Organic waste is an important biomass resource. In this study the definition of coefficients for the calculation of all the secondary 10 forestry residue categories was improved and established according to literature reviewing of previous reports between 1982 and 2017. The rationality of value-taking for each coefficient in previous literature was examined by tracing to its citation sources. The irrational, or unstudied coefficients were assessed using the data in previous reports or questionnaire surveys. Lastly, values of the coefficient for the calculation of woody nursery residue, forest woody pruning residue, wood logging residue, firewood, wood bucking residue, wood handling residue, waste wood, banana and pineapple plant residue, bamboo processing residue and waste bamboo were reasonably assessed in this study.

In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbβ, the PGRN gene in HEK293 (Rev-erbβ-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbβ and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbβ-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbβ on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbβ enhanced the regulation of Rev-erbβ on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbβ-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbβ on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbβ in transcriptional regulation remains to be further studied.

To establish a simple, quick and effective method to get a large amount of spider toxin JZTX-26 (35 aa) and JZTX-51 (27 aa) with 3 disulfide bonds each, the mature peptides coding gene fragments were constructed and fused with maltose-binding protein (MBP) tag in an Escherichia coli expression vector pMAL-p2x. The recombinant constructs pMAL-jz26 and pMAL-jz51 were transformed and cultured in E. coli TB1 and BL21 (DE3). After being induced by isopropyl-β-d-thiogalactoside (IPTG), the periplasmic proteins were purified by amylose affinity chromatography and analyzed by SDS-PAGE. The fusion proteins were digested with factor X, and purified by Sizes-Exclusion chromatography and Reversed Phase HPLC. Molecular weights of the purified peptides were obtained by using a MALDI-TOF-TOF mass spectrometer, which were consistent with the theoretical molecular weights. Five milligram of target protein could be purified from 1 L of culture medium. The results indicate that the peptides with three disulfide bonds can be expressed by using the prokaryotic expression system with MBP tag. Our findings suggest the possibility of genetic engineering to obtain large amount of spider peptide toxins.

To observe the migration of human amniotic mesenchymal stem cells (hAMSCs) labeled with PKH26 in the endometrium of rats intrauterine adhesion. hAMSCs were isolated, identified and labeled with PKH26 to detect the biological characteristics of the cells. Rat intrauterine adhesion models were established using mechanical and infective method and PKH26-labeled hAMSCs were transplanted through the tail vein. The distribution of PKH26 labeled hAMSCs in the endometrium of rats were observed with the fluorescence confocal microscope. The results showed that PKH26 stain had no significant effect on cell activity, cycle, apoptosis and so on. PKH26-labeled positive cells were mainly distributed in injured endometrium of rats. It shows that the PKH26 labeling technique is a safe and effective method for tracing the human amniotic mesenchymal stem cells in the treatment of intrauterine adhesions.

In order to provide a basic theory for the materials of repairing central nervous system injury, we have studied the growth and differentiation of neural stem cells (NSCs) on poly (L-lysine) modified silk fibroin film. First, we used poly (L-lysine) to modify silk fibroin film and confirmed by UV-vis and 1H NMR spectra. Then NSCs were isolated and seeded on the silk fibroin film (Silk group), poly (L-lysine) (PLL group) and poly (L-lysine) modified Silk fibroin film (Silk-PIL group). The proliferation of NSCs was evaluated by Cell Counting Kit-8 (CCK-8) assay on days 1, 3, 5 and 7 after seeding. Immunofluorescence was used to analyze the differentiation of NSCs at the 7th day. The levels of apoptosis were detected by Western blotting and TUNEL. The mRNA level of brain derived neurotrophic factor (BDNF) was identified by real-time PCR. UV-vis and 1H NMR spectra confirmed that poly (L-lysine) was successfully grafted onto the silk fibroin film. From the 3rd day after seeding to the 7th day, the CCK-8 test showed that proliferation rate of NSCs in the Silk-PIL was significantly higher than Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Immunofluorescence staining displayed that more NSCs in Silk-PIL group were differentiated into neuron compared with Silk group (P<0.05), however, there was no significant difference compared with PLL group (P>0.05). The number of NSCs differentiated into astrocytes was not significantly different between the three groups. Western blotting and TUNEL test presented that the degree of apoptosis of NSCs in the Silk-PIL group was significantly lower than Silk group (P<0.05). RT-PCR exhibited that mRNA level of brain derived neurotrophic factor (BDNF) of NSCs was higher in Silk-PIL group compared with Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Thus, poly (L-lysine) modified silk fibroin film could promote the proliferation of NSCs and reduce NSCs apoptosis. Furthermore, it also can enhance the differentiation of NSCs into neurons. It is expected to become a new type of tissue engineering scaffold carrying NSCs to repair central nervous system injury.

To test the therapeutic effect of recombinant fusion polypeptide hEGF-AWRK6 (EK) on burn infection of model mice. EK6 was expressed and purified with Escherichia coli expression system, and the Ⅱ degree burns and Pseudomonas aeruginosa infection model mouse were established. Experiment group was treated with EK (30 mg/L), and the control group was treated with PBS, gentamicin (30 mg/L), burn ointment (10 mg/L). The wound healing rate and colony count were calculated. Wound and surrounding skin were taken for HE staining and collagen western-blot analysis, and the wound pathological changes were observed after 10 days of drug delivery. The results showed that fusion peptide EK was successfully expressed and purified with significant antibacterial activities against Pseudomonas aeruginosa. Compared to the control group, the colony count (CFU) of the wound surface in EK mouse had a remarkable decrease (P<0.01) and healing rate had a significant increase in group EK6 (P<0.01). Pathological analysis result showed that compared to the control group, wound dermal cells in group EK arranged regularly, had more hair growth and a faster epithelization. These results indicated that the fusion peptide EK would be a good candidate for the drug development for the treatment of burning wounds.

The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.

Integrins are cell adhesion receptors, which consists of several transmembrane glycoproteins. They are widely distributed on the cell surface and involved in signal transduction pathways. As a heterodimer, each integrin is composed of one α subunit and one β subunit. Integrins are mainly expressed on lepidopteran hemocytes and involved in cell immune response. The full-length cDNA sequence of BmIntegrin β2 was obtained by PCR and RACE, including 2 434 bp. BmIntegrin β2 was predicted to be a transmembrane protein. The BmIntegrin β2 expression profile was detected by qRT-PCR at L4D3 or L5D3 larval stage, and it was highly expressed in hemocyte and hematopoietic organ. Anti-BmIntegrin β2 polyclonal antibody was generated following prokaryotic expression, protein purification and animal immunization, which is highly specific and effective for recognizing BmIntegrin β2 protein through Western blotting. The results of plasmatocytes adhesion experiment showed that BmIntegrin β2 plays an important role on the adhesion and spreading of plasmatocytes to foreign surfaces. This study provides a foundation for further research of the biological function of BmIntegrin β2 gene.

Corynebacterium glutamicum is the main industrial strain to produce L-valine by microbial fermentation. In this study, a low L-alanine producing C. glutamicum strain VWB-2 was constructed by knocking out the alanine aminotransferase encoding gene alaT in a high L-valine producing strain VWB-1. Meanwhile, a site-directed mutagenesis (ilvBN₁ (M13)) was done on the regulatory subunit of acetohydroxyacid synthase (ilvBN), a key enzyme in the L-valine synthesis pathway. Furthermore, the overexpression of the genes involved in the biosynthesis of L-valine, the mutated ilvBN₁ (M13), the acetohydroxy acid isomerase coding genes ilvC, the dihydroxy-acid dehydratase coding gene ilvD and branched-chain amino acid aminotransferase coding gene ilvE, could all promote the L-valine production of VWB-1 by strengthening the carbon flow towards L-valine. With the overexpression of the branched chain amino acid transporter coding gene brnFE and its regulator lrp₁, the L-valine producing capability of VWB-1 was further enhanced. The finally obtained engineered strain VWB-2/pEC-XK99E-ilvBN₁ (M13)CE-lrp₁-brnFE could produce 461.4 mmol/L L-valine in a 5 L fermentor with a sugar acid conversion rate of 0.312 g/g glucose.

Riemerella anatipestifer is a pathogen that mainly infects ducks, gooses, turkeys and other birds, causing septicemia and serositis. At present, the function of R. anatipestifer genes are studied by gene deletion and complementation. However, the shuttle plasmid pLMF03 used at present is inefficient for conjugation. Moreover, less restriction enzyme site can be used for cloning. It is not able to use for all the genes complementation. To solve this disadvantage, the conjugative transfer site, R. anatipestifer replication initiation gene, high expression promoter and a number of enzyme cutting sites were cloned into the plasmid pPM5, to generate the new shuttle plasmid pFY02. The shuttle plasmid pFY02 was stable in R. anatipestifer and had a high conjugative transfer efficiency. The R. anatipestifer tonB2 mutant strain could be complemented by shuttle plasmid pFY02 expressing tonB2, indicating that the shuttle plasmid can be used to the complementation of R. anatipestifer. Taken together, the new shuttle plasmid pFY02 constructed in this study replenishes the genetic tool for complementation.