Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs.

Monitoring the prevalence of antimicrobial resistance genes (ARGs) is vital for addressing the global crisis of antibiotic-resistant bacterial infections. Despite its importance, the characterization of ARGs and microbiome structures, as well as the identification of indicators for routine ARG monitoring in pig farms, are still lacking, particularly concerning variations in antimicrobial exposure in different countries or regions. Here, metagenomics and random forest machine learning were used to elucidate the ARG profiles, microbiome structures, and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe. Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs ( P<0.05). ANT(6)-Ib, APH(3')-IIIa, and tet(40) were identified as shared core ARGs between the two pig populations. Furthermore, the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions. Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs, respectively. Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100% and 98.7%, respectively. Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy ( r=0.72-0.88). Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs. The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

[Temporal and Spatial Variation Characteristics of Methane, Carbon Dioxide, and Nitrous Oxide Concentrations and the Influencing Factors in Small Aquaculture Pond].

As an important source of greenhouse gases, the changes in greenhouse gas concentrations of aquaculture ponds are not only the basis for accurate quantification of greenhouse gases emissions but are also important for identifying their influencing factors. The spatial and temporal variation characteristics of CH4, CO2, and N2O concentrations and the influencing factors in a typical small aquaculture pond in the Yangtze River Delta were analyzed based on the headspace equilibrium-gas chromatograph method. Except in spring, the concentrations of CH4, and N2O appeared high at noon or afternoon and were influenced by water temperature. Impacted by water temperature and aquatic plant photosynthesis, the concentrations of CO2 were high in the morning when photosynthesis was weak. The concentrations of CH4 and CO2 were the highest in autumn and the lowest in winter. The mean concentrations of CH4 in autumn and winter were 176.34 nmol·L-1 and 32.75 nmol·L-1, respectively, which were mainly affected by air temperature, water temperature, and dissolved oxygen. The average CO2 concentrations in autumn and winter were 134.37 µmol·L-1 and 23.10 µmol·L-1, respectively, and were mainly affected by aquatic vegetation photosynthesis and pH. N2O concentration was the highest in summer and the lowest in winter, with mean values of 97.05 nmol·L-1 and 19.41 nmol·L-1, respectively, which were mainly affected by air temperature and water temperature. In terms of the vertical spatial variations of the three greenhouse gases, the concentration of CH4decreased with water depth in summer, and the concentration differences between the surface layer and the bottom and middle layers were 71.28 nmol·L-1 and 42.80 nmol·L-1, respectively. The concentration of CH4 increased with water depth in autumn, and the concentration difference between the bottom layer and surface layer was 163.94 nmol·L-1. The CO2 concentration increased with water depth in summer and autumn. The concentration differences between the bottom and surface concentrations were 18.69 µmol·L-1 and 29.90 µmol·L-1, respectively. N2O concentration showed no obvious change in the vertical direction. For the horizontal variations, the concentrations of CH4, CO2, and N2O in the feeding area in summer and in chicken manure in spring were approximately 1.34-1.98 times and 1.95-2.42 times those in other areas, respectively, and the concentrations of N2O and CO2 in spring and summer were approximately 1.13-1.26 times and 1.39-1.74 times those in other areas.

Enzyme-free and triple-amplified electrochemical sensing of 8-hydroxy-2'-deoxyguanosine by three kinds of short pDNA-driven catalyzed hairpin assemblies followed by a hybridization chain reaction.

A sensitive and enzyme-free electrochemical aptasensor was constructed for the sensing of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). In the process of constructing the aptasensor, triple signal amplification strategies were introduced to enhance the sensitivity. First, every aptamer/pDNA complex immobilized on magnetic beads could release three kinds of pDNAs when 8-OH-dG was introduced, which caused three-fold magnification of the target. Second, the released three kinds of pDNAs initiated catalyzed hairpin assembly between two hairpin DNAs (HP1 and HP2) on a gold electrode. Meanwhile, the three kinds of pDNAs were released again by a strand displacement reaction to obtain the next catalyzed hairpin assembly. Third, the emerging toehold of HP2 further induced a hybridization chain reaction (HCR) between two hairpin DNAs (HP3 and HP4), forming a long double-stranded DNA concatemer on the surface of the electrode. Finally, [Ru(NH3)6]3+, an electroactive cation, was adsorbed onto the long dsDNA concatemer by electrostatic interactions and consequently, an electrochemical signal was generated. Under this triple signal amplification, a low detection limit down to 24.34 fM has been obtained for 8-OH-dG determination, which is superior to those of most previously reported methods.

Ultrasensitive electrochemiluminescence aptasensor for 8-hydroxy-2'-deoxyguanosine detection based on target-induced multi-DNA release and nicking enzyme amplification strategy.

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a principal stable marker of DNA oxidative damage. Sensitive and specific detection of 8-OH-dG is of great importance for early disease diagnosis. In this paper, we developed an electrochemiluminescence aptasensor for 8-OH-dG detection based on target induced multi-DNA release and nicking enzyme signaling amplification strategy. First, three kinds of short DNAs were aligned on the aptamers immobilized on the magnetic beads. In the presence of 8-OH-dG, the aptamer recognized and specifically bound with 8-OH-dG, leading to the release of three kinds of short DNAs and three-fold signal amplification. Then the released short DNAs hybridized with ferrocence (Fc) labeled hairpin DNA (Fc-HP) immobilized on the gold electrode to form a double strand DNA. Subsequently, nicking endonuclease (Nt.AlwI) recognized the asymmetric sequence in the dsDNA and cleaved the substrate strand (Fc-HP) into two parts, one fragments containing Fc would leave the surface of electrode. Based on the quenching effect of Fc on the electrochemiluminescence (ECL) of Ru(bpy)32+/TPA, a signal-on ECL aptasensor was developed. At the same time, three kinds of short DNAs were released again and reused to initiate the repeated cycles of hybridization-cleavage. Under double signal amplification, this aptasensor achieved a low detection of 25 fM and a wide linear range from 100 fM to 10 nM for 8-OH-dG. Besides, the amount of 8-OH-dG in urine samples derived from different people were determined with satisfactory results.

A sensitive electrochemical aptasensor for Mucin 1 detection based on catalytic hairpin assembly coupled with PtPdNPs peroxidase-like activity.

In this work, an ultrasensitive aptasensor for the detection of Mucin 1 (MUC1) was presented based on the target-induced catalytic hairpin assembly combined with excellent mimic peroxidase performance of PtPd bimetallic nanoparticles (PtPdNPs). Traditionally, the cyclic reuse of target protein was achieved by protein conversion with enzyme cleavage or polymerization, which is costly and complex. However, in this work, it can be performed by simple strand displacement. In addition, PtPdNPs, a mimic peroxidase, was used a probe to catalyze the oxidation of tetramethylbenzidine (TMB) by H2O2, leading to the electrochemical signal amplification. With this ingenious design, the prepared aptasensor for MUC1 detection showed a favorable linear response from 100 fg mL-1 to 1 ng mL-1 and a relatively low detection limit of 16 fg mL-1. The proposed biosensor possessed acceptable stability, selectivity and reproducibility for MUC1 assay. Additionally, the fabricated aptasensor has been successfully applied to detect MUC1 in serum samples with satisfactory results. This new strategy supplied one efficient approach to improve signal amplification, which also open an avenue for sensitivity enhancement in targets detection.

Aptamer based electrochemical assay for protein kinase activity by coupling hybridization chain reaction.

The present work reported a simple, lable-free and sensitive electrochemical method for the detection of protein kinase A (PKA) activity. This method was based on the specific recognition of aptamer and the aptamer-induced hybridization chain reaction (HCR) amplification strategy. The aptasensor was constructed by immobilizing capture probe on a gold electrode via an Au-S bond. When adenosine triphosphate (ATP) aptamer was introduced, its one terminus hybridized with capture probe and the other hybridized with the complementary region of an auxiliary probe, which other region triggered HCR between two hairpin DNA (H1 and H2) to form a long DNA concatamer. At last a large number of electroactive methyle blue (MB) molecules were assembled on the dsDNA concatamer, which generated a significantly amplified electrochemical signal. In the presence of ATP, the HCR would not be performed because the aptamer specifically bond to ATP and the electrochemical response would decrease. However, when ATP and PKA coexisted, the electrochemical response would recovery because that ATP had been translated into ADP by PKA. So the activity of PKA could be effectively monitored according to the change of electrochemical signal. Based on the HCR amplification strategy, the aptasensor showed a wide linear range (4 - 4 ×105 U L-1) and a low detection limit (1.5 U L-1) for the detection of PKA. Furthermore, the method was applied to study the inhibitory effect of H-89 on PKA activity. The developed aptasensor was also used to the analysis of drug-induced PKA activity in cell lysates, indicating the potential application of the developed method in the fields of clinical diagnostics and discovery of new targeted drugs.