Hydrogel nanoparticle encapsulated plasmid as a suitable gene delivery system

To facilitate the delivery of genetic material, the use of appropriate carriers such as polymers is necessary. Nanoparticles comprising of chitosan-alginate polymers were formed through pregel preparation method. Chi/Alg nanoparticles had a mean Z-Average diameter of 161.8 nm and mean zeta 29.3 mV, respectively. The ability of plasmid-complex in preventing DNA migration showed Chi/Alg nanoparticles have great capacity to maintain plasmid. The efficiency of nanoparticles for transfection of pEGFP-N1 plasmid in the cultured HEK 293 cells was measured by flow cytometry. Cell viability assays indicated that nanoparticles had no toxic effect on HEK 293 cells after 4 or 24 h. Our suitable candidate for gene delivery would be alg/chi nanoparticles.Для облегчения доставки генетического материала необходимо использование подходящих носителей, таких как полимеры. Наночастицы, состоящие из хитозан-альгинатных полимеров, были получены методом подготовки прегеля. Chi/Alg наночастицы имели средний диаметр 161.8 нм (Z-Average) и средний zeta-потенциал 29.3 mV. Отсутствие миграции ДНК во время электрофореза комплексов плазмиды с наночастицами показало, что Chi/Alg наночастицы могут удерживать плазмидную ДНК внутри комплекса. Эффективность наночастиц для трансфекции плазмиды pEGFP-N1 в культивируемые клетки HEK 293 была измерена с помощью жидкостной цитометрии. Тесты на жизнеспособ-ность клеток показали, что наночастицы не имели токсичного эффекта на клетки HEK 293 через 4 ч или 24 ч. Наночастицы Alg/Chi являются подходящим кандидатом для доставки генов

miR-192 overexpression effect on DHFR and TYMS in acute lymphoblastic leukemia

Purpose: Non-coding RNAs play a critical role in gene regulation in cancer cells. Reduced expression of microRNA-192 has been detected in many cancers. Here, we investigated the role of miR-192 in Dihydrofolate reductase (DHFR) and Thymidylate Synthase (TYMS) expression level and in an acute lymphoblastic leukemia cell line. Basic procedures: 20 patients diagnosed with acute lymphoblastic leukemia (ALL) were studied for the level of (micro RNA-192) miR-192, DHFR and TYMS expression level by qRT-PCR. NALM-6 cells were transduced using recombinant lentiviruses for overexpression of miR-192 and its backbone, then the relative changes in DHFR and TYMS genes expression were studied by qRT-PCR. DHFR Protein level changes were analyzed by Western blotting. Main findings: ALL patients with relapse, experience lower levels of miR-192 and higher levels of DHFR and TYMS in comparison with treated patients. Overexpression of miR-192 in NALM-6 cells resulted in decreased expression of DHFR and TYMS. Moreover, the protein level of DHFR was decreased after overexpression of miR-192. Principal conclusions: These results suggest the tumor suppression effects of miR-192 and its correlation with decreased DHFR and TYMS level in acute lymphoblastic leukemia cells for the first time. © 202

Cloning, Expression and Characterization of Zebra Fish Ferroportin in Hek 293T Cell Line

Background: Ferroportin (Fpn), a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms.Methods: To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP-N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of Fpn-EGFP protein in Hek 293T cells.Results: The expression was confirmed by appearance of fluorescence in Hek 293 T cells. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids.Conclusion: The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies