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Glycogen is a predominant carbohydrate reserve in various organisms, which provides energy for different life activities. Glycogen synthase kinase 3 (GSK3) is a central player that catalyzes glucose and converts it into glycogen. In this study, a GSK3 gene was identified from the D. citri genome database and named DcGSK3. A reverse transcription quantitative PCR (RT-qPCR) analysis showed that DcGSK3 was expressed at a high level in the head and egg. The silencing of DcGSK3 by RNA interference (RNAi) led to a loss-of-function phenotype. In addition, DcGSK3 knockdown decreased trehalase activity, glycogen, trehalose, glucose and free fatty acid content. Moreover, the expression levels of the genes associated with chitin and fatty acid synthesis were significantly downregulated after the silencing of DcGSK3. According to a comparative transcriptomics analysis, 991 differentially expressed genes (DEGs) were identified in dsDcGSK3 groups compared with dsGFP groups. A KEGG enrichment analysis suggested that these DEGs were primarily involved in carbon and fatty acid metabolism. The clustering analysis of DEGs further confirmed that chitin and fatty acid metabolism-related DEGs were upregulated at 24 h and were downregulated at 48 h. Our results suggest that DcGSK3 plays an important role in regulating the chitin and fatty acid metabolism of D. citri.

Chitin synthase is a critical enzyme that catalyzes N-acetylglucosamine to form chitin, which plays an important role in the growth and development of insects. In this study, we identified a chitin synthase gene (CHS) with a complete open reading frame (ORF) of 3180 bp from the genome database of Diaphorina citri, encoding a protein of 1059 amino acid residues with the appropriate signature motifs (EDR and QRRRW). Reverse transcription-quantitative PCR (RT-qPCR) analysis suggested that D. citri CHS (DcCHS) was expressed throughout all developmental stages and all tissues. DcCHS had the highest expression level in the integument and fifth-instar nymph stage. Furthermore, the effects of diflubenzuron (DFB) on D. citri mortality and DcCHS expression level were investigated using fifth-instar nymph through leaf dip bioassay, and the results revealed that the nymph exposed to DFB had the highest mortality compared with control group (Triton-100). Silencing of DcCHS by RNA interference resulted in malformed phenotypes and increased mortality with decreased molting rate. In addition, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) also revealed corresponding ultrastructural defects. Our results suggest that DcCHS might play an important role in the development of D. citri and can be used as a potential target for psyllid control.

Chitin deacetylase (CDA) is a chitin degradation enzyme that strictly catalyzes the deacetylation of chitin to form chitosan, which plays an important role in regulating growth and development, as well as the immune response. In this study, a chitin deacetylase 3 gene (CDA3) was identified with a complete open reading frame (ORF) of 1362 bp from the genome database of Diaphorina citri, encoding a protein of 453 amino acids. Spatiotemporal expression analysis suggested that D. citri CDA3 (DcCDA3) had the highest expression level in the integument and third-instar nymph stage. Furthermore, DcCDA3 expression level can be induced by 20-hydroxyecdysone (20E). Injection of Escherichia coli and Staphylococcus aureus induced the upregulation of DcCDA3 in the midgut, while DcCDA3 was downregulated in the fat body. After silencing DcCDA3 by RNA interference, there was no influence on the D. citri phenotype. In addition, bactericidal tests showed that recombinant DcCDA3 inhibited gram-positive bacteria, including S. aureus and Bacillus subtilis (B. subtilis). In conclusion, our results suggest that DcCDA3 might play an important role in the immune response of D. citri.

Chitin is one the main components of the insect cuticle, and chitin synthase (CHS) is an important enzyme required for chitin formation. CHS has been characterized in various insect species, but the structure and biochemical properties in Spodoptera litura have not been determined. In this study, we identified two CHS genes, SlCHS1 and SlCHS2, which encode proteins with 1565 and 1520 amino acid residues, respectively. Transcriptional analysis suggested that SlCHS1 has a high expression level in the integument whereas SlCHS2 showed the highest expression level in the midgut. During S. litura growth and development, SlCHS1 and SlCHS2 were both predominantly expressed in the fourth-instar larval stage. In addition, the expression of SlCHS1 and SlCHS2 could be induced by 20-hydroxyecdysone (20E). Silencing of SlCHS1 by RNA interference significantly inhibited the pupation and molting of S. litura larvae (RNAi), while knockdown of SlCHS2 had no significant effects on the S. litura phenotype. These results may provide a new molecular target for control of S. litura.

Huanglongbing (HLB) is a systemic plant disease caused by ‘Candidatus Liberibacter asiaticus (CLas)’ and transmitted by Diaphorina citri. D. citri acquires the CLas bacteria in the nymph stage and transmits it in the adult stage, indicating that molting from the nymph to adult stages is crucial for HLB transmission. However, the available D. citri reference genomes are incomplete, and gene function studies have been limited to date. In the current research, PacBio single-molecule real-time (SMRT) and Illumina sequencing were performed to investigate the transcriptome of D. citri nymphs and adults. In total, 10,641 full-length, non-redundant transcripts (FLNRTs), 594 alternative splicing (AS) events, 4522 simple sequence repeats (SSRs), 1086 long-coding RNAs (lncRNAs), 281 transcription factors (TFs), and 4459 APA sites were identified. Furthermore, 3746 differentially expressed genes (DEGs) between nymphs and adults were identified, among which 30 DEGs involved in the Hippo signaling pathway were found. Reverse transcription–quantitative PCR (RT-qPCR) further validated the expression levels of 12 DEGs and showed a positive correlation with transcriptome data. Finally, the spatiotemporal expression pattern of genes involved in the Hippo signaling pathway exhibited high expression in the D. citri testis, ovary, and egg. Silencing of the D. citri transcriptional co-activator (DcYki) gene significantly increased D. citri mortality and decreased the cumulative molting. Our results provide useful information and a reliable data resource for gene function research of D. citri.

Trehalose-6-phosphate synthase (TPS) plays an important role in the synthesis of trehalose. In the current study, a TPS gene was obtained from Diaphorina citri, and named as DcTPS1 which encoded a protein of 833 amino acid residues. Real-time quantitative PCR (qPCR) analysis revealed that DcTPS1 had the highest expression level in the midgut and fifth-instar nymph stage. Knockdown of DcTPS1 by RNA interference (RNAi) induced an abnormal phenotype and increased mortality and malformation rate with a decreased molting rate. In addition, silencing of DcTPS1 significantly inhibited D. citri chitin metabolism and fatty acid metabolism, while the expression levels of fatty acid decomposition-related genes were downregulated. Furthermore, comparative transcriptomics analysis revealed that 791 differentially expressed genes (DEGs) were upregulated and 678 DEGs were downregulated when comparing dsDcTPS1 groups with dsGFP groups. Bioinformatics analysis showed that upregulated DEGs were mainly involved in oxidative phosphorylation, whereas downregulated DEGs were mainly attributed to the lysosome and ribosome. These results indicated that DcTPS1 played an important role in the growth and development of D. citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important transmission vector of the citrus greening disease Candidatus Liberibacter asiaticus (CLas). The D. citri midgut exhibits an important tissue barrier against CLas infection. However, the molecular mechanism of the midgut response to CLas infection has not been comprehensively elucidated. In this study, we identified 778 differentially expressed genes (DEGs) in the midgut upon CLas infection, by comparative transcriptome analyses, including 499 upregulated DEGs and 279 downregulated DEGs. Functional annotation analysis showed that these DEGs were associated with ubiquitination, the immune response, the ribosome, endocytosis, the cytoskeleton and insecticide resistance. KEGG enrichment analysis revealed that most of the DEGs were primarily involved in endocytosis and the ribosome. A total of fourteen DEG functions were further validated by reverse transcription quantitative PCR (RT-qPCR). This study will contribute to our understanding of the molecular interaction between CLas and D. citri.

Purpose The aim of this study was to evaluate the efficacy and safety of adalimumab (ADA) for Crohn’s disease. Methods Electronic databases, including PubMed, Embase, the Cochrane Library, and the Science Citation Index, were searched to retrieve relevant trials. We estimated pooled estimates of the odds ratio (OR) and relevant 95 % confidence interval (CI) using fixed effects model or random effects model as appropriate. Results Six randomized placebo-controlled studies met the selection criteria. Short-term clinical response/remission and long-term remission were better in the ADA groups than in the control groups ( P  < 0.05), both in anti-TNF-naive patients and in subjects who lost their response and/or became intolerant to infliximab (IFX). And ADA was also effective for patients who were previously treated with IFX, and its efficacy in infliximab-exposed patients was probably less than in infliximab-naive patients. In patients with active Crohn’s disease (CD), ADA therapy was more effective than placebo for obtaining complete fistula closure. In comparison with placebo, ADA does not increase the risk of serious adverse events. Conclusions ADA appears to be effective in achieving short-term clinical response/remission, long-term remission, and complete fistula healing in CD, including patients not manageable with IFX, and appears to have a favorable safety profile. A longer duration of follow-up and a larger number of patients are required to better assess the safety profile of ADA in CD.

Aim： To study the molecular mechanisms underlying α-tocopheryl succinate （α-TOS）-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand （hrTRAIL） act synergically to induce cell death of erbB2-expressing breast cancer cells. Methods： The annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAMTM NF-κB p65 kit was used to assess NF-κB activation. Results： α-TOS （100 pmol/L） significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-KB. α-TOS （50 and 100 μmol/L） inhibited the expression of Flice-like inhibitory protein （FLIP） and cellular inhibitor of apoptosis protein 1 （c-IAP1） in erbB2-positive cells, α-TOS （100 pmoVL） inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2, α-TOS （50 pmol/L） and hrTRAIL （30 mg/mL） acted synergically to induce apoptosis in breast cancer cells, α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB. Conclusion： Our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.

to study the molecular mechanisms underlying α-tocopheryl succinate (α-TOS)-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand (hrTRAIL) act synergically to induce cell death of erbB2-expressing breast cancer cells. the annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAM(TM) NF-κB p65 kit was used to assess NF-κB activation. α-TOS (100 μmol/L) significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-κB. α-TOS (50 and 100 μmol/L) inhibited the expression of Flice-like inhibitory protein (FLIP) and cellular inhibitor of apoptosis protein 1 (c-IAP1) in erbB2-positive cells. α-TOS (100 μmol/L) inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2. α-TOS (50 μmol/L) and hrTRAIL (30 mg/mL) acted synergically to induce apoptosis in breast cancer cells. α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB . our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.