Construction and analysis of the immune effect of two different vaccine types based on Vibrio harveyi VgrG.

Vibrio harveyi poses a significant threat to fish and invertebrates in mariculture, resulting in substantial financial repercussions for the aquaculture sector. Valine-glycine repeat protein G (VgrG) is essential for the type VI secretion system's (T6SS) assembly and secretion. VgrG from V. harveyi QT520 was cloned and analyzed in this study. The localization of VgrG was determined by Western blot, which revealed that it was located in the cytoplasm, secreted extracellularly, and attached to the membrane. The effectiveness of two vaccinations against V. harveyi infection-a subunit vaccine (rVgrG) and a DNA vaccine (pCNVgrG) prepared with VgrG was evaluated. The findings indicated that both vaccines provided a degree of protection against V. harveyi challenge. At 4 weeks post-vaccination (p.v.), the rVgrG and pCNVgrG exhibited relative percent survival rates (RPS) of 71.43% and 76.19%, respectively. At 8 weeks p.v., the RPS for rVgrG and pCNVgrG were 68.21% and 72.71%, respectively. While both rVgrG and pCNVgrG elicited serum antibody production, the subunit vaccinated fish demonstrated significantly higher levels of serum anti-VgrG specific antibodies than the DNA vaccine group. The result of qRT-PCR demonstrated that the expression of major histocompatibility complex (MHC) class Iα, tumor necrosis factor-alpha (TNF-α), interferon γ (IFNγ), and cluster of differentiation 4 (CD4) were up-regulated by both rVgrG and pCNVgrG. Fish vaccinated with rVgrG and pCNVgrG exhibited increased activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme. These findings suggest that VgrG from V. harveyi holds potential for application in vaccination.

Molecular Mechanism of D1135E-Induced Discriminated CRISPR-Cas9 PAM Recognition.

The off-target effects of Streptococcus pyogenes Cas9 (SpCas9) pose a significant challenge to harness it as a therapeutical approach. Two major factors can result in SpCas9 off-targeting: tolerance to target DNA-guide RNA (gRNA) mismatch and less stringent recognition of protospacer adjacent motif (PAM) flanking the target DNA. Despite the abundance of engineered SpCas9-gRNA variants with improved sensitivity to target DNA-gRNA mismatch, studies focusing on enhancing SpCas9 PAM recognition stringency are quite few. A recent pioneering study identified a D1135E variant of SpCas9 that exhibits much-reduced editing activity at the noncanonical NAG/NGA PAM sites while preserving robust on-target activity at the canonical NGG-flanking sites (N is any nucleobase). Herein, we aim to clarify the molecular mechanism by which this single D1135E mutation confers on SpCas9 enhanced specificity for PAM recognition by molecular dynamics simulations. The results suggest that the variant maintains the base-specific recognition for the canonical NGG PAM via four hydrogen bonds, akin to that in the wild type (WT) SpCas9. While the noncanonical NAG PAM is engaged to the two PAM-interacting arginine residues (i.e., R1333 and R1335) in WT SpCas9 via two to three hydrogen bonds, the D1135E variant prefers to establish two hydrogen bonds with the PAM bases, accounting for its minimal editing activity on the off-target sites with an NAG PAM. The impaired NAG recognition by D1135E SpCas9 results from the PAM duplex displacement such that the hydrogen bond of R1333 to the second PAM base is disfavored. We further propose a mechanistic model to delineate how the mutation perturbs the noncanonical PAM recognition. We anticipate that the mechanistic knowledge could be leveraged for continuous optimization of SpCas9 PAM recognition specificity toward high-precision demanding applications.

An established kidney cell line from humpback grouper (Cromileptes altivelis) and its susceptibility to bacteria and heavy metals.

Humpback grouper (Cromileptes altivelis), one kind of commercial fish with considerable economic value, has been recognized as a promising candidate for mariculture. In the wake of the development of aquaculture industry, the breeding density of C. altivelis has increased gradually, which gave rise to the occurrence of various pathogenic diseases. In our research, we established a new kidney cell line (designated as CAK) from humpback grouper and evaluated its susceptibility to bacteria and heavy metals. The results of our study showed that the optimal growth temperature was 26 °C, and optimal medium was L-15 supplemented with 20% fetal bovine serum (FBS). The sequencing of 18S rRNA gene indicated that CAK cell line was derived from C. altivelis. Chromosome analysis showed that the number of chromosome in CAK was 48. After being transfected of pEGFP-N3 plasmid, high transfection efficiency of CAK was observed, suggesting the potential to be used for the study of foreign functional genes. Moreover, the bacterial susceptibility results revealed that CAK cells were sensitive to Vibrio harveyi and Edwardsiella tarda, especially V. harveyi. Meanwhile, three heavy metals (Hg, Cu, and Cd) had toxic effects on the CAK cells with a dose-dependent manner. To sum up, the CAK cell line might be an ideal tool in vitro for analyzing the function of exogenous genes, bacterial susceptibility, and toxicity assay of heavy metals.

[Pollution Characteristics and Risk Assessment of PAHs in the Soil of Wild Forsythia Suspensa in Shanxi].

Shanxi is one of the main producing areas of Forsythia suspensa in China. In order to explore the safety of the soil in the areas where Forsythia suspensa grows,70 surface (0-25 cm) soil samples were collected from the main growing areas of F. suspensa in the eastsouth of Shanxi Province in July 2017. The concentration and composition characteristics of 16 polycyclic aromatic hydrocarbons (PAHs) in the sample soils were analyzed using chemical extraction and gas chromatography-mass spectrometry (GC-MS). The diagnostic ratio method was used to determine the source of PAHs in the areas. The potential ecological risk was assessed by using the method of calculating the equivalent carcinogenic concentration of benzo[a]pyrene. The results showed that the average concentration of total PAHs (Σ16PAHs) in all of the soil samples was 1.85 µg·g-1, which was dominated by three ring number PAHs, accounting for 76.7% of the total PAHs. The detection rates of phenanthrene (Phe) and anthracene (Ant) were both 100% of all the sample sites. The soil PAHs in the wild F. suspensa growing areas mainly originated from coal, biomass burning, and motor vehicle exhaust emissions, which resulted from air transport and sedimentation pathways. In all of the sample sites, the concentration of Σ16PAHs the limit standard level (0.2 µg·g-1) of Maliszewska-Kordybach for agricultural soil pollution and exceeded the soil heavy pollution level limit value (1.0 µg·g-1) in 41.4% of the sample sites. The concentration of BaP was above the risk control standard for soil contamination of agricultural land (0.55 µg·g-1) in 10% of all the soil samples. A total of 11.4% of the sample soil ΣBaPeq16PAHs and ΣBaPeq8BPAHs exceeded the agricultural soil screening value (0.55 µg·g-1). These results indicate that the contamination of PAHs was at a detectable level in the soil of wild F. suspensa growing in Shanxi, and thus their potential ecological risks should not be ignored. It is necessary to enhance the research regarding these areas to ensure the safe production of medicinal plants.