Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.

Fluorescent competitive assay for melamine using dummy molecularly imprinted polymers as antibody mimics

A fluorescent competitive assay for melamine was first developed utilizing dummy molecularly imprinted polymers（DMIPs） as artificial antibodies. This method is based on the competition between fluorescent substances and the unlabeled analyte for binding sites in synthesized DMIPs and the decreased binding of fluorescent substances to DMIPs due to increased concentrations of melamine in the solutions. DMIPs for melamine were synthesized under a hot water bath in the presence of the initiator azobisisobutyronitrile（AIBN） using 2,4-diamino-6-methyl-1,3,5-triazine（DAMT） as a dummy template, methacrylic acid（MAA） as a functional monomer, and ethylene glycol dimethacrylate（EGDMA） as a crosslinking agent. The adsorption capacity and selectivity of DMIPs for melamine were evaluated by the isothermal adsorption curve and Scatchard analysis. The evaluation results showed that the synthesized DMIPs had specific recognition sites for melamine and the maximum adsorption amount was 1 066.33 μg g^（-1）. Later, 5-（4,6-d ichlorotriazinyl） amino fluorescein（DTAF） with a triazine ring, which s lightly resembles m elamine, w as selected as the fluorescent substance. The fluorescent competitive assay using DMIPs as t he antibody mimics was finally established by selecting and optimizing the reaction solvents, DMIPs amount, DTAF concentration, and incubation time. The optimal detection system showed a linear response w ithin range of 0.05-40 mg L^（-1） and the limit of detection（LOD） was 1.23 μg L^（-1）. It was successfully applied to the detection of melamine in spiked milk samples wi th satisfactory recoveries（71.9 to 86.3%）. According to the comparative analysis, the result of optimized fluorescent competitive assay re vealed excellent agreement with the HPLC-MS/MS result for melamine.

The Fluorescence Immunochromatographic Strips for Natamycin and Its Application in Food Safety Detection

A fluorescence immunochromatographic strip was developed in this study for natamycin detection in food. The results showed that the best amount of labeled antibody was 10 μg, for every 50 μl of fluorescent microspheres with a 2.5%(w/v) concentration. This labeled antibody was diluted for 10 times, and the diluted solution was dispensed into conjugate pad at the amount of 3 μl/cm. The concentrations of natamycin labeled BSA for test line and goat anti-mouse IgG for control line were 2.0 and 1 mg/ml, respectively, which performed best. With the best conditions, the limit of detection was 1 ng/ml, the linearity ranged from 2 to 100 ng/ml, the recovery was about 80% to 120%, and the CV was below 23%.

Characterization of the chemosensory protein EforCSP3 and its potential involvement in host location by Encarsia formosa

Chemosensory proteins(CSPs) perform several functions in insects.This study performed the gene expression,ligand-binding,and molecular docking assays on the EforCSP3 identified in the parasitoid wasp Encarsia formosa,to determine whether EforCSP3 functions in olfaction,especially in host location and host preference.The results showed that EforCSP3 was highly expressed in the female head,and its relative expression was much higher in adults than in other developmental stages.The fluorescence binding assays suggested that the EforCSP3 exhibited high binding affinities to a wide range of host-related volatiles,among which dibutyl phthalate,1-octene,β-elemene,and tridecane had the strongest binding affinity with EforCSP3,besides α-humulene and β-myrcene,and should be assessed as potential attractants.Protein structure modeling and molecular docking predicted the amino acid residues of EforCSP3possibly involved in volatile binding.α-Humulene and β-myrcene attracted E.formosa in a previous study and exhibited strong binding affinities with EforCSP3 in the current study.In conclusion,EforCSP3 may be involved in semiochemical reception by E.formosa.

Ratiometric fluorescence probe for accurate detection of Concanavalin A by coupling fluorescent microsphere with boric acid functionalized carbon dots

Accurate and sensitive strategies for Concanavalin A(Con A)sensing are conducive to the better cognition of various important biological and physiological processes.Here,by designing dextran-functionalized fluorescent microspheres(DxFMs)and boric acid-modified carbon dots(BCDs)as recognition unit and built-in signal reference respectively,a ratiometric fluorescent detection platform was proposed for Con A detection with high reliability.In this protocol,the BCDs/DxFMs precipitation was formed due to the covalent interactions between cis-diol of DxFMs and boronic acid groups of BCDs,thus only fluorescence of BCDs could be detected in the supernatant.When Con A was presented,it could bind to DxFMs through its carbohydrate recognition ability and suppress the subsequent assembly between DxFMs and BCDs,leading to the simultaneous capture of DxFMs and BCDs fluorescence in the supernatant.Since the BCDs content was superfluous,their fluorescence intensities were basically constant in all cases.Based on the unchanged BCDs fluorescence signal and target-dependent DxFMs fluorescence signal in supernatant,the ratiometric detection of Con A was realized.Under optimized conditions,this ratiometric fluorescent platform displayed a linear detection range from 0.125μg/mL to 12.5μg/mL with a detection limit of 0.089μg/mL.Moreover,satisfied analytical outcomes for Con A detection in serum samples were obtained,manifesting huge application potential of this ratiometric fluorescent platform in clinical diagnosis.

A Fluorescent Cell-Based Technique for Monitoring Efflux of MRP4

Background: Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. Aim: The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). Methods: DH5α competent E. coli cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. Results: The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. Conclusion: The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.

Structure,Binding Characteristics,and 3D Model Prediction of a Newly Identified Odorant-Binding Protein from the Cotton Bollworm,Helicoverpa armigera (Hübner)

The full-length sequence of the odorant binding protein 5 gene,HarmOBP5,was obtained from an antennae cDNA library of cotton bollworm,Helicoverpa armigera （Hübner）.The cDNA contains a 444 bp open reading frame,encoding a protein with 147 amino acids,namely HarmOBP5.HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography.SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics.Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles,including （E）-β-farnesene,ethyl butyrate,ethyl heptanoate,and acetic acid 2-methylbutyl ester.Based on three-dimensional （3D） model of AaegOBP1 from Aedes aegypti,a 3D model of HarmOBP5 was predicted.The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H.armigera.This study provides clues for better understanding physiological functions of OBPs in H.armigera and other insects.

Development of a Label-Free Colorimetric Aptasensor with Rationally Utilized Aptamer for Rapid Detection of Okadaic Acid

Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis.In this study,a label-free col-orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish.To exploit the binding capability of the anti-OA aptamer,the inherent molecular recognition mechanism of aptamer and OA was studied,based on molecular docking,fluorescent assay,and biolayer interferometry.Consistent results showed that the stem-loop near the 3’terminal of the aptamer exhibit dominate binding capacity.Based on the revealed recognition information,the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride(PDDA)for the development of the label-free colorimetric aptasensor,in which the 3’terminal was thoroughly exposed to OA.The aptasensor provided robust performance with a linear detection range of 100-1200 nmol L-1,a limit of detection of 41.30 nmol L-1,recovery rates of 91.6%-106.2%,as well as a high selectivity towards OA in shellfish samples.The whole detection process can be completed within 1 h.To our best knowledge,this is the first time that the anti-OA aptamer was thoroughly studied,and a label-free colorimetric aptasensor was rationally designed in this way.This study not only provides a rapid detection method for highly sensitive and specific detection of OA,but also serves as a reference for the design of efficient aptasensors in the future.

Fluorescence polarization assay improves the rapid detection of human brucellosis in China

Background:Brucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella.Early diagnosis is the key to preventing,treating,and controlling brucellosis.Fluorescence polarization immunoassay(FPA)is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen-antibody interaction.However,there is no report on FPA-based detection of human brucellosis in China.Therefore,this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China.

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.

Interactions among SARS-CoV accessory proteins revealed by bimolecular fluorescence complementation assay

The accessory proteins(3a, 3b, 6, 7a, 7b, 8a, 8b, 9b and ORF14), predicted unknown proteins(PUPs) encoded by the genes, are considered to be unique to the severe acute respiratory syndrome coronavirus(SARS-Co V) genome. These proteins play important roles in various biological processes mediated by interactions with their partners. However, very little is known about the interactions among these accessory proteins. Here, a EYFP(enhanced yellow fluorescent protein) bimolecular fluorescence complementation(BiFC) assay was used to detect the interactions among accessory proteins. 33 out of 81 interactions were identified by BiFC, much more than that identified by the yeast two-hybrid(Y2H)system. This is the first report describing direct visualization of interactions among accessory proteins of SARS-CoV. These findings attest to the general applicability of the BiFC system for the verification of protein-protein interactions.

Enzyme-free photothermally amplified fluorescent immunosorbent assay(PAFISA)for sensitive cytokine quantification

Cytokine monitoring has attracted great attention due to its significance in the diagnosis and treatment of many diseases,such as tumors,microbial infections,and immunological diseases.Enzyme-linked immunosorbent assay(ELISA)is one of the most popular methods in cytokine detection,ascribing to the lavish signal amplification methods in the ELISA platform.In addition to classical enzymes,other signal amplifiers such as fluorescent probes,artificial nano-enzymes,and photothermal reagents have been applied to reduce the detection limit and produce more sensitive ELISA kits.Due to the accumulative effect of heat,photothermal reagents are promising materials in the signal amplification of ELISA.However,the lack of efficient photothermal generation material at an aggregate scale may delay the further development of this area.In this contribution,based on an efficient organic photothermal aggregate material,an enzyme-free photothermally amplified fluorescent immunosorbent assay system consisting of an assay microfluidic chip and detecting platform was developed.The photothermal nanoparticles with highly efficient photothermal conversion by harvesting energy via excited-state intramolecular motions and enlarging molar absorptivity were successfully prepared.The detection concentration at 50 pg/mL of interleukin-2 was achieved,realizing a signal improvement of detection limits by 20-fold compared to that of previously reported photothermal ELISA.The microscopic imaging integrated with plane sweeping technology provided high spatial resolution and precision,indicating the potential of achieving high throughput profiling at the microscale.Moreover,as an alternative excitation source,light-emitting diode not only provided a more affordable and miniaturized detection system but also revealed the great feasibility of intramolecular motion-induced photothermy nanoparticles for biological analyses.

A hydroxyl radical detection system using gas expansion and fast gating laser-induced fluorescence techniques

An OH radical measurement instrument based on Fluorescence Assay by Gas Expansion（FAGE）has been developed in our laboratory.Ambient air is introduced into a low-pressure fluorescence cell through a pinhole aperture and irradiated by a dye laser at a high repetition rate of 8.5 k Hz.The OH radical is both excited and detected at 308 nm using A-X（0,0）band.To satisfy the high efficiency needs of fluorescence collection and detection,a 4-lens optical system and a self-designed gated photomultiplier（PMT）is used,and gating is actualized by switching the voltage applied on the PMT dynodes.A micro channel photomultiplier（MCP）is also prepared for fluorescence detection.Then the weak signal is accumulated by a photon counter in a specific timing.The OH radical excitation spectrum range in the wavelength of 307.82–308.2 nm is detected and the excited line for OH detection is determined to be Q1（2）line.The calibration of the FAGE system is researched by using simultaneous photolysis of H2O and O2.The minimum detection limit of the instrument using gated PMT is determined to be 9.4×10~5molecules/cm^3,and the sensitivity is 9.5×10^（-7）cps/（OH·cm^（-3））,with a signal-to-noise ratio of 2 and an integration time of 60 sec,while OH detection limit and the detection sensitivity using MCP is calculated to be 1.6×10~5molecules/cm^3and 2.3×10^（-6）cps/（OH·cm^（-3））.The laboratory OH radical measurement is carried out and results show that the proposed system can be used for atmospheric OH radical measurement.

Interactomic study on interaction between lipid droplets and mitochondria

An increasing body of evidence shows that the lipid droplet,a neutral lipid storage organelle,plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria.However,the cellular functions and molecular mechanisms of the interaction remain ambiguous.Here we present data from transmission electron microscopy,fluorescence imaging,and reconstitution assays,demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro.Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae,we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes.The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75%of the interactions detected.Interestingly,interactions between 3 pairs of lipid metabolic enzymes were detected.Collectively,these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes,and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.

Identification and expression profiles analysis of odorant-binding proteins in soybean aphid,Aphis glycines(Hemiptera:Aphididae)

The soybean aphid,Aphis glycines,is an extreme specialist and an important invasive pest that relies on olfaction for behaviors such as feeding,mating,and foraging.Odorant-binding proteins(OBPs)play a vital role in olfaction by binding to volatile compounds and by regulating insect sensing of the environment.In this work we used rapid amplification of complementary DNA ends technology to identify and characterize 10 genes encoding A.glycines OBPs(AglyOBPs)belonging to 3 subfamilies,including 4 classic OBPs,5 Plus-C OBPs,and one Minus-C OBP.Quantitative real-time polymerase chain reaction demonstrated variable specific expression patterns for the 10 genes based on developmental stage and aphid tssue sampled.Expression levels of 7 AglyOBPs(2,3,4,5,7,9,and 10)were highest in the 4th instar,indicating that the 4th nymphal instar is an important developmental period during which soybean aphids regulate feeding and search for host plants.Tissue-specific expression results demonstrated that AglyOBP2,7,and 9 exhibited significantly higher expression levels in antennae.Meanwhile,ligand-binding analysis of5 OBPs demonstrated binding of AglyOBP2 and AglyOBP3 to a broad spectrum of volatiles released by green leaf plants,with bias toward 6-to 8-carbon chain volatiles and strong binding of AglyOBP7 to trans-B-farnesene.Taken together,our findings build a foundation of knowledge for use in the study of molecular olfaction mechanisms and prov ide insights to guide future soybean aphid research.

Relationship between serum HER2 extracellular domain levels, tissue HER2 expression, and clinico-pathological parameters in early stage breast cancer

Background Measurement of human epidermal growth factor receptor 2 （HER2） protein in the serum of metastatic breast cancer patients has previously been reported, but there are no consistent data to support the clinical utility of serum HER2 extracellular domain for patients with early stage breast cancer. We aimed to evaluate the correlation between serum extracellular domain levels and tissue HER2 expression, and analyzed their relationship with clinico-pathological parameters in patients with early stage disease. Methods A prospective study was conducted on 232 breast cancer patients with stage I-III prior to treatment. Preoperative serum samples were measured by enzyme-linked immunosorbent assay. Tissue HER2 status was analyzed by immunohistochemistry and fluorescence in situ hybridization assays. Results The median serum extracellular domain concentration was 6.8 ng/ml. The best diagnostic cut-off value was 7.4 ng/ml, with 62.9% sensitivity and 85.3% specificity. High serum extracellular domain levels were reported in 89 patients （38.3%）, and HER2-positive expression was observed in 77 patients （33.2%）. Multivariate analysis showed that elevated serum extracellular domain correlated with postmenopausal status （P 〈0.001）, high histological grade （P 〈0.001）, negativity of both estrogen （P=0.012） and progesterone receptors （P 〈0.001）, and high levels of carcinoembryonic antigen 153 （P=-0.048）. Conclusions We recommend that 7.4 ng/ml should be used as the cut-off value when evaluating serum extracellular domain levels in early stage of breast cancer. Patients with high serum extracellular domain levels have a certain clinico- pathological characteristics, may provide a basis for clinical practice.