Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment <em>In Vitro</em>

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Wuhan University of Science and Technology

Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities. 

Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR.

Cytoskeletal filamin A (FLNA) is an important protein involved in multiple cellular processes. Previous studies have shown that FLNA can promote or inhibit cancer growth and development; however, the mechanisms underlying these events are not fully understood. Here we show that, in both 293T and SaOS2 cells, knockdown of FLNA significantly enhanced transcription of RNA polymerase (pol) III-transcribed genes except for a subset of tRNA genes. In contrast, re-expression of FLNA in an FLNA-deficient melanoma cell line (A7) repressed transcription of all pol III-transcribed genes, suggesting that FLNA inhibits pol III transcription in a cell type-specific manner. Chromatin immunoprecipitation assays revealed that the repression of pol III gene transcription by FLNA correlates with the decreased occupancy of the RNA pol III transcription machinery at promoters. Immunofluorescence microscopy and coimmunoprecipitation assays revealed that FLNA can associate with the RNA pol III transcription machinery through its actin-binding domain within nuclei. Mechanistic analysis revealed that FLNA suppresses pol III gene transcription by confining the recruitment of the RNA pol III transcription machinery at the promoters of the genes that are sensitive to the alteration of FLNA expression. These findings not only extend the understanding of FLNA function in cells but also provide novel insights into the mechanism by which FLNA represses cell proliferation.

Cytoskeletal Filamin A Differentially Modulates RNA Polymerase III Gene Transcription in Transformed Cell Lines.

Mammalian cells are very important experimental materials and widely used in biological and medical research fields. It is often required that mammalian cells are transported from one laboratory to another to meet with various researches. Conventional methods for cell shipment are laborious and costive despite of maintaining high viability. In this study we aimed to develop a simple and low-cost method for cell shipment by investigating the viabilities of different cell lines treated at different temperatures. We show that the viability of mammalian cells incubated at 1°C or 5°C significantly reduced when compared with that at 16°C or 22°C. Colony formation assays revealed that preservation of mammalian cells at 1°C or 5°C led to a poorer recovery than that at 16°C or 22°C. The data from proliferation and apoptotic assays confirmed that M2 cells could continue to proliferate at 16°C or 22°C, but massive death was caused by apoptosis at 1°C or 5°C. The morphology of mammalian cells treated under hypothermia showed little difference from that of the untreated cells. Quantitative RT-PCR and alkaline phosphatase staining confirmed that hypothermic treatment did not change the identity of mouse embryonic stem cells. A case study showed that mammalian cells directly suspended in culture medium were able to be shipped for long distance and maintained a high level of viability and recovery. Our findings not only broaden the understanding to the effect of hypothermia on the viability of mammalian cells, but also provide an alternative approach for cell shipment.

The analysis of viability for mammalian cells treated at different temperatures and its application in cell shipment.

RNA polymerase II (pol II) is required for the transcription of all protein-coding genes and as such represents a major enzyme whose activity is tightly regulated. Transcriptional initiation therefore requires numerous general transcriptional factors and cofactors that associate with pol II at the core promoter to form a pre-initiation complex. Transcription factor IIA (TFIIA) is a general cofactor that binds TFIID and stabilizes the TFIID-DNA complex during transcription initiation. Previous studies showed that TFIIA can make contact with the DNA sequence upstream or downstream of the TATA box, and that the region bound by TFIIA could overlap with the elements recognized by another factor, TFIIB, at adenovirus major late core promoter. Whether core promoters contain a DNA motif recognized by TFIIA remains unknown. Here we have identified a core promoter element upstream of the TATA box that is recognized by TFIIA. A search of the human promoter database revealed that many natural promoters contain a TFIIA recognition element (IIARE). We show that the IIARE enhances TFIIA-promoter binding and enhances the activity of TATA-containing promoters, but represses or activates promoters that lack a TATA box. Chromatin immunoprecipitation assays revealed that the IIARE activates transcription by increasing the recruitment of pol II, TFIIA, TAF4, and P300 at TATA-dependent promoters. These findings extend our understanding of the role of TFIIA in transcription, and provide new insights into the regulatory mechanism of core promoter elements in gene transcription by pol II.

A transcription factor IIA-binding site differentially regulates RNA polymerase II-mediated transcription in a promoter context-dependent manner.

Silica columns from PCR purification and gel extraction kits are widely used in laboratories worldwide to assist in gene cloning. However, the use of these columns can generate plastic waste that has an environmental impact due to their one-off design and massive consumption. Thus, it is important to develop a novel method that can reduce the utilization of silica columns but not affect research efficiency. In this study, various chemical and nonchemical reagents were used to eliminate residual DNA within used columns from PCR purification and gel extraction kits. We show that phosphoric acid is the most effective reagent among those tested to remove DNA contamination from used columns. Columns regenerated using 1 M phosphoric acid have a DNA purification capability that is comparable to that of fresh columns. We demonstrate that silica columns can be regenerated and reused a minimum of five times. The lab-made buffers are compatible with the regenerated columns for DNA purification, and DNA that is prepared with the regenerated columns can be used for gene cloning without affecting the gene cloning efficiency. Thus, the use of this novel method greatly reduces the production of laboratory waste and benefits numerous laboratories worldwide.

Rapid Regeneration and Reuse of Silica Columns from PCR Purification and Gel Extraction Kits.

One of the conventional methods to study the effects of gene mutations is that gene mutants are transfected into mammalian cells, and the dominant effects of gene mutants in the cells are examined. However, the result obtained using this method is not always satisfactory due to the interference of endogenous expression. Whether there is a better method to investigate the effects of gene mutations in cells remains to be examined. In the present study, a novel dual expression lentiviral vector was constructed using a shRNA-expressing lentiviral vector and combined techniques. Using this dual expression system, the vectors expressing both transcription factor IIA γ (TFIIAγ) shRNA and HA-TFIIAγ or its mutants were generated, and the effects of  gene mutations on transcription and protein-DNA interaction were investigated. We show that the transfection of the vector expressing TFIIAγ shRNA and  fusion gene was able to silence the expression of endogenous  gene but not affect that of exogenous  fusion gene in either transiently transfected cells or stable cell lines. Mutations in the conservative domain between AA and AA in TFIIAγ inhibit the activities of promoters and endogenous gene expression, and reduce TFIIAγ binding to AdML core promoter compared with wild-type (WT) TFIIAγ. ChIP-qPCR data suggest that the TFIIAγ N63A mutant inhibits insulin-like growth factor 2 (IGF2) transcription by reducing the recruitments of TFIIAγ, polymerase II (Pol II), TATA box-binding protein (TBP), and TBP associated factor 1 (250 kDa) (TAF1) at its promoter. Our study provides a novel method that is used to investigate the effects of gene mutations at the cellular level.

A novel method to investigate the effects of gene mutations at the cellular level using a dual expression lentiviral vector.

Neural differentiation from embryonic stem cells (ESCs) is an excellent model for elucidating the key mechanisms involved in neurogenesis, and also provides an unlimited source of progenitors for cell-based nerve regeneration. However, the existing protocols such as small molecule substances, 3D matrix, co-culture technique and transgenic method, are complicated and difficult to operate, thus are limited by laboratory conditions. Looking for an easy-to-operate protocol with easily gained material and high induction efficiency has always been a hot issue in neuroscience research.

An optimized method for neuronal differentiation of embryonic stem cells in vitro.

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing -silencing shRNAs. Both  promoter 4 (P4) and  promoter 2 (P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating  and  expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription,  and  expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both  and  were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects P4 and P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling  and  gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both P4 and P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.

The transcription factor Sp1 modulates RNA polymerase III gene transcription by controlling  and  expression in human cells.

Arsenic in fine air particulate matter (PM) has been identified as an important factor responsible for the morbidity of lung cancer, which has increased sharply in many regions of China. Some reports in China have shown that arsenic in the air exceeds the ambient air quality standard value, while long-term airborne arsenic concentrations in central China and human exposure via inhalation of PM-bound arsenic (inhalable airborne PM) have not been well characterized. In this study, 579 outdoor air PM samples from Wuhan, a typical city in central China, were collected from 2015 to 2017, and arsenic was measured by inductively coupled plasma-mass spectrometry. Personal exposure to PM-bound arsenic via inhalation and urinary arsenic concentration were also measured. The concentrations of arsenic in PM were in the range of 0.42-61.6 ng/m (mean 8.48 ng/m). The average concentration of arsenic in 2015 (10.7 ng/m) was higher than that in 2016 (6.81 ng/m) and 2017 (8.18 ng/m), exceeded the standard value. The arsenic concentrations in spring and winter were higher than those in summer and autumn. No significant differences (p > 0.05) were found among different sites. The daily intake of arsenic inhalation based on PM samples collected by personal samplers (median, 10.8 ng/m) was estimated. Urban residents inhaled higher levels of PM-bound arsenic than rural residents. Daily intake of arsenic via inhalation accounted for a negligible part (< 1%) of the total daily intake of arsenic (calculated based on excreted urinary arsenic); however, potential associations between the adverse effects (e.g., lung adenocarcinoma) and inhaled PM-bound arsenic require more attention, particularly for those who experience in long-term exposure. This study is the first report of a 3-year temporal trend of airborne PM-bound arsenic in central China.

Temporal trend of arsenic in outdoor air PM in Wuhan, China, in 2015-2017 and the personal inhalation of PM-bound arsenic: implications for human exposure.

Shasha Zhao

College of Life Science and Health

Wuhan University of Science and Technology

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Shasha Zhao

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}College of Life Science and Health

Wuhan University of Science and Technology

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

College of Life Science and Health