Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway.

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS-DNA interactions, affecting the production of the STING activator 2'3'-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.

DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.

[Bioavailability and Ecological Risk Assessment of Cadmium in the Sea-Land Interaction Sediments of the Pearl River Delta].

Cadmium (Cd) pollution poses a threat to human health, and research on Cd bioavailability as well as its ecological risk assessment can help prevent and mitigate Cd hazards. The enrichment characteristics and variability of Cd were investigated in sea-land interaction soil fractions and the associated environmental and ecological risks were evaluated using the accumulation index (Igeo), potential ecological hazard index (Er), and risk assessment coding (RAC). The results showed that:① The Cd content of miscellaneous fill material was typically lower than 0.3 mg·kg-1 and that of plain full was higher than 0.3 mg·kg-1. The Cd content of marine sediment was significantly higher than that of continental sediments, averaging 0.36 and 0.10 mg·kg-1, respectively. The concentration of Cd in marine sediments buried at shallow depths (<5 m) was generally higher than at greater depths (>5 m). ② There was a moderate correlation between Cd and CEC in artificial fill (Q4ml; correlation coefficient=0.52, P<0.05). There was a weak correlation between Cd and organic matter in the marine sediments (correlation coefficient=0.49, P<0.05). Total cadmium and the physical and chemical properties of soil had a significant influence on the fraction of soil cadmium. ③ The Igeo of artificially fill and marine sediment was dominated by the relationship 1 < Igeo < 2, which indicated a moderate level of pollution. The Er of artificial fill and marine sediment was mainly 80 < Er < 160, indicating a high potential ecological hazard. Soil acid-extractable Cd accounted for more than 50% of the total Cd in each drill hole, which generally indicated a very high potential ecological risk. These results provide a basis for environmental and agricultural decision-making and provide theoretical guidance for soil pollution investigations and remediation.

[Distribution Characteristics and Influencing Factors of Organophosphorus Pesticides in Typical Soil Environment of Jianghan Plain].

In order to study the distribution characteristics of organophosphorus pesticides (OPPs)in the soil of Jianghan plain,78 profile-soil samples from various depth and 7 surface-soil samples were collected in September,2015 in established groundwater monitoring field site, Jianghan plain. The OPPs concentrations were determined by gas chromatography with nitrogen-phosphorus detector(GC-NPD). The results showed that OPPs were widespread in our studied area. The OPPs concentrations of suface-soil samples ranged from 89.80 ng·g-1 to 193.85 ng·g-1,with an average of 140.05 ng·g-1, whereas the OPPs concentrations of profile-soil samples ranged from 19.81 ng·g-1 to 138.28 ng·g-1,with an average of 40.99 ng·g-1. The main ingredients of OPPs in surface and profile soil samples were methamidophos, omethoate, diazinon and quinalphos,and the residual amount of 10 kinds of OPPs had posed a threat to agricultural products according to the America soil pesticide residue limits standards. The horizontal distribution of OPPs concentration in profile soil followed the order of nearby river farm area > nearby river area > farm area, namely GS1-1 > GS4 > GS2 > GS3. while the vertical distribution mostly decreased at first and then increased with increasing depth. The distribution characteristics of OPPs were also influenced by many factors, such as application amount of OPPs, the adsorption and desorption actions of soil, vertical movement of groundwater, the terrain environment in the study area, and the concentration of soil organic matters.

[Pollution characteristics and distribution of polycyclic aromatic hydrocarbons and organochlorine pesticides in groundwater at Xiaodian Sewage Irrigation Area, Taiyuan City].

Sewage irrigation has been widely used in areas of water shortage in northern China, and it may introduce organic contaminants into groundwater. To characterize the organic contaminants in groundwater in sewage irrigation area, the Xiaodian sewage irrigation area in Shanxi Province was chosen as the case study area. A total of 16 groundwater samples (13 from shallow aquifer, 3 from deep aquifer) were collected. Polycyclic aromatic hydrocarbons (PAHs) were determined by gas chromatography-mass spectrometry (GC-MS) and organochlorine pesticides (OCPs) were ainalyzed by gas chromatography-electron capture detection (GC-ECD). The results showed that the concentrations of PAHs ranged from 13.98 to 505.89 ng x L(-1) with an average concentration of 115.67 ng x (L)(-1). The 2 and 3 ring-PAHs were the main components, while naphthalene and phenanthrene were most frequently detected. The concentrations of OCPs were in the range of 13.91-103.23 ng x L(-1) with an average concentration of 40.99 ng x L(-1), while alpha-HCH, delta-HCH, o,p'-DDD, Aldrin, Endosulfan-sulfate and HCB were most frequently detected. Overall, shallow aquifers appeared more contaminated with these pollutants than deep aquifers. In the area, the order of the organic contaminants concentration in groundwater was: East Main Channel < Beizhang Drainage < Taiyu Drainage, which indicated the quality of groundwater was influenced by the sewage irrigation.

Experimental degradation of nonylphenol (endocrine disruptor) by using ultraviolet irradiation in the presence of hydrogen peroxide.

The photodegradation of nonylphenol under UV irradiation was investigated in an aqueous solution containing hydrogen peroxide (H2O2) as the photochemically active substance was investigated. Influences of pH, the concentration of hydrogen peroxide, and the initial concentration of nonylphenol were studied. The following conditions were determined as optimal for nonylphenol removal: pH 1, H2O2 concentration of 40 mg/L, and an initial nonylphenol concentration of 15 of mg/L. Under these conditions, the removal of nonylphenol reached about 80% after 2 hours of irradiation. Identification of degradation products was done by gas chromatography-mass spectrometry, and analyses revealed Benzaldehyde, 4-methoxy and Diisobutyl phthalate as the main products of nonylphenol photodegradation.

The source apportionment of polycyclic aromatic hydrocarbons (PAHs) in the topsoil in Xiaodian sewage irrigation area, North of China.

31 topsoil samples were collected by grid method in Xiaodian sewage irrigation area, Taiyuan City, North of China. The concentrations of 16 kinds of polycyclic aromatic hydrocarbons (PAHs) were determined by gas chromatograph coupled with mass spectrum. Generally speaking, the distribution order of PAHs in the area is: those with five and six rings > those with four rings > those with two and three rings. Source apportionment shows a significant zonation of the source of PAHs: the civil coal pollution occurred in the north part, the local and far factory pollution happened in the middle area and the mixed pollution sources from coal and wood combustion, automotive emission, presented in the south area. The distribution of PAHs has a definite relationship with the sewage water flow and soil adsorption. The related coefficient between PAHs and physicochemical property showed there was a negative correlation between pH, silt, clay and PAHs while there was a positive correlation between total organic carbon, sand and PAHs.

[Influence of organochlorine pesticides in wastewater on the soil along the channel].

Nine profile soil samples and two sewage water samples were collected from Xiaodian sewage irrigation area in Taiyuan city, concentrations of organochlorine pesticides (OCPs) were determined by the gas chromatography coupled with electron capture detector (GC-ECD) to analyze the influence of the leakage of sewage water. The result shows that OCPs in sewage water were mainly composed of HCHs. Concentrations of DDTs and other organochlorine pesticides were very low or out of the detection limit. Concentrations of sigmaOCPs and HCHs in eight profiles near irrigation channels to some extend decreased with the increasing of the linear distance off the channel, which shows influences of the leakage of sewage water on the soil nearby. Concentrations of HCHs clearly decreased with the increasing of soil depth in most profile soils. For the horizontal direction, concentrations of HCHs also decreased with the increasing of the linear distance off the channel. The correlation between HCHs and TOC was positive, but no correlation between pH and HCHs was found.

[Vertical distribution and possible sources of polycyclic aromatic hydrocarbon in sewage area soil].

Nine profile soil samples were collected from Xiaodian sewage irrigation area, Taiyuan city, China. The concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were analyzed by gas chromatography equipped with a mass spectrometry detector (GC/ MS). The rank order of the average concentrations of PAHs in the 0-10 cm upper soil layer was background area < swamp area < groundwater irrigation area < sewage irrigation area. The concentrations of PAHs in most profile soils decreased with the increase of depth, and the PAHs were mainly accumulated in the surface soil layer (0-40 cm). 4-6 rings of PAHs were mainly accumulated in the 0-50 cm soil layer, and the accumulation capacity in groundwater-irrigation area was better than that in sewage irrigation area. The correlation between different rings of PAHs and TOC was positive (r(max) = 0.791, P = 0), and the same situation was found for PAHs and sand (r(max) = 0. 882, P = 0). The correlation between PAHs and pH was negative (r(min) = -0.1, P = 0.702). The main source of PAHs in the surface soil layer (0-40 cm) of study area was coal combustion. There were two pollution ways of PAHs in soil, one was settled into soil directly, the other was first settled into water and absorbed on the surface of solid particles, and then got enrichment in soil as irrigation water flew.

[Variation characteristics of relative molecular mass of different fractions of dissolved organic matter from landfill leachate during photocatalytic degradation].

Gel permeation chromatography was applied to analyze the variation characteristics of molecular mass of DOM six fractions during photocatalytic degradation. The results showed that most of DOM with average relative molecular mass was below 10(4). Large molecular fractions higher than 10(4) mainly containing HIB and HIN, accounted for nearly 20% of total DOM. Relative molecular mass of other fractions was mainly from 10(3) to 10(4). Fractions with relative molecular mass less than 500 were little. Most of DOM in leachate was yellow humic acid with moderate relative molecular mass and that is difficult to be biologically degraded. Relative molecular mass distribution of DOM fraction in different effluent broadened gradually during photocatalytic process, while polydispersity distribution coefficient (D) increased gradually. Relative molecular mass of HOB, HIB, HIA and HON decreased notably after photocatalytic treatment, especially HOB which relative molecular mass decreased from initial (4-25) x 10(3) to (0.4-1) x 10(3) after 72 h degradation. On the contrary, relative molecular mass of HOA and HIN tended to increase, and the more obvious is HOA which relative molecular mass increased from (2-20) x 10(3) of raw leachate to (20-50) x 10(3) of 72 h effluent. RID peak signal intensity of each DOM fraction decreased with the treatment time, which illustrated that the concentration decreased. As a whole, all fractions of DOM can be photocatalytically transformed efficiently.

[3D-EEM fluorescence characteristics of different fraction of dissolved organic matter in landfill leachate].

Three-dimensional excitation emission matrix fluorescence spectroscopy (3D-EEM) was applied to analyze the fluorescence characterization of dissolved organic matter in landfill leachate. The results showed that fulvic-like, tryptophan-like and humic acids-like matters were the main compounds in landfill leachate. A number of UV fulvic-like matters led to ineffective biodegradation of leachate. HOA contained more UV fulvic-like and less Vis fulvic-like, by contrary to HIA; HIN was mainly composed of UV fulvic-like and Vis fulvic-like matters; HOB, HIB and HIN had more intense fluorescence signal, whose fluorescence peaks occurred in the region of fulvic-like, tryptophan-like and humic acids-like. This was similar to the original leachate as well as HON which had relatively weak fluorescence signal. Moreover, different fractions had different fluorescence signal intensity in different regions. HOB and HIB had more intense signal in the region of UV fulvic-like. HIN had more intense signal in the region of UV fulvic-like and Vis fulvic-like. HON had moderately intense signal in the whole region. HOA and HIA had relatively weak fluorescence signal.

[Vertical distribution and source analysis of organochlorine pesticides in sewage irrigation area, Taiyuan city].

Nine profile soil samples were collected from Xiaodian sewage irrigation area in Taiyuan city, and the concentrations of organochlorine pesticides (OCPs) were determined by gas chromatography coupled with electron capture detector (GC-ECD) to analyze the vertical distribution. The results showed that the OCPs were mainly accumulated in the surface soil layer (0-30 cm) with the maximum concentration of 98.56 ng x g(-1), and HCHs, DDTs, endosulfans and methoxychlor were the predominant contaminants compared with other pesticides in the surface soil, which accounted for 85.1% in total OCPs. The concentrations of OCPs were clearly decreased with the increasing of the depth to the top layer in the most profile soils. Beta-HCH and DDE (sum of o,p'-DDE and p,p'-DDE) were the main contaminants in HCH pesticides and DDT pesticides, respectively. Composition analysis indicated that new DDT sources might be introduced into the groundwater irrigation and swamp area recently, and the main source of HCHs and DDTs was the residual of history use in other areas. Most profile soils were defined silt loam in study area. The correlations between the concentration of sigma OCPs and the total organic carbon (TOC) were positively significant in sewage irrigation area and groundwater irrigation area, but they were not significantly correlated in swamp area and background area.