Molecular mechanism of sex pheromone perception in male Mythimna loreyi revealed by in vitro system.

BACKGROUND: Mythimna loreyi is an important agricultural pest with a sensitive sex pheromone communication system. To clarify the pheromone binding proteins (PBPs) and pheromone receptors (PRs) involved in sex pheromone perception is important for both understanding the molecular olfactory mechanism and developing a new pest control strategy in M. loreyi. RESULTS: First, the electroantennogram (EAG) assay showed that male M. loreyi displayed the highest response to the major sex pheromone component Z9-14:Ac, and higher responses to two minor components, Z7-12:Ac and Z11-16:Ac. Second, the fluorescence competition binding assay showed that PBP1 bound all three pheromones and other tested compounds with high or moderate affinity, while PBP2 and PBP3 each bound only one pheromone component and few other compounds. Third, functional study using the Xenopus oocyte system demonstrated that, of the six candidate PRs, PR2 was weakly sensitive to the major pheromone Z9-14:Ac, but was strongly sensitive to pheromone analog Z9-14:OH; PR3 was strongly and specifically sensitive to a minor component Z7-12:Ac; PR4 and OR33 were both weakly sensitive to another minor component, Z11-16:Ac. Finally, phylogenetic relationship and ligand profiles of PRs were compared among six species from two closely related genera Mythimna and Spodoptera, suggesting functional shifts of M. loreyi PRs toward Spodoptera PRs. CONCLUSION: Functional differentiations were revealed among three PBPs and six PRs in sex pheromone perception, laying an important basis for understanding the molecular mechanism of sex pheromone perception and for developing new control strategies in M. loreyi. © 2023 Society of Chemical Industry.

Ionotropic Receptor IR75q.2 Mediates Avoidance Reaction to Nonanoic Acid in the Fall Armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae).

Ionotropic receptors (IRs) play an important role in olfaction, but little is known in nondrosophila insects. Here, we report in vitro and in vivo functional characterization of IR75q.2 in the invasive moth pest Spodoptera frugiperda. First, 13 IRs (including four coreceptor IRs) were found specifically or highly expressed in adult antennae. Second, these IRs were tested for responding profiles to 59 odorants using the Xenopus oocyte expression system, showing that only SfruIR75q.2 responded to 8-10C fatty acids and their corresponding aldehydes, with SfruIR8a as the only coreceptor. Third, the three acids (especially nonanoic acid) showed repellent effects on moth's behavior and oviposition, but the repellence significantly reduced to the insects with IR75q.2 knockout by CRISPR/Cas9. Taken together, our study reveals the function of SfruIR75q.2 in perception of acid and aldehyde odorants and provides the first in vivo evidence for olfactory function of an odor-specific IR in Lepidoptera.

Electrochemical skin conductance and heart rate variability in patients with non-dialysis chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is very common now and associates with high overall and cardiovascular mortality. Numerous studies have reported that Heart rate variability (HRV) could also be used to detect cardiovascular autonomic dysfunction (CAD). We investigated the association of electrochemical skin conductance (ESC) of EZSCAN results with HRV in non-dialysis CKD patients. METHODS: In a cross-sectional study, we enrolled 248 prevalent non-dialysis CKD patients. Patients underwent a 24-h Holter (CB-2302-A, Bio Instrument, China). A time domain analysis of HRV was performed, and the following parameters were obtained: SDNN, SDANN, rMSSD, pNN50. EZSCAN device (Impeto Medical, Paris, France) measures ESC values of each participants. Mean global skin conductance computed as 0.5 * (reflecting (right + left hand)/2 + (right and left foot)/2). Log transforms data into a normal distribution for statistical analysis. RESULTS: There were 142 males and 106 females included in the present study. Patients' age was 56.6±17.08 years. Logarithm(Log) (global ESC) was independently predicted by age (P<0.01), hypertension history, estimated Glomerular filtration rate (eGFR) and log SDNN (P<0.05). While log SDANN, rMSSD and pNN50 were not independent predictors for log (global ESC). CONCLUSION: Increased global ESC significantly associated with elevated HRV, specifically SDNN in non-dialysis CKD patients. This suggested that global ESC may appear to be an important predictor of CAD, and even could be used as a cardiovascular risk factor in non-dialysis CKD patients.

[Polymorphisms of 35 Insertion/deletion Loci and Genetic Structure of the Han Population in Shaanxi Province].

Objective To evaluate the genetic polymorphisms and forensic efficiencies of 35 deletion/insertion polymorphism(DIP)loci and explore the genetic structure of the Han population in Shaanxi province.Methods Blood samples of 305 unrelated healthy individuals of the Han population in Shaanxi province were collected.And the allelic frequencies and forensic parameters of 35 DIP loci were calculated and analyzed based on their genotyping results by a self-developed amplification system.The genetic relationship of the Han population in Shaanxi province with the reference populations was explored by molecular variance analyses,phylogenetic tree reconstruction,multidimensional scale analyses,principal component analyses,and STRUCTURE analyses.Results The combined power of discrimination and probability of exclusion of the 35 DIP loci in the Han population in Shaanxi province were 0.999 999 999 999 991 119 and 0.9991,respectively.Population genetic analyses indicated that the Han population in Shaanxi province shared relatively closer genetic relationships with those in other regions of China.Conclusions The 35 DIP loci possessed high polymorphisms and could be used as an effective tool for forensic identification of individuals of the Han population in Shaanxi province.Furthermore,the 35 DIP loci could provide basic data for the genetic analysis of the Han population in Shaanxi province.

Different binding properties of two general-odorant binding proteins in Athetis lepigone with sex pheromones, host plant volatiles and insecticides.

Athetis lepigone (Alep) is a polyphagous pest native to Europe and Asia that has experienced major outbreaks in the summer maize area of China since 2011 and has shown evidence of resistance to some insecticides. Insect olfaction is crucial for recognition of sex pheromones, host plant volatiles and even insecticides, in which two general-odorant binding proteins (GOBPs) play important roles. To elucidate the functions of GOBPs in A. lepigone, we first expressed the two AlepGOBP proteins in the E. coli expression system. Then, the results of fluorescence competitive binding assays demonstrated that the high binding affinity of AlepGOBP2 with sex pheromones [(Z)-7-dodecenyl acetate (Z7-12:Ac), Ki = 0.65 µM; (Z)-9-tetradecenyl acetate (Z9-14:Ac), Ki = 0.83 µM], two maize plant volatiles [Ocimene, Ki = 9.63 µM; (E)-ß-Farnesene, Ki = 4.76 µM] and two insecticides (Chlorpyrifos Ki =5.61 µM; Phoxim, Ki = 4.38 µM). However, AlepGOBP1 could only bind Ocimene (Ki = 13.0 µM) and two insecticides (Chlorpyrifos Ki =4.46 µM; Phoxim, Ki = 3.27 µM). These results clearly suggest that AlepGOBP1 and AlepGOBP2 differentiate among odorants and other ligands. The molecular docking results further revealed different key residues involved in the ligand binding of AlepGOBPs. In summary, this study provides a foundation for exploring the olfactory mechanism of A. lepigone and identified two potential target genes for the development of highly effective insecticides in the future.

A Δ9 desaturase (SlitDes11) is associated with the biosynthesis of ester sex pheromone components in Spodoptera litura.

Sex pheromone biosynthesis in moths relies on the activity of multiple enzymes, including Δ9 desaturase, which plays an important role in catalyzing desaturation at the Δ9 position of the carbon chain. However, the physiological function of moth Δ9 desaturase has not been elucidated in vivo. In this study, we used the CRISPR/Cas9 system to knockout the Δ9 desaturase gene (SlitDes11) of Spodoptera litura to analyze its role in sex pheromone biosynthesis. First, through the direct injection of SlitDes11-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, gene editing was induced in around 30% of eggs 24 h after injection and was induced in 20.8% of the resulting adult moths. Second, using a sibling-crossing strategy, insects with mutant SlitDes11 (bearing a premature stop codon) were selected, and homozygous mutants were obtained in the G5 generation. Third, pheromone gland extracts of adult female homozygous SlitDes11 mutants were analyzed using Gas chromatography (GC). The results showed that titers of all three ester sex pheromone components; Z9, E11-14:Ac, Z9,E12-14:Ac, and Z9-14:Ac; were reduced by 62.40%, 78.50%, and 72.50%, respectively. This study provides the first direct evidence for the role of SlitDes11 in sex pheromone biosynthesis in S. litura, and indicates the gene could be as potential target to disrupt sexual communication in S. litura for developing a new pollution-free insecticide.

Characteristics of peripheral blood CD4+CD25+ regulatory T cells and related cytokines in severe atopic dermatitis.

Regulatory T cells (Tregs) have been suggested to play a role in the pathogenesis of atopic dermatitis (AD). However, alterations in the ability of Tregs remain to be determined. To investigate the expression of various surface receptors on CD4(+)CD25(high) regulatory T cells and to investigate their capacity for inhibiting the proliferation of CD4(+) CD25(-) effector T cells (Teffs). Peripheral blood samples were obtained from 15 patients with severe atopic dermatitis (AD) and 20 control subjects. FACs was then carried out to analyze the expression levels of FoxP3, CD152 (CTLA-4), CD39, CD73, CD223 (LAG-3), CCR4, CCR5, and CCR10 on Tregs. The proliferative responses of Teffs were assessed in the absence or presence of autologous Tregs and the TGF-ß1 and IL-10 levels in the culture supernatant and sera were detected by enzyme-linked immunosorbent assay (ELISA). The CD152, CD39, CD73, CCR4, and CCR5 expression levels on Tregs were higher in patients with severe AD than in the controls. Tregs showed an attenuated suppressive function of the proliferation of autologous Teffs in severe AD. The concentrations of IL-10 and TGF-ß in the culture supernatants of Tregs were lower in the AD group than in the control. The attenuated ability of Tregs to suppress Teff proliferation may be responsible for the autoimmune reaction of severe AD.

Ginsenosides Rb1 and Rg1 Stimulate Melanogenesis in Human Epidermal Melanocytes via PKA/CREB/MITF Signaling.

Reduced or defective melanin skin pigmentation may cause many hypopigmentation disorders and increase the risk of damage to the skin triggered by UV irradiation. Ginsenosides Rb1 and Rg1 have many molecular targets including the cAMP-response element-binding protein (CREB), which is involved in melanogenesis. This study aimed to investigate the effects of ginsenosides Rb1 and Rg1 on melanogenesis in human melanocytes and their related mechanisms. The effects of Rb1 and Rg1 on cell viability, tyrosinase activity, cellular melanin content and protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF), and activation of CREB in melanocytes were assessed. Results showed that Rb1 or Rg1 significantly increased cellular melanin content and tyrosinase activity in a dose-dependent manner. By contrast, the cell viability of melanocytes remained unchanged. After exposure to Rb1 or Rg1, the protein levels of tyrosinase, MITF, and phosphorylated CREB were significantly increased. Furthermore, pretreatment with the selective PKA inhibitor H-89 significantly blocked the Rb1- or Rg1-induced increase of melanin content. These findings indicated that Rb1 and Rg1 increased melanogenesis and tyrosinase activity in human melanocytes, which was associated with activation of PKA/CREB/MITF signaling. The effects and mechanisms of Rb1 or Rg1 on skin pigmentation deserve further study.

Simultaneous stereoselective analysis of naftopidil and O-desmethyl naftopidil enantiomers in rat feces using an online column-switching high-performance liquid chromatography method.

A novel online column-switching chiral high-performance liquid chromatography method was developed and validated for the simultaneous determination of naftopidil (NAF) and its O-desmethyl metabolites (DMN) enantiomers in rat feces. Direct and multiple injections of supernatant from rat feces homogenate were allowed through the column-switching system. Analyte extraction was performed on the Capcell Pak mixed-functional column by acetonitrile-phosphate buffer (pH 7.4; 10 mm; 8:92, v/v) flowing at 1 mL/min. Separation of NAF and DMN enantiomers was achieved on the Chiralpak IA column by methanol-acetonitrile-acetate buffer (pH 5.3; 5 mm; 45:33:22, v/v/v) flowing at 0.5 mL/min. The analytes were measured with a fluorescence detector at 290 nm (λ(ex)) and 340 nm (λ(em)). The validated method showed a good linearity [22.5-15,000 ng/mL for (+)-/(-)-NAF; 35-25,000 ng/mL for (+)-/(-)-DMN] and the lowest limits of quantification for NAF and DMN enantiomers were 22.5 and 35 ng/mL, respectively. Both intra- and inter-day variations were <10%. The assay was successfully applied to the fecal excretion of NAF and DMN enantiomers in rat after single oral administration of (±)-NAF. Nonstereoselective excretion of (+)- and (-)-NAF was found in feces, while stereoselective excretion of (+)- and (-)-DMN was observed with higher excretion levels of (+)-DMN, indicating that there may exist stereoselective metabolism for NAF enantiomers.

Inhibition of ABA-induced stomatal closure by fusicoccin is associated with cytosolic acidification-mediated hydrogen peroxide removal.

BACKGROUND: Fusicoccin (FC), a fungal phytotoxin produced by Fusicoccum amygdale, causes the inhibition of ABA-induced stomatal closure. The mechanism of inhibition is remaining unclear. We analyzed the role of hydrogen peroxide (H2O2) and relationship between H2O2 removal and cytosolic pH changes during inhibition of ABA-induced stomatal closure by FC. RESULTS: According to the results, ABA treatment induced H2O2 production and stomatal closure, but FC inhibited the effects of ABA on these two parameters. Treatment with catalase (CAT) and NADPH oxidase inhibitor diphenylene iodonium (DPI) mimicked the effect of FC. These data suggest that inhibition of ABA effect by FC is related to the decrease of H2O2 levels in guard cells. Furthermore, similar to CAT, FC not only suppressed stomatal closure and H2O2 levels in guard cells treated with exogenous H2O2, but also reopened the stomata which had been closed by ABA and reduced the level of H2O2 that had been produced by ABA, indicating that FC causes H2O2 removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomatal aperture and H2O2 levels in guard cells treated with exogenous H2O2 and had been closed by ABA, and both FC and butyric acid reduced cytosolic pH in guard cells of stomata treated with H2O2 and had been closed by ABA, which demonstrate that cytosolic acidification mediates FC-induced H2O2 removal. CONCLUSION: These results suggest that FC causes cytosolic acidification in guard cells, then induces H2O2 removal and reduces H2O2 levels in guard cells, finally inhibits stomatal closure induced by ABA.

Curcumin inhibits melanogenesis in human melanocytes.

Plant derived compounds, as potentially safe and effective skin lightening agents (SLAs), have attracted great attention from many researchers. Curcumin is a plant-derived polyphenol, which has been reported to suppress melanogenesis in B16 melanoma cells. However, little is known about whether curcumin affects melanogenesis in cultured human melanocytes. In addition, the molecular mechanism for the antimelanogenic effects of curcumin remains largely unknown. The present study assessed the effects of curcumin on melanin synthesis, cellular tyrosinase activity, the expression of melanogenesis-related proteins (microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein 1 and 2 (TRP-1, TRP-2)), and activation of melanogenesis-regulating signals including phosphatidylinositol 3-kinase (PI3K)/Akt/ glycogen synthase kinase 3 (GSK 3ß), extracellular signal-regulated kinase (ERK) and p38 MAPK in human melanocytes. The results showed that the melanin content and tyrosinase activity, as well as the expression of melanogenesis-related proteins in human melanocytes, were significantly inhibited by curcumin in a dose dependent manner. In addition, PI3K/Akt/ GSK 3ß, ERK and p38 MAPK were activated by curcumin, while inhibitors of these signals attenuated the inhibitory effects of curcumin on melanogenesis. These results suggest that curcumin inhibits melanogenesis in human melanocytes through activation of Akt/GSK 3ß, ERK or p38 MAPK signaling pathways.

Detection of mutant p53 protein in workers occupationally exposed to benzidine.

To investigate the expression of mutant p53 protein in workers occupationally exposed to benzidine, we detected mutant p53 protein by immuno-PCR assay in the serum of 331 benzidine-exposed healthy workers, while we classified exfoliated urothelial cells in urine samples with Papanicoloau's grading (PG). The Papanicoloau's grading classified exfoliated urothelial cells of the subjects from grade I (normal cells) to grade III (suspicious malignant cells). The subjects were also divided into high, medium and low exposure groups according to the exposure intensity index. The results revealed that mutant p53 protein in the medium and high exposure groups were significantly higher than the in low exposure group (p<0.05), and in PG II and III were significantly higher than in the PG I (p<0.05). There was no significant differences among Papanicoloau's gradings strata in the low exposure group on the incidence and quantity of mutant p53 protein. In the medium and high exposure groups, the incidence and/or quantity of mutant p53 protein in the stratum of PG II and/or III were significantly higher than that of PG I (p<0.05). Detection of mutant p53 protein in conjunction with benzidine exposure level and Papanicoloau's gradings of exfoliated urothelial cells could provide more information to help us elevate surveillance efficiency and diagnose bladder cancer in the early period.

[Expression of protein p53 in workers occupationally exposed to benzidine and bladder cancer patients.].

OBJECTIVE: To study expression of mutant p53 protein in workers occupationally exposed to benzidine and bladder cancer patients. METHODS: Mutant p53 protein in serum from the workers occupationally exposed to benzidine and bladder cancer patients were determined with Immuno-PCR, while exfoliated urothelial cells in the urine samples were classified with Papanicolau grading. RESULTS: Positive rate of mutant p53 protein increased with the exposed intensity index in workers occupationally exposed to benzidine. The positive rate of mutant p53 protein in bladder cancer patients (83.3%) was significantly higher than that in the group 1 of exposed intensity index. The average scanning integrals of PCR amplified band in the group of bladder cancer patients and group 2 of exposed intensity index were both higher than that in the group 1 significantly. Workers in the groups of different exposed intensity indices were further stratified according to Papanicolau grades. In the group 2 of exposed intensity index, the average scanning integrals of PCR amplified band in the stratum of Papanicolau grade II and III were significantly higher than that in the strata of Papanicolau grade I. And in the group 3 of exposed intensity index, the positive rate of mutant p53 protein in the strata of Papanicolau grade III was higher than that in the strata of Papanicolau grade I significantly. CONCLUSION: The increase of exposed intensity may not only result in the positive rate of mutant p53 protein, but also the quantity of mutant p53 protein in serum within the low range of benzidine exposure. Once the exposed intensity was beyond that spectrum, the positive rate of mutant p53 protein in serum and the average scanning integrals of PCR amplified band were no longer enhanced with the increase of exposed intensity. There was tight correlation between Papanicolau grade of exfoliated urothelial cells and the positive rate or the quantity of mutant p53 protein for the higher benzidine exposure intensity.