Fork coupling directs DNA replication elongation and termination.

DNA replication is initiated at multiple loci to ensure timely duplication of eukaryotic genomes. Sister replication forks progress bidirectionally, and replication terminates when two convergent forks encounter one another. To investigate the coordination of replication forks, we developed a replication-associated in situ HiC method to capture chromatin interactions involving nascent DNA. We identify more than 2000 fountain-like structures of chromatin contacts in human and mouse genomes, indicative of coupling of DNA replication forks. Replication fork interaction not only occurs between sister forks but also involves forks from two distinct origins to predetermine replication termination. Termination-associated chromatin fountains are sensitive to replication stress and lead to coupled forks-associated genomic deletions in cancers. These findings reveal the spatial organization of DNA replication forks within the chromatin context.

Cohesin maintains replication timing to suppress DNA damage on cancer genes.

Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks and 147 translocation hotspot genes, co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor disruption. However, damage-induced chromosomal translocations coincide with the asymmetrically distributed Okazaki fragments of DNA replication, suggesting that RAD21 depletion causes replication stresses evidenced by the slower replication speed and increased stalled forks. Mechanistically, approximately 30% of the human genome exhibits an earlier replication timing after RAD21 depletion, caused by the early initiation of >900 extra dormant origins. Correspondingly, most translocation hotspot genes lie in timing-altered regions. Therefore, we conclude that cohesin dysfunction causes replication stresses induced by excessive DNA replication initiation, resulting in gross DNA damages that may promote tumorigenesis.

A Sensitive Response Index Selection for Rapid Assessment of Heavy Metals Toxicity to the Photosynthesis of Chlorella pyrenoidosa Based on Rapid Chlorophyll Fluorescence Induction Kinetics.

Heavy metals as toxic pollutants have important impacts on the photosynthesis of microalgae, thus seriously threatening the normal material circulation and energy flow of the aquatic ecosystem. In order to rapidly and sensitively detect the toxicity of heavy metals to microalgal photosynthesis, in this study, the effects of four typical toxic heavy metals, chromium (Cr(VI)), cadmium (Cd), mercury (Hg), and copper (Cu), on nine photosynthetic fluorescence parameters (φPo, ΨEo, φEo, δRo, ΨRo, φRo, FV/FO, PIABS, and Sm) derived from the chlorophyll fluorescence rise kinetics (OJIP) curve of microalga Chlorella pyrenoidosa, were investigated based on the chlorophyll fluorescence induction kinetics technique. By analyzing the change trends of each parameter with the concentrations of the four heavy metals, we found that compared with other parameters, φPo (maximum photochemical quantum yield of photosystem II), FV/FO (photochemical parameter of photosystem II), PIABS (photosynthetic performance index), and Sm (normalized area of the OJIP curve) demonstrated the same monotonic change characteristics with an increase in concentration of each heavy metal, indicating that these four parameters could be used as response indexes to quantitatively detect the toxicity of heavy metals. By further comparing the response performances of φPo, FV/FO, PIABS, and Sm to Cr(VI), Cd, Hg, and Cu, the results indicated that whether it was analyzed from the lowest observed effect concentration (LOEC), the influence degree by equal concentration of heavy metal, the 10% effective concentration (EC10), or the median effective concentration (EC50), the response sensitivities of PIABS to each heavy metal were all significantly superior to those of φRo, FV/FO, and Sm. Thus, PIABS was the most suitable response index for sensitive detection of heavy metals toxicity. Using PIABS as a response index to compare the toxicity of Cr(VI), Cd, Hg, and Cu to C. pyrenoidosa photosynthesis within 4 h by EC50 values, the results indicated that Hg was the most toxic, while Cr(VI) toxicity was the lowest. This study provides a sensitive response index for rapidly detecting the toxicity of heavy metals to microalgae based on the chlorophyll fluorescence induction kinetics technique.

Sevoflurane participates in the protection of rat renal ischemia-reperfusion injury by down-regulating the expression of TRPM7.

INTRODUCTION: To investigate the protective effect of sevoflurane preconditioning on renal ischemia-reperfusion injury (renalischemiareperfusionmodel, RIRI) and its related mechanism. METHODS: Eighty healthy adult male SD rats were randomly divided into control group (Sham group), model group (RIRI group), sevoflurane pretreatment group (Sev group) and TRPM7 inhibitor combined with sevoflurane pretreatment group (T + Sev group), 20 animals in each group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, and the levels of creatinine and urea nitrogen in each group were detected. Deoxyribonucleic acid terminal transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect renal cell apoptosis, and Western blottingwas used to detect the expression of apoptotic proteins cleaved-caspase-3, bax, Bcl-2, and TRPM7 in renal tissue; Detection of oxidative stress-related index levels in renal tissue and levels of inflammatory factors in renal tissue and serum. RESULTS: Compared with the Sham group, the renal tissue pathological damage was aggravated, the levels of creatinine and blood urea nitrogen were increased, and the apoptosis was increased in the RIR group and the Sev group. Death, malondialdehyde (MDA) levels and inflammatory factors were increased, and superoxide dismutase (SOD) levels were decreased (all p < .05); The scores, apoptosis rate, MDA level, and relative expression of inflammatory factor levels were decreased, and SOD levels were increased (all p < .05). Compared with the Sev group, the renal tissue pathological damage in the T + Sev group was aggravated, creatinine, blood urea nitrogen levels increased, apoptosis increased, apoptosis-related proteins cleaved-caspase-3, bax, Bcl-2 showed increased apoptosis, malondialdehyde (MDA) levels, inflammatory factor levels increased, ultrahigh The levels of oxide dismutase (SOD) were decreased (all p < .05). CONCLUSIONS: Therefore, we believe that sevoflurane is involved in the protection of rat renal ischemia-reperfusion injury by downregulating the expression of TRPM7.

PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair.

The repair products of double-stranded DNA breaks (DSBs) are crucial for investigating the mechanism underlying DNA damage repair as well as evaluating the safety and efficiency of gene-editing; however, a comprehensively quantitative assay remains to be established. Here, we describe the step-by-step instructions of the primer extension-mediated sequencing (PEM-seq), followed by the framework of data processing and statistical analysis. PEM-seq presents a full spectrum of repair outcomes for both genome-editing-induced and endogenous DSBs in mouse and human cells. For complete details on the use and execution of this profile, please refer to Gan et al. (2021), Yin et al. (2019), Liu et al. (2021a), and Zhang et al. (2021).

C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency.

In activated B cells, activation-induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C-terminal peptide. In immunodeficient-patient cells expressing mutant AID lacking its C-terminus, a catalytically active AID-delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID-delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID-delC proteins form condensates both in vivo and in vitro, dependent on its N-terminus and on a surface arginine-rich patch. Co-expression of AID-delC and wild-type AID leads to an unbalanced nuclear AID-delC/AID ratio, with AID-delC proteins able to trap wild-type AID in condensates, resulting in a dominant-negative phenotype that could contribute to immunodeficiency. The co-condensation model of mutant and wild-type proteins could be an alternative explanation for the dominant-negative effect in genetic disorders.

A new method for detecting mixed bacteria based on multi-wavelength transmission spectroscopy technology.

Previously, we successfully realized the identification of a single species of bacteria based on the multi-wavelength transmission spectrum of bacteria. The current research is focused on realizing the spectral analysis of mixed bacteria. Principal component analysis-Monte Carlo (PCA-MC) model was developed for the implementation of spectral separation of mixed bacteria by obtaining the ratio of components. And, the separated spectrum was regarded as the model input of the neural network concentration inversion model to obtain the concentration of each bacteria in the mix. Mean relative errors in component analysis of mixing S.aureus with K.pneumoniae, mixing S.aureus with S.typhimurium twice, mixing K.pneumoniae with S.typhimurium are 3%, 2%, 3.9% and 6.1%, respectively. The coefficient of determination (R2) of validation set and test set are 0.9947 and 0.9954 in concentration inversion model. The results show that this method can quickly and accurately determine the component ratio and concentration information in the mixed bacteria. A new method was proposed to separate the spectrum of mixed bacteria effectively and measure its concentration quickly, which makes a big step forward in the detection and online monitoring of waterborne microbial contamination based on multi-wavelength transmission spectroscopy.

Rapid and Sensitive Detection of Water Toxicity Based on Photosynthetic Inhibition Effect.

To achieve rapid and sensitive detection of the toxicity of pollutants in the aquatic environment, a photosynthetic inhibition method with microalgae as the test organism and photosynthetic fluorescence parameters as the test endpoint was proposed. In this study, eight environmental pollutants were selected to act on the tested organism, Chlorella pyrenoidosa, including herbicides (diuron, atrazine), fungicides (fuberidazole), organic chemical raw materials (phenanthrene, phenol, p-benzoquinone), disinfectants (trichloroacetonitrile uric acid), and disinfection by-products (trichloroacetonitrile). The results showed that, in addition to specific PSII inhibitors (diuretic and atrazine), other types of pollutants could also quickly affect the photosynthetic system. The photosynthetic fluorescence parameters (Fv/Fm, Yield, α, and rP) could be used to detect the effects of pollutants on the photosynthetic system. Although the decay rate of the photosynthetic fluorescence parameters corresponding to the different pollutants was different, 1 h could be used as an appropriate toxicity exposure time. Moreover, the lowest respondent concentrations of photosynthetic fluorescence parameters to diuron, atrazine, fuberidazole, phenanthrene, P-benzoquinone, phenol, trichloroacetonitrile uric acid, and trichloroacetonitrile were 2 µg·L-1, 5 µg·L-1, 0.05 mg·L-1, 2 µg·L-1, 1.0 mg·L-1, 0.4 g·L-1, 0.1 mg·L-1, and 2.0 mg·L-1, respectively. Finally, diuron, atrazine, fuberidazole, and phenanthrene were selected for a comparison of their photosynthetic inhibition and growth inhibition. The results suggested that photosynthetic inhibition could overcome the time dependence of growth inhibition and shorten the toxic exposure time from more than 24 h to less than 1 h, or even a few minutes, while, the sensitivity of the toxicity test was not weakened. This study indicates that the photosynthetic inhibition method could be used for rapid detection of the toxicity of water pollutants and that algae fluorescence provides convenient access to toxicity data.

RAG2 abolishes RAG1 aggregation to facilitate V(D)J recombination.

RAG1 and RAG2 form a tetramer nuclease to initiate V(D)J recombination in developing T and B lymphocytes. The RAG1 protein evolves from a transposon ancestor and possesses nuclease activity that requires interaction with RAG2. Here, we show that the human RAG1 aggregates in the nucleus in the absence of RAG2, exhibiting an extremely low V(D)J recombination activity. In contrast, RAG2 does not aggregate by itself, but it interacts with RAG1 to disrupt RAG1 aggregates and thereby activate robust V(D)J recombination. Moreover, RAG2 from mouse and zebrafish could not disrupt the aggregation of human RAG1 as efficiently as human RAG2 did, indicating a species-specific regulatory mechanism for RAG1 by RAG2. Therefore, we propose that RAG2 coevolves with RAG1 to release inert RAG1 from aggregates and thereby activate V(D)J recombination to generate diverse antigen receptors in lymphocytes.

Transcription shapes DNA replication initiation to preserve genome integrity.

BACKGROUND: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage. RESULTS: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage. CONCLUSION: RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

Transdermal iontophoresis delivery system for terazosin hydrochloride: an in vitro and in vivo study.

This study aimed to construct a transdermal iontophoresis delivery system for terazosin hydrochloride (IDDS-TEH), which included a positive and negative electrode hydrogel prescription. Intact guinea pig skin was used as a model for the skin barrier function, and the current intensity, terazosin hydrochloride (TEH) concentration, pH, competitive salt, and transdermal enhancer properties were studied. The blood drug concentration was determined in Sprague-Dawley (SD) rats using HPLC, and the antihypertensive effects of IDDS-TEH were evaluated in spontaneously hypertensive rats (SHRs). The results showed that the steady-state penetration rate of TEH increased (from 80.36 µg·cm-2·h-1 to 304.93 µg·cm-2·h-1), followed by an increase in the current intensity (from 0.10 mA·cm-2 to 0.49 mA·cm-2). The pH values also had a significant influence on percutaneous penetration. The blood concentration of IDDS-TEH was significantly higher (p < .05) than with passive diffusion, which could not be detected. The main pharmacokinetic parameters of the high current group (0.17 mA·cm-2) and the low current group (0.09 mA·cm-2) were AUC0-t: 5873.0 ng·mL-1·h and 2493.7 ng·mL-1·h, respectively. Meanwhile, the pharmacodynamic results showed that IDDS-TEH significantly decreased the blood pressure of SHRs compared with the TEH hydrogel without loading current. Therefore, TEH could be successfully delivered by the transdermal iontophoresis system in vitro and in vivo, and further clinical studies should be explored to develop a therapeutically useful protocol.

Artificial neural networks combined multi-wavelength transmission spectrum feature extraction for sensitive identification of waterborne bacteria.

Present research is focused on the rapid and accurate identification of bacterial species based on artificial neural networks combined with spectral data processing technology. The spectra of different bacterial species in the logarithmic growth phase were obtained. Model input features were extracted from the raw spectra using signal processing techniques, including normalization, principal component analysis (PCA) and area-based feature value extraction. The identification models based on artificial neural network of back propagation neural networks (BPNN), generalized regression neural networks (GRNN) and probabilistic neural networks (PNN) were developed using the extracted features in order to ascertain whether the different species of bacteria could be differentiated. The performance of developed models and its corresponding signal processing techniques is tested by the recognition accuracy of validation set and test set, and model error. The maximum recognition accuracy of normalized spectrum combined with BPNN was 95.5% (error: 10%, test accuracy: 100%). The total recognition accuracy of PCA-reduced features (200-400 nm) combined with GRNN resulted in 96.3%~96.8% (error: 3.3%~6.7%, test accuracy: 97.5%~100%). While the overall recognition accuracy of area-based features combined with GRNN reached 97.3% with test accuracy of 100% (model error: 5.0%). Choosing of model and signal processing techniques has a positive influence on improving classification accuracy, so as to make it possible to realize the rapid detection and online monitoring of waterborne microbial contamination.

Molecularly imprinted polymer coated Mn-doped ZnS quantum dots embedded in a metal-organic framework as a probe for selective room temperature phosphorescence detection of chlorpyrifos.

As one of the most widely used organophosphorus pesticides, chlorpyrifos (CPF) is toxic to humans. However, the rapid, effective and sensitive detection of CPF is still a challenge. In this paper, a novel molecularly imprinted phosphorescent sensor with a core-shell structure (Mn:ZnS QDs@ZIF-8@MIP) using Mn:ZnS quantum dots (QDs) as phosphorescent emitters was prepared for the highly sensitive and selective detection of CPF, and a simple and rapid room-temperature phosphorescence (RTP) detection method for CPF was proposed. For the prepared Mn:ZnS QDs@ZIF-8@MIP, Mn:ZnS QDs had good phosphorescence emission characteristics, ZIF-8 as support materials was used to improve the dispersibility of Mn:ZnS QDs, and molecularly imprinted polymer (MIP) on the surface of ZIF-8 was used to improve the selectivity of Mn:ZnS QDs for CPF. Under the optimal response conditions, the RTP intensity of Mn:ZnS QDs@ZIF-8@MIP showed a rapid response to CPF (less than 5 min), the RTP intensity ratio of P 0/P had a good linear relationship with the concentration of CPF in the range of 0-80 µM, and the detection limit of this method was 0.89 µM with the correlation coefficient of 0.99. Moreover, this simple and rapid method has been successfully used to detect CPF in real water samples with satisfactory results, and the recoveries ranged from 92% to 105% with a relative standard deviation of less than 1%. This method combines the advantages of phosphorescence emission and molecular imprinting, and greatly reduces the potential interferences of competitive substances, background fluorescence and scattered light, which opens up a broad prospect for the highly sensitive and selective detection of pollutants in water based on molecularly imprinted phosphorescent sensors.

Simultaneous determination of nitrate, chemical oxygen demand and turbidity in water based on UV-Vis absorption spectrometry combined with interval analysis.

In this paper, a new method for simultaneous determination of nitrate, COD and turbidity in water based on UV-Vis absorption spectrometry combined with interval analysis was studied. By analyzing the spectral absorption characteristics of nitrate, COD, and turbidity standard solutions and the mixtures of them, the absorption spectra in the range of 225-260 nm, 260-320 nm and 320-700 nm were selected as the characteristic spectra of nitrate, COD and turbidity, respectively. Multiplicative scatter correction was employed to compensate turbidity of the absorption spectra of the mixture solutions in the wavelength range of 225-320 nm. Then, the spectra after turbidity compensation in the range of 225-260 nm was compensated for COD using the method of spectral difference. The original spectra in the range of 320-700 nm, the turbidity compensated spectra in the range of 260-320 nm, and the COD compensated spectra in the range of 225-260 nm were analyzed by PLS algorithm in order to calculate the concentrations of nitrate, COD and turbidity in the mixture solutions. The results showed that this method could simultaneously and accurately determine the concentrations of nitrate, COD and turbidity. After interval analysis, all the correlation coefficients (R2) between the predicted values and the true values of nitrate, COD and turbidity were higher than 0.9, and root mean square error (RMSE) of predicted values were between 0.696 and 2.337.

Preconcentration with Chlorella vulgaris combined with energy dispersive X-ray fluorescence spectrometry for rapid determination of Cd in water.

Freshwater green algae Chlorella vulgaris was selected as an adsorbent, and a simple, rapid, economical and environmentally friendly method for the detection of heavy metal Cd in water samples based on preconcentration with C. vulgaris combined with energy dispersive X-ray fluorescence (EDXRF) spectrometry was proposed.  Chlorella vulgaris could directly and rapidly adsorb Cd2+ without any pretreatment, and the maximum adsorption efficiency could be obtained when the contact time was 1 min with an optimal pH of 10. The obtained Cd-enriched thin samples after preconcentration with C. vulgaris by suction filtration of reaction solution had very good uniformity, which could be directly measured by EDXRF spectrometry, and the net integral fluorescence intensity of Cd Kα characteristic peak had a very good linear relationship with the initial concentration of Cd in the range of 0.703-74.957 µg ml-1 with a correlation coefficient of 0.9979. When the Cd thin samples with a Cd-enriched region of 15.1 mm in diameter were formed by the developed preconcentration method with suction filtration of 10 ml reaction solution, the detection limit of this method was 0.0654 µg ml-1, which was lower than the maximum allowable discharge concentration of Cd in various industrial wastewaters. The proposed method was simple to operate, and could effectively remove the influence of matrix effect of water samples and effectively improve the sensitivity and stability of EDXRF spectrometry directly detecting heavy metals in water samples, which was successfully applied to detect Cd in real water samples with satisfactory results, and the recoveries ranged from 94.80% to 116.94%. Moreover, this method can be applied to the rapid detection and early warning of excessive Cd in discharged industrial wastewaters. This work will provide a methodological basis for the development of rapid and online monitoring technology and instrument of heavy metal pollutants in water.

The Deubiquitinase USP38 Promotes NHEJ Repair through Regulation of HDAC1 Activity and Regulates Cancer Cell Response to Genotoxic Insults.

The DNA damage response (DDR) is essential for maintaining genome integrity. Mounting evidence reveals that protein modifications play vital roles in the DDR. Here, we show that USP38 is involved in the DDR by regulating the activity of HDAC1. In response to DNA damage, USP38 interacted with HDAC1 and specifically removed the K63-linked ubiquitin chain promoting the deacetylase activity of HDAC1. As a result, HDAC1 was able to deacetylate H3K56. USP38 deletion resulted in persistent focal accumulation of nonhomologous end joining (NHEJ) factors at DNA damage sites and impaired NHEJ efficiency, causing genome instability and sensitizing cancer cells to genotoxic insults. Knockout of USP38 rendered mice hypersensitive to irradiation and shortened survival. In addition, USP38 was expressed at low levels in certain types of cancers including renal cell carcinoma, indicating dysregulation of USP38 expression contributes to genomic instability and may lead to tumorigenesis. In summary, this study identifies a critical role of USP38 in modulating genome integrity and cancer cell resistance to genotoxic insults by deubiquitinating HDAC1 and regulating its deacetylation activity. SIGNIFICANCE: This study demonstrates that USP38 regulates genome stability and mediates cancer cell resistance to DNA-damaging therapy, providing insight into tumorigenesis and implicating USP38 as a potential target for cancer diagnosis.

Lung cancer cells release high mobility group box 1 and promote the formation of neutrophil extracellular traps.

Lung cancer is the leading cause of cancer-associated mortality. Tumor-associated neutrophils represent a large portion of inflammatory cells within the lung tumor microenvironment. However, the roles of neutrophil extracellular traps (NETs) in lung cancer remain unclear. In the present study, it was identified that Lewis lung carcinoma cells actively released the danger-associated molecular pattern protein high mobility group box 1 (HMGB1). Furthermore, HMGB1 in lung cancer cell supernatants promoted the formation of neutrophil extracellular traps (NETs), which was dependent on Toll-like receptor 4 (TLR4). The downstream molecules of TLR4, including myeloid differentiation factor 88, TIR-domain-containing adapter-inducing interferon-ß, p38 mitogen-activated protein kinases (p38 MAPKs) and extracellular signal-regulated kinases (ERKs), were activated during the formation of NETs. In addition, inhibition of p38 MAPKs or ERKs significantly decreased NETs. Morphine, an additional ligand for TLR4, aggravated the NETs induced by lung cancer cells. The present study revealed novel mechanisms in tumor-associated NET formation.

Optimal chlorophyll fluorescence parameter selection for rapid and sensitive detection of lead toxicity to marine microalgae Nitzschia closterium based on chlorophyll fluorescence technology.

Heavy metal pollution as one of the most serious pollution problems of marine environment, seriously threatens the safety of marine organism and human health, and will lead to potential risks for the marine ecological environment. In order to develop a rapid and sensitive toxicity detection method for marine heavy metals, in this study, marine diatom Nitzschia closterium was used as the test organism, and the effects of different concentrations of lead (Pb) on the five chlorophyll fluorescence parameters of N. closterium including the maximum photochemical quantum yield of PSII (Fv/Fm), the effective quantum yield of PSII photochemical energy conversion (ΦPSII), the effective absorption cross section of PSII photochemistry (σPSII'), the relative electron transfer rate of PSII (rP), and the PSII electron flux per unit volume (JVPII) at different exposure times were investigated based on chlorophyll fluorescence technology. By comparing with the photosynthetic activity fluorescence parameter Fv/Fm which is commonly used for toxicity analysis of pollutants using algae as test organisms, the optimal chlorophyll fluorescence parameter that could rapidly and sensitively determine Pb toxicity to N. closterium was selected. The results indicate that all the five chlorophyll fluorescence parameters of Fv/Fm, ΦPSII, σPSII', rP and JVPII showed good dose-response relationships with Pb within 8 h exposure time, and they all could be used as endpoints to rapidly determine Pb toxicity to N. closterium. Among the five chlorophyll fluorescence parameters, JVPII was the most sensitive fluorescence parameter for detecting the toxicity of Pb to N. closterium within 6 h exposure. And for JVPII, the median effective concentration (EC50) values of Pb at 2, 4 and 6 h were 0.329, 0.068 and 0.040 mmol L-1, respectively. However, when the exposure time was 8 h, ΦPSII was the most sensitive fluorescence parameter for the toxicity detection of Pb, and the EC50 value of Pb at 8 h was 0.038 mmol L-1. This study will provide an important basis for the development of a rapid and sensitive detection method for the biological toxicity of marine heavy metals, and those results will be helpful for ecological risk assessment in marine environment.

Extracellular RNAs from lung cancer cells activate epithelial cells and induce neutrophil extracellular traps.

Neutrophil infiltration is frequently observed in lung cancer tissues. Extracellular RNAs (exRNAs) may facilitate tumor progression. The present study investigated the cross­talk of tumor exRNAs and neutrophil extracellular traps (NETs) in lung cancer. Lewis lung carcinoma (LLC) cells were cultured with the deprived sera. And the cell culture supernatants (CCS) were analyzed in vitro and in vivo. The results revealed that exRNAs from lung cancer CCS promoted the inflammatory cytokine interleukin­1ß and reduced the vascular cell adhesion molecule­1 expression in lung epithelial cells. Lung cancer CCS­treated epithelial cells induced the production of NETs. By contrast, NETs reduced the tight junction protein claudin­5 in epithelial cells. Furthermore, NETs caused the necrosis of epithelial cells, which resulted in the release of exRNAs. In mice, lung cancer cells instilled in the lung recruited neutrophils and initiated NETs. In patients with lung cancer, NETs were also observed. These results suggested that exRNAs in the cell culture supernatant may indirectly induce NETs and contribute to lung cancer oncogenesis.

Optimizing genome editing strategy by primer-extension-mediated sequencing.

Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.