Molecular Characterization, Expression Patterns and Binding Properties of Two Pheromone-Binding Proteins from the Oriental Fruit Moth, Grapholita molesta(Busck)

Insect pheromone-binding proteins （PBPs） play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors （ORs）. However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identiifed from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were signiifcantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding afifnities to two sex pheromone components, E8-12：Ac and Z8-12：Ac, as well as weaker binding afifnities to Z8-12：OH and 12：OH. GmolPBP2 also bound some ordinary odor molecules. However, the afifnity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth.

Mechanism of Competition between Nutlin3 and p53 for Binding with Mdm2

The tumour suppressor p53 is a transcription factor that regulates multiple biological functions including metabolism, DNA repair, cell cycle arrest, apoptosis and senescence. About half of human cancers show a normal TP53 gene and aberrant overexpression of Mdm2. This fact promotes a promising cancer therapeutic strategy by inhibiting the interactions between p53 and Mdm2. Various inhibitors have been designed to achieve this novel approach for cancer therapy. However, the detailed competition mechanism between these inhibitors and the p53 molecule in their binding process to Mdm2 is still unclear. We investigate this competition mechanism between Nutlin3 and p53 using molecular dynamics simulations. It is found that Nutlin3 binds faster than the p53 molecule to Mdm2 to prevent p53 binding to Mdm2 when Nutlin3 and p53 have equal distance from Mdm2. Nutlin3 can also bind to the p53-Mdm2 complex to disturb and weaken the interactions between p53 and Mdm2. Consequently, p53 cannot bind to Mdm2 and its tumour suppression function is reactivated. These results provide the detailed competition mechanism between Nutlin3 and p53 in their binding to Mdm2. Because the binding site of most other inhibitors to Mdm2 is the same as Nutlin3, therefore this competition mechanism can extend to most inhibitors which target the p53-Mdm2 interaction.

Chromosome-level genome assembly of Cylas formicarius provides insights into its adaptation and invasion mechanisms

Cylasformicarius is one of the most important pests of sweet potato worldwide, causing considerable ecological and economic damage.This study improved the effect of comprehensive management and understanding of genetic mechanisms by examining the functional genomics of C. formicarius.Using Illumina and PacBio sequencing, this study obtained a chromosome-level genome assembly of adult weevils from lines inbred for 15 generations.The high-quality assembly obtained was 338.84 Mb, with contig and scaffold N50 values of 14.97 and 34.23 Mb, respectively.In total, 157.51 Mb of repeat sequences and 11 907 protein-coding genes were predicted.A total of 337.06 Mb of genomic sequences was located on the 11 chromosomes, accounting for 99.03%of the total length of the associated chromosome.Comparative genomic analysis showed that C. formicarius was sister to Dendroctonus ponderosae, and C. formicarius diverged from D. ponderosae approximately 138.89 million years ago (Mya).Many important gene families expanded in the C. formicarius genome were involved in the detoxification of pesticides, tolerance to cold stress and chemosensory system.To further study the role of odorant-binding proteins (OBPs) in olfactory recognition of C. formicarius, the binding assay results indicated that Cfor OBP4–6 had strong binding affinities for sex pheromones and other ligands.The high-quality C. formicarius genome provides a valuable resource to reveal the molecular ecological basis, genetic mechanism, and evolutionary process of major agricultural pests;it also offers new ideas and new technologies for ecologically sustainable pest control.

Investigation of the interaction between two phenylethanoid glycosides and bovine serum albumin by spectroscopic methods

Two phenylethanoid glycosides, acteoside and forsythoside B, were first isolated from the traditional Chinese herb Callicarpa peii H.T. Chang. The interaction between the two phenylethanoid glycosides and bovine serum albumin （BSA） was investigated by fluorescence, UV vis absorbance and circular dichroism （CD）. The results showed that the quenching mechanism in the drug-BSA binary systems was a combination of static quenching and non- radiative energy transfer. Displacement experiments confirmed that the drug bound to the site I of BSA. UVvis and CD measurements indicated that the binding of the drug to BSA induced conformational changes in BSA.

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera (Hübner)

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera （Hvbner） was obtained from antennal eDNA libraries and expressed in Escherichia coll. The real time quantitative PCR （RT-qPCR） results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isobomeol, nerolidol, 2- nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38 （αl）, 40-48 （α2）, 62-72 （α3）, 80-96 （α4）, 98-108 （α5）, and 116-119 （α6）, two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trpl01, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.

Molecular and in vitro biochemical assessment of chemosensory protein 10 from brown planthopper Nilaparvata lugens at acidic pH

Chemosensory proteins(CSPs)are important molecular components of the insect olfactory system,which are involved in capturing,binding,and transporting hydrophobic odour molecules across the sensillum in sensillar lymph in regulating insect behavior.This protein family(CSPs)is also involved in many other systems that are not linked to olfactory receptors in olfactory sensilla.The brown planthopper(BPH)is a monophagous pest of rice that causes damage by sucking phloem sap and transmitting a number of diseases caused by viruses.In this study,fluorescence competitive binding assay and fluorescence quenching assay at acidic p H were performed as well as homology modelling to describe the binding affinity of Nlug CSP10.Fluorescence competitive binding assay(FCBA)demonstrated that Nlug CSP10 bound strongly to nonadecane,farnesene,and 2-tridecanone at acidic p H.The results of FCBA indicated that Nlug CSP10 bound different ligands at the physiological p H(5.0)of the bulk sensillum lymph.Fluorescence quenching assay demonstrated that Nlug CSP10 generated a stable complex with 2-tridecanone,while two ligands nonadecane and farnesene collided due to molecular collisions.The interaction of selected ligands with the modelled structure of Nlug CSP10 was also analyzed,which found the key amino acids(Gln23,Gln24,Gln25,Asn27,Met33,Ser34,Ile35,Tyr36,Asn42,Met43,Val45,Asn46,Asn93,Arg96,Ala97,Lys99,and Ala100)in Nlug CSP10 that were involved in binding of volatile compounds.The present study contributes to the binding profile of Nlug CSP10 that promotes the development of behaviorally active ligands based on BPH olfactory system.

The role of chemosensory protein 10 in the detection of behaviorally active compounds in brown planthopper, Nilaparvata lugens

Chemosensory proteins(CSPs)play important roles in insects’chemoreception,although their specific functional roles have not been fully elucidated.In this study,we conducted the developmental expression patterns and competitive binding assay as well as knock‐down assay by RNA interference both in vitro and in vivo to reveal the function of NlugCSP10 from the brown planthopper(BPH),Nilaparvata lugens(Stål),a major pest in rice plants.The results showed that NlugCSP10 messenger RNA was significantly higher in males than in females and correlated to gender,development and wing forms.The fluorescence binding assays revealed that NlugCSP10 exhibited the highest binding affinity with cis‐3‐hexenyl acetate,eicosane,and(+)‐β‐pinene.Behavioral assay revealed that eicosane displayed attractant activity,while cis‐3‐hexenyl acetate,similar to(+)‐β‐pinene significantly repelled N.lugens adults.Silencing of NlugCSP10,which is responsible for cis‐3‐hexenyl acetate binding,significantly disrupted cis‐3‐hexenyl acetate communication.Overall,findings of the present study showed that NlugCSP10 could selectively interrelate with numerous volatiles emitted from host plants and these ligands could be designated to develop slow‐release mediators that attract/repel N.lugens and subsequently improve the exploration of plans to control this insect pest.

Host-guest inclusion for enhancing anticancer activity of pemetrexed against lung carcinoma and decreasing cytotoxicity to normal cells

Advanced chemotherapy strategies are in urgent demand for improving anticancer efficacy.Herein,a water-soluble pillar[6]arene(WP6 A)was used to load chemotherapeutic agent pemetrexed(PMX)by forming direct host-guest inclusion,which is beneficial for decreasing cytotoxicity of PMX on BEAS-2 B cells.NMR and florescence titration served to confirm the complexation between WP6 A and ATP with higher affinity[(5.67?0.31)x 10^(5)L/mol],favoring competitive replacement of PMX.Complexation ATP by WP6 A effectively prevented ATP from being hydrolyzed in presence of alkaline phosphatase.The formed host-guest complex was further used to block the efflux pump by cutting off energy source from ATP hydrolysis,which was accompanied with releasing PMX to produce synergistic enhancement of anticancer performance towards A549 cells.This supramolecular strategy would also be extended to other clinical chemotherapeutic agents and it was expected to provide salutary profits for cancer patients.

Fractionation and fixation of rare earth elements in soils:Effect of spiking with lanthanum,cerium,and neodymium chlorides

Industrial and agricultural activities lead to the release of rare earth elements(REEs)in wastewater and aquatic ecosystems,and their accumulation in soils.However,the behavior of REEs in soils remains somewhat unclear.In the present work the fractionation and fixation of REEs in soddy-podzolic and chernozem soils spiked with La,Ce,and Nd chlorides were studied using dynamic(continuous flow)extraction,which allows natural conditions to be mimicked and artefacts to be minimised.The eluents applied are aimed to dissolve exchangeable,specifically sorbed,bound to Mn oxides,bound to metal-organic complexes,and bound to amorphous and poorly ordered Fe/Al oxides fractions extractable by 0.05 mol/L Ca(NO_(3))2,0.43 mol/L CH_(3)COOH,0.1 mol/L NH_(2)OH·HCl,0.1 mol/L K_(4)P_(2)O_(7) at pH 11,and 0.1 mol/L(NH4)_(2)C_(2)O_(4) at pH 3.2,respectively.It is found that the fixations of added La,Ce,and Nd in the form of metal-organic complexes is predominant for both types of soils:35%-38%in soddy-podzolic soil and 50%-79%in chernozem.The fixation of added elements in the first three fractions(exchangeable,specifically sorbed,and bound to Mn oxides)is significant for soddy-podzolic soil(5%-25%).For chernozem,the relative contents of added Ce and Nd in these fractions are nearly negligible.Only the content of exchangeable La is notable,about 5%.Adding any of three elements(La,Ce,or Nd)at the level of100 mg/kg to an initial sample results in changing the fractionation and bioaccessibility of other REEs present in soil.Their contents increase in the first three fractions and decrease in fifth(oxalate extractable)fraction for both soddy-podzolic soil and chernozem.The main difference is the behavior of REEs in pyrophosphate extractable fraction.For soddy-podzolic soil,adding La,Ce,or Nd results in decreasing the contents of other REEs associated with organic matter.For chernozem,on the contrary,the contents of REEs in the form of metal-organic complexes slightly increase.These processes may be attributed to competitive binding of elements and soil properties;they must be taken into account when assessing the environmental risks of soil pollution with REEs.