GPI-anchored glutathione S-transferase as marker allows affinity sorting of transfection-positive cells.

Cell transfection efficiency is still a limiting factor in gene function research. A method that allows isolation and enrichment of the transfection-positive cells is an effective solution. Here, we report a transfection-positive cell sorting system that utilizes GPI-anchored GST (Glutathione S-transferase) as a plasmid marker. The Glutathione S-transferase fusion protein will be expressed and displayed on the cell surface through GPI anchor, and hence permits the positive cells to be isolated using Glutathione (GSH) Magnetic Beads. We prove that the system works efficiently in both the adherent Lenti-X 293T cells and the suspension K-562 cells. The affinity cell sorting procedure efficiently enriched positive cells from 20% to 98% in K-562 cells. The applications in gene knockdown and overexpression experiments in K-562 cells dramatically enhanced the extent of gene alteration, with the gene knockdown efficiency increasing from 7% to 60% and the gene overexpression level rising from 47 to 253 times. This Glutathione S-transferase affinity transfection-positive cell sorting method is simple and fast to operate, large-instrument free, low cost, and hence possesses great potential in gene function study in vitro.

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag.

Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.

Effects of Bacillus subtilis and nanohydroxyapatite on the metal accumulation and microbial diversity of rapeseed (Brassica campestris L.) for the remediation of cadmium-contaminated soil.

This study investigated the effects of the co-application of Bacillus subtilis and nanohydroxyapatite (NHAP) on plant growth, soil cadmium (Cd) dynamics, and the microbiological characteristics (such as enzyme activity and bacterial species richness) of the rhizosphere soil. Rapeseed was used as a model plant in pot experiments. Different concentrations of B. subtilis and 0.5% NHAP were applied alone and in combination to Cd-contaminated soil. The Cd contents in soils and plants as well as the rhizospheric microorganism diversity were assessed. The addition of B. subtilis or NHAP alone increased the soil Cd content and decreased the plant Cd content, while their co-application more effectively increased the soil and plant Cd contents than either treatment alone. B. subtilis and NHAP reduced the plant Cd content by 43.15-57.04% compared with that in the control. Rhizosphere community richness and bacterial diversity were significantly increased after co-application of B. subtilis and NHAP. Co-application of B. subtilis and NHAP effectively promoted rapeseed growth and improved Cd-contaminated soil remediation.

Effect of nanohydroxyapatite on cadmium leaching and environmental risks under simulated acid rain.

Cadmium (Cd) contamination in soil is a global environmental pollution issue. Nanohydroxyapatite (NHAP) has been used in soil remediation to immobilize cadmium in contaminated soils. However, the effect of acid rain on the export of cadmium from topsoil and its behavior in deep soil and leachate is unclear. In this study, column experiments and development of parsimonious model were performed to estimate Cd leaching behavior from topsoil and environmental risk of groundwater after 0.5% NHAP remediation. Four leaching events were performed and total Cd, different fractions of Cd determined by sequential extraction procedure and pH were determined for each leaching. The results show that with the export of Cd in topsoil, the total Cd concentration in soil at different depths had the following vertical distributions: 0-5 cm > 5-10 cm > 10-15 cm > 15-20 cm. NHAP treatment increased the soil pH and decreased Cd leaching loss by 56.45% compared to the control, and the results fit the parabolic diffusion model. With sequential extraction it was observed that NHAP application increased the residual fraction of cadmium in soil. After leaching, there was a positive correlation between soil pH and Cd concentration with regards to the exchangeable, reducible, oxidizable and residual Cd fractions. The parabolic diffusion model showed that Cd-contaminated soil with NHAP remediation is harmless to humans after sufficient remediation duration, whereas the resultant concentrations from the CK treatment could be toxic. The results indicate that nanohydroxyapatite could significantly reduce the bioavailability of cadmium and the environmental risk. However, the release of Ca and P from the dissolution of NHAP should be carefully studied as this will impact the mobilization of Cd or colloid Cd, and high leaching of P may result in P-induced eutrophication risk.

Influence of Nano-Hydroxyapatite on the Metal Bioavailability, Plant Metal Accumulation and Root Exudates of Ryegrass for Phytoremediation in Lead-Polluted Soil.

Lead is recognized as one of the most widespread toxic metal contaminants and pervasive environmental health concerns in the environment. In this paper, the effects of nano-hydroxyapatite (NHAP) on remediation in artificially Pb-contaminated soils and ryegrass were studied in a pot experiment. The addition of NHAP decreased the water- and acid-soluble, exchangeable, and reducible fractions of Pb, extracted using the Community Bureau of Reference (BCR) method, whilst greatly increasing the residual fraction of Pb. Oxidizable Pb was increased slightly. No significant increase in soil pH was caused by the application of NHAP. Compared to conditions without NHAP, the addition of NHAP decreased the Pb content in ryegrass shoots and roots by 13.19-20.3% and 2.86-21.1%, respectively. Therefore, the application of NHAP reduced the mobility and bioavailability of Pb in the soil. In addition, the application of NHAP improved the fresh weight of shoots and roots, and promoted the growth of ryegrass. NHAP played a positive role in stimulating ryegrass to secrete tartaric acid.

Nano-hydroxyapatite alleviates the detrimental effects of heavy metals on plant growth and soil microbes in e-waste-contaminated soil.

The crude recycling activities of e-waste have led to the severe and complex contamination of e-waste workshop topsoil (0-10 cm) by heavy metals. After nano-hydroxyapatite (NHAp) application in June 2013, plant and soil samples were obtained in November 2013, December 2013, March 2014 and June 2014. The results showed that NHAp effectively reduced the concentration of CaCl2-extractable Pb, Cu, Cd, and Zn in the topsoil and significantly reduced the metal content in ryegrass and also increased the plant biomass compared with that of the control. Moreover, the concentrations of CaCl2-extractable metals in the soil decreased with increasing NHAp. NHAp application also increased the activities of soil urease, phosphatase and dehydrogenase. Moreover, the soil bacterial diversity and community structure were also altered after NHAp application. Particularly, Stenotrophomonas sp. and Bacteroides percentages were increased. Our work proves that NHAp application can alleviate the detrimental effects of heavy metals on plants grown in e-waste-contaminated soil and soil enzyme activities, as well as soil microbial diversity.

Instrument development and validation of the stroke pre-hospital delay behavior intention scale in a Chinese urban population.

BACKGROUND: Several stroke impairment scales are currently available for stroke patients but none of them provide information regarding the pre-stroke behavioral intentions of high-risk stroke patients and their relatives. This study's objective was to generate and validate a new written tool, the Stroke Pre-hospital Delay Behavior Intention (SPDBI) scale. It is suitable for use with high-risk stroke patients and their relatives to predict the likelihood of pre-hospital delay. METHODS: From a review of related studies, we formulated a prototype scale. We interviewed ten stroke patients in a semi-structured iterative process that included interviews with experts, high-risk patients, and their family members. Then, we pretested and filtered items. We next used a large sample size and factor analysis to determine the scale's structure. Finally, we checked the reliability and validity of the scale. RESULTS: We identified five sub-domains (stroke warning signs, non-treatment justification, symptom attributions, habitual response style, and emergency system use). The SPDBI demonstrated good internal consistency and test-retest reliability (Cronbach's α =0.808; Intraclass Correlation Coefficient [ICC] =0.797). CONCLUSIONS: This SPDBI scale is a reliable, and valid measure of the likeliness of pre-hospital delay in high-risk stroke patients and their family members. It may provide scientific assessment for targeted health education intervention.

Development and validation of the Pre-hospital Stroke Symptoms Coping Test.

BACKGROUND AND PURPOSE: Measures of specific knowledge of coping with pre-hospital stroke symptoms can help educate high-risk patients and family caregivers. This study aimed to develop and validate the Pre-hospital Stroke Symptoms Coping Test (PSSCT). MATERIALS AND METHODS: Reliability and validity were analyzed using multiple data sources. The Delphi expert consultation method was applied to assess the test's surface validity and content validity index. The final edition of the 19-item PSSCT contained 3 sections assessing coping with typical symptoms and symptoms associated with vomiting and twitching. Its psychometric properties were investigated in a community sample of 300 high-risk patients and family members. RESULTS: The PSSCT was readily accepted by participants. It demonstrated adequate surface validity and content validity, and good internal consistency (KR20 = 0.822) and test-retest reliability (0.769), with difficulty (P) and degree of differentiation (D) ranges of 0.28-0.83 and 0.15-0.66, respectively. It was also able to distinguish between individuals who had/had not experienced a stroke. Experienced individuals scored significantly higher overall and on coping with typical symptoms and twitching (P<0.01). CONCLUSIONS: The PSSCT can practically and directly assess critical knowledge regarding coping with pre-hospital stroke symptoms and has good reliability and validity.