Transcriptome changes of Apis mellifera female embryos with fem gene knockout by CRISPR/Cas9.

The sex of honey bees is decided by a regulatory cascade comprising of csd, fem and Amdsx. In order to further identify other genes involved in sex determination and differentiation of honey bees in the early stages of embryo development, the CRISPR/Cas9 method was used to knock out fem gene in the embryonic stage of diploid western honey bees, and RNA-seq was used to analyze gene expression changes in the embryo after fem knockout. Finally, we found that the bees had undergone gender changes due to fem knockout. A total of 155 differentially expressed genes (DEGs) were obtained, with 48 up-regulated and 107 down-regulated DEGs in the mutant group compared to the control group. Of them, many genes are related to sex development or differentiation. In addition, 1502 differentially expressed alternative splicing events (DEASEs) related to 1011 genes, including the main honey bee sex-determining genes csd, tra2, fem, and Amdsx, were identified between the mutant group and control group, indicating that fem regulates alternative splicing of a large number of downstream genes. Our results provide valuable clues for further investigating the molecular mechanism of sex determination and differentiation in honey bees.

Apelin/APJ-Manipulated CaMKK/AMPK/GSK3ß Signaling Works as an Endogenous Counterinjury Mechanism in Promoting the Vitality of Random-Pattern Skin Flaps.

A random-pattern skin flap plays an important role in the field of wound repair; the mechanisms that influence the survival of random-pattern skin flaps have been extensively studied but little attention has been paid to endogenous counterinjury substances and mechanism. Previous reports reveal that the apelin-APJ axis is an endogenous counterinjury mechanism that has considerable function in protecting against infection, inflammation, oxidative stress, necrosis, and apoptosis in various organs. As an in vivo study, our study proved that the apelin/APJ axis protected the skin flap by alleviating vascular oxidative stress and the apelin/APJ axis works as an antioxidant stress factor dependent on CaMKK/AMPK/GSK3ß signaling. In addition, the apelin/APJ-manipulated CaMKK/AMPK/GSK3ß-dependent mechanism improves HUVECs' resistance to oxygen and glucose deprivation/reperfusion (OGD/R), reduces ROS production and accumulation, maintained the normal mitochondrial membrane potential, and suppresses oxidative stress in vitro. Besides, activation of the apelin/APJ axis promotes vascular migration and angiogenesis under relative hypoxia condition through CaMKK/AMPK/GSK3ß signaling. In a word, we provide new evidence that the apelin/APJ axis is an effective antioxidant and can significantly improve the vitality of random flaps, so it has potential be a promising clinical treatment.

Effect of hydraulic retention time, ZVI concentration, and Fe2+ concentration on autotrophic denitrification efficiency with iron cycle bacterium strain CC76.

The immobilized reactor of iron-reducing bacteria and zero-valent iron (ZVI) integrated system was established. This study has shown that the effects of hydraulic retention times (9, 11, 13 h), ZVI concentrations (2, 4, 6, 8 mg/L), and Fe2+ concentrations (5, 10, 15 mg/L) on the denitrification characteristics of iron cycle bacterium strain CC76. The results show that the longer the HRT is, the stronger ability of bacteria to remove nitrate. When ZVI concentration was 4 mg/L and the Fe2+ concentration is 15 mg/L, the removal efficiency of nitrate was the highest, reaching the maximum value of 93.02% (1.07 mg/L/h). Since increasing ZVI concentration in a certain range can not only promote chemical reduction but also make use of strain CC76 as an electron donor. Also, the abundance of strain CC76 decreased with the increase of ZVI concentration, which indicated that adding a low concentration of ZVI could reduce the inhibitory effect on bacteria. Hypothesis analysis of principal components showed that a low concentration of ZVI is beneficial to increase nitrate removal rate. Community structure analysis indicated that strain CC76 and related bacteria were the most abundant bacteria in the reactor.

The performance and mechanism of simultaneous removal of fluoride, calcium, and nitrate by calcium precipitating strain Acinetobacter sp. H12.

A calcium precipitating and denitrifying bacterium H12 was used to investigate the F- removal performance and mechanism. The results showed that the strain H12 reduced 85.24% (0.036 mg·L-1·h-1) of F-, 62.43% (0.94 mg·L-1·h-1) of Ca2+, and approximately 100% of NO3- over 120 h in continuous determination experiments. The response surface methodology analysis demonstrated that the maximum removal efficiency of F- was 88.98% (0.062 mg·L-1·h-1) within 72 h under the following conditions: the initial Ca2+ concentration of 250.00 mg·L-1, pH of 7.50, and the initial C4H4Na2O4·6H2O concentration of 800.00 mg·L-1. The scanning electron microscopy images, the X-ray photoelectron spectroscopy, and X-ray diffraction results suggested the following removal mechanism of F-: (1) the bacteria, as the nucleation site, were encapsulated by bioprecipitation to form biological crystal seeds; (2) Biological crystal seeds adsorbed F- to form Ca5(PO4)3F and CaF2; (3) Under the induction of bacteria, calcium, fluoride and phosphate coprecipitated to form Ca5(PO4)3F and CaF2. In addition, the gas chromatography data indicated that F- had little or no effect on the gas composition during denitrification, and the fluorescence spectroscopy analysis also proved that the extracellular polymeric substance (protein) is the site of bioprecipitation nucleation.

High-Efficiency CRISPR/Cas9-Mediated Gene Editing in Honeybee (Apis mellifera) Embryos.

The honeybee (Apis mellifera) is an important insect pollinator of wild flowers and crops, playing critical roles in the global ecosystem. Additionally, the honeybee serves as an ideal social insect model. Therefore, functional studies on honeybee genes are of great interest. However, until now, effective gene manipulation methods have not been available in honeybees. Here, we reported an improved CRISPR/Cas9 gene-editing method by microinjecting sgRNA and Cas9 protein into the region of zygote formation within 2 hr after queen oviposition, which allows one-step generation of biallelic knockout mutants in honeybee with high efficiency. We first targeted the Mrjp1 gene. Two batches of honeybee embryos were collected and injected with Mrjp1 sgRNA and Cas9 protein at the ventral cephalic side and the dorsal posterior side of the embryos, respectively. The gene-editing rate at the ventral cephalic side was 93.3%, which was much higher than that (11.8%) of the dorsal-posterior-side injection. To validate the high efficiency of our honeybee gene-editing system, we targeted another gene, Pax6, and injected Pax6 sgRNA and Cas9 protein at the ventral cephalic side in the third batch. A 100% editing rate was obtained. Sanger sequencing of the TA clones showed that 73.3% (for Mrjp1) and 76.9% (for Pax6) of the edited current-generation embryos were biallelic knockout mutants. These results suggest that the CRISPR/Cas9 method we established permits one-step biallelic knockout of target genes in honeybee embryos, thereby demonstrating an efficient application to functional studies of honeybee genes. It also provides a useful reference to gene editing in other insects with elongated eggs.

[The differential expression of the human lung carcinoma cells infected with high pathogenic avian influenza virus A/Anhui/1/2005 (H5N1)].

OBJECTIVE: To identify genes in human cells infected with high pathogenic avian influenza viruses H5N1. METHODS: The lung carcinoma cells line A549 was infected with H5N1 and H1N1, respectively. We harvested the infected cells at the different time points after infection and screened the genes with differential expression via microarray technology. The candidate genes were selected and confirmed by quantitative real-time PCR. RESULTS: The spectrum of genes with the differential expression in the cells infected with H5N1 was obtained and 16 candidate genes were identified in the cellular apoptosis pathway, mTOR pathway, and the cellular immunity as well. CONCLUSIONS: Our results suggest that H5N1 exert a stronger impact on eliciting apoptosis of infected cells than the common influenza virus H1N1.

[Generation of neutralizing recombinant human antibodies for targeting highly pathogenic avian influenza A (H5N1) virus].

Two human Fab antibodies against avian influenza A (H5N1) virus were obtained by panning a H5N1 patient-derived antibody phage library using purified virions of the H5N1 patient isolate A/Anhui/1/2005 and HA protein of the H5N1 reference viruse A/Viet Nam/1203/2004. After testing the binding properties and antiviral function to H5N1 virus, the selected Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell system. Both mAbs, AVFluIgG01 and AVFluIgG03, bound to HA in immunofluorescence assay (IFA) without cross-reaction with the other substypes of influenza A viruses (H1N1, H3N2). The cross-reactivity of the two antibodies for different strains of H5N1 was tested in vitro by micro-neutralization assays. In vitro, mAb AVFluIgG01 potently neutralized not only the selected well-characterized Clade 2 H5N1 viruses isolated from mainland of China except A/Guangdong/1/2006, but also the Clade 1 representative isolate A/Viet Nam/1203/2004; and AVFluIgG03 neutralized all the selected Clade 2 H5N1 viruses isolated from mainland of China, but had no neutralizing activity with the Clade 1 H5N1 virus A/Viet Nam/1203/2004. The results bring new prospect for the prophylaxis or treatment of H5N1 virus infection and may provide a clue for novel vaccine development.

[Generation of Chicken Germ-line Chimeras by Transferring PGCs and Their Identification by AFLP.].

PGCs (Primordial germ cells) were isolated from the blood of 51~56 h hatching Shiqiza chicken embryos by Ficoll density gradient centrifugation. The PGCs were injected into 2.5 d hatching embryos of H breed chicken to produce germ-line chimeras. AFLP checking method was established to identify chicken germline chimeras. Eight germ-line H-S chimera embryos were identified among 20 developing H breed embryos.