REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.</p

Genetically Engineered Lung Cancer Cells for Analyzing Epithelial–Mesenchymal Transition

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological process that also exerts important roles in disease progression. Two forms of cellular plasticity are epithelial&ndash;mesenchymal transition (EMT) and its inverse process, mesenchymal&ndash;epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in vivo in the future

Profiling Glioblastoma Cases with an Expression of DCX, OLIG2 and NES

Glioblastoma (GBM) remains the leading cause of cancer-related deaths with the lowest five-year survival rates among all of the human cancers. Multiple factors contribute to its poor outcome, including intratumor heterogeneity, along with migratory and invasive capacities of tumour cells. Over the last several years Doublecortin (DCX) has been one of the debatable factors influencing GBM cells&rsquo; migration. To resolve DCX&rsquo;s ambiguous role in GBM cells&rsquo; migration, we set to analyse the expression patterns of DCX along with Nestin (NES) and Oligodendrocyte lineage transcription factor 2 (OLIG2) in 17 cases of GBM, using immunohistochemistry, followed by an analysis of single-cell RNA-seq data. Our results showed that only a small subset of DCX positive (DCX+) cells was present in the tumour. Moreover, no particular pattern emerged when analysing DCX+ cells relative position to the tumour margin. By looking into single-cell RNA-seq data, the majority of DCX+ cells were classified as non-cancerous, with a small subset of cells that could be regarded as glioma stem cells. In conclusion, our findings support the notion that glioma cells express DCX; however, there is no clear evidence to prove that DCX participates in GBM cell migration

Kaiso Protein Expression Correlates with Overall Survival in TNBC Patients

Triple-negative breast cancers (TNBCs) are histologically heterogenic invasive carcinomas of no specific type that lack distinctive histological characteristics. The prognosis for women with TNBC is poor. Regardless of the applied treatments, recurrences and deaths are observed 3–5 years after the diagnosis. Thus, new diagnostic markers and targets for personalized treatment are needed. The subject of our study—the Kaiso transcription factor has been found to correlate with the invasion and progression of breast cancer. The publicly available TCGA breast cancer cohort containing Illumina HiSeq RNAseq and clinical data was explored in the study. Additionally, Kaiso protein expression was assessed in formalin-fixed and paraffin-embedded tissue archive specimens using the tissue microarray technique. In this retrospective study, Kaiso protein expression (nuclear localization) was compared with several clinical factors in the cohort of 103 patients with TNBC with long follow-up time. In univariate and multivariate analysis, high Kaiso protein but not mRNA expression was correlated with better overall survival and disease-free survival, as well as with premenopausal age. The use of radiotherapy was correlated with better disease-free survival (DFS) and overall survival (OS). However, given the heterogeneity of TNBC and context-dependent molecular diversity of Kaiso signaling in cancer progression, these results must be taken with caution and require further studies

Evaluation of influence of stretching therapy and ergonomic factors on postural control in patients with chronic non-specific low back pain

[b]Introduction and objectives. [/b]The vertical orientation of the body in the upright standing position is maintained by keeping the body’s centre of gravity (COG) upright, above the base of support, by a dynamic interplay of visual, vestibular, and somatosensory control systems. The objectives of this study were: to compare the postural control strategy between people with and without low back pain (LBP), to estimate the influence of the stretching therapy on the postural control strategy, and to discover the relationship between the restriction of spine mobility and occurrence of some ergonomic factors. [b]Materials and methods.[/b] The study consisted of 32 patients with LBP and 25 healthy controls. Postural characteristics of the subjects were measured with the use of a computerized force platform. The software programme filters and measures COG sway velocity in different conditions. Additional measurements and tests were conducted in patients after stretching therapy. Based on survey research, all individuals were selected and evaluated from the aspect of ergonomics. [b]Results[/b]. The results of the COG sway velocity vary under the testing conditions. From the aspect of ergonomic attitude and influence of the rehabilitation, results varied in the groups. [b]Conclusions[/b]. Ergonomic factors are often accompanied by the appearance of LBP. The restrictions within the musculoskeletal system cause disorders in muscle synergies, which is expressed by an increase in the angular velocity of the COG. In patients with chronic back pain syndrome, selected stretching therapy techniques improves the range of motion of the spine and reduces pain