Generation of anti-idiotypic antibodies mimicking Cry2Aa toxin from an immunized mouse phage display library as potential insecticidal agents against Plutella xylostella.

This study tried to generate anti-idiotypic antibodies (Ab2s) which mimic Cry2Aa toxin using a phage-display antibody library (2.8 × 107 CFU/mL). The latter was constructed from a mouse immunized with F (ab')2 fragments digested from anti-Cry2Aa polyclonal antibodies. The F (ab')2 fragments and Plutella xylostella (P. xylostella) brush border membrane vesicles (BBMV) were utilized as targets for selection. Eight mouse phage-display single-chain variable fragments (scFvs) were isolated and identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing after four rounds of biopanning. Among them, M3 exhibited the highest binding affinity with F (ab')2, while M4 bound the best with the toxin binding region of cadherin of P. xylostella (PxCad-TBR). Both of these two fragments were chosen for prokaryotic expression. The expressed M3 and M4 proteins with molecular weights of 30 kDa were purified. The M4 showed a binding affinity of 29.9 ± 2.4 nM with the PxCad-TBR and resulted in 27.8 ± 4.3 % larvae mortality against P. xylostella. Computer-assisted molecular modeling and docking analysis showed that mouse scFv M4 mimicked some Cry2Aa toxin binding sites when interacting with PxCad-TBR. Therefore, anti-idiotypic antibodies generated by BBMV-based screening could be useful for the development of new bio-insecticides as an alternative to Cry2Aa toxin for pest control.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody.

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC20 to IC80 was 1.11 µg/mL - 60.70 µg/mL, and an IC50 of 10.65 µg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.

Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

Epitopes prediction for microcystin-LR by molecular docking.

Microcystin-LR (MC-LR) is one of the most worldwide harmful cyanobacterial toxins. A lots of antibodies against MC-LR have been generated and characterized. However, the knowledge about the epitopes of MC-LR was still limited. The objective of this study was to analyze the epitopes of MC-LR and demonstrate the binding mode of MC-LR with its antibody. The variable genes of a mouse hybridoma cell line (Mab5H1-3B3) raised against MC-LR have been cloned and assembled in a single chain variable fragment (scFv), and then soluble expressed in E.coli BL21. Based on the scFv, the IC50 and IC10 for MC-LR were determined to be 7.45 nM and 0.30 nM by competitive ELISA. And the scFv also showed 115% and 112% cross-reactivities to MC-RR and MC-YR, and 59% to MC-LA. By molecular docking, the binding mode between MC-LR and its scFv was demonstrated. A hydrogen bond interaction was observed between the carbonyl group of Adda5 residue of MC-LR and its scFv, and the guanidyl group of Arg4 residue and phenyl group of Adda5 residue of MC-LR were also involved in the interaction. These predicted epitopes were supported by antibody cross-reactivity data. By comparing the antibody informatics of MC-LR scFv with its predicted paratopes, VH-CDR1 was crucial for MC-LR binding, and its specificity could be tuned by engineering in Vκ-CDR1 and Vκ-CDR3. These information would be useful for the hapten design for microcystins or improving the properties of MC-LR scFv in vitro.

Construction and characterization of a class-specific single-chain variable fragment against pyrethroid metabolites.

Pyrethroids are insecticides that are widely used in rural and urban areas worldwide. After entering the environment, pyrethroids are rapidly metabolized or degraded by various biological or abiotic methods. In this study, a single-chain variable fragment (scFv) which could simultaneously detect three pyrethroid metabolites was constructed based on a hybridoma raised against 3-phenoxybenzoic acid (3-PBA). By molecular docking, it showed that there were hydrogen bonds, hydrophobic interactions, CH-π interaction, and cation-π interaction between 3-PBA and its scFv. All the contact residues contributing to hydrogen bonds are located in VH-CDR2 or its neighboring region, and two of them were mutants of the closest germline sequence. Based on competitive ELISA, the half maximal inhibitory concentration (IC50) of the scFv for 3-PBA, 3-phenoxybenzaldehyde (PBAld), and 3-phenoxybenzyl alcohol (PBAlc) were calculated to be 0.55, 0.59, and 0.63 µgmL-1, respectively. The scFv also showed 23.91%, 13.41%, 1.15%, 1.00%, and 0.56% cross-reactivity with phenothrin, deltamethrin, fenvalerate, beta-cypermethrin, and fenpropathrin. The broad specificity of the scFv may be due to its hapten design. The scFv could be employed in class-specific immunoassays for pyrethroid metabolites with phenoxybenzyl (PB) group. It is also potentially used for characterizing degradation of pyrethroids or detecting PBAlc (PBAld) alone, and the detection results should be confirmed by other selective methods. KEY POINTS: • A scFv which can simultaneously detect 3-PBA, PBAlc, and PBAld was constructed. • Antibody informatics and binding mode of the scFv were obtained. • The reason for its broad specificity was discussed. • It could be used to monitor single or multi-pyrethroid metabolites with PB group.

Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library.

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL-1 and the medium inhibition of control (IC50) was 0.73 ng mL-1. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%-96.6% and with a coefficient of variation (CV) among 2.0%-8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.

Development of competitive ELISA for the detection of bovine serum albumin using single-chain variable fragments.

Soluble anti-bovine serum albumin (BSA) single-chain variable fragments (scFvs) were expressed in E. Coli. HB2151. The antigen-binding equilibrium dissociation constant of the scFvs was determined to be 2.9 × 10-8 M by surface plasmon resonance analysis. A competitive ELISA for the detection of BSA was developed using the antibody fragment above. The limits of detection (I10) and I50 were 0.002 and 0.74 µg/ml respectively, with a recovery between 87.8 and 119.2% in spiked milk samples. The assay has the potential to be used to detect concentration of BSA in milk or other matrix instead of the ELISA based on traditional antibodies.

An in vivo quantitative Raman-pH sensor of arterial blood based on laser trapping of erythrocytes.

We report on a continuous and non-invasive approach in vivo to monitor arterial blood pH based on the laser trapping and Raman detection of single live erythrocytes. A home-built confocal laser tweezers Raman system (LTRS) is applied to trace the live erythrocytes at different pH values of the extracellular environment to record their corresponding Raman changes in vitro and in vivo. The analysis results in vitro show that when the extracellular environment pH changes from 6.5 to 9.0, the Raman intensity ratio (R1603, 1616 = I1603/I1616) of single erythrocytes decrease regularly; what is more, there is a good linear relationship between these two variables, and the linearity is 0.985, which is also verified successfully via in vivo Raman measurements. These results demonstrate that the Raman signal of single live erythrocytes is possible as a marker of the extracellular pH value. This in vivo and quantitative Raman-pH sensor of arterial blood will be an important candidate for monitoring the acid-base status during the treatment of ill patients and in some major surgeries because of its continuous and non-invasive characters.

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 µg/ml, respectively, with a dynamic range between 0.19 and 2.04 µg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 µg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.

Gaining insights into the responses of individual yeast cells to ethanol fermentation using Raman tweezers and chemometrics.

Saccharomyces cerevisiae is the most common microbe used for the industrial production of bioethanol, and it encounters various stresses that inhibit cell growth and metabolism during fermentation. However, little is currently known about the physiological changes that occur in individual yeast cells during ethanol fermentation. Therefore, in this work, Raman spectroscopy and chemometric techniques were employed to monitor the metabolic changes of individual yeast cells at distinct stages during high gravity ethanol fermentation. Raman tweezers was used to acquire the Raman spectra of individual yeast cells. Multivariate curve resolution-alternating least squares (MCR-ALS) and principal component analysis were employed to analyze the Raman spectra dataset. MCR-ALS extracted the spectra of proteins, phospholipids, and triacylglycerols and their relative contents in individual cells. Changes in intracellular biomolecules showed that yeast cells undergo three distinct physiological stages during fermentation. In addition, heterogeneity among yeast cells significantly increased in the late fermentation period, and different yeast cells may respond to ethanol stress via different mechanisms. Our findings suggest that the combination of Raman tweezers and chemometrics approaches allows for characterizing the dynamics of molecular components within individual cells. This approach can serve as a valuable tool in investigating the resistance mechanism and metabolic heterogeneity of yeast cells during ethanol fermentation.

PM2.5-induced premature senescence in HUVECs through the SIRT1/PGC-1α/SIRT3 pathway.

Vascular endothelial cell senescence plays a pivotal role in the development of atherosclerosis. Recent studies have demonstrated that ambient fine particulate matter (PM2.5) induces stress-induced premature senescence (SIPS) in vascular endothelial cells. However, the precise mechanisms underlying this process remain to be fully elucidated. Cellular senescence is closely associated with reactive oxygen species (ROS), and emerging research has established a strong connection between the SIRT1/PGC-1α/SIRT3 signaling pathway and the antioxidant system in vascular endothelial cells. In this study, we aimed to investigate the impact of PM2.5 on vascular endothelial cell senescence and to elucidate the underlying mechanisms. Our findings revealed that PM2.5 exposure led to an increase in senescence-associated ß-galactosidase (SA-ß-gal) activity and the expression of the cell cycle-blocking proteins P53/P21 and P16 in human umbilical vein endothelial cells (HUVECs). Flow cytometry analysis demonstrated an elevated proportion of cells arrested in the G0/G1 phase after PM2.5 exposure. In addition, PM2.5-induced cellular senescence was attributed to the disruption of the cellular antioxidative defense system through the SIRT1/PGC-1α/SIRT3 signaling pathway. The expression of cellular senescence markers was reduced after targeted scavenging of mitochondrial ROS using MitoQ. Moreover, treatment with SRT1720, a SIRT1-specific activator, upregulated the SIRT1/PGC-1α/SIRT3 signaling pathway, restored the antioxidant system, and attenuated the expression of cellular senescence markers. Taken together, our results suggest that PM2.5 downregulates the SIRT1/PGC-1α/SIRT3 signaling pathway, resulting in impaired antioxidant defenses in HUVECs. This, in turn, allows for the accumulation of ROS, leading to inhibition of endothelial cell cycle progression and the onset of stress-induced senescence in HUVECs.

Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective and broader detection methods for commonly used Cry toxins. Using ligand blot and bio-layer interferometry, we confirmed that a recombinant toxin-binding fragments derived from Helicoverpa armigera cadherin-like protein (HaCad-TBR) could broadly bind Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab with the affinity of 0.149, 0.402, 120, and 4.12 nM, respectively. Based on the affinity results, a novel receptor-antibody sandwich assay broadly detecting Cry1A and Cry2 toxins was developed by using HaCad-TBR as capture molecules, and anti-Cry1A/Cry2A polyclonal antibodies (pAbs) as the detection antibodies. The detection limit (LOD) for Cry1Ab, Cry1Ab, Cry2Aa, and Cry2Ab were 5.30, 5.75, 30.83 and 13.70 ng/mL. To distinguish Cry1A and Cry2A toxins in a singular test, anti-Cry1A pAbs and anti-Cry2A pAbs were labelled with different quantum dots (QDs). The LOD for the four toxins by receptor-QDs-pAbs sandwich assay were calculated to be 1.36, 4.71, 17.48, and 7.54 ng/mL, respectively. The two developed methods were validated by spiked rice and corn samples, suggesting they may potentially be used in monitoring and quantifying Cry toxins in food and environment.

Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin.

The widespread use of Bacillus thuringiensis toxins as insecticides has brought about resistance problems. Anti-idiotypic nanobody approaches provide new strategies for resistance management and toxin evolution. In this study, the monoclonal antibody generated against the receptor binding region Domain II of Cry2Aa toxin was used as a target to screen materials with insecticidal activity. After four rounds of screening, anti-idiotypic nanobody 1C12 was obtained from the natural alpaca nanobody phage display library. To better analyze the activity of 1C12, soluble 1C12 was expressed by the Escherichia coli BL21 (DE3). The results showed that 1C12 not only binds the midgut brush border membrane vesicles (BBMV) of two lepidopteran species and cadherin CR9-CR11 of three lepidopteran species but also inhibits Cry2Aa toxins from binding to CR9-CR11. The insect bioassay showed that soluble 1C12 caused 25.65% and 23.61% larvae mortality of Helicoverpa armigera and Plutella xylostella, respectively. Although 1C12 has low insecticidal activity, soluble 1C12 possesses the ability to screen a broad-spectrum recognition of the toxin binding region of lepidopteran cadherins and can be used for the identification of the toxin binding region of other lepidopteran cadherins and the subsequent evolution of Cry2Aa toxin. The present study demonstrates a new strategy to screen for the production of novel insecticides.

Self-expansion full information optimization strategy: Convenient and efficient method for near infrared spectrum auto-analysis.

An essential step in the application of near infrared spectroscopy technology is the spectrum preprocessing. A reasonable implementation of it ensures that the effective spectral information is correctly extracted and, also that the model's accuracy is increased. However, some analysts' research still uses the manual approach of trial and error, particularly those less skilled ones. Previous papers have provided preprocessing optimization algorithms for NIR, but there are still some problems that need to be resolved, such as the unwieldy sequence determination of preprocessing method or, the fluctuated optimization outcomes or, lack of sufficient statistical information. This research suggests a spectrum auto-analysis methodology named self-expansion full information optimization strategy, a new powerful open-source technique for concurrently addressing all of these above issues simultaneously. For the first time in the field of chemometrics, this algorithm offers a reliable and effective automatic near infrared auto-modelling method based on the statistical informatics. With the aid of its built-in modules, such as information generators, spectrum processors, etc., it is able to fully search the common preprocessing techniques, which is determined by Monte Carlo cross validation. Then the final ensemble calibration model is built by employing the optimized preprocessing schemes, along with the wavelength variables screening algorithm. The optimization strategy can offer the user objective useful statistics information created throughout the modeling process to further examine the model's effectiveness. The results demonstrate that the suggested method can easily and successfully extract spectrum information and develop calibration models by putting it to the test on two groups of actual near-infrared spectral data. Additionally, this optimization strategy can also be applied to other spectrum analysis areas, such Raman spectroscopy or infrared spectroscopy, by changing a few of its parameters, and has extraordinary application value.

Development of a micro-Raman system for in vivo studying the mechanism of laser biological effects.

The laser irradiation on organism will produce a series of biological effects, which can be used for basic medical research, diagnosis and treatments of diseases. However, the mechanism of this biological effects is still unclear. As a sensitive molecular monitoring technique, Raman spectroscopy has became a very popular detection method in biomedical research especially in vivo study. In this paper, we present a compact and flexible micro-Raman system for in vivo studying the mechanism of laser biological effects. The system has the two functions of laser induction and Raman measurement, which can realize the micro-area radiation of laser and simultaneously collect the corresponding Raman spectra in vivo. The detection method provided by this home-built system is able to deepen the understanding of laser biological effects mechanism at molecular level, so it is expected that the system is significant for the treatments and diagnosis of diseases in the near future.

Identifying the Epitopes of Bacillus thuringiensis Cry2Aa Toxin Involved in Cadherin Interaction by a Monoclonal Antibody.

Antibodies are a useful tool for assistance to map the binding epitopes in Bacillus thuringiensis Cry toxins and their receptors, and even determine how receptors promote toxicity. In this work, a monoclonal antibody (mAb-1D2) was produced by the hybridoma cell line raised against Cry2Aa toxins, with a half inhibition concentration (IC50) of 9.16 µg/mL. The affinity constant of two recombinant toxin-binding fragments derived from Helicoverpa armigera and Plutella xylostella cadherin-like protein (HaCad-TBR or PxCad-TBR) to Cry2Aa toxin was measured to be 1.21 µM and 1.24 µM, respectively. Competitive ELISA showed that mAb-1D2 competed with HaCad-TBR or PxCad-TBR binding to Cry2Aa. Meanwhile, the toxicity of the Cry2Aa toxin to the H. armigera and P. xylostella larvae were greatly reduced when the toxin was mixed with mAb-1D2, which indicated that cadherin may play an important functional role in the toxicity of Cry2Aa. After transforming mAb-1D2 to a single-chain variable fragment (scFv), the hot spot residues of Cry2Aa with 1D2-scFv, PxCad-TBR, and HaCad-TBR were analyzed by molecular docking. It was demonstrated that the hot spot residues of Cry2Aa involving with 1D2-scFv interaction were mainly in Domain II, and some residues in Domain I. Moreover, mAb-1D2 and the two cadherin fragments shared the common hot spot residues on Cry2Aa, which could explain mAb-1D2 inhibited Cry2Aa binding with cadherin fragments. This monoclonal antibody could be a useful tool for identifying the binding epitopes between Cry2Aa and cadherin, and even assist to analyze the roles of cadherin in Cry2Aa toxicity.

LINC00518 affects the proliferation, invasion and migration of cutaneous malignant melanoma cells via miR-526b-3p/EIF5A2 axis.

OBJECTIVE: To investigate the mechanism of LINC00518 affecting the proliferation, invasion, and migration of cutaneous malignant melanoma (CMM) cells via miR-526b-3p/EIF5A2 axis. METHODS: qRT-PCR was performed to measure the expression of LINC00518, miR-526b-3p, and EIF5A2 in CMM tissues from 40 patients. Si-LINC00518, pcDNA-LINC00518, miR-526b-3p mimic, miR-526b-3p inhibitor, si-EIF5A2, and their corresponding negative controls were transfected alone or co-transfected into CMM cells A375 and A2058. The expression of LINC00518, miR-526b-3p and EIF5A2 in A375 and A2058 cells was measured. Cell proliferation was tested by CCK-8 assay and EdU assay. Cell invasion and migration were detected by Transwell and scratch tests, respectively. The binding between LINC00518 and miR-526b-3p, and the binding between miR-526b-3p and EIF5A2 were verified by dual-luciferase reporter and RNA pull-down assays. RESULTS: LINC00518 and EIF5A2 were up-regulated and miR-526b-3p was down-regulated in CMM tissues and cells. CMM patients with highly expressed LINC00518 showed decreased survival time than those with lowly expressed LINC00518. Transfection of si-LINC00518, miR-526b-5p mimic or si-EIF5A2 weakened the proliferative, migratory, and invasive abilities of melanoma cells, while transfection of miR-526b-5p inhibitor or pcDNA-LINC00518 enhanced the progression of melanoma cells. Moreover, the proliferative, migratory, and invasive potentials of melanoma cells were decreased after co-transfection of si-EIF5A2 and pcDNA-LINC00518 compared with cells transfected with pcDNA-LINC00518 alone. LINC00518 bound to miR-526b-3p and miR-526b-3p targeted EIF5A2. LINC00518 negatively regulated miR-526b-3p expression but positively regulated EIF5A2. Furthermore, EIF5A2 expression was negatively associated with miR-526b-3p expression. CONCLUSION: LINC00518 encourages CMM through the miR-526b-3p/EIF5A2 axis in terms of cell proliferation, invasion, and migration.

Screening and identification of vancomycin anti-idiotypic antibodies for against Staphylococcus aureus from a human phage display domain antibody library.

Staphylococcus aureus is a common food-borne pathogenic microorganism that poses a serious threat to food quality and safety, and can do harm to human health. In the past, researchers relied on antibiotics to control Staphylococcus aureus, though very effective, yet it was also worrying in the aspect of bio-safety. In fact, anti-idiotypic antibody (Anti-Id) shows its potential to mimic some of the structural and biological functions of antigens. Therefore, in this study, based on Anti-Id theory and technology, we expect to obtain the vancomycin Anti-Id which can mimic vancomycin against Staphylococcus aureus from a human phage display domain antibody library. After four rounds of bio-panning, a total of 18 positive Anti-Ids were obtained. Among them, two Anti-Ids named Anti-Id-2C12 and Anti-Id-1F5 were identified as "ß" type Anti-Ids, and afterwards they were selected for gene cloning and protein expression in prokaryotic expression system. As a result, a concentration of purified proteins with 568.6 µg/mL (Anti-Id-2C12) and 602.3 µg/mL (Anti-Id-1F5) were successfully obtained, and their minimum inhibitory concentration (MIC) values for Staphylococcus aureus were 125 and 200 µg/mL, respectively. As they are human heavy-chain domain antibodies, which were theoretically harmless to humans, they have the potential application value as preservatives in food and edible agricultural products.