Assessment of the neuroprotective effect of Cocos nucifera L. oil on learning and behavior impairment in ovariectomized rats.

Objective: The current study aimed to investigate whether Cocos nucifera L. oil (CO) is effective on menopause-related memory dysfunction in ovariectomized (OVX) rats. Materials and Methods: Fifty healthy female Wistar rats were randomly selected and classified into five groups as control, OVX rats, and three OVX groups of rats which received three different doses (100, 200, and 400 mg/kg/day) of CO for five consecutive weeks by gavage. To assess the effect of CO, neurobehavioral tests such as Morris water maze (MWM) and Passive avoidance (PA) were done and then the animals were sacrificed to remove cortical and hippocampal tissues for biochemical analysis. Results: In both behavioral tests including MWM and PA, treatment with CO particularly two higher doses of 200, and 400 mg/kg demonstrated significant improvement in comparison with OVX group. Furthermore, antioxidant biomarkers such as total thiol content, catalase and superoxide dismutase (SOD) activities were significantly higher in the OVX-CO groups versus the OVX group. On the contrary, malondialdehyde (MDA) concentration as an oxidative stress biomarker was remarkably lower in the OVX-CO200 and 400 mg groups than the OVX group. Conclusion: The present study demonstrated the significant improvement of CO on learning and memory impairment induced by ovariectomy. Although the exact mechanism needs further investigation, it might have occurred due to the anti-oxidative effect of CO.

The genetic factors contributing to the risk of cleft lip-cleft palate and their clinical utility.

Cleft lip and cleft palate (CL/P) are among the most common congenital malformations in neonates and have syndromic or nonsyndromic forms. Nonsyndromic forms of malformation are being reported to be associated with chromosomal DNA modification by teratogenic exposure and to complex genetic contributions of multiple genes. Syndromic forms are shown to be related to chromosomal aberrations or monogenic diseases. There is a growing body of data illustrating the association of several genes with risk of developing this malformation, including genetic defects in T-box transcription factor-22 (TBX22), interferon regulatory factor-6 (IRF6), and poliovirus receptor-like-1 (PVRL1), responsible for X-linked cleft palate, cleft lip/palate-ectodermal dysplasia syndrome, and Van der Woude and popliteal pterygium syndromes, respectively. Genetic variants in MTR, PCYT1A, ASS1, SLC 25A13, GSTM1, GSTT1, SUMO1 BHMT1, and BHMT2 are being reported to be linked with CL/P risk. The etiology of nonsyndromic CLP is still remained to be unknown, although mutations in candidate genes have been found. Here, we provide an overview about the potential variants to be associated with CL/P for identification of the relative risk of CLP with respect to the basis of genetic background and environmental factors (e.g., dietary factors, alcohol use).

Investigating Efficacy of Three DNA-Aptamers in Targeted Plasmid Delivery to Human Prostate Cancer Cell Lines.

Selection of targeted and efficient carriers to deliver drugs and genes to cells and tissues is still a major challenge and to overcome this obstacle, aptamers conjugated to nanoparticles have been broadly examined. To assess whether polycation of aptamers can improve plasmid delivery efficacy, we investigated the effect of three DNA-aptamers (AS1411, WY-5a, and Sgs-8) conjugated to branched polyethylenimine (b-PEI; MW ∼25 kDa) with different combinations of gene (plasmid) for delivery to prostate cancer cell lines (DU145 and PC3). According to transfection assessments, the dual conjugation of aptamers (AS:WY) with b-PEI produced the best results and increased the efficiency of plasmid delivery to up to three folds compared to unmodified PEI. Surprisingly, triple aptamer arrangement not only reduced transfection ability but also showed cytotoxicity. While our results demonstrated potential synergistic effects of AS1411 and WY-5a aptamers for gene delivery, it is important to note that the present evidence relies on the aptamer and cell types.

Synthesis and characterization of polyethyleneimine-terminated poly(ß-amino esters) conjugated with pullulan for gene delivery.

Cationic polymers endowed with a flexible system for condensing DNA, are regarded as effective materials for gene delivery. The synthesis of poly(ß-amino esters) (pBAEs) based on 1,4-butanediol diacrylate-ethanolamine monomer (1.2:1 molar ratio) and 1,4-butanediol diacrylate-ethylene diamine (1:2 molar ratio) was carried out and modification with 1800 Da polyethyleneimine (PEI) at different weight ratios (3 and 1) as well as conjugation with pullulan in various weight ratios of (0.0625, 0.125, 0.25, and 1) was performed. Gel-retardation assay demonstrated that the synthesized polymers were able to condense DNA at low carrier/plasmid (C/P) ratios. The polyplexes with ratio 3 of PEI (pß1/PEI3) were restricted in all C/P ratios and the polyplexes of pß1/PEI3/pull0.125 were condensed at C/P ratios higher than 0.5. The particle size at C/P was approximately about 200 nm with a positive surface charge. The presence of the pullulan in the structure of the synthesized pBAEs could be effective in reducing toxicity of the base polymer. Highest metabolic activity was dedicated to C/P2 of pß2/PEI3/pull0.125 with 80.6% viability. Furthermore, the most efficient gene reporter delivery was seen at C/P ratio of 6 in pß2/PEI3/pull0.125 nanoparticles. Therefore, pullulan grafting could enhance the cellular response of cells in terms of cytotoxicity and transfection efficiency.

Biosensors, microfluidics systems and lateral flow assays for circulating microRNA detection: A review.

MicroRNAs (miRNAs) are short RNA sequences found in eukaryotic cells and they are involved in several diseases pathogenesis including different types of cancers, metabolic and cardiovascular disorders. Thus, miRNAs circulating in serum, plasma, and other body fluids are employed as biomarkers for diagnostic and prognostic purposes and in assessment of drug response. Thus, various methods have been developed for detection of miRNAs including northern blotting, reverse transcriptase polymerase chain reaction (RT-PCR), next-generation sequencing, microarray, and isothermal amplification that are recognized as traditional methods. Considering the importance of early diagnosis and treatment of miRNAs-related diseases, development of simple, one-step, sensitive methods is of great interest. Nowadays developing technologies including lateral flow assay, biosensors (optical and electrochemical) and microfluidic systems which are simple fast responding, user-friendly, and are enabled with visible detection have gained considerable attention. This review briefly discusses miRNAs detection' methods, with a particular focus on lateral flow assay, biosensors, and microfluidic systems as novel and practical procedures.

Biological Properties, Current Applications and Potential Therapeautic Applications of Brevinin Peptide Superfamily.

The Brevinin peptides are antimicrobial agents obtained from frog skin secretions. Brevinin-2R has attracted many attentions due to its very low hemolytic activity, cationic property, and high affinity to cancer cells. Moreover, it has shown little toxicity against normal mammalian cells, while having killed several tumor cell lines by activation of lysosome-mitochondrial death pathway. In this review, we introduced the Brevinin superfamily with a focus on its therapeutic applications. Next, some unique properties of Brevinins were briefly discussed, including their ability to stimulate insulin secretion, dendritic cell maturation, and wound healing. In this context, we also provide information about the decoration of nanoparticles, such as cerium nano-oxide, by Brevinins. Finally, we addressed their potential for anti-tumor and drug design applications.

Viral vector mimicking and nucleus targeted nanoparticles based on dexamethasone polyethylenimine nanoliposomes: Preparation and evaluation of transfection efficiency.

Non-viral vectors such as polymers and liposomes have been used as gene delivery systems to overcome intrinsic problems of viral vectors, but transfection efficiency of these vectors is lower than viral vectors. In the present study, we tried to design non-viral gene delivery vectors that mimic the viral vectors using the benefits of both cationic liposomes and cationic polymer vectors along with targeting glucocorticoid receptors to enhance cellular trafficking of vectors. Cationic liposomes containing DOTAP and cholesterol were prepared by thin-film hydration following extrusion method. Dexamethasone mesylate was synthesized and then conjugated to polyethylenimine through a one-step reaction. A novel gene delivery system, Lipopolyplex was developed by premixing liposome and different molecular weight of bPEI-Dexa as carriers followed by addition of plasmid at three different carrier/pDNA (C/P) weight ratios. The resulted complexes were characterized for their size, zeta potential and ability of DNA condensation. Transfection efficiency of vectors in neuro2A was determined by Luciferase reporter gene assay. Also, the toxicity of gene carriers was investigated in this cell line. Mean particle size of prepared complexes was less than 200 nm and there was no significant difference in their size by increasing the molecular weight of PEIs. All complexes had positive surface charge. Complete condensation of DNA was occurred at C/P ratio of one for all complexes. Lipopolyplexes were more efficient than polyplexes and lipoplexes alone and transfection efficiency was improved by adding dexamethasone. The complexes containing liposome, PEI 10 kDa and dexamethasone (PEI10:Lipo:Dexa(0.05)) had the highest transfection activity about 40-fold and 3.6-fold in comparison with PEI10 and PEI10:Lipo, respectively. Furthermore, the non-viral vectors described in this study showed low cytotoxicity. The results of this study confirmed that PEI in combination with liposome forms lipopolyplex with low toxicity and enhanced transfection efficiency. Moreover, using dexamethasone, in combination with lipopolyplex might be useful to increase the gene delivery potential of these lipopolyplexes.

Aptamer-targeted delivery of Bcl-xL shRNA using alkyl modified PAMAM dendrimers into lung cancer cells.

RNAi-based gene therapy has been recently considered as a promising approach against cancer. Targeted delivery of drug, gene or therapeutic RNAi-based systems to tumor cells is one of the important issues in order to reduce side effects on normal cells. Several strategies have been developed to improve the safety and selectivity of cancer treatments including antibodies, peptides and recently aptamers with various attractive characteristics including higher target specificity, affinity and reduced toxicity. Here we described a novel targeted delivery platform comprising modified PAMAM with 10-bromodecanoic acid (10C) and 10C-PEG for improvement of transfection efficiency, AS1411 aptamer for targeting nucleolin ligand on target cancer cells and shRNA plasmid for specific knockdown of Bcl-xL protein. Modified vector could significantly improve the transfection efficiency even after covalent or non-covalent aptamer binding compared to the non-targeted vector in A549 cells. The results of gene silencing and apoptosis assay indicated that our targeted shRNA delivery system could efficiently down-regulate the Bcl-xL expression up to 25% and induce 14% late apoptosis in target cancer cells with strong cell selectivity. This study proposed a novel targeted non-viral system for shRNA-mediated gene-silencing in cancer cells.

PAMAM-pullulan conjugates as targeted gene carriers for liver cell.

Targeted nano-carriers are highly needed to promote nucleic acid delivery into the specific cell for therapeutic approaches. Pullulan as a linear carbohydrate has an intrinsic liver targeting property interacting with asialoglycoprotein receptor (ASGPR) found on liver cells. In the present study, we developed polyamidoamine (PAMAM)-pullulan conjugates and investigated their targeting activity in delivering gene into liver cells. The particle size, zeta potential, buffering capacity and ethidium bromide exclusion assays of the conjugates were evaluated. The cytotoxicity and transfection efficiency of new derivatives were assessed following in vitro transfection of HepG2 (receptor positive) and N2A (receptor negative) cell lines. Size of conjugated polymers ranged between 118 and 184 nanometers and their cytotoxicity were similar to PAMAM. Among six produced nanocarriers, G4PU4 and G5PU4 enhanced transfection efficiency in HepG2 cells compared to unmodified PAMAM. Therefore, the PAMAM-pullulan derivatives seem to improve delivery of nucleic acids into the liver cells expressing asialoglycoprotein receptor with minimal transfection in non-targeted cells.

Gene delivery to neuroblastoma cells by poly (l-lysine)-grafted low molecular weight polyethylenimine copolymers.

Polyethylenimine (PEI) and poly (l-lysine) (PLL) are among the most investigated non-viral gene carriers. However, both polymers contain deficiencies that restrict their applications. In the present study, we synthesized PLL-alkyl-PEI conjugates via 6-carbon alkyl linker and investigated their possible advantages in gene delivery. Four PLL copolymers were synthesized with different molecular weights and ratios of PEI. The physiochemical properties of synthesized conjugates such as size, zeta potential, DNA condensation ability, buffering capacity and cytotoxicity were investigated. Renilla luciferase assay was employed to evaluate the gene transfection efficiency of pDNA-polymer to Neuro2A cell line. DNA condensation and particle size measurements showed that new PLL-PEI conjugates could form polyplexes in nano-scale size in the range of 99-122 nm and were able to condense DNA at low concentration. While cytotoxicity reduced in some groups, the transfection efficiency increased about 2.8 and 4 fold as compared to the unmodified PEI 1.8 kDa and 10 kDa, respectively. The results of the present study showed that the chemical modifications of PEI with PLL could significantly improve transfection efficiency and PLP10-10% shows the most promise as a new gene carrier.

Preparation and evaluation of polyethylenimine-functionalized carbon nanotubes tagged with 5TR1 aptamer for targeted delivery of Bcl-xL shRNA into breast cancer cells.

In this study, single-walled carbon nanotubes (SWCNTs) were covalently attached to poly(ethylene glycol) (PEG) and polyethylenimine (PEI) 10 kDa, or its derivatives, to fabricate efficient carriers for gene delivery. PEI 10 kDa was modified by alkylcarboxylation of its primary amines with a series of ω-bromo-alkylcarboxylic acids to provide a range of vectors with increased lipophilicity. PEI 10 kDa or its alkylcarboxylate derivatives were conjugated to SWCNT-PEG to develop vectors possessing effective DNA condensation ability which can interact with cell membrane via both nano-needle mechanism and electrostatic interactions produced by SWCNT and PEI, respectively. The results demonstrated that SWCNT-PEG-PEI and SWCNT-PEG-derivatives of PEI could condense DNA into particle size less than 150 nm with positive surface charges between 6.3-30.8 mV. To improve the antitumor efficacy, we developed a targeted gene delivery system using a 5 TR1 aptamer. The most efficient vector, which was prepared by attachment of SWCNT-PEG to modified PEI 10 kDa with 10-bromodecanoic acid (10%), showed 8.5-10 folds enhancement in transfection activity at C/P ratio 6 as compared to the gold standard PEI 25 kDa at C/P ratio of 0.8. We also showed that the selected polyplex could efficiently and selectively transfer plasmid shRNA to MUC1 positive cells.

Cellular delivery of shRNA using aptamer-conjugated PLL-alkyl-PEI nanoparticles.

Introduction of an efficient gene delivery vector is still the main challenge of gene therapy. Both polyethylenimine (PEI) and poly(l-lysine) (PLL) comprise disadvantages which limited their application. To explore whether their deficiencies could be compensated by preparing copolymers consisting of both PLL and PEI, we generated several combinations of PLL-alkyl-PEI copolymers conjugated to aptamer and evaluated their both gene delivery efficiency and down-regulation of Bcl-XL, an anti-apoptotic gene, in lung cancer cell line. PLL was conjugated to either 10% or 50% of PEI by grafting different percentages of PEI to alkylated-PLL as core. The properties of modified polymers including size, surface charge density, DNA condensation ability, buffering capacity and cytotoxicity were evaluated. According to transfection results, aptamer conjugated PLL-alkyl-10%-PEI (PLPE8%) was selected for further gene silencing study by plasmid shRNA. Decrease in Bcl-XL gene expression was estimated by both RT-PCR and western-blot experiments. The obtained results revealed that the new copolymers had appropriate nano-scale size (117-128 nm) even after aptamer conjugation (168-183 nm). Moreover, they exhibited increased transfection efficiencies by up to 1.8-5 folds and acceptable cytotoxicity. The apoptosis was induced in transfected cells by shRNA-aptamer-copolymer due to the down-regulation of mRNA and protein levels. This study suggested a new vector for targeted non-viral gene delivery with high transfection efficiency in lung cancer or pulmonary systems.

Expression of e-cadherin, leukemia inhibitory factor and progesterone receptor in mouse blastocysts after ovarian stimulation.

OBJECTIVE: The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. MATERIALS AND METHODS: In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student's t-test (one sample t-test). RESULTS: Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05). CONCLUSION: Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from the natural cycle. The results observed in this study have shown the possibility of an unfavorable effect on implantation and pregnancy rate.