Cytology and Genetics最新文献

Abstract

The objective of this study was to investigate the optimization of electroporation of hepatocyte nuclear factor 1 alpha (Hnf-1α) in murine mesenchymal stem cells (mBM-MSCs). mBM-MSCs were phenotypically observed and confirmed by positive expression of stemness markers with differentiation capacity into osteocytes. Hnf-1α plasmid DNA was transfected via Neon electroporation system into the mBM-MSCs. The cells were maintained in a complete DMEM medium. Following single 0.5 μg Hnf-1α electroporation the differences in viability of mBM-MSCs were statistically insignificant at 24 h, 72 h, and post-21 days. Fluorescence imaging of turbo green fluorescence protein (tGFP) was detected for the efficiency of transfection. The transfection efficiency was detected at parameters of 1000 pulse voltage (V), 10 pulse width (ms), and at 3 pulse number at 24 h (***p-value < 0.001, 66.5 ± 12.2%) in mBM-MSCs. The efficiency of transfected 0.5 μg Hnf-1α was decreased at 72 h (40.2 ± 10.9%) and 21 days (31.7 ± 5%). 250 μg/mL G418 Sulfate was used for the selection of Hnf-1α transfected positive cells. TaqMan-qRT-PCR results of independent experiments revealed significant fold differences in Hnf-1α expression with above mentioned defined parameters. Therefore, 0.5 μg Hnf-1α plasmid into 2.5 × 10⁵ mBM-MSCs with a pulse voltage of 1000 V, pulse width of 10 ms, and pulse number of 3, was optimized, which was not reported before. These parameters can be considered for transfection with cell viability of 65–96% from 24 h to 21 days and 60–70% transfection efficiency after 24 h. Hence, this optimized procedure with efficient transfection rates can be applied for further gene functions and differentiation studies in the liver, pancreas, kidney, intestine, and for other tissues in specialized niches.

Morphological features of angiogenesis processes in Lewis lung carcinoma (LLC) regrowth tumors were investigated and analyzed during their independent development and under the influence of cryopreserved mesenchymal stem cells of the human placenta (hP-MSC) under different administration conditions. In LLC tumors, an earlier (day 1) onset of angiogenesis was revealed, with pronounced extramedullary hematopoiesis and the formation of unstructured provascular structures. It was shown that, in general, hP-MSC provides a more structurally perfect angiogenesis in the tumor, with the formation on the 15th day of vascular formations surrounded by a cell wall formed from the cells of the tumor itself. Different morphological forms of neoangiogenesis were detected both in LLC tumors that develop independently, and with different methods of hP-MSC administration—with simultaneous inoculation with tumor cells and with systemic (intravenous) administration. However, more active neoangiogenesis in LLC tumors was noted with systemic intravenous administration of hP-MSC.

A polymorphism (g.2141C>G) of the growth hormone (GH) gene was studied in the populations of cows of the Ukrainian red-spotted dairy, Montbeliard breeds, and crossbreeds obtained from the crossing of Ukrainian red-spotted dairy cows with Montbeliard bulls, and the analysis of reproductive traits of individuals with different genotypes was conducted. Genotyping of the growth hormone gene was carried out using a PCR–RFLP method. Both L and V alleles were detected in the experimental cow populations. The ratio of the frequencies of LL/LV/VV genotypes did not differ from the theoretically expected one for each of the experimental groups (χ² = 0.91, p < 0.05; χ² = 1.41, p < 0.05; χ² = 1.66, p < 0.05, respectively). In the experimental populations of the Montbeliard breed and crossbred animals, the VV genotype frequency was 0.03 and 0.06, respectively; no VV genotype was detected in the population of the Ukrainian red-spotted breed. According to the traits of reproductive function, the cows of the experimental groups with the LL genotype variant had better indices as compared with their peers with the LV genotype: an earlier age of first insemination, lower index of insemination, and shorter intercalving period. According to the live weight at birth and growth dynamics in each experimental group of cows, the carriers of the LV genotype had an advantage. Regardless of the genotype, higher indices of live weight at birth and average daily growths were established in animals of the crossbred origin as compared with purebreds. The established polymorphism of the growth hormone gene (SNP g.2141C>G) indicates a feasibility of further studies and prospect of using it as a genetic marker of reproductive ability in marker-assisted selection of dairy cattle direction of productivity.

BIN1 gene is the second highest associated locus to Alzheimer’s disease (AD). Association between genetic ancestry and susceptibility to AD have been found for APOE, the principal genetic risk factor for AD. However, there is a lack of studies on this relationship for BIN1, which is the aim of this work, focused in four Latin American populations. The individual proportions of genetic ancestry were estimated by using a SNP panel previously proposed and optimized for Latin American populations. Three models were set: a risk allele dominant model, a non-risk allele dominant model and a codominant model. The Poisson regression analysis was applied to the risk allele accumulation and logistic regresìsion to a SNP by SNP model. The Poisson regression models tests showed that the accumulation of risk alleles was associated to ancestry as follow: an increasing risk effect for African in both codominance and dominant models, and a protective effect for Native-American for the risk allele dominant model. In the logistic regression analyses, association to individual genetic ancestry proportions for both models was found for rs17014923 and rs6743470, with negative association for Native American ancestry and positive for African and European. rs744373, showed an opposite pattern: positive association for Native-American ancestry and negative for European. Finally, positive association for African ancestry in Peruvian population at rs17014923. These results suggest an overall protective effect for Native-American ancestry and a risk effect for African ancestry in the Latin American sample, even if some particular SNPs shows an opposite pattern.

Abstract

Protein kinases represent one of the largest eukaryotic enzyme superfamilies. However, only a few can directly phosphorylate tubulin and contribute to the modulation of the “tubulin code.” The authors previously confirmed the structural and functional homology of the plant protein kinase IREH1 and members of the mammalian MAST kinase family. Their participation in the regulation of the microtubule system in plant and animal cells was also experimentally confirmed. At the same time, the direct contribution of MAST/IRE to the “tubulin code” remains unclear. In the current study, based on bioinformatical and structural biology methods, the possibility of such an interaction was evaluated. The target sites of MAST/IRE-phosphorylation of tubulin were predicted based on similarity to the generalized specific profiles. Two potential MAST/IRE specific sites, conserved in human and Arabidopsis tubulins were selected: Thr73 (80) exists in most isotypes of α-tubulin and Ser115 was found in the majority of human and plant isotypes of β-tubulin. It was predicted that phosphorylation of the first site can affect the assembly of α/β-tubulin heterodimer, and phosphorylation of the second may affect the interaction between neighboring protofilaments of microtubules. The last site Ser433, was found in both γ-tubulin isotypes of A. thaliana, but it was absent in mammals. The external position of Ser433 in plant γ-tubulin allows for suggesting that phosphorylation of this amino acid can affect the structure of the γTuRC complex but it does not affect inner contacts of γTuSC and their interaction in the ring.

Transcription factors govern various functions in the cell such as proliferation, repair, regenerative programs, etc. Currently, there are no available drugs on the market or subjected to clinical trials that can combat cardiac remodeling. Understanding the role of transcription factors in cardiac remodeling may open the door to developing agents that can reverse structural remodeling of the heart and prevent heart failure. Recent studies shed light on the function of transcription factors involved in cardiac remodeling. Pharmacological modulation of signaling involving transcription factors may present as a novel mechanism for improving cardiac metabolism, promoting cardiac cell survival, etc. Various transcription factor-targeting agents were tested in animal models and showed promising results. Nevertheless, despite significant advances, the role of transcription factors in cardiac remodeling presents an extremely unexplored area. The main goal of this literature review was to summarize the latest advances made in our understanding of the role of transcription factors in cardiac remodeling and their potential to be used as molecular therapy targets.

Inositol polyphosphate 5-phosphatases (5Tase) are the important enzymes of the phosphatidylinositol (PI) signaling pathway and have been found to play important roles in plant growth, development, and stress responses. Most of the Arabidopsis genes encoding Inositol polyphosphate 5-phosphatases have been characterised. However, promoters of genes encoding Inositol polyphosphate 5-phosphatases of plants have not been characterized so far. Here, we report the characterization of Arabidopsis thaliana SALK mutant lines having T-DNA insertion in the upstream intergenic region of the Inositol polyphosphate 5-phosphatases8 (At5Tas8) gene. The location of T-DNA insertion in the SALK line was confirmed by PCR, and plant homozygous and hemizygous for the T-DNA insertion were identified. The homozygous plants were observed for the morphological difference as well as for the root phenotype. However, no significant morphological differences were observed in the mutant and wild-type plants. The expression analysis using qRT-PCR revealed a similar level of At5Tase8 transcript in the mutant and wild-type plants suggesting T-DNA insertion lies beyond the At5Tase 8 promoter. In silico analysis of the 3000 bp sequence upstream of the translation start site covering the T-DNA insertion site has revealed the presence of potential cis-regulatory elements for heat, light, drought, salt, sugar, and hormone. Besides, most predicted cis-elements were located downstream of the T-DNA insertion site, further supporting that the promoter of At5Tase8 lies within the 2738 bp sequence upstream of the translation start site. Further study is required to delineate the At5Tase8 promoter using promoter-reporter fusion in the transgenic Arabidopsis.

Abstract

Among a vast number of polymorphisms in the wheat genome, of special interest are those that are markers for loci under selection. Such loci include storage protein loci, which directly determine the level of bread-making quality, and disease resistance genes. In this study we analyzed diversity of functional markers (seven storage protein loci and three disease resistance genes) in the group of East European winter common wheat genotypes (cultivars and lines of Poltava breeding). Using the UPGMA or NJ methods, the cultivars and lines were divided into two clusters differing in the Glu-1 quality score. The cultivars of cluster 1 with the lower mean Glu-1 quality score predominantly carried the combination of alleles Glu-B1c, Glu-D1a, and Gli-B1e, as well as the resistance allele of Lr34, whereas those of cluster 2 with the higher quality score mainly had the alleles Glu-B1u, Glu-D1d, and Gli-B1b. In the sample of Poltava cultivars, a high correlation between the Glu-1 quality score and the sedimentation value (0.84) was observed. On average for two years, the group of cultivars from cluster 1 showed higher grain protein content and some spike traits in comparison with the means of the sample from cluster 2. Significant associations of Lr34 alleles and alleles at some storage protein loci were revealed, in particular, the association of the resistance allele Lr34R and the allele Glu-D1a in both cultivar and line groups, indicating the adaptive value of such a combination for local soil and climate conditions.

We obtained the full-length cDNA clone of KAP26.1 in Liaoning cashmere goat, and then we further investigated biological functions of KAP26.1. First, we discovered KAP26.1 was specifically expressed in internal root sheath of skin hair follicles by semi-quantitative reverse transcription and polymerase chain reaction (semi-quantitative RT-PCR), hybridization in situ; immunohistochemistry revealed KAP26.1 was located in the internal and external root sheaths. Next, quantitative real-time polymerase chain reaction (qRT-PCR) results showed relative KAP26.1 expression quantity was significantly different between primary and secondary follicles during anagen, catagen, and remarkably increased during telogen. Moreover, after inhibiting Noggin expression, we found relative KAP26.1 expression quantity significantly declined; after KAP26.1 overexpression, we found relative Noggin expression quantity highly significantly declined. Finally, we found MT played a positive role in KAP26.1 and KAP26.1 expression; FGF5 and IGF-1 palyed a negative role in KAP26.1 and blocked the degradation of KAP26.1. The results revealed KAP26.1 played an important role in regulating fine hair development.

Abstract

Currently, there are approximately 6500 species of viruses known in the world, among which more than 1500 are plant viruses. Most of them are capable of causing epiphytoties, which lead to decreased yields, reduced product quality, and sometimes put valuable commercial varieties or even entire plant species at risk of extinction. The global spread of viruses leads to the need to strengthen phytosanitary and quarantine restrictions, which requires additional financial costs. Understanding of viral biology and the principles of its propagation is a key factor in the formation of strategies and methods for combating these pathogens. Among the newest approaches are the genetic engineering technologies. Their use made it possible to create a number of plant varieties with increased resistance to viruses. However, the problem of creating virus-resistant plants still remains one of the most urgent since viruses acquire the ability to bypass defense mechanisms with time and there is a need to obtain new resistant varieties. There are several main approaches for obtaining of transgenic plants with increased resistance to viruses. They are based on RNA interference, resistance associated with viral capsid proteins, RNA-satellites, antisense RNAs, replicases, RNA-dependent RNA polymerase, the action of ribonucleases, ribosome-inactivating proteins, hammerhead ribozymes, miRNAs, plant antibodies, etc. One of the approaches to creating virus-resistant plants is the use of ribonuclease genes. The genes encoding ribonucleases have different origin and belong to a wide range of hosts: bacteria, fungi, plants, and animals. In particular, extracellular ribonucleases are able to cut nonspecifically molecules of viral RNA in apoplast that allows for creating plants with increased resistance to various plant viruses. This review is focused on the study of various genetic engineering approaches and the prospects of their use for the creation of virus-resistant plants. Emphasis is placed on the study of heterologous ribonuclease genes influence.