Diversity in TAF Proteomics: Consequences for Cellular Differentiation and Migration

Development is a highly controlled process of cell proliferation and differentiation driven by mechanisms of dynamic gene regulation. Specific DNA binding factors for establishing cell- and tissue-specific transcriptional programs have been characterised in different cell and animal models. However, much less is known about the role of “core transcription machinery” during cell differentiation, given that general transcription factors and their spatiotemporally patterned activity govern different aspects of cell function. In this review, we focus on the role of TATA-box associated factor 4 (TAF4) and its functional isoforms generated by alternative splicing in controlling lineage-specific differentiation of normal mesenchymal stem cells and cancer stem cells. In the light of our recent findings, induction, control and maintenance of cell differentiation status implies diversification of the transcription initiation apparatus orchestrated by alternative splicing

Graphene-augmented nanofiber scaffolds demonstrate new features in cells behaviour

Three-dimensional (3D) customized scaffolds capable to mimic a native extracellular matrix open new frontiers in cells manipulation and advanced therapy. The major challenge is in a proper substrate for in vitro models on engineered scaffolds, capable to modulate cells differentiation. Here for the first time we demonstrate novel design and functionality of the 3D porous scaffolds of aligned, self-assembled ceramic nanofibers of ultra-high anisotropy ratio (∼10 7), augmented into graphene shells. This unique hybrid nano-network allows an exceptional combination of selective guidance stimuli of stem cells differentiation, immune reactions variations, and local immobilization of cancer cells, which was not available before. The scaffolds were shown to be able to direct human mesenchymal stem cells (important for stimulation of neuronal and muscle cells) preferential orientation, to suppress major inflammatory factors, and to localize cancer cells; all without additions of specific culture media. The selective downregulation of specific cytokines is anticipated as a new tool for understanding of human immune system and ways of treatment of associated diseases. The effects observed are self-regulated by cells only, without side effects, usually arising from use of external factors. New scaffolds may open new horizons for stem cells fate control such as towards axons and neurites regeneration (Alzheimer's disease) as well as cancer therapy development.Peer reviewe

Biomechanical features of graphene-augmented inorganic nanofibrous scaffolds and their physical interaction with viruses

Nanofibrous substrates and scaffolds are widely being studied as matrices for 3D cell cultures, and disease models as well as for analytics and diagnostic purposes. These scaffolds usually comprise randomly oriented fibers. Much less common are nanofibrous scaffolds made of stiff inorganic materials such as alumina. Well-aligned matrices are a promising tool for evaluation of behavior of biological objects affected by micro/nano-topologies as well as anisotropy. In this work, for the first time, we report a joint analysis of biomechanical properties of new ultra-anisotropic, self-aligned ceramic nanofibers augmented with two modifications of graphene shells (GAIN scaffolds) and their interaction of three different viral types (influenza virus A, picornavirus (human parechovirus) and potato virus). It was discovered that nano-topology and structure of the graphene layers have a significant implication on mechanical properties of GAIN scaffolds resulting in non-linear behavior. It was demonstrated that the viral adhesion to GAIN scaffolds is likely to be guided by physical cues in dependence on mutual steric factors, as the scaffolds lack common cell membrane proteins and receptors which viruses usually deploy for transfection. The study may have implications for selective viral adsorption, infected cells analysis, and potentially opening new tools for anti-viral drugs development.Peer reviewe

GLI2 cell-specific activity is controlled at the level of transcription and RNA processing: Consequences to cancer metastasis

AbstractHigh activity of GLI family zinc finger protein 2 (GLI2) promotes tumor progression. Removal of the repressor domain at the N terminus (GLI2∆N) by recombinant methods converts GLI2 into a powerful transcriptional activator. However, molecular mechanisms leading to the formation of GLI2∆N activator proteins have not been established. Herein we report for the first time that the functional activities of GLI2 are parted into different protein isoforms by alternative promoter usage, selection of alternative splicing, transcription initiation and termination sites. Functional studies using melanoma cells revealed that transcriptional regulation of GLI2 is TGFbeta-dependent and supports the predominant production of GLI2∆N and C-terminally truncated GLI2 (GLI2∆C) isoforms in cells with high migratory and invasive phenotype. Taken together, these results highlight the role of transcription and RNA processing as major processes in the regulation of GLI2 activity with severe impacts in cancer development

Graphene-Augmented Nanofiber Scaffolds Trigger Gene Expressions Switching of Four Cancer Cell Types

Three-dimensional (3D) customized scaffolds are anticipated in providing new frontiers in cells manipulation and advanced therapy methods. Here we demonstrate the application of hybrid 3D porous scaffolds, representing networks of highly aligned self-assembled ceramic nano-fibers, for culturing four types of cancer cells. Ultra-high aspect ratio (~~107) of graphene augmented fibers of tailored nano-topology is shown as an alternative tool to substantially affect cancerous gene expression, eventually due to differences in local biomechanical features of the cell-matrix interactions. Here we report a clear selective up- and down-regulation of groups of markers for breast cancer (MDA-MB231), colorectal cancer (CaCO2), melanoma (WM239A) and neuroblastoma (Kelly) depending on only fibers orientation and morphology without application of any other stimulus. Changes in gene expression are also revealed for Mitomycin C treatment of MDA-MB231, making the scaffold a suitable platform for testing of anticancer agents. This allows an opportunity for selective »clean» guidance to deep understanding mechanisms of cancer cells progressive growth and tumor formation without possible side effects by manipulation with the specific markers.Peer reviewe

Alternative Splicing Targeting the hTAF4-TAFH Domain of TAF4 Represses Proliferation and Accelerates Chondrogenic Differentiation of Human Mesenchymal Stem Cells

<div><p>Transcription factor IID (TFIID) activity can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. Alternative splicing of TFIID subunits is often the result of external stimulation of upstream signaling pathways. We studied tissue distribution and cellular expression of different splice variants of TFIID subunit <i>TAF4</i> mRNA and biochemical properties of its isoforms in human mesenchymal stem cells (hMSCs) to reveal the role of different isoforms of TAF4 in the regulation of proliferation and differentiation. Expression of <i>TAF4</i> transcripts with exons VI or VII deleted, which results in a structurally modified hTAF4-TAFH domain, increases during early differentiation of hMSCs into osteoblasts, adipocytes and chondrocytes. Functional analysis data reveals that TAF4 isoforms with the deleted hTAF4-TAFH domain repress proliferation of hMSCs and preferentially promote chondrogenic differentiation at the expense of other developmental pathways. This study also provides initial data showing possible cross-talks between TAF4 and TP53 activity and switching between canonical and non-canonical WNT signaling in the processes of proliferation and differentiation of hMSCs. We propose that TAF4 isoforms generated by the alternative splicing participate in the conversion of the cellular transcriptional programs from the maintenance of stem cell state to differentiation, particularly differentiation along the chondrogenic pathway.</p></div

Expression of TAF4 splice variants and isoforms in human MSCs differentiated into adipocytes, osteoblasts or chondrocytes and treated with <i>TAF4</i> RNAi.

<p>(<b>A</b>) RT-PCR analysis of different individual hMSCs clones (hMSCs 1–5) using <i>TAF4_v1a</i> (full length) specific primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074799#pone.0074799.s002" target="_blank">Table S2</a>). Expression of <i>GAPDH</i> mRNA is shown at the bottom. (<b>B</b>) Expression of <i>TAF4</i> ASVs encoding proteins with compromised hTAF4-TAFH domain is dominant in differentiated hMSCs. RT-PCR analysis using <i>TAF4</i> ASV-specific primers was performed 7 days after induction of differentiation of hMSCs towards adipo-, osteo- and chondrogenic lineages. <i>GAPDH</i> mRNA expression was used for the normalization. (<b>C</b>) Expression of <i>TAF4</i> ASVs following treatment of human MSCs with control and hTAF4-TAFH-domain targeting <i>TAF4</i> siRNAs. Cells were treated with <i>TAF4</i> or control siRNAs at the indicated time points and RT-PCR analysis performed using <i>TAF4</i> ASV-specific primers. Analysis of <i>GAPDH</i> mRNA expression was used for normalization. (<b>D</b>) siRNA-mediated silencing targeting <i>TAF4</i> exons V or VI induces changes in the expression of TAF4 protein isoforms as detected at 48 h post-treatment using Western blot analysis. The asterisk indicates the canonical form of TAF4 protein with the molecular weight of 135 kDa, two asterisks indicate TAF4_v2 isoform with a calculated molecular weight of about 73 kDa.</p

Microbiological, chemical, and sensorial characterisation of commercially available plant-based yoghurt alternatives

Consumer demand for plant-based dairy alternatives has increased rapidly during the past few years and the market has been saturated with a wide variety of alternative products. The general aim of this study was to broaden the understanding of the composition and characteristics of currently commercially available plant-based yoghurt alternatives focusing especially on the content of live bacteria. The bacterial composition, including the content of live bacteria in yoghurt alternatives, was evaluated using metagenetic sequencing of the 16S rRNA gene amplicons in combination with the novel PMAxx treatment approach. The content of organic acids, sugars, and volatiles was measured, and descriptive sensory analysis was carried out to comprehensively describe the products. While the main ingredient (soya, oat, coconut, or lupin) determined the general characteristics of the product, significant differences were observed in both chemical and microbiological composition and sensorial attributes even among the yoghurt alternatives made from the same plant ingredient

Absolute quantification of viable bacteria abundances in food by next-generation sequencing

Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota