Dual RNA Sequencing of Beauveria bassiana-Infected Spodoptera frugiperda Reveals a Fungal Protease with Entomopathogenic and Antiphytopathogenic Activities

Insect pests and phytopathogens significantly impact crop yield and quality. The fall armyworm (FAW) Spodoptera frugiperda and the phytopathogen Fusarium graminearum cause substantial economic losses in crops like barley and wheat. However, the entomopathogen Beauveria bassiana shows limited efficacy against FAW, and its antiphytopathogenic activities against F. graminearum remain unclear. Here, dual RNA sequencing was performed to identify differentially expressed genes in B. bassiana-infected FAW larvae. We found that the BbAorsin gene was significantly upregulated at 36 and 48 h post-infection. BbAorsin encodes a serine-carboxyl protease and is mainly expressed in blastospores and hyphae. Overexpression of BbAorsin in B. bassiana ARSEF2860 enhanced virulence against Galleria mellonella and FAW larvae and inhibited F. graminearum growth. The recombinant BbAorsin protein induced apoptosis and necrosis in FAW hemocytes and inhibited F. graminearum spore germination. These findings shed light on transcriptomic mechanisms governing insect–pathogen interactions, which could aid in developing dual-functional entomopathogens and anti-phytopathogens

CCL22 and TGF-β1 expression in BC.

<p>(A) Negative expression of TGF-β1 in BC (×200). (B) Weak expression of TGF-β1 in BC (×200). (C) Strong expression of TGF-β1 in BC (×200). (D) Negative expression of CCL22 in BC (×200). (E) Weak expression of CCL22 in BC (×200). (F) Strong expression of CCL22 in BC (×200).</p

Prognostic significance of CCL22 Expression in BC.

<p>Kaplan–Meier curves of overall survival (OS) (A) and progression-free survival (PFS) (B) for CCL22 expression in BC to show the association of CCL22 expression with reduced OS (P<0.0001) and PFS (P<0.0001). P values were calculated by the log-rank test.</p

Expression of both CCL22 and Foxp3 ⁺Tregs infiltration in the tumor bed was associated with BC prognosis.

<p>Kaplan-Meier curves are shown for overall survival (OS) (A) and progression-free survival (PFS) (B) was stratified by expressions of two factors to divide the patients into three subsets, CCL22^-Foxp3^low+ group, CCL22^-Foxp3^high+/CCL22⁺Foxp3^low+ group, and CCL22⁺Foxp3^high+ group. The CCL22⁺Foxp3^high+ group was associated with both shorter OS (P=0.001) and PFS (P=0.001) than CCL22^-Foxp3^high+/CCL22⁺Foxp3^low+ group, and CCL22^-Foxp3^low+ group.</p

Syndecan binding protein expression in normal breast and breast cancer tissue.

*<p>, the <i>P</i> value was calculated by Mann–Whitney <i>U</i>-test.</p

Two Monoclonal Antibodies Recognising aa 634-668 and aa 1026-1055 of NogoA Enhance Axon Extension and Branching in Cultured Neurons

<div><p>In a previous study, we generated two monoclonal antibodies (mAbs) in mice, aNogoA-N and aNogo-66 mAb, which were raised against recombinant N-terminal fragments of rat NogoA and Nogo-66, respectively. When compared with the commercial rabbit anti-rat NogoA polyclonal antibody (pAb), which can specifically recognise NogoA, the two mAbs were also specific for the NogoA antigen in immunofluorescence histochemical (IHC) staining and Western blot (WB) analysis. Serial truncations of NogoA covering the N-terminal region of NogoA (aa 570–691) and Nogo-66 (aa 1026–1091) were expressed in <i>E. coli</i>. The epitopes recognised by aNogoA-N and aNogo-66 are located in the aa 634–668 and aa 1026–1055 regions of NogoA, respectively. Both mAbs remarkably enhanced the axon growth and branching of cultured hippocampal neurons <i>in vitro</i>. These results suggest that the antibodies that bind to aa 634–668 and aa 1026–1055 of NogoA may have stimulatory effects on axon growth and branching. Additionally, the two mAbs that we generated are specific for NogoA and significantly block NogoA function. In conclusion, two sites in NogoA located within aa 634–668 and aa 1026–1055 are recognised by our two antibodies and are novel and potentially promising targets for repair after central nervous system (CNS) injury.</p></div

Construction of SDCBP silenced breast cancer cells.

<p>(<b>A</b>) Syndecan binding protein (SDCBP) and negative control (NC) short-hairpin RNA (shRNA) expression constructs were transiently transfected into human embryonic kidney 293T cells and the holoproteins in cell lysates were western-blotted for SDCBP shRNA selection. Digits 397, 523, 611 and 987 represent initiation site of four candidate target sequences selected in SDCBP mRNA (NM_001007067) and (*) represent the target sequence we selected as SDCBP shRNA. (<b>B</b>) MDA-MB-231 cells with SDCBP silenced were selected by Western Blot analysis. (*) represented the clone we selected as the MDA-MB-231-SDCBP shRNA cell. (<b>C</b>) BT-549 cells with SDCBP silenced were selected by Western Blot analysis. (*) represented the clone we selected as the BT-549-SDCBP shRNA cell.</p

Western blot analysis of the levels of GAP43 expression on the fifth and seventh days after primary hippocampal neuron culture <i>in vitro</i>.

<p>(n = 6 wells per condition). A: Hippocampal neurons were grown on 100 pmol NogoA FC (aa 1026–1090) substrate for the control group; hippocampal neurons were grown on NogoA FC (aa 1026–1090) and treated with aNogo66 mAb for the Nogo66 mAb group; B: Data are expressed as the level of the GAP43-positive band in the control group and the Nogo66 mAb group on the fifth day or seventh day. ^*<i>P</i><0.05. C: Hippocampal neurons grown on the NogoA FC (aa 544–725) substrate were the control group; hippocampal neurons grown on NogoA FC (aa 544–725) and treated with aNogoA-N mAb were the aNogoA-N mAb group; D: Data are expressed as the level of GAP43-positive substance in the control group and the aNogoA-N mAb group on the fifth day or seventh day. ^*<i>P</i><0.05.</p

aNogo66 mAb and aNogoA-N mAb reduce the inhibition exerted by the targeted Nogo-A region on axon outgrowth and branching.

<p>A: Dissociated rat E18.5 hippocampal neurons were cultured on the control substrate PLL (Normal group). B, C, D: Hippocampal neurons cultured on 100 pmol NogoA FC (aa 1026–1090); E, F, G: Hippocampal neurons cultured on 100 pmol NogoA FC (aa 544–725); C, F: Treated with aNogo66 mAb; D, G: treated with aNogoA-N mAb. I: Statistical analysis of the axon growth is expressed as the mean ± SEM of each group in each separate experiment. J: Statistical analysis of the number of axonal branch points is expressed as the mean ± SEM of each group in each separate experiment (^**<i>P</i><0.01, C group vs. B or D group; ^**<i>P</i><0.01, G group vs. E or F group; ^*<i>P</i><0.05, C group vs. B or D group; ^*<i>P</i><0.05, G group vs. E or F group; scale bars  = 100 µm)</p

Identification of the epitopes recognised by the two mAbs by Western blot.

<p><b>A</b>: Several peptide epitopes from NogoA were produced. A library of six Nogo deletion constructs was made, and GST-tagged recombinant proteins were designed. <b>B</b>: Lysates of induced BL21 bacteria with recombinant plasmids were analysed using Coomassie brilliant blue staining (lanes from left to right: lysates of induced BL21 bacteria with recombinant plasmids GST-ΔNogoA-Na, GST-ΔNogoA-Nb, GST-ΔNogoA-Nc, GST-ΔNogoA-Nd, GST-ΔNogo66a, and GST-ΔNogo66b; lane 7: lysate of induced BL21 bacteria with empty pGEX-4T-1 vector; lane 8: lysates of non-induced BL21 bacteria alone). <b>C</b>: Recombinant proteins identified by WB. Anti-GST antibody was used to detect the fragments (lanes from left to right: the recombinant proteins GST-ΔNogoA-Na, GST-ΔNogoA-Nb, GST-ΔNogoA-Nc, GST-ΔNogoA-Nd, GST-ΔNogo66a, and GST-ΔNogo66b; lane 7: GST protein; lane 8: non-induced). <b>D</b>: The epitope recognised by the aNogoA-N mAb (lanes from left to right: the recombinant proteins GST-ΔNogoA-Na, GST-ΔNogoA-Nb, GST-ΔNogoA-Nc, GST-ΔNogoA-Nd and GST protein). <b>E</b>: The epitope recognised by the aNogo66 mAb (lanes from left to right: the recombinant proteins GST-ΔNogo66a and GST-ΔNogo66b and GST protein).</p