Proteomic analysis of plasma in healthy adults receiving recombinant vaccinia virus provides novel insights into HIV-1 neutralizing antibodies.

Human immunodeficiency virus (HIV) infection is still a global public health issue, and the development of an effective prophylactic vaccine inducing potent neutralizing antibodies remains a significant challenge. This study aims to explore the inflammation-related proteins associated with the neutralizing antibodies induced by the DNA/rTV vaccine. In this study, we employed the Olink chip to analyze the inflammation-related proteins in plasma in healthy individuals receiving HIV candidate vaccine (DNA priming and recombinant vaccinia virus rTV boosting) and compared the differences between neutralizing antibody-positive (nab + ) and -negative(nab-) groups. We identified 25 differentially expressed factors and conducted enrichment and correlation analysis on them. Our results revealed that significant expression differences in artemin (ARTN) and C-C motif chemokine ligand 23 (CCL23) between nab+ and -nab- groups. Notably, the expression of CCL23 was negatively corelated to the ID50 of neutralizing antibodies and the intensity of the CD4+ T cell responses. This study enriches our understanding of the immune picture induced by the DNA/rTV vaccine, and provides insights for future HIV vaccine development.

DNA vaccine prime and replicating vaccinia vaccine boost induce robust humoral and cellular immune responses against MERS-CoV in mice.

As of December 2022, 2603 laboratory-identified Middle East respiratory syndrome coronavirus (MERS-CoV) infections and 935 associated deaths, with a mortality rate of 36%, had been reported to the World Health Organization (WHO). However, there are still no vaccines for MERS-CoV, which makes the prevention and control of MERS-CoV difficult. In this study, we generated two DNA vaccine candidates by integrating MERS-CoV Spike (S) gene into a replicating Vaccinia Tian Tan (VTT) vector. Compared to homologous immunization with either vaccine, mice immunized with DNA vaccine prime and VTT vaccine boost exhibited much stronger and durable humoral and cellular immune responses. The immunized mice produced robust binding antibodies and broad neutralizing antibodies against the EMC2012, England1 and KNIH strains of MERS-CoV. Prime-Boost immunization also induced strong MERS-S specific T cells responses, with high memory and poly-functional (CD107a-IFN-γ-TNF-α) effector CD8+ T cells. In conclusion, the research demonstrated that DNA-Prime/VTT-Boost strategy could elicit robust and balanced humoral and cellular immune responses against MERS-CoV-S. This study not only provides a promising set of MERS-CoV vaccine candidates, but also proposes a heterologous sequential immunization strategy worthy of further development.

Amino acid substitution of the membrane-proximal external region alter neutralization sensitivity in a chronic HIV-1 clade B infected patient.

The membrane-proximal external region (MPER) represents a highly conserved region of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (env) targeted by several broadly neutralizing antibodies (bnAbs). In this study, we employed single genome amplification to amplify 34 full-length env sequences from the 2005 plasma sample of CBJC504, a chronic HIV-1 clade B infected individual. We identified three amino acid changes (N671S, D674N, and K677R) in the MPER. A longitudinal analysis revealed that the proportion of env sequences with MPER mutations increased from 26.5 % in 2005 to 56.0 % in 2009, and the sequences with the same mutation clustered together. Nine functional pseudoviruses were generated from the 34 env sequences to examine the effect of these mutations on neutralizing activity. Pseudoviruses carrying N674 or R677 mutations demonstrate increased sensitivity to autologous plasma and monoclonal antibodies 2F5, 4E10, and 10E8. Reverse mutations were performed in env including N674, R677, D659, and S671/N677 mutations, to validate the impact of the mutations on neutralizing sensitivity. Neutralization assays indicated that the N671S mutation increased neutralization sensitivity to 2F5 and 10E8. The amino acid R at position 677 increased viral resistance to 10E8, whereas N enhanced viral resistance to 4E10 and 10E8. It has been proposed that critical amino acids in the extra-MPER and the number of potential N-like glycosylation sites (PNGSs) in the V1 loop may have an impact on neutralizing activity. Understanding the mutations and evolution of MPER in chronically infected patients with HIV-1 is crucial for the design and development of vaccines that trigger bnAbs against MPER.

Dual oxidase 2 (duox 2) participates in the intestinal antibacterial innate immune responses of Procambarus clarkii by regulating ROS levels.

Dual oxidase (Duox) a member of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) family can induce the production of reactive oxygen species (ROS). In vertebrates, the duox gene was indicated to be associated with the mucosal immunity. The roles of the duox gene in invertebrates were mainly studied in insects for the function of maintaining intestinal flora balance. In recent years, some studies have reported that Duox is involved in regulating the production of ROS and plays an important role in defending against the intestinal pathogen infection. However, the molecular mechanism has not been fully illuminated. In this study, a duox 2 involved in the production of H2O2 was identified for the first time in P. clarkii. Mature Pc-Duox 2 is a 7-transmembrane protein molecule that includes PHD, FAD, and NAD domains. Pc-duox 2 was mainly expressed in hemocytes and intestinal tissue. Its expression levels were obviously upregulated after intramuscular or oral infection with V. harveyi. In the RNAi assay, the upregulated trends of H2O2 and total ROS levels in crayfish intestine were significantly suppressed when Pc-duox 2 was knocked down. Compared with the slightly affected SOD activity, the upregulated CAT activity was suppressed more obviously in the crayfish intestine. Furthermore, Pc-duox 2 had an important effect on the maintenance of the structural stability of crayfish the intestine. Further research revealed that the knockdown of Pc-duox 2 could cause an obvious suppression in the upregulated levels of Toll signalling pathway-related genes, including Pc-toll 1, Pc-toll 3, Pc-dorsal, Pc-ALF 5, Pc-crustin 1, and Pc-lysozyme. Ultimately, these changes triggered the accelerated death of crayfish. Overall, we speculated that Pc-duox 2 played an important role in antibacterial innate immunity in the crayfish intestine by regulating the total ROS level.

Sequential heterologous immunization with COVID-19 vaccines induces broader neutralizing responses against SARS-CoV-2 variants in comparison with homologous boosters.

The recently prevalent variants of concerns (VOCs) of SARS-CoV-2 belong to Omicron variants which display increased transmissibility and evade from immune protection generated by vaccines and/or natural infections. Better immunization strategies should be explored to induce broader immune responses against evolving SARS-CoV-2 variants. Here, we used inactivated vaccines derived from ancestral (Wu), Delta (Del) and Omicron (Omi) strains to immunize mice with homologous booster (3 × Wu, 3 × Del and 3 × Omi) or heterologous sequential booster (Wu/Del/Omi and Omi/Wu/Del) to evaluate their responses against two pre-Omicron (Wu and Del) and four Omicron variants. Even though neutralization responses against Wu and Del variants were similar in heterologous and homologous immunization groups, heterologous immunization groups induced significantly stronger neutralizing antibody against BA.1 (4.1-11 folds higher) and BA.2 (4.7-14.2 folds higher) than those of homologous immunization groups. While homologous immunization only induced strong neutralizing responses to either pre-Omicron variants (Wu and Del) in 3 × Wu and 3 × Del groups or to Omicron variants (BA.1 and BA.2) in 3 × Omi group, heterologous immunization groups induced strong and broader neutralizing responses to both pre-Omicron (Wu, Del) and Omicron variants (BA.1 and BA.2). Homologous and heterologous immunization groups elicited similar antigen-specific T cell (IFN-γ+) and B cell responses. Compared with homologous immunization, heterologous immunization could induce stronger plasma cell responses, which have the potential to generate broader and stronger neutralizing antibodies. However, neither heterologous nor homologous immunization groups induced strong neutralizing antibody against variants with bigger genetic deviation, such as BA.4/5 or BF.7, only weak neutralizing responses were induced. Surveillance on SARS-CoV2 variants evolution and immunization strategy are needed to explore better vaccines with broader and stronger neutralizing antibodies against post pandemic COVID-19.

Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii.

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.

Neutralization Sensitivity and Evolution of Virus in a Chronic HIV-1 Clade B Infected Patient with Neutralizing Activity against Membrane-Proximal External Region.

The membrane-proximal external region (MPER) is a promising HIV-1 vaccine target owing to its linear neutralizing epitopes and highly conserved amino acids. Here, we explored the neutralization sensitivity and investigated the MPER sequences in a chronic HIV-1 infected patient with neutralizing activity against the MPER. Using single-genome amplification (SGA), 50 full-length HIV-1 envelope glycoprotein (env) genes were isolated from the patient's plasma at two time points (2006 and 2009). The neutralization sensitivity of 14 Env-pseudoviruses to autologous plasma and monoclonal antibodies (mAbs) was evaluated. Env gene sequencing revealed that the diversity of Env increased over time and four mutation positions (659D, 662K, 671S, and 677N/R) were identified in the MPER. The K677R mutation increased the IC50 values of pseudoviruses approximately twofold for 4E10 and 2F5, and E659D increased the IC50 up to ninefold for 4E10 and fourfold for 2F5. These two mutations also decreased the contact between gp41 and mAbs. Almost all mutant pseudoviruses were resistant to autologous plasma at both the earlier and concurrent time points. Mutations 659D and 677R in the MPER decreased the neutralization sensitivity of Env-pseudoviruses, providing a detailed understanding of MPER evolution which might facilitate advances in the design of HIV-1 vaccines.

Influence of nano-Fe3O4 biochar on the methanation pathway during anaerobic digestion of chicken manure.

Volatile fatty acids and ammonia nitrogen (AN) accumulate during anaerobic digestion (AD) of high N substrates, such as chicken manure (CM), causing decreases in methane yield. Previous research found that the addition of nano-Fe3O4 biochar can alleviate the inhibition caused by acids and ammonia and increase methane production. The mechanism of enhanced methane production in nano-Fe3O4 biochar-mediated AD of CM was explored in depth in this study. The results showed the lowest AN concentration in the control and nano-Fe3O4 biochar addition groups were 8,229.0 mg/L and 7,701.5 mg/L, respectively. Methane yield of volatile solids increased from 92.0 mL/g to 219.9 mL/g in the nano-Fe3O4 biochar treatment, which was attributed to the enrichment of unclassified Clostridiales and Methanosarcina. The mechanism of nano-Fe3O4 biochar in AD of CM under high AN level was to improve methane production by promoting syntrophic acetate oxidation and facilitating direct electron transfer between microorganisms.

Synthesis of aryloxyacetamides from arylboronic acids and 2-bromoacetonitrile promoted by alkaline solutions of hydrogen peroxide.

A novel and metal catalyst-free synthesis of aryloxyacetamides from the corresponding arylboronic acids and 2-bromoacetonitrile promoted by alkaline solutions of hydrogen peroxide has been developed involving an oxidation-reduction of eco-friendly H2O2 with simultaneous reaction ipso-hydroxylation of arylboronic acid and hydration of the nitrile. This protocol is compatible with sensitive substituents attached to the arylboronic acid and provides desired products in moderate to good yields in pure water.

Alcoholysis kinetics and mechanism studies of ethyl levulinate production from ball milled corn stover.

Alcoholysis of ball-milled biomass over catalysts with Brønsted and Lewis acid sites provides an efficient and sustainable scheme to produce versatile biobased chemicals under mild conditions; however, optimizing the process parameters is challenged by the complexity of reaction pathways and the multiplicity of ball milling and combination catalyst gains. To address these challenges, we present kinetic analysis of ethyl levulinate (EL) production from ball-milled corn stover catalyzed by Brønsted (B) acidic ionic liquid [Bmim-SO3H][HSO4] (SO3H-IL) and Lewis (L) acidic Al2(SO4)3. Product analysis shows that cellulosic substrates can form EL either through the intermediate ethyl-d-glycopyranoside (EDGP) or levoglucosenone (LGO), with the former leading the alcoholysis reaction. Kinetics results reveal that ball milling accelerates the reaction rate by promoting the formation of EDGP and LGO from cellulose. Pure SO3H-IL gives high selectivity towards EDGP from ball-milled corn stover and promotes the LGO production, whereas addition of Al2(SO4)3 substantially facilitates their further conversion to EL. Our findings contribute to the rational design of efficient catalytic strategies for sustainable and profitable biorefinery.

Oxygen dynamics, organic matter degradation and main gas emissions during pig manure composting: Effect of intermittent aeration.

To investigate the effect of intermittent aeration on oxygen dynamics, organic matter degradation and main gas emissions, a lab-scale pig manure composting experiment was conducted with intermittent aeration (I_A, 30-min on and 30-min off) and continuous aeration (C_A). Although aeration volume and oxygen supply of I_A was only half of C_A, I_A could obviously enhance the oxygen utilization efficiency by 96.67 % and reduce energy dissipation for aeration by 50.87 %. Based on the comprehensive analysis of total organic matter, total carbon, total nitrogen, cellulose, hemicellulose and lignin contents, there was no significant difference in organic matter degradation between I_A and C_A (p > 0.05). Moreover, a reduction of 21.71 %, 38.93 %, 44.40 % and 62.19 % of CH4, N2O and the total GHG emission equivalent as well as NH3 emissions was realized, respectively, in I_A compared with C_A. Therefore, adopting intermittent aeration was a useful strategy and choice for high-efficiency, high-quality and environment-friendly composting.

Dopamine receptor (DAR) and dopa decarboxylase (DDC) mediate hepatopancreas antibacterial innate immune reactions in Procambarus clarkii.

Dopa is decarboxylated by dopa decarboxylase (DDC) to form dopamine, which is a significant signaling molecule in the neuroendocrine system. The dopamine receptor (DAR) is an important transmembrane receptor responsible for receiving extracellular signals in the DAR-mediated signaling pathway. In the present study, the expression patterns of Pc-dar were investigated after bacterial challenge. The obviously changed expression patterns showed Pc-dar was related to the antibacterial innate immunity. Endogenous Pc-DDC enzymatic activities were obviously downregulated after Pc-ddc dsRNA injection. The expression level of Pc-dar mRNA was obviously upregulated after bacterial injection when the expression level of Pc-ddc was knocked down. In addition, the upregulation trend of endogenous Pc-DDC enzymatic activities was obviously restrained after bacterial stimulation when Pc-ddc was knocked down. Finally, melanization was downregulated in crayfish hemolymph compared with the dsGFP injection group. In the RNAi assay, the results of qRT-PCR showed that Toll (TLRs) signaling pathway-related genes were activated in the early stages of bacterial stimulation when Pc-ddc was knocked down. Four tested ROS-related antioxidant enzyme genes were significantly upregulated after bacterial challenge compared with the dsGFP injection group. The above results indicated that Pc-DDC and Pc-DAR play important mediating roles in the neuroendocrine immune (NEI) system of crayfish.

Interleukin enhancer binding factor 2 (IEBF 2) was involved in the regulation of the antibacterial immune reactions in fresh water crayfish, Procambarus clarkii.

Although interleukin and interleukin analogues which play important immunomodulatory roles in mammals have not yet been reported in invertebrates, interleukin enhancer binding factor (IEBF) which acts as a transcription factor has been recently studied in several crustaceans and it may be involved in innate immune defence against pathogens. In this study, an IEBF 2 homologue was identified in the fresh water crayfish, Procambarus clarkii. The significantly changed expression levels of Pc-iebf 2 after bacterial challenge revealed the possibility of its participation in defence against bacterial infection. The results of an RNAi assay showed that the crayfish survival rate was obviously decreased after dsIEBF 2 injection, compared with the control groups. And S. aureus proliferation was obviously enhanced at 24 and 48 h post bacterial injection, when Pc-iebf 2 was knocked down. The possible molecular mechanisms for the innate immune regulation functions of Pc-IEBF 2 were also investigated. We speculated that Pc-IEBF 2 plays an important role in defending against bacterial infection in crayfish. It could regulate some innate immune responses by affecting the Toll signalling pathway, melanisation, and cell apoptosis.

A comprehensive assessment on bioavailability, leaching characteristics and potential risk of polycyclic aromatic hydrocarbons in biochars produced by a continuous pyrolysis system.

Biochar application as a soil amendment has attracted worldwide attention. Nevertheless, polycyclic aromatic hydrocarbons (PAHs) formed during biochar production might enter into ecosystems and threaten human health after application to soil. Continuous pyrolysis systems tend to cause an accumulation of PAHs in biochar owing to short residence time and rapid cooling. This study conducted a comprehensive assessment regarding potential risk of PAHs in biochars produced by a continuous pyrolysis system based on bioavailability, leaching behavior, toxic equivalent quantity, health risk and phytotoxicity of PAHs. Results showed that the concentrations of total PAHs in biochars were in the range of 93.40-172.40 mg/kg, exceeding the European Biochar Certificate standard. 3-rings PAHs were the predominant groups. The percentages of total freely dissolved and leachable PAHs were lower than 1%. RH contained the least bioavailable and leachable PAHs concentration and phytotoxicity compared with CS and PS, which might attribute to the characteristic of three biochars. CS and PS were acidic and exhibited high levels of DOC and VFAs, while RH was strongly alkaline and presented greater aromaticity and higher surface area, which might have resulted in high adsorptive capacity and decreased bioavailability of PAHs. When the biochar application rate was higher than 0.6 t/ha, the incremental lifetime cancer risk value for human exposure to biochar-borne PAHs through the biochar-amended soil was over 10-6, suggesting carcinogenic risks. Germination index values of biochars ranged from 25.66 to 88.95%. Phytotoxicity mainly was caused by bioavailable PAHs and dissolved organic compounds. Overall, these findings highlighted that although the percentage of bioavailable PAHs was low, the potential health risk and phytotoxicity of PAHs in biochars produced by a continuous pyrolysis system was of a great concern. High biochar application rates should be avoided without processing both for soil safety and human health.

Anaerobic co-digestion of corn stover and wastewater from hydrothermal carbonation.

This study aimed to investigate the interactions between wastewater of hydrothermal carbonation (W-HTC) and corn stover (CS) during anaerobic co-digestion. The results showed the maximum cumulative methane production of co-digestion was 280.7 ± 3.2 mL/g VS, and it increased by 5.84% and 10.69% compared with mono-digestion of CS and W-HTC, respectively. Increasing the HTC temperature and excess addition of W-HTC inhibits early and middle stage of co-digestion due to toxic organic inhibitors, and the negative effect of phenols is substantially more than furans. The microbial analysis illustrated the addition of W-HTC can promote the growth of Clostridia and Bacteroidia. The growth of Methanomassiliicoccus and Methanosarcina was more vigorous in most of co-digestions, which was positively correlated with methane production. The study concluded methanogenesis can be enhanced by the co-digestion of W-HTC and CS, which provide optimization of process conditions and some reaction mechanism for application of W-HTC in anaerobic digestion.

Molecular characterization and expression analysis of tumor necrosis factor receptor-associated factor 6 (traf6) like gene involved in antibacterial innate immune of fresh water crayfish, Procambarus clarkii.

In invertebrates, innate immunity was the crucial defending pattern against pathogenic microorganisms. For the past few years, Toll or Toll like receptors (TLRs) signaling pathway was studied extensively in crustaceans. Among the components of Toll or Toll like receptors (TLRs) signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6) acted as an important cytoplasmic adaptor, which was conserved from Drosophila to human. In this study, a new traf6 like gene was cloned from hepatopancreas of P. clarkii. After challenged respectively by S. aureus or E. ictaluri, the expression profiles were studied. And the results showed that the mRNA transcript of Pc-traf6 like gene was up-regulated significantly in the hemocytes, hepatopancreas, gills, and intestine of crayfish. After Pc-traf6 like gene was knocked down, the expression levels of transcription factor (Dorsal) and some crucial immunity effectors (ALF 3, Lysozyme 1, Lectin 1, and Crustin 2) in TLRs signaling pathway were dramatically suppressed. Simultaneously, the survival rate of crayfish challenged respectively by S. aureus or E. ictaluri was significantly decreased in RNAi assay. All these results indicated that Pc-traf6 like gene played an important role in regulating the expression of downstream effectors in the TLRs signaling pathway of crayfish.

Sensitivity programmable ratiometric electrochemical aptasensor based on signal engineering for the detection of aflatoxin B1 in peanut.

Accurately monitoring of aflatoxin B1 (AFB1), the most hazardous mycotoxin in agricultural products, is essential for the public health, but various testing demands (e.g. detection range, sensitivity) for different samples can be challenging for sensors. Here, we developed a sensitivity-programmable ratiometric electrochemical aptasensor for AFB1 analysis in peanut. Thionine functionalized reduced graphene oxide (THI-rGO) served as reference signal generator, ferrocene-labelled aptamer (Fc-apt) output the response signal. During analysis, the formation of Fc-apt-AFB1 complex led to its stripping from the electrode and faded the current intensity of Fc (IFc), while the current intensity of THI (ITHI) was enhanced. And ratiometric detection of AFB1 was achieved by using the current intensity ratio (ITHI/IFc) as quantitative signal. Compared with ratiometric strategies that highly rely on the labelled aptamers, the proposed strategy could regulate the value of ITHI/IFc by changing the modification of Fc-apt. And the detection sensitivity was found to be closely related to ITHI/IFc. Under the optimal conditions, the fabricated aptasensor with a dynamic range from 0.05-20 ng mL-1 and a detection limit of 0.016 ng mL-1 for AFB1 analysis. Besides, it exhibited excellent selectivity, reliability and reproducibility. The proposed sensitivity-programmable biosensor can be applied to detect various aptamer-recognized mycotoxins in agricultural sensing.

Effect of pyrolysis temperature on characteristics, chemical speciation and environmental risk of Cr, Mn, Cu, and Zn in biochars derived from pig manure.

The comprehensive analysis of environmental risk for heavy metals in pig manure was essential for optimization of pyrolysis conditions and scientific utilization of pig manure biochars as soil amendment. However, in previous studies, the selected pyrolysis temperature points were limited and temperature interval was large, it's was difficult to accurately verify the effect of pyrolysis temperature on chemical speciation and environmental risk of heavy metals. Therefore, in this study, pig manure was pyrolyzed at 300-700 °C with a small interval of 50 °C to study the effect of pyrolysis temperature on characteristics and environmental risk of Cr, Mn, Cu and Zn in pig manure biochar. Results indicated that the characteristics of biochars (>500 °C) were relatively stable. The biochar obtained at 700 °C exhibited the largest surface area (8.28 m2 g-1) and pore volume (25.17 m3 kg-1), secondly is the biochar derived at 500 °C. The total percentages of exchangeable and acid fraction and reducible fraction decreased from 16.98% to 9.43% for Cr, 85.60% to 65.55% for Mn, 57.26% to 10.61% for Cu, 37.90% to 13.78% for Zn, respectively, suggesting that exchangeable and acid fraction and reducible fraction of Cr, Mn, Cu and Zn in pig manure were transformed into oxidizable and residual fractions after pyrolysis. The leaching rates, risk assessment code and potential ecological risk index values significantly decreased after pyrolysis and presented lower value at 500 and 700 °C. Biochars derived at 300-700 °C conditions posed no phytotoxicity with germination index >80%. Correlation analyses revealed that larger surface area, pore volume and pH values of biochars may help to immobilize heavy metals and reduce bioavailability. These findings demonstrated that bioavailability and toxicity of Cr, Mn, Cu and Zn in pig manure biochar were greatly reduced after pyrolysis and the optimum temperature was 500 °C considering energy cost.

Ratiometric electrochemical aptasensor for ultrasensitive detection of Ochratoxin A based on a dual signal amplification strategy: Engineering the binding of methylene blue to DNA.

A novel ratiometric electrochemical aptasensor was developed for Ochratoxin A (OTA) detection based on the binding of methylene blue (MB) to DNA with a dual signal amplification strategy. The formation of dsDNA structures between ferrocene-labeled complementary DNA (Fc-cDNA), the OTA aptamer, and complementary helper DNA (hDNA) caused Fc away from the electrode, and allowed dsDNA to bind with a certain amount of MB. Here, a small oxidation current of Fc (IFc) and a large oxidation current of MB (IMB) were obtained. In the presence of OTA, its specific recognition with the aptamer induced the release of aptamer and hDNA from the electrode and subsequently the formation of hairpin structure for cDNA, which caused Fc close to the electrode and a weaker binding ability with MB. Then, an increased IFc and a decreased IMB were obtained. Based on this principle, OTA could be accurately quantified by measuring the ratiometric signal of IFc/IMB. Herein, the dual signal amplification strategy of the introduction of hDNA and the binding with MB after the OTA recognition was exploited to amplify the response signal. The obtained aptasensor showed a linear detection range from 10 pg mL-1 to 10 ng mL-1 and a detection limit of 3.3 pg mL-1. The aptasensor was successfully applied to determine OTA in wheat, and the results were validated through HPLC-MS. Furthermore, by changing the target aptamers, this strategy could be universally used for the determination of various mycotoxins, showing promising potential applications for mycotoxins monitoring in agricultural products and foods.

A new antimicrobial peptide isoform, Pc-crustin 4 involved in antibacterial innate immune response in fresh water crayfish, Procambarus clarkii.

The main advantage of antimicrobial peptides (AMPs) used as the effectors in the innate immunity system of invertebrates is that the high specificity is not indispensable. And they play important roles in the systemic defenses against microbial invasion. In this study, a new full-length cDNA of the crustins molecule was identified in red swamp crayfish, P. clarkii (named Pc-crustin 4). The ORF of Pc-crustin 4 contained 369 bp which encoded a protein of 122 amino acids, with a 20-amino-acid signal peptide sequence. On the base of the classification method established by Smith et al., Pc-crustin 4 belonged to Type Ⅰ crustin molecule. The Pc-crustin 4 transcripts were expressed in hemocytes at relatively high level, and relatively low level in hepatopancreas, gills, and intestine in normal crayfish. After respectively challenged with S. aureus or E. ictaluri, the expression levels of Pc-crustin 4 showed up-regulation trends at different degrees in the hemocytes, hepatopancreas, gills, and intestine tissues. Besides, the results of liquid antibacterial assay showed that rPc-crustin 4 inhibited obviously the growth of S. aureus and E. ictaluri. The results of bacteria binding assay showed that rPc-crustin 4 could bind strongly to S. aureus and E. ictaluri. Finally, RNAi assay was performed to study the immunity roles of Pc-crustin 4 in crayfish in vivo. Taken together, Pc-crustin 4 is an important immunity effector molecule, which plays crucial roles in defending against bacterial infection in crayfish.