Manifestation of agronomically valuable traits in the progeny of a sorghum mutant carrying the genetic construct for RNA silencing of the γ-kafirin gene.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T2-T4 generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T2, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T3 and T4 plants, the genetic construct was present in 2-4 copies. In T3, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5-7 %) was selected.

High-throughput cell optoporation system based on Au nanoparticle layers mediated by resonant irradiation for precise and controllable gene delivery.

The development of approaches based on genetically modified cells is accompanied by a constant intensive search for new effective and safe delivery systems and the study of existing ones. Recently, we developed a new plasmonic nanoparticle layers-mediated optoporation system that can be proposed for precisely controlled, high-performance laser transfection compatible with broad types of cells and delivered objects of interest. The main goal of the present study is to demonstrate the broad possibilities and advantages of our system for optoporation of several mammalian cells, classified as "easy-to-transfect" cells, namely HeLa and CHO lines, and "hard-to-transfect" cells, namely A431 and RAW 264.7 cells. We show the efficient delivery of various sized cargo molecules: from small molecular dyes propidium iodide (PI) with molecular mass 700 Da, control plasmids (3-10 kb) to fluorophore-labeled dextranes with masses ranging from 10 kDa up to 100 kDa. The performance of optoporation was investigated for two types of laser sources, 800-nm continuous-wave laser, and 1064-nm ns pulsed laser. We provided a comparative study between our system and commercial agent Lipofectamine for transient transfection and stable transfection of HeLa cells with plasmids encoding fluorescent proteins. The quantitative data analysis using flow cytometry, Alamar blue viability assay, and direct fluorescence microscopy revealed higher optoporation efficacy for hard-to-transfect A431 cells and Raw 264.7 cells than lipofection efficacy. Finally, we demonstrated the optoporation performance at the single-cell level by successful delivering PI to the individual CHO cells with revealed high viability for at least 72 h post-irradiation.

[Hormonal and genetic causes of cryptorchidism].

Cryptorchidism is the most frequent congenital disorders of the reproductive system, is present in 2-3% of term newborn boys. Genes involved in embryonic testicular migration are known but their role in cryptorchidism development are not investigated enough. Genetical causes of cryptorchidism are identified in 5-7% of patients. The article contains data on the role of insulin-like peptide 3 and its receptor, anti-Müllerian hormone, gonadotropins, androgens in embryonic testicular migration. INSL3 and AMH are presented as markers of testicular dysfunction associated with cryptorchidism. Hypogonadotropic hypogonadism is also associated with cryptorchidism and can be diagnosed based on it. Results of modern investigations determine the necessary of hormonal and genetical examination of patients with isolated cryptorchidism to detect causes of cryptorchidism and manage of patients.

Colorimetric and dynamic light scattering detection of DNA sequences by using positively charged gold nanospheres: a comparative study with gold nanorods.

We introduce a new genosensing approach employing CTAB (cetyltrimethylammonium bromide)-coated positively charged colloidal gold nanoparticles (GNPs) to detect target DNA sequences by using absorption spectroscopy and dynamic light scattering. The approach is compared with a previously reported method employing unmodified CTAB-coated gold nanorods (GNRs). Both approaches are based on the observation that whereas the addition of probe and target ssDNA to CTAB-coated particles results in particle aggregation, no aggregation is observed after addition of probe and nontarget DNA sequences. Our goal was to compare the feasibility and sensitivity of both methods. A 21-mer ssDNA from the human immunodeficiency virus type 1 HIV-1 U5 long terminal repeat (LTR) sequence and a 23-mer ssDNA from the Bacillus anthracis cryptic protein and protective antigen precursor (pagA) genes were used as ssDNA models. In the case of GNRs, unexpectedly, the colorimetric test failed with perfect cigar-like particles but could be performed with dumbbell and dog-bone rods. By contrast, our approach with cationic CTAB-coated GNPs is easy to implement and possesses excellent feasibility with retention of comparable sensitivity--a 0.1 nM concentration of target cDNA can be detected with the naked eye and 10 pM by dynamic light scattering (DLS) measurements. The specificity of our method is illustrated by successful DLS detection of one-three base mismatches in cDNA sequences for both DNA models. These results suggest that the cationic GNPs and DLS can be used for genosensing under optimal DNA hybridization conditions without any chemical modifications of the particle surface with ssDNA molecules and signal amplification. Finally, we discuss a more than two-three-order difference in the reported estimations of the detection sensitivity of colorimetric methods (0.1 to 10-100 pM) to show that the existing aggregation models are inconsistent with the detection limits of about 0.1-1 pM DNA and that other explanations should be developed.