Disease-linked mutations in Munc18-1 deplete synaptic Doc2.

Heterozygous de novo mutations in the neuronal protein Munc18-1/STXBP1 cause syndromic neurological symptoms, including severe epilepsy, intellectual disability, developmental delay, ataxia and tremor, summarized as STXBP1 encephalopathies. Although haploinsufficiency is the prevailing disease mechanism, it remains unclear how the reduction in Munc18-1 levels causes synaptic dysfunction in disease as well as how haploinsufficiency alone can account for the significant heterogeneity among patients in terms of the presence, onset and severity of different symptoms. Using biochemical and cell biological readouts on mouse brains, cultured mouse neurons and heterologous cells, we found that the synaptic Munc18-1 interactors Doc2A and Doc2B are unstable in the absence of Munc18-1 and aggregate in the presence of disease-causing Munc18-1 mutants. In haploinsufficiency-mimicking heterozygous knockout neurons, we found a reduction in Doc2A/B levels that is further aggravated by the presence of the disease-causing Munc18-1 mutation G544D as well as an impairment in Doc2A/B synaptic targeting in both genotypes. We also demonstrated that overexpression of Doc2A/B partially rescues synaptic dysfunction in heterozygous knockout neurons but not heterozygous knockout neurons expressing G544D Munc18-1. Our data demonstrate that STXBP1 encephalopathies are not only characterized by the dysfunction of Munc18-1 but also by the dysfunction of the Munc18-1 binding partners Doc2A and Doc2B, and that this dysfunction is exacerbated by the presence of a Munc18-1 missense mutant. These findings may offer a novel explanation for the significant heterogeneity in symptoms observed among STXBP1 encephalopathy patients.

Integrated transcriptome sequencing and RNA interference reveals molecular changes in Diaphorina citri after exposure to validamycin.

Validamycin has been widely used as a specific competitive inhibitor of trehalase. In our previous research, validamycin significantly inhibited trehalase activity and chitin synthesis in Diaphorina citri, resulting in abnormal phenotypes. However, the mechanism of validamycin's action on D. citri remains unclear. Here, using a comparative transcriptome analysis, 464 differentially expressed genes (DEGs) in D. citri were identified after validamycin treatment. A Gene Ontology enrichment analysis revealed that these DEGs were mainly involved in "small molecule process", "structural molecule activity" and "transition metal ion binding". DEGs involved in chitin metabolism, cuticle synthesis and insecticide detoxification were validated by reverse transcription quantitative polymerase chain reaction. The RNA interference of D. citri chitinase-like protein ENO3 and D. citri cuticle protein 7 genes significantly affected D. citri molting. Moreover, the recombinant chitinase-like protein ENO3 exhibited a chitin-binding property, and an antimicrobial activity against Bacillus subtilis. This study provides a first insight into the molecular changes in D. citri after exposure to validamycin and identifies two effective RNA interference targets for D. citri control.

Improved permeability and antifouling properties of polyvinyl chloride ultrafiltration membrane via blending sulfonated polysulfone.

To improve the permeability and antifouling properties of polyvinyl chloride (PVC) ultrafiltration (UF) membrane, amphiphilic sulfonated polysulfone (SPSF) was introduced into PVC matrix. Three types of PVC/SPSF blend membranes containing different SPSF with the sulfonation degree (SD) of 20%, 30%, and 50% were fabricated by non-solvent induced phase separation (NIPS) process. The excellent compatibility between PVC and SPSF was confirmed by differential scanning calorimetry (DSC). Surface chemical compositions, morphology, roughness, charge, hydrophilicity, permeability and antifouling properties of the pristine PVC membrane and the PVC/SPSF blend membranes were systematically compared and characterized. Due to the improved hydrophilicity and endowed negative charge, the blend membrane showed high water permeability (i.e. 880 L m-2h-1 bar-1), high bovine serum albumin (BSA) rejection (i.e. 95.7%), and high flux recovery ratio (i.e. 96%), which outperformed ever reported and commercialized PVC membranes. Furthermore, the permeability and rejection properties of PVC/SPSF UF membranes were maintained after soaking in acidic and alkaline solutions for 30 days, indicating their outstanding acid and alkali tolerance. Therefore, SPSF was expected as a potential versatile modifier for fabricating high performance UF membranes.

Identification and Functional Analysis of Two Chitin Synthase Genes in the Common Cutworm, Spodoptera litura.

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.

Dearomative [3 + 2] cycloaddition reaction of nitrobenzothiophenes with nonstabilized azomethine ylides.

A highly diastereoselective dearomative [3 + 2] 1,3-dipolar cycloaddition reaction of nitrobenzothiophenes with an in situ-generated nonstabilized azomethine ylides has been developed. The transformation provides a series of functionalized fused tricyclic benzo[4,5]thieno[2,3-c]pyrroles in good yields (up to 92%) under mild reaction conditions. In addition, a gram-scale experiment and the synthetic transformation of the cycloadduct further highlighted the synthetic utility. The relative configurations of the typical products were clearly confirmed by X-ray crystallography.

Immune Functional Analysis of Chitin Deacetylase 3 from the Asian Citrus Psyllid Diaphorina citri.

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.

Silencing of the Chitin Synthase Gene Is Lethal to the Asian Citrus Psyllid, Diaphorina 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.

Identification and functional analysis of an iron-binding protein, ferritin heavy chain subunit, from the swallowtail butterfly, Papilio xuthus.

Ferritin, which is ubiquitous among all living organisms, plays a crucial role in maintaining iron homeostasis, immune response, and detoxification. In the present research, we identified an iron-binding protein, ferritin heavy chain subunit, from Papilio xuthus and named PxFerHCH. The complete complementary DNA of PxFerHCH was 1,252 bp encoding a sequence of 211 amino acids, which includes an iron-responsive element. Phylogenetic analysis showed that PxFerHCH is clustered with Manduca sexta and Galleria mellonella ferritin heavy chain subunits. Expression levels of PxFerHCH in various tissues were analyzed by reverse transcription quantitative polymerase chain reaction, and the results exhibited that PxFerHCH was expressed in all tissues with the highest expression in the fat body. The relative expression level of PxFerHCH in response to bacterial (Escherichia coli and Staphylococcus aureus) challenges sharply increased by about 12 hr postinfection (hpi) and then decreased at 24 hpi. In addition, the iron-binding capacity and antioxidation activity of recombinant PxFerHCH protein were also investigated. These results reveal that PxFerHCH might play an important role in defense against bacterial infection.

Potential roles of insect Tropomyosin1-X1 isoform in the process of Candidatus Liberibacter asiaticus infection of Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the transmitting vector of Candidatus Liberibacter asiaticus (CLas), which causes citrus disease Huanglongbing (HLB). In recent years, control of HLB has been achieved by reducing the vector population. In the present study, we identified an isoform of D. citri tropomyosin (herein designated as DcTm1-X1). DcTm1-X1 was down-regulated in CLas-infected ACPs compared with uninfected ACPs. Bioinformatics analysis revealed that the full-length DcTm1-X1 is 2955 bp and encodes a protein of 284 amino acids with a deduced molecular weight of 32.15 kDa. Phylogenetic tree analysis suggested that DcTm1-X1 shares a high amino acid identity with its homolog in Acyrthosiphon pisum. Higher DcTm1-X1 expression levels were found in the leg of the psyllid by reverse transcription quantitative PCR (RT-qPCR). According to Blue Native PAGE analysis and mass spectrometric analysis, DcTm1-X1 interacts with citrate synthase (CS) and V-type proton ATPase subunit B-like (VAT). In addition, knockdown of DcTm1-X1 by RNA interference (RNAi) significantly increased the mortality rate of nymphs and the infection rate of CLas at different time points. Taken together, our results show that DcTm1-X1 might play an important role in response to CLas, but also lay a foundation for further research on the functions of DcTm1-X1.

Mycoplasmas infection in male HIV/AIDS patients in Jiangsu, China.

Mycoplasmas are widely distributed among animals, plants, and human. The four species namely, Mycoplasmas genitalium(Mg), Mycoplasmas fermentans(Mf), Mycoplasmas pentrans(Mpe), Mycoplasmas pirum(Mpi) are also called AIDS-associated mycoplasmas due to their involvement in the development and outcome of AIDS. To investigate the infection prevalence of Mg, Mf, Mpe and Mpi among male HIV/AIDS patients in Jiangsu Province and to analyze the relationship between pathogenic mycoplasmas and cellular immune function of them. First void urine and venous blood samples were collected and epidemiology questionnaires were administered after informed consent. Nested PCR was performed to determine the infection of Mg, Mf, Mpe and Mpi while ELISA assay was applied to detect interleukin-2(IL-2), interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α). SAS 9.0 software was applied to analyze the data. A total of 713 HIV/AIDS patients were recruited in this study. The overall infection rates of Mg, Mf, Mpe and Mpi are 27.9%, 9.7%, 1.0% and 18.4% respectively. Generally, the infection rates of Mg(χ(2) = 10.311, P = 0.006) and Mpi were declined as the CD4+ cell counts increased, while Mf infection was higher in CD4+ T cell>350/µl group. The levels of cytokines are different with the variance of mycoplasmas infection. Mycoplasma infection among male HIV/AIDS patients is associated with changes in cellular immune response (cytokines). However, the affect of mycoplasmas on the immune function is complex, further studies are still required to elucidate whether mycoplasmas interact with HIV by interfering host immune system.

[Analysis on Mycoplasma pirum infection in male HIV/AIDS patients and related 16S rRNA genes in Jiangsu province].

OBJECTIVE: To investigate the prevalence of Mycoplasma pirum (Mpi) in male HIV infected patients, and to identify the 16S rRNA gene of Mpi. METHODS: The first void urine of male HIV/AIDS patients in Jiangsu province was collected for Mpi detection. Purified 16S rRNA gene PCR production was sequenced for analysis on its identification, homogeneity and phylogenetic tree. P1 protein sequence of Mpi was analyzed by Vector NTI Advance 11.0 to calculate the coded amino acid sequence. Homogeneity analysis was conducted between the theoretical amino acid sequence of Mpi and other Mycoplasmas. RESULTS: The prevalence of Mpi in male HIV/AIDS patients was 21.5% while the Mpi prevalence rates in different age groups were significantly different (χ² Mpi = 124.63, P < 0.01). The homogeneity of 18 strains of Mpi was higher than 90%. CONCLUSION: The Mpi prevalence seemed much higher than the results from previous detection on HIV/AIDS patients, suggesting that more attention should be paid on AIDS treatment. More bioinformatic research on gene/nucleotide sequence analysis and forecast should be carried out to identify the molecular characteristics of Mpi.