Identification and functional study of AA11 family polysaccharide monooxygenase genes in filamentous fungus Podospora anserina.

The lytic polysaccharide monooxygenase (LPMO) in the auxiliary active protein family (AA family) catalyzes the oxidative depolymerization of various refractory carbohydrates including cellulose, chitin and starch. While accumulating studies investigate the enzymology of LPMO, the research on the inactivation of LPMO genes has been rarely explored. In this study, five LPMO genes PaLPMO11A (Pa_4_4790), PaLPMO11B (Pa_1_5310), PaLPMO11C (Pa_2_7840), PaLPMO11D (Pa_2_8610) and PaLPMO11E (Pa_3_9420) of the AA11 family in the filamentous fungus Podospora anserina were knocked out by homologous recombination. Single mutants ΔPaLPMO11A (ΔA), ΔPaLPMO11B (ΔB), ΔPaLPMO11C (ΔC), ΔPaLPMO11D (ΔD) and ΔPaLPMO11E (ΔE) were constructed, and then all polygenic mutants were constructed via genetic crosses. The differences in the growth rate and sexual reproduction between wild type and mutant strains were observed on different carbon source media. The alteration of oxidative stress and cellulose degradation ability were found on DAB and NBT staining and cellulase activity determination. These results implicated that LPMO11 genes play a key role in the growth, development, and lignocellulose degradation of P. anserina. The results showed that the spore germination efficiency, growth rate and reproductive capacity of mutant strains including ΔBΔCΔE, ΔAΔBΔCΔE, ΔAΔCΔDΔE and ΔAΔBΔCΔDΔE was significantly decreased on different cellulose carbon sources and the remaining strains have no difference. The reduced utilization of various carbon sources, the growth rate, the spore germination rate, the number of fruiting bodies, the normal fruiting bodies, the shortened life span and the ability to degrade cellulose were found in strains which all five genes in the PaLPMO11 family were deleted. However, the strain still had 45% cellulase activity compared to wild type. These results suggest that LPMO11 genes may be involved in the growth and development, sexual reproduction, senescence and cellulose degradation of P. anserina. This study provides information for systematically elucidating the regulatory mechanism of lignocellulose degradation in filamentous fungus P. anserina.

Ionizable drug delivery systems for efficient and selective gene therapy.

Gene therapy has shown great potential to treat various diseases by repairing the abnormal gene function. However, a great challenge in bringing the nucleic acid formulations to the market is the safe and effective delivery to the specific tissues and cells. To be excited, the development of ionizable drug delivery systems (IDDSs) has promoted a great breakthrough as evidenced by the approval of the BNT162b2 vaccine for prevention of coronavirus disease 2019 (COVID-19) in 2021. Compared with conventional cationic gene vectors, IDDSs can decrease the toxicity of carriers to cell membranes, and increase cellular uptake and endosomal escape of nucleic acids by their unique pH-responsive structures. Despite the progress, there remain necessary requirements for designing more efficient IDDSs for precise gene therapy. Herein, we systematically classify the IDDSs and summarize the characteristics and advantages of IDDSs in order to explore the underlying design mechanisms. The delivery mechanisms and therapeutic applications of IDDSs are comprehensively reviewed for the delivery of pDNA and four kinds of RNA. In particular, organ selecting considerations and high-throughput screening are highlighted to explore efficiently multifunctional ionizable nanomaterials with superior gene delivery capacity. We anticipate providing references for researchers to rationally design more efficient and accurate targeted gene delivery systems in the future, and indicate ideas for developing next generation gene vectors.

[Generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells with adeno-associated virus expressing α-fetoprotein].

OBJECTIVE: To investigate the generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells (DC) with recombinant adeno-associated virus expressing α-fetoprotein (rAAV-AFP). METHODS: Peripheral blood mononuclear cells were isolated from healthy volunteers. Adherent peripheral blood mononuclear cells were transduced with AAV-AFP and cultured in the presence of granulocyte macrophage colony stimulating factor and interleukin-4 to generate dendritic cells. MTS assay was used to measure the ability of DC transduced with AAV-AFP (AAV-AFP + DC) to stimulate the proliferation of T cell. The phenotype and AFP protein expression of DC and the secretion of IFN (interferon)-γ and IL (interleukin)-4 by T cells were detected by flow cytometry. The killing efficacy of cytotoxic T lymphocytes (CTL) activated by AAV-AFP + DC against AFP positive hepatocellular carcinoma cell lines was detected by lactate dehydrogenase (LDH) release assay. RESULTS: AAV-AFP + DC expressed HLAI (97.12%), HLAII (97.32%), CD80 (38.94%), CD83 (60.84%) and CD86 (98.14%). AFP was secreted by 81.2% of AAV-AFP + DC. And it could stimulate effectively the proliferation of T cell. 19.84% of CD4(+)T cells and 18.65% of CD8(+)T cells activated by AAV-AFP + DC produced IFN-γ but not IL-4 and showed distinct killing activities against AFP positive hepatocellular carcinoma cell lines HepG2 (56.45%) and BEL7402 (78.84%). CONCLUSION: AAV-AFP + DC can elicit distinct antitumor responses against AFP positive hepatocellular carcinoma cell lines so as to provide a basis for further researches on the clinical application of AAV-AFP + DC in the treatment of hepatocellular carcinoma.

Circular RNA circ-PVT1 contributes to paclitaxel resistance of gastric cancer cells through the regulation of ZEB1 expression by sponging miR-124-3p.

Gastric cancer (GC) is the fifth most commonly diagnosed malignancy. Paclitaxel (PTX) is an effective first-line chemotherapy drug in GC treatment, but the resistance of PTX attenuates the therapeutic effect. Circular RNA circ-PVT1 can exert the oncogenic effect in GC. But the function of circ-PVT1 involved in PTX resistance of GC is still unknown. In the present study, the expression levels of circ-PVT1, miR-124-3p and ZEB1 in PTX-resistant GC tissues and cells were detected by quantitative real-time polymerase chain reaction (RT-qPCR). PTX resistance in PTX-resistant cells was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The protein levels of Zinc finger E-box binding homeobox 1 (ZEB1), P-glycoprotein (P-gp) and glutathione S-transferase (GST-π) were detected by Western blot assay. Cell apoptosis and invasion were measured in PTX-resistant cells by flow cytometry and transwell invasion assays, severally. The interaction between miR-124-3p and circ-PVT1 or ZEB1 was predicted by starBase software, and then verified by the dual-luciferase reporter assay. The role of circ-PVT1 in PTX resistance of GC in vivo was measured by xenograft tumor model. Our results showed that circ-PVT1 expression was up-regulated in PTX-resistant GC tissues and cells. Circ-PVT1 down-regulation enhanced PTX sensitivity in PTX-resistant GC cells by negatively regulating miR-124-3p. ZEB1 served as a direct target of miR-124-3p. Circ-PVT1 enhanced ZEB1 expression by sponging miR-124-3p. Circ-PVT1 knockdown increased PTX sensitivity of GC in vivo. Taken together, our studies disclosed that circ-PVT1 facilitated PTX resistance by up-regulating ZEB1 mediated via miR-124-3p, suggesting an underlying therapeutic strategy for GC.