Effects of µ-Conotoxin GIIIB on the cellular activity of mouse skeletal musculoblast: combined transcriptome and proteome analysis.

µ-Conotoxin GIIIB (µ-CTX GIIIB) is a polypeptide containing three disulfide bridges, produced by the sea snail Conus geographus. This study was aimed to explored the cytotoxic effects of µ-CTX GIIIB on mouse skeletal musculoblast (Sol8). Sol8 cells were exposed to ouabain and veratridine to establish the cell injury model, and then treated with µ-CTX GIIIB. CCK-8 was adopted to evaluate the cytotoxicity of µ-CTX GIIIB. Then, proteomics and transcriptome were conducted, and the explore the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) affected by µ-CTX GIIIB were found. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to investigate the affected signaling pathways. µ-CTX GIIIB increased the cell survival rate of injured Sol8 cells. We found and identified 1,663 DEGs and 444 DEPs influenced by µ-CTX GIIIB. 106 pairs of correlated DEGs and DEPs were selected by combining transcriptome and proteome data. The results of KEGG and GO analysis showed that µ-CTX GIIB affected the cell cycle, apoptosis, DNA damage and repair, lipid metabolism and other biological processes of Sol8 cells. µ-CTX GIIIB could affected cell cycle regulation, DNA damage repair, and activation of tumor factors, with potential carcinogenic effects. Our results provide an important basis for the study of in vitro toxicity, the mechanism of toxicity and injury prevention by µ-CTX GIIIB.

Outlook on the Security and Potential Improvements of CRISPR-Cas9.

Gene editing technology is regarded as a good news to save patients with genetic diseases because of its significant function of specifically changing genetic information. From zinc-finger proteins to transcription activator-like effector protein nucleases gene editing tools are constantly updated. At the same time, scientists are constantly developing a variety of new gene editing therapy strategies, in order to promote gene editing therapy from various aspects and realize the maturity of the technology as soon as possible. In 2016, CRISPR-Cas9-mediated CAR-T therapy was the first to enter the clinical trial stage, indicating that the use of CRISPR-Cas system as the blade of the genetic lancet to save patients is officially on the schedule. The first challenge to achieve this exciting goal is to improve the security of the technology. This review will introduce the gene security issues faced by the CRISPR system as a clinical treatment tool, the current safer delivery methods and the newly developed CRISPR editing tools with higher precision. Many reviews summarize the means of improving the security of gene editing therapy and the comprehensive delivery method, while few articles focus on the threat of gene editing to the genomic security of the treatment target. Therefore, this review focuses on the risks brought by gene editing therapy to the patient genome, which provides a broader perspective for exploring and improving the security of gene editing therapy from two aspects of delivery system and CRISPR editing tools.

Oral administration of Synechococcus sp. PCC7942 trans-vp19 and trans-vp (19+28) genes improve the immune and antioxidant capacity in Procambarus clarkii under white spot syndrome virus stress.

This study aimed to evaluate antioxidant capacity and protection from white spot syndrome virus (WSSV) challenge of Procambarus clarkii fed trans-vp19 and trans-vp (19 + 28) genes of Synechococcus sp. PCC7942 (Syn7942). P. clarkii were fed transgenic cyanobacteria continuously for 7 days, and then infected with WSSV after 12 h starvation. The daily mortality in each group was measured for 10 days and hepatopancreas and muscle of P. clarkii were examined for enzymes phenoloxidase (PO) activity, catalase (CAT) activity, glutathione peroxidase (GSH-px) activity, and malondialdehyde (MDA) concentration after immunization and viral challenge at different times. Compared with the WSSV-infected crayfish in positive control group (challenge and no vaccination) and wild type group (challenge, feeding wild-type Syn7942), vp19 group (challenge, feeding Syn7942 trans-vp19 gene) and vp (19 + 28) group [challenge, feeding Syn7942 trans-vp (19 + 28) genes] significantly improved the survival rate from 0% to 60% and 56.7%, respectively. Consistently, significantly greater PO, CAT, and GSH-px activity and significantly lower MDA concentration in the vp19 and vp (19 + 28) groups compared to the control group. These results demonstrate that the trans-vp19 and trans-vp (19 + 28) gene of Syn7942 significantly facilitated the immune and antioxidant capacity of crayfish. Therefore, the trans-vp19 and trans-vp (19 + 28) genes of Syn7942 could provide protection for crayfish as an anti-WSSV oral medication.

Regulation of pepc gene expression in Anabaena sp. PCC 7120 and its effects on cyclic electron flow around photosystem I and tolerances to environmental stresses.

Since pepc gene encoding phosphoenolpyruvate carboxylase (PEPCase) has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregulated (forward) or downregulated (reverse) pepc gene in Anabaena sp. PCC 7120. Results from real-time quantitative polymerase chain reaction (RT-qPCR), Western blot and enzymatic analysis showed that PEPCase activity was significantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I (PSI) activity was inhibited, as reflected by Y(I), and Y(NA) was elevated, and dark reduction of P700(+) was stimulated, indicating enhanced cyclic electron flow (CEF) around PSI in the reverse mutant. Additionally, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.

Recombinant expression and functional analysis of a Chlamydomonas reinhardtii bacterial-type phosphoenolpyruvate carboxylase gene fragment.

To investigate the function of a bacterial-type phosphoenolpyruvate carboxylase (PEPC2) derived from photosynthetically-grown Chlamydomonas reinhardtii, a fragment of the pepc2 gene was cloned and expressed in Escherichia coli. After optimal induction for 6 h, PEPC activity in the reverse mutant was lower than wild type (0.9 vs. 1.7 U/mg protein), and soluble protein was also lower than wild type (119 vs. 186 mg/g dry wt). In contrast, the total lipid content was increased from 56 (in wild type) to 71 mg/g dry wt, despite the growth rate being slightly diminished. The changes in PEPC activity, soluble protein and total lipid in the forward mutant were the opposite (2.4 U/mg, 230 mg/g, and 44 mg/g dry wt, respectively). Together, these data indicate that PEPC may function as a metabolic pivot in the regulation of protein and lipid accumulation in this alga.

Variations of morphology and photosynthetic performances of Ulva prolifera during the whole green tide blooming process in the Yellow Sea.

Since 2007, the world's largest macroalgal blooms have occurred along the coastal area of the Yellow Sea for 6 consecutive years. In 2012, shipboard surveying and satellite remote sensing were used to monitor the whole blooming process. The blooms originated in Rudong sea area of the South Yellow Sea where bloom patches were of dark green and filamentous thalli were the dominant morphology. The scale of the blooms reached its peak size in Rizhao sea area of the North Yellow Sea, and decreased promptly and became insignificant in Qingdao coast where the blooms turned yellow, mostly with air sac blades. Meanwhile, vegetative cells of the green tide algae changed into cytocysts gradually from which germ cells were released as the blooms drifted northward. Additionally, chlorophyll contents and fluorescence activity of free-floating thalli in the North Yellow Sea were both significantly lower than that in the South Yellow Sea. Those studies presented here contributed to increasing our understanding about how the green tide declined gradually in the North Yellow Sea.

Biosorption of heavy metals onto nonliving Laminaria japonica.

In this paper, study of the biosorption of Cd(2+) and Pb(2+) by nonliving Laminaria japonica in a batch adsorption system is described. The content of acidic sites and the dissociation constant of carboxylic acid functional groups (metal-binding site) of L. japonica were experimentally determined by conductometric and potentiometric titrations and theoretically predicated by using monodentate and bidentate binding models. The models are based on the monodentate or bidentate binding reactions of bivalent metal ions to acidic sites. The acidic site content and carboxylic acid dissociation constants determined are 1.25 and 0.18 mmol L(-1), respectively. The results showed that the bidentate adsorption model fits well the biosorption of bivalent metal ions onto L. japonica with the bidentate binding constants for Cd(2+) and Pb(2+) being 5.72 × 10(3) and 6.24 × 10(4) L mol(-1), respectively. The adsorption process of L. japonica followed the pseudo-second-order kinetics.

[Bioremediation of river water quality by consecutively adjustable submerged vegetation net].

A series of consecutively adjustable submerged vegetation nets were constructed in a polluted shallow river with a length of about 200 m and nearby the water resource protection area of Taihu Lake in East China, forming an aquatic vegetation consisted of submerged plant species Cabomba caroliniana, Vallisneria natans, Elodea nuttallii, Hydrilla verticillata, and Potamogeton crispus. The water quality indices including total nitrogen (TN), ammonium nitrogen (NH4(+)-N), nitrite nitrogen (NO2(-)-N), nitrate nitrogen (NO3(-)-N), total phosphorus (TP), and phosphate (PO4(3-)-P) were monitored, and the bioremediation effect of the vegetation nets was evaluated. After setting up the vegetation nets, the Secchi depth (SD) of the river changed from 0.5 m to 1.7-1.8 m, and the TN and TP concentrations 15 and 20 days after the nets constructed decreased by 35.6% and 66.3%, and 29.4% and 63.2%, respectively. After five months, the concentrations of NH4(+)-N, NO2(-)-N, NO3(-)-N, TN, TP, and PO4(3-)-P decreased by 92.4%, 76.8%, 72.7%, 73.9%, 90.5%, and 92.0%, respectively. This study showed that consecutively adjustable submerged vegetation net could be a potential approach for treating polluted river waters, particularly for the bioremediation of polluted small landscape shallow water bodies.

[Bioremediation efficiency of applying Daphnia magna and submerged plants: a case study in Dishui Lake of Shanghai, China].

From April 2007 to January 2008, a bioremediation experiment was conducted in a diversion channel of D-port pilot area of Dishui Lake (the channel length is 950 m, and its water volume is 10000 m3). Daphnia magna was first introduced to filter the high biomass of phytoplankton and other particulate organic matter, and then, five submerged plant species Elodea canadensis, Vallisneria spiralis, Hydrilla verticillata, Potamogeton lucens, and Potamogeton crispus were transplanted. Water samples were collected monthly to monitor the water quality and to investigate the bioremediation efficiency. Ten months monitoring data showed that in the remediation area, the water body's total nitrogen (TN), ammonium nitrogen (NH4(+)-N), nitrate nitrogen (NO3(-)-N), nitrite nitrogen (NO2(-)-N), total phosphorus (TP), and reactive phosphate (PO4(3-)-P) concentrations and chemical oxygen demand (COD) were significantly lower (P < 0.01), dissolved oxygen (DO) was increased by 50.4%, and the Secchi depth (SD) reached to an average of 3.4-3.7 m. Overall, the water quality was up to grades II or III of state water quality standards for surface water. In March 2008, the established submerged plant community was used to test its effectiveness in improving the eutrophicated water body from Dishui Lake, and the results showed that after 7-day treatment, except biological oxygen demand (BOD), the TN, TP, NO3(-)-N, NO2(-)-N, NH4(+)-N, and PO4(3-)-P concentrations and COD of the eutrophicated water were all decreased significantly, the DO was increased by 17.98%, and the SD was increased by 30 cm. The present study demonstrated the effectiveness of introducing D. magna and transplanting submerged plants in improving the water quality of Dishui Lake.

In vivo protective effect of Porphyra yezoensis polysaccharide against carbon tetrachloride induced hepatotoxicity in mice.

To study the protective effect and possible mechanism of Porphyra yezoensis polysaccharide (PYP) in hepatotoxicity mice, acute liver injury was successfully induced by injecting 0.2% carbon tetrachloride (CCl(4)) intraperitoneally. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and liver homogenate, content of malondialdehyde (MDA), activities of total superoxide dismutase (T-SOD) in liver were measured by biochemical methods. Liver index was calculated and pathological changes of the liver tissue were observed microscopically. PYP was found to significantly decrease the activities of ALT and AST (P<0.05), to remarkably lower the liver indexes and MDA level in hepatical tissues in mice (P<0.05), and to upregulated the lower T-SOD level in liver homogenate (P<0.01). Furthermore, histologic examination showed that PYP could attenuate and the extent of necrosis, reduce the immigration of inflammatory cells. PYP plays a protective action against hepatotoxicity induced by CCl(4) in mice, and its mechanisms may be related to free radical scavenging, increasing SOD activities and anti-lipid peroxide.

Establishment and cultivation of cell line HB with high temperature resistance and fast growth in Porphyra yezoensis.

A new breeding technology with cell culture in Porphyra yezoensis was studied, to establish a system of fast breeding with cell engineering in Porphyra. By means of somatic cell isolation and multiple clone technique, 4 pure cell-lines (HA, HB, HC, HD) have been established in Porphyra yezoensis. With purely culturing the cell seedlings and conchocelis filaments of the cell lines, their growth rate and resistance to higher temperature were measured. Among cell line HA, HB, HC, HD, HB was the best for resisting higher temperature (at 19 degrees C, 21 degrees C, 23 degrees C, 25 degrees C). Also the growth rate of HB was faster than others. In 1998-2000, the HB was cultivated at sea field of Haifeng in Qidong County, Jiangsu Province. The yield of HB was higher than that of local cultivar. So the HB might be a good cell line for both resisting higher temperature and faster growth. It showed the breeding with cell culture was a fast breeding method.

Expression of Choline Oxidase Gene (codA) Enhances Salt Tolerance of the Tobacco.

The codA gene for choline oxidase, which converts choline into betaine. This enzyme, cloned from a soil bacterium Arthrobactor globiformis, has been transferred and expressed in tobacco by the Agrobatcterium-method transformation through the binary plasmid pGAH/codA. The pGAH/codA carried with Km(R) and Hyg(R),and coding sequence for a transit peptide from Rubisco small subunit gene (rbcS) was inserted between 35 S promoter and codA, so that the COD could be introduced into the chloroplast by this transit peptide. The transformed plants were screened on the medium containing the Km and Hyg. PCR, Western and gold immunolocalization tests showed that the codA gene has integrated into the tobacco DNA genome and its protein was expressed and the mature peptide has gone into the chloroplasts by the transit peptide. The results of salt-tolerance measuring for transgenic plants showed that the transgenic plants were more tolerance to the salt than the control plants. The young transgenic plants (1.0--1.5 cm) could survive at 400 mmol/L NaCl MS medium for more than 30 days. From them the higher tolerance plant T4-400 was obtained, which could grow at the 300 mmol/L NaCl MS medium well. The transgenic plants (6--8cm) could grow normally at the 400 mmol/L NaCl MS medium while wild plants failed to do so. So the transferred plant with codA enhanced its tolerance to the salt stress.