the essential oil and lower risk in occurrence of resistance in larvae, use of clove EO is preferred as larvicide in comparison with eugenol, against An. stephensi.

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation.

Towards DNA methylation detection using biosensors.

DNA methylation, a stable and heritable covalent modification which mostly occurs in the context of a CpG dinucleotide, has great potential as a biomarker to detect disease, provide prognoses and predict therapeutic responses. It can be detected in a quantitative manner by many different approaches both genome-wide and at specific gene loci, in various biological fluids such as urine, plasma, and serum, which can be obtained without invasive procedures. The current, classical methods are effective in studying DNA methylation patterns, however, for the most part; they have major drawbacks such as expensive instruments, complicated and time consuming protocols as well as relatively low sensitivity, and high false positive rates. To overcome these obstacles, great efforts have been made toward the development of reliable sensor devices to solve these limitations, providing sensitive, fast and cost-effective measurements. The use of biosensors for DNA methylation biomarkers has increased in recent years, because they are portable, simple, rapid, and inexpensive which offers a straightforward way to detect methylated biomarkers. In this review, we give an overview of the conventional techniques for the detection of DNA methylation and then will focus on recent advances in biosensor based methylation detection that eliminate bisulfite conversion and PCR amplification.

Induction of pancreatic ß cell gene expression in mesenchymal stem cells.

Transdifferentiattion potential of mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) has been suggested recently. In our recent works, we demonstrated the high performance of mouse neonate pancreas extract (MPE) in the production of functional IPCs from carcinoma stem cells. In this study, MPE was used to generate IPCs from MSCs without any genetic manipulation. To this end, bone marrow MSCs were isolated and characterized. In order to differentiate, MSCs were induced by selection of nestin-expressing cells and treatment with 100 µg/mL MPE. Morphological features of the differenti-ated cells were confirmed by dithizone staining. Immunoreactivity to insulin receptor beta, proinsulin, insulin, and C-peptide was observed by immunoflourescence. We also quantified glucose-dependent insulin production and secretion by ELISA. Real-time PCR indicated the expressions of ß cell-related genes, PDX-1, INS1, INS2, EP300, and CREB1, in IPC cells. Possible pathways governed by CREB1, EP300, and PDX-1 transcription factors in differentiation of MSCs to IPCs were determined based on Gene Set Enrichment (GSE) approach at P = 0.05. Pathway discovery highlighted the negative regulatory effects of MIR124-2, HDAC5 protein, REST, and NR0B2 transcription factors on expression of CREB1, EP300, and PDX-1 and inhabitation of IPC differentiations. In contrast, a crosstalk between FOXA2 and TCF7L2 transcription factors, DNA-PK complex, KAT2B protein positively interacting with PDX-1, CREB1, EP300 resulted in the induction of IPC and following insulin production. In conclusion, we report an efficient, simple, and easy method for production of functional IPCs from MSCs by MPE treatment.

Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium.

The ability of embryonal carcinoma )EC (stem cells to generate insulin-producing cells (IPCs) is still unknown. We examined the trophic effects of pancreas-conditioned medium (PCM) on in vitro production of IPCs. Initially, P19 EC cells were characterized by the expression of stem cell markers, Oct3/4, Sox-2 and Nanog. To direct differentiation, P19-derived embryoid bodies (EBs) were induced by selection of nestin-positive cells and treatment with different concentrations of PCM. Morphological studies documented the presence of islet-like cell IPCs clusters. The differentiated cells were immunoreactive for ß cell-specific proteins, including insulin, proinsulin, C-peptide and insulin receptor-ß. The expression of genes related to pancreatic ß cell development and function (PDX-1, INS1, INS2, EP300 and CREB1) was confirmed by qPCR. During differentiation, the expression of EP300 and CREB1 increased by 2.5 and 3.1 times, respectively. In contrast, a sharp decrease in the expression of Oct3/4, Sox-2 and Nanog by 4, 1.5 and 1.5 times, respectively, was observed. The differentiated cells were functionally active, synthesizing and secreting insulin in a glucose-regulated manner. Network prediction highlighted crosstalk between PDX-1 transcription factor and INS2 ligand in IPC generation and revealed positive regulatory effects of EP300, CREB1, PPARA, EGR, KIT, GLP1R, and PKT2 on activation of PDX-1 and INS2. This is the first report of the induction of IPC differentiation from EC cells by using neonate mouse PCM. Since P19 EC cells are widely available, easily cultured without feeders and do not require special growth conditions, they would provide a valuable tool for studying pancreatic ß cell differentiation and development. Copyright © 2016 John Wiley & Sons, Ltd.

Efficient and simple production of insulin-producing cells from embryonal carcinoma stem cells using mouse neonate pancreas extract, as a natural inducer.

An attractive approach to replace the destroyed insulin-producing cells (IPCs) is the generation of functional ß cells from stem cells. Embryonal carcinoma (EC) stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1-2 weeks old mouse pancreas extract (MPE). Since, mouse pancreatic islets undergo further remodeling and maturation for 2-3 weeks after birth, we hypothesized that the mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 µg/ml). The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic ß cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 µg/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1) during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic ß cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid production of IPCs for transplantation.

Effects of selegiline, a monoamine oxidase B inhibitor, on differentiation of P19 embryonal carcinoma stem cells, into neuron-like cells.

Selegiline, the irreversible inhibitor of monoamine oxidase B (MAO-B), is currently used to treat Parkinson's disease. However, the mechanism of action of selegiline is complex and cannot be explained solely by its MAO-B inhibitory action. It stimulates gene expression, as well as expression of a number of mRNAs or proteins in nerve and glial cells. Direct neuroprotective and anti-apoptotic actions of selegiline have previously been observed in vitro. Previous studies showed that selegiline can induce neuronal phenotype in cultured bone marrow stem cells and embryonic stem cells. Embryonal carcinoma (EC) cells are developmentally pluripotene cells which can be differentiated into all cell types under the appropriate conditions. The present study was carried out to examine the effects of selegiline on undifferentiated P19 EC cells. The results showed that selegiline treatment had a dramatic effect on neuronal morphology. It induced the differentiation of EC cells into neuron-like cells in a concentration-dependent manner. The peak response was in a dose of selegiline significantly lower than required for MAO-B inhibition. The differentiated cells were immunoreactive for neuron-specific proteins, synaptophysin, and ß-III tubulin. Stem cell therapy has been considered as an ideal option for the treatment of neurodegenerative diseases. Generation of neurons from stem cells could serve as a source for potential cell therapy. This study suggests the potential use of combined selegiline and stem cell therapy to improve deficits in neurodegenerative diseases.

Stable suppression of gene expression by short interfering RNAs targeted to promoter in a mouse embryonal carcinoma stem cell line.

RNA interference (RNAi) can induce gene silencing via two pathways: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). The mediators of gene inactivation in both pathways are 21-bp small interfering RNAs (siRNAs) generated from longer double-stranded RNA (dsRNA). PTGS involves siRNA-mediated targeting and degradation of mRNA. However, siRNAs induce TGS via DNA methylation at the targeted promoter. Synthetic siRNAs can induce loss of gene activity comparable to long dsRNA. The limitation of this method is that the transfected synthetic siRNA works for only a few days. In this study, we tested the RNAi response to siRNA (PTGS pathway) by using a plasmid containing an enhanced green fluorescent protein (eGFP) gene as a target as well as a plasmid creates siRNA transcript, in a form of a hairpin, against eGFP gene. To investigate TGS pathway via RNAi, we also used a plasmid creates hairpin siRNA transcript against pgk-1 promoter. The data presented here indicated long-lasting inhibition in expression of eGFP and puromycin genes, both under the control of the murine Pgk-1 promoter. However, Southern blot analysis showed no methylation in pgk-1 promoter.

Enhancement of RNA interference effect in P19 EC cells by an RNA-dependent RNA polymerase.

BACKGROUND: RNA interference (RNAi) is a phenomenon uses double-stranded RNA (dsRNA) to specifically inhibit gene expression. The non-specific silencing caused by interferon response to dsRNA in mammalian cells limits the potential of utilizing RNAi to study gene function. Duplexes of 21-nucleotide short interfering dsRNA (siRNA) inhibit gene expression by RNAi. In some organisms, siRNA can also function as a primer converting mRNA into dsRNA that are further cleaved to produce more siRNA. This activity involves the enzyme RNA-dependent RNA polymerase (RdRP). There are no known RdRP involved in RNAi in mammals. By using an RdRP from Caenorhabditis elegance named ego-1, investigators intend to enhance RNAi effect in mammalian cells. The aims of this project were: 1) to investigate the efficiency of siRNA to enhanced green fluorescent protein (eGFP) gene silencing and 2) to enhance the RNAi effect. METHODS: We used a vector-based siRNA to target eGFP. Also we used a vector expressing ego-1 to test for a possible amplification effect of RNAi. The expression of eGFP in the cells was detected by using fluorescent microscopy, flowcytometry and Western-blotting. RESULTS: Transfection of the plasmid into P19 cells significantly decreased eGFP fluorescence. In addition, eGFP protein was reduced. Preliminary data suggested that the presence of ego-1 enhanced the RNAi effect. CONCLUSION: The results indicated that use of hairpin siRNA expression vectors for RNAi is a promising method to inhibition of gene expression in mammalian cells. Also, introducing RdRP enzyme to mammalian cells might amplify the RNAi effect in the cells.