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RNotion Transfection Reagent _1 ml

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[#2201500] RNotion Transfection Reagent _1 ml

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(1) Polyethylene glycol and octa-arginine dual-functionalized nanographene oxide: an optimization for efficient nucleic acid delivery.[TOP]

Pubmed ID :29757340
Publication Date : //
The successful application of nucleic acid-based therapy for the treatment of various cancers is largely dependent on a safe and efficient delivery system. A dual-functionalized graphene oxide (GO)-based nanocarrier with the conjugation of aminated-polyethylene glycol (PEG-diamine) and octa-arginine (R8) for the intracellular delivery of nucleic acids is proposed. The functionalized sites are covalently co-conjugated and the PEG : R8 molar ratio is optimized at 10 : 1 to achieve a hydrocolloidally stable size of 252 ± 2.0 nm with an effective charge of +40.97 ± 1.05 and an amine-rich content of 10.87 ± 0.4 μmol g-1. The uptake of the nanocarrier in breast cancer cell lines, MCF-7 and MDA-MB 231, is investigated. The siRNA and pDNA condensation ability in the presence and absence of enzymes and the endosomal buffering capacity, as well as the intracellular localization of the gene/nanocarrier complex are also evaluated. Furthermore, the delivery of functional genes associated with the nanocarrier is assessed using c-Myc protein knockdown and EGFP gene expression. The effective uptake of the nanocarrier by the cells shows superior cytocompatibility, and protects the siRNA and pDNA against enzyme degradation while inhibiting their migration with N : P ratios of 10 and 5, respectively. The co-conjugation of PEG-diamine and the cationic cell-penetrating peptide (CPP) into the GO nanocarrier also provides a superior internalization efficacy of 85% in comparison with a commercially available transfection reagent. The c-Myc protein knockdown and EGFP expression, which are induced by the nanocarrier, confirm that the optimized PEG-diamine/R8-functionalized GO could effectively deliver pDNA and siRNA into the cells and interfere with gene expression.

Authors : Imani Rana, Prakash Satya, Vali Hojatollah, Faghihi Shahab,

(2) Photoluminescent Cationic Carbon Dots as efficient Non-Viral Delivery of Plasmid SOX9 and Chondrogenesis of Fibroblasts.[TOP]

Pubmed ID :29728593
Publication Date : //
With the increasing demand for higher gene carrier performance, a multifunctional vector could immensely simplify gene delivery for disease treatment; nevertheless, the current non- viral vectors lack self-tracking ability. Here, a type of novel, dual-functional cationic carbon dots (CDs), produced through one-step, microwave-assisted pyrolysis of arginine and glucose, have been utilized as both a self-imaging agent and a non-viral gene vector for chondrogenesis from fibroblasts. The cationic CDs could condense the model gene plasmid SOX9 (pSOX9) to form ultra-small (10-30 nm) nanoparticles which possessed several favorable properties, including high solubility, tunable fluorescence, high yield, low cytotoxicity and outstanding biocompatibility. The MTT assay indicated that CDs/pSOX9 nanoparticles had little cytotoxicity against mouse embryonic fibroblasts (MEFs) compared to Lipofectamine2000 and PEI (25 kDa). Importantly, the CDs/pSOX9 nanoparticles with tunable fluorescence not only enabled the intracellular tracking of the nanoparticles, but also could successfully deliver the pSOX9 into MEFs with significantly high efficiency. Furthermore, the CDs/pSOX9 nanoparticles-mediated transfection of MEFs showed obvious chondrogenic differentiation. Altogether, these findings demonstrated that the CDs prepared in this study could serve as a paradigmatic example of the dual-functional reagent for both self-imaging and effective non-viral gene delivery.

Authors : Cao Xia, Wang Jianping, Deng Wenwen, Chen Jingjing, Wang Yan, Zhou Jie, Du Pan, Xu Wenqian, Wang Qiang, Wang Qilong, Yu Qingtong, Spector Myron, Yu Jiangnan, Xu Ximing,

(3) [Downregulation of PTTG1 expression inhibits the proliferation and invasiveness and promotes the apoptosis of human prostate cancer LNCaP-AI cells].[TOP]

Pubmed ID :29723450
Publication Date : //
To investigate the effects of down-regulation of PTTG1 expression on the proliferation, invasiveness and apoptosis of androgen-independent human prostate cancer LNCaP-AI cells and their sensitivity to androgen antagonists.

Authors : Cao Xi-Liang, Wei Yang-Yang, Song Xiao-Ming, Lu Ke-Quan, Yu Wen-Chao, Chen Yong-Qiang, Liu Yong-Liang, Gao Jiang-Ping,

(4) Metformin induces autophagy and G0/G1 phase cell cycle arrest in myeloma by targeting the AMPK/mTORC1 and mTORC2 pathways.[TOP]

Pubmed ID :29554968
Publication Date : //
Metformin is a commonly used drug for the treatment of diabetes. Accumulating evidence suggests that it exerts anti-tumor effects in many cancers, including multiple myeloma (MM); however, the underlying molecular mechanisms have not been clearly elucidated.

Authors : Wang Yan, Xu Wenbin, Yan Zixun, Zhao Weili, Mi Jianqing, Li Junmin, Yan Hua,

(5) [Homocysteine induces calcium overload in neonatal rat atrial cells through activation of sodium current and CaMKⅡδ].[TOP]

Pubmed ID :29495239
Publication Date : //
To investigate the effect and related mechanism of homocysteine (Hcy) on calcium overload in neonatal rat atrial cells (NRICs). NRICs were assigned to 9 groups after culture for 3 days: (1) control group; (2) Hcy group (0, 50, 100, 200, 500 μmol/L for 48 hours); (3) antioxidant group (NAC, 10 μmol/L for 24 hours); (4) Hcy+NAC group (500 μmol/L Hcy for 48 hours, then treated with 10 μmol/L NAC for 24 hours); (5) calcium/calmodulin dependent protein kinase Ⅱδ (CaMKⅡδ) inhibitor group (KN-93, 3 μmol/L KN-93 for 5 hours); (6) specific sodium current inhibitor group (ELE, 1 μmol/L ELE for 5 hours); (7) Hcy+KN-93 group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 for 5 hours); (8) Hcy+ELE group (500 μmol/L Hcy for 48 hours, then treated with 1 μmol/L ELE for 5 hours; (9) Hcy+KN-93+ELE group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 and 1 μmol/L ELE for 5 hours). Moreover, NRICs were also treated with CaMKⅡδ-siRNA lentivirus, and Nav1.5-siRNA lentivirus, negative lentivirus carrier containing green fluorescent protein (GFP) for 24 hours. The MOI values of the three groups were 10. Infection efficiency of lentivirus was determined by observing the percentage of GFP fluorescence under inverted fluorescence microscope after transfection for 24 hours, and cultured regularly with simultaneous Puro screening, then cells were grouped as Hcy+CaMKⅡδ-siRNA group, Hcy+Nav1.5-siRNA group and Hcy+negative group. The concentration of Ca(2+) in NRICs ([Ca(2+)]i) of various groups was detected through Fluo-4/AM fluorescence probe, then 2', 7'- two chlorofluorescein diacetate (DCFH-DA) was used as a probe to detect reactive oxygen species (ROS) in NRICs by flow cytometry. The malondialdehyde (MDA) was detected by the activity of superoxide dismutase (SOD) and xanthine oxidase was detected by thiobarbituric acid colorimetry. The protein and mRNA expression level of CaMKⅡδ and Nav1.5 in NRICs were detected by Western blot and quantitative real-time PCR. (1) ROS, MDA and SOD were similar between NAC group and control group, ROS and MDA were significantly increased, while SOD was significantly reduced in Hcy group in a concentration-dependent manner. (2) [Ca(2+)]i: The level of [Ca(2+)]i was (155.57+7.25), (187.43+13.07), (248.98+27.22) and (307.36+15.09) nmol/L in 50, 100, 200 and 500 μmol/L Hcy groups, which was significantly higher than that in the control group ((123.18+7.24) nmol/L, 0.01). In addition, the level of [Ca(2+)]i in Hcy+NAC group ((222.87+23.71)nmol/L) was significantly lower than that in Hcy 500 μmol/L group ((305.15+39.45) nmol/L, 0.05), while [Ca(2+)]i level was similar between NAC group and the control group. (3) The protein expression of CaMKⅡδ and Nav1.5 was significantly upregulated in Hcy groups than in the control group. The protein expression level of CaMKⅡδ-Thr287 was significantly lower in NAC group than in Hcy 500 μmol/L group (0.01), however, there was no significant difference on the protein expression levels of CaMKⅡδ-Thr287 and Nav1.5 between NAC group and control group (all 0.05). (4) The protein expression levels of CaMKⅡδ-Thr287 and the concentration of [Ca(2+)]i were significantly lower in Hcy+KN-93 group and Hcy+KN-93+ELE group than in Hcy 500 μmol/L group (0.05). [Ca(2+)]i concentration was significantly lower in Hcy+KN-93 group, Hcy+ELE group and KN-93+ELE+Hcy group than in Hcy 500 μmol/L group (0.05). (5) The mRNA and protein expression levels of CaMKⅡδ and Nav1.5 in each group infected with lentivirus: the GFP expression was ideal post lentivirus transfection for 24 hours (up to 90%), which was significantly lower in the CaMKⅡδ-siRNA group and Nav1.5-siRNA group than in the negative infection group (all 0.05), which was similar between negative infection group and control group (0.05). Moreover, the mRNA and protein expression levels of CaMKⅡδ and CaMKⅡδ-Thr287 was significantly lower in Hcy+Nav1.5-siRNA group than in Hcy+negative infection group (0.05). The protein and mRNA levels of Nav1.5 were similar between Hcy+CaMKⅡδ-siRNA group and Hcy+negative infection group (0.05). Hcy can induce calcium overload in NRICs by increasing oxidative stress, upregulating the sodium channel protein, and activating the late sodium current and phosphorylating CaMKⅡδ.

Authors : Han L, Dong Q B, Wei Y C, Zheng A C, Li J X, Hong K, Wu Y Q, Cheng X S,

(6) Insights into the influences of carboxymethyl-β-cyclodextrin on DNA formulations characteristics and gene transfection efficiency.[TOP]

Pubmed ID :29484997
Publication Date : //
Gene therapy is an expanding field and it can treat genetic and acquired diseases. It was found that formulations with DNA: CM-β-CD (Carboxymethyl-beta-cyclodextrin): Pluronic-F127 1:3:3 and 1:3 DNA: CM-β-CD are the most stable formulations indicating high incorporation of DNA within CM-β -CD. Gel electrophoresis revealed DNA with low CM-β -CD concentration has formed a more stable complex. Samples 1:3 DNA: CM-β-CD and 1:3:3 DNA: CM-β-CD: Pluronic-127 show no DNA fragment suggesting good condensation of DNA inside cyclodextrin cavity. This was confirmed by fluorescence data where fluorescence intensity was reduced for samples DNA: CM-β-CD 1:3. Overall the findings showed that Carboxymethyl-beta-cyclodextrin (as a novel non-viral gene vector) was able to provide condensation and protection to the DNA, with and without Pluronic-F127, at low concentration. pDNA/CM-β-CD complex has not just shown to be able to transfect COS 7 and SH-SY5Y cell lines but it gave a higher transfection efficiency than that produced by the TransIT-LT1 commercial transfection reagent.

Authors : Elsana Hassan, Mysina Svetlana, Elkordy Emal Ali, Carr-Wilkinson Jane, Elkordy Amal Ali,

(7) Functionalized Asymmetric Bola-Type Amphiphiles for Efficient Gene and Drug Delivery.[TOP]

Pubmed ID :29462991
Publication Date : //
The studies of bolaamphiphile-based nanoparticles as delivery vectors are still rudimentary and under development. In this study, several asymmetric bolaamphiphiles containing lysine and another moiety with special functions, such as pH-sensitive or cell-targeting property, were designed and synthesized. The potentials of these bolaamphiphile-based nanoparticles as versatile vectors for both nucleic acids and chemical drugs were studied. With the presence of 1,2-dioleoyl--glycero-3-phosphoethanolamine (DOPE), these amphiphiles could be prepared into bolasomes, which showed good DNA binding ability and could condense plasmid DNA into nanoparticles with appropriate size and surface potential. , which has a pH-sensitive histidine on one head, exhibited higher transfection efficiency than the symmetric counterpart and comparable efficiency to commercially available transfection reagent. Mechanism studies confirmed that the bolaplexes formed from might induce the highest cellular uptake and the best endosomal escape ability. On the other hand, these bolaamphiphiles also exhibited good drug loading ability. The self-assembly vesicles could efficiently encapsulate the hydrophobic anti-cancer drug doxorubicin (DOX) in aqueous solution with high drug loading content and encapsulation efficiency. Confocal laser scanning microscopy (CLSM) experiment and cell viability assay exhibited a controlled release of the drug with the assistance of bolasomes. It was shown that such bolaamphiphiles have great potential as nano-vectors for both drug and gene or their co-delivery.

Authors : Huang Zheng, Zhao Dong-Mei, Deng Xuan, Zhang Ji, Zhang Yi-Mei, Yu Xiao-Qi,

(8) Characterization of DNA Condensation by Conformationally Restricted Dipeptides and Gene Delivery.[TOP]

Pubmed ID :29372986
Publication Date : //
A wide variety of non-viral vectors have been developed for gene delivery in past few decades but find limited applications mainly due to lower encapsulation, endosomal entrapment, high toxicity and low transfection efficiency. In this work, we explored plasmid DNA binding ability of several low molecular weight dipeptides containing α,β-dehydrophenylalanine (ΔF) and found that an arginine containing dipeptide, arginine-α,β-dehydrophenylalanine (R-ΔF) condensed pEGFP-N1 plasmid into positively charged spherical nanoparticles of size 250–275 nm. Single molecule techniques showed that R-ΔF interacted with the plasmid DNA in a dose dependent manner which was accompanied by a decrease in diffusion time of the plasmid DNA as well as release of the bound fluorophore. The plasmid DNA in R-ΔF-plasmid complex (R-ΔF-Pl) was stable against DNase action. A pH dependent release of the plasmid DNA from R-ΔF-Pl was observed and the released plasmid DNA retained its natural conformation at endosomal pH, as evidenced from time correlated single photon counting. R-ΔF-Pl was biocompatible and showed ready uptake in HEK 293T cells. Transfection assays using reporter plasmids for green fluorescent protein (GFP), luciferase enzyme and chloramphenicol acetyltransferase (CAT) showed R-ΔF mediated gene delivery both in the presence and absence of serum in the medium. Ease of synthesis, homogenous assembly and biocompatibility balanced with significant expression of gene of interest make R-ΔF a potential vector for development for in vivo application.

Authors : Khatri Anjali, Mishra Aseem, Chauhan Virander Singh,

(9) Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.[TOP]

Pubmed ID :29329339
Publication Date : //
Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying β-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater β-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the β-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Authors : Granger Bruce L,

(10) [Impact of PRDM1 gene inactivation on C-MYC regulation in diffuse large B-cell lymphoma].[TOP]

Pubmed ID :29325247
Publication Date : //
To investigate the role of PRDM1 gene inactivaion in the regulation of C-MYC in diffuse large B-cell lymphoma (DLBCL), and to explore the correlation of its immunophenotype and prognosis. 100 cases paraffin-embedded DLBCL tissues were collected from January 2009 to December 2015 at the First Affiliated Hospital of Xinjiang Medical University along with 20 cases of reactive proliferative lymph nodes as control. Immunohistochemical methods were used to detect the expression of CD20, CD10, MUM1, Ki-67, bcl-6, PRDM1/Blimp1, C-MYC and PAX5 protein. The tumors were classified into two subtypes according to Hans classification.The expression of PRDM1 and C-MYC gene in tumor group and control group was detected by reverse transcription PCR (RT-PCR) and the relationship between PRDM1 and C-MYC gene was analyzed.OCI-LY1 (GCB subtype) and OCI-LY3 (non-GCB subtype) cell lines were transfected with small interfering RNA by cationic liposome reagent transfection, and the expression of C-MYC in the transfected cell lines was detected by RT-PCR and Western blot. The Kaplan-Meier method was used to analyze the prognostic significance of PRDM1/Blimp1 and C-MYC at protein and mRNA levels. There were 27 cases of GCB subtype and 73 cases of non-GCB subtype according to Hans classification. The positive expression of Blimp1 in DLBCL group and proliferative lymph nodes in control group was seen in 26(26.0%) and 20 cases(100%), respectively. There were 58 cases with high expression of PRDM1 at mRNA level, including 22 cases of GCB subtype and 36 cases non-GCB subtype, and the difference was statistically significant (=0.004). There were differences in PRDM1 gene expression between the two immunological subtypes, serum lactate dehydrogenase (serum LDH) level, presence of B symptoms, tumor primary sites and other clinical pathological parameters, while C-MYC expression was different in gender, IPI score, and serum LDH levels. Upon PRDM1/Blimp1 gene silencing in the two cell lines, C-MYC protein and gene expression were up-regulated in the transfection group, compared with the blank control group and negative control group by reverse transcription PCR and Western blot analyses. Moreover, PRDM1 expression was significantly associated with C-MYC(χ(2)=7.648, =0.006) at mRNA level. The up-regulation of C-MYC gene expression induced by PRDM1 inactivation in DLBCL may play an important role for the development of DLBCL.PRDM1 protein and mRNA are associated with immunophenotyping and PRDM1 mRNA is a marker of poor prognosis.

Authors : Zhang X Y, Ma Z P, Cui W L, Pang X L, Chen R, Wang L, Zhang W, Li X X,