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In this study, temperature-responsive anti-aflatoxin B1 (AFB1) nanoantibodies were obtained by fusion expression of elastin-like polypeptides (ELPs) of different lengths with nanoantibodies. The recombinant expression vectors pET30a-G8-ELP20, pET30a-G8-ELP40, pET30a-G8-ELP60 and pET30a-G8-ELP80 were synthesized by recursive directional ligation (RDL) and transformed into Escherichia coli BL21 (DE3) cells, separately. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the four fusion proteins were expressed in the form of inclusion bodies in the bacterial precipitate. The inclusion bodies were dissolved in denaturing buffer and refolded by dilution, inverse transition cycling (ITC) purification, dialysis or column refolding. SDS-PAGE analysis showed that the purity of the four proteins obtained achieved using the four refolding methods was similar. After refolding by ITC, the highest yield of 83%, 90.7%, 89.5%, and 88.3% was obtained for the four fusion proteins, respectively. The half maximal inhibitory concentration (IC50) of G8-ELP80 obtained by dilution refolding was the lowest (4.35 ng/mL), as determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). The phase transition temperatures (Tt) of nanoantibodies tagged with the different ELPs, as determined by the turbidity method, were 45, 38, 32 and 28 ℃, respectively. Circular dichroism (CD) spectroscopy showed that the main secondary structures of the four fusion proteins were β-sheet and β-turn. This study provides a basis for subsequent AFB1 detection and analysis.

Nanoluciferase (Nluc), the smallest luciferase known, was used as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection of the aflatoxin B1 in cereal. Nanobody (clone G8) against aflatoxin B1 was fused with nanoluciferase and cloned into a pET22b expression vector, and then transformed into Escherichia coli. The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography, yielding a biologically active fusion protein. The fusion protein G8-Nluc retained binding properties of the original nanobody. Concentration of the coelenterazine substrate and buffer composition were also optimized to provide high intensity and long half-life of the luminescent signal. The G8-Nluc was used as a detection antibody to establish a competitive bioluminescent ELISA for the detection of aflatoxin B1 in cereals successfully. Compared to classical ELISA, this novel assay showed more than 20-fold improvement in detection sensitivity, with an IC50 value of 0.41 ng/mL and linear range from 0.10 to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can be completed in one step within 2 h, from sample preparation to data analysis. These results suggest that nanobody fragments fused with nanoluciferase might serve as useful and highly sensitive dual functional reagents for the development of rapid and highly sensitive immunoanalytical methods.

Background: Meat originating from the spent hen is an important source of poultry meat production; however, multiple factors cause the decline in the meat quality of spent hens. Chinese herbs have been widely used as medicine for a long time to prevent diseases and as nutrient supplements to improve the product quality. This experiment explored the effects of adding 1.0% Chinese herbal formula (CHF, including 0.30% Leonurus japonicus Houtt., 0.20% Salvia miltiorrhiza Bge., 0.25% Ligustrum lucidum Ait., and 0.25% Taraxacum mongolicum Hand.-Mazz.) for 120 d to the spent hens' diet through metabolomics, network pharmacology, and microbiome strategies. Results: The results indicated that CHF supplementation improved the meat quality by reducing drip loss (P < 0.05), b* value (P = 0.058), and shear force (P = 0.099) and increasing cooked meat percentage (P = 0.054) and dry matter (P < 0.05) of breast muscle. The addition of CHF improved the nutritional value of breast muscle by increasing (P < 0.05) the content of C18:2n-6, n-6/n-3 polyunsaturated fatty acids (PUFA), total PUFA, PUFA-to-saturated fatty acids (SFA) ratio, and hypocholesterolemic-to-hypercholesterolemic ratio, and tending to increase serine content (P = 0.069). The targeted metabolomics analysis revealed that the biosynthesis of SFA, linoleic acid metabolism, fatty acid degradation, fatty acid elongation, and fatty acid biosynthesis pathways were enriched by CHF supplementation. Furthermore, the network pharmacology analysis indicated that CHF was closely associated with oxidative stress and lipid metabolism. The CHF supplementation increased the glutathione peroxidase level (P < 0.05) and upregulated gene expression related to the Nrf2 pathway (including HO-1, P < 0.05; Nrf2, P = 0.098; CAT, P = 0.060; GPX1, P = 0.063; and SOD2, P = 0.052) and lipid metabolism (including PPARγ, P < 0.05; SREBP1, P = 0.059; and CPT1A, P = 0.058). Additionally, CHF supplementation increased Firmicutes and decreased Bacteroidetes, Spirochaetes, and Synergistetes abundances (P < 0.05), which may contribute to better meat quality. Conclusions: Our results suggest that CHF supplementation improved the quality and nutritional value of meat, which will provide a theoretical basis for the utilization of CHF as a feed additive in spent hens' diets. [ABSTRACT FROM AUTHOR]

The bivalent fear of evaluation model proposes that fear of negative evaluation (FNE) and fear of positive evaluation (FPE) are distinct but related constructs, and that social anxiety arises when they are elevated. This represents a variable-centered perspective. However, a recent review suggested that individuals may be affected by unique combinations of FNE and FPE because they have different functions, mechanisms, and outcomes. Such patterns can be revealed by taking a person-centered approach. To explore this, the current study employed latent profile and k-means clustering analyses in four independent samples (total n = 2,913) to identify subgroups based on FNE and FPE levels. Between-group levels of social anxiety were further examined. Results demonstrated that participants could be classified into four groups: (1) high bivalent fears of evaluation group, (2) high FNE group, (3) high FPE group, and (4) low fears of evaluation group. Importantly, significant differences in social anxiety levels were found among these groups. Our findings extend the bivalent fear of evaluation model to a person-centered perspective and enable clinicians to tailor interventions that specifically address distinct underlying fears of evaluation. This may facilitate more effective treatment for individuals with social anxiety. [ABSTRACT FROM AUTHOR]

To improve the immunoassay performance of mycotoxin anti-idiotype nanobody and to realize mimicry of bispecific detected antigens, in this study, anti-idiotype nanobody against zearalenone (ZEN) and deoxynivalenol (DON) were used, and the SpyCatcher/SpyTag tags were fusion-expressed with N28 and Z6, respectively. Based on the in vitro self-assembly, the construction study of bivalent and bispecific anti-idiotype nanobody of N28 and Z6 were carried out. The results showed that the prokaryotic soluble expression of four SpyCatcher/SpyTag labeled fusion anti-idiotype nanobody, N28-SpyCatcher, N28-SpyTag, Z6-SpyCatcher, and Z6-SpyTag were achieved. The IC50 of the competition inhibition standard curves established based on Z6-SpyCatcher and Z6-SpyTag were 0.18 and 0.20 ng/mL, respectively. Their sensitivities were significantly higher than that of the IC50 of monomeric Z6 (0.28 ng/mL). The fusion of the SpyCatcher tag with N28 was favorable for the enhancement of the sensitivity of the N28 assay, with the IC50 value decreased from 94.39 to 42.33 ng/mL, whereas the fusion of SpyTag label with N28 inhibited its binding to DON monoclonal antibody. In addition, bivalent and bispecific anti-idiotype nanobody Z6-Z6, N28-28, and N28-Z6, Z6-N28 of Z6 and N28 were successfully prepared based on the in vitro self-assembly property of SpyCatcher/SpyTag under the simple reaction condition of 4 °C for 12 h. The characterization results of the ELISA showed that the bivalent anti-idiotype nanobody retained their binding properties to the corresponding antibodies. The binding properties of the corresponding antibodies, in which N28-Z6 also achieved the binding activity with ZEN and DON antibodies simultaneously, and Z6-N28 only retained the binding activity with ZEN antibody because the binding of SpyTag tag to N28 would hinder its binding to DON antibody. In conclusion, the present study realized a simple and rapid preparation of ZEN/DON bivalent and bispecific antiidiotype nanobody based on SpyCatcher/SpyTag and suggested that the SpyCatcher tag had a facilitating effect on the immunoassay performance enhancement of ZEN/DON anti-idiotype nanobody. [ABSTRACT FROM AUTHOR]

Lipase, a green biocatalyst, finds extensive application in the food sector. Enhancing the thermal stability of lipase presents both challenges and opportunities within the food industry. This research employed multiple rounds of cross-screening using tools like FoldX and I-Mutant 3.0 to strategically design mutations for Rhizomucor miehei lipase (RML), resulting in eight unique single-point mutation designs. E230I, N120M, and N264M have been confirmed experimentally to be potential combination mutation candidates. The resulting triple mutant N120M/E230I/N264M showed a higher thermal stability, with an optimum temperature of 55 °C, 10 °C higher than that of the wild-type RML. The half-life was extended from 46 to 462 min at 50 °C. Furthermore, the catalytic activity of N120M/E230I/N264M on camphor tree seed oil increased by 140% to 600 U/mg, which aids in the production of novel structured lipids. Using molecular docking and molecular dynamics simulations, we analyzed the molecular mechanism of enhanced thermal stability. This study validated the efficacy and dependability of computer-aided design to generate heat-resistant RML mutants and indicated that RML N120M/E230I/N264M lipase can be used as an effective biocatalyst for fat processing in the food industry. [ABSTRACT FROM AUTHOR]

Human decision-making can be irrational, as in the case of the gambler's fallacy (GF). Converging evidence from behavioral and functional neuroimaging studies has suggested that a hyperactive cognitive system and a hypo-active affective system contribute to the false world model that generates the GF. However, the neuroanatomical basis underlying the GF remains unclear. The current study addressed this issue by collecting high-resolution magnetic resonance anatomical images from a large sample of 350 healthy Chinese adults. Univariate voxel-based morphometry (VBM) analysis suggested that the gray matter volume (GMV) in the anterior cingulate cortex (ACC) and medial temporal lobe (MTL) (two regions of the cognitive system) showed negative correlations with the degree of GF, while the GMV in the striatum and orbitofrontal cortex (OFC; two regions of the affective system) showed positive correlations. Further multivariate VBM analysis showed that the GMV in these regions could potentially predict the degree of GF. Moreover, a mediation analysis suggested that the GMV in MTL, ACC, and OFC mediated the relationships between the cognitive abilities or affective decision-making performance and the GF. Results of our study help us to understand the potential neural bases of the cognitive system's constructive role and the affective system's destructive role in decision making.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B1 (AFB1) was developed using an AFB1-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (Tt) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB1 as well as phase transition when the temperature rose above Tt. To establish the optimal conditions for cleaning AFB1, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody–ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods. [ABSTRACT FROM AUTHOR]

There is a keen interest in identifying specific brain regions that are related to individual differences in true and false memories. Previous functional neuroimaging studies showed that activities in the hippocampus, right fusiform gyrus, and parahippocampal gyrus were associated with true and false memories, but no study thus far has examined whether the structures of these brain regions are associated with short-term and long-term true and false memories. To address that question, the current study analyzed data from 205 healthy young adults, who had valid data from both structural brain imaging and a misinformation task. In the misinformation task, subjects saw the crime scenarios, received misinformation, and took memory tests about the crimes an hour later and again after 1.5 years. Results showed that bilateral hippocampal volume was associated with short-term true and false memories, whereas right fusiform gyrus volume and surface area were associated with long-term true and false memories. This study provides the first evidence for the structural neural bases of individual differences in short-term and long-term true and false memories.

Previous correlational imaging studies have implicated the dorsolateral prefrontal cortex (DLPFC) in decision making. Using High-Definition Transcranial Direct Current Stimulation (HD-tDCS), the present study directly investigated the causal role of the DLPFC in performing the Iowa Gambling Task (IGT) and the Inter-Temporal Choice (ITC) task. Three experiments were conducted: Experiment 1 (N = 41) to study the left DLPFC, Experiment 2 (N = 49) to study the right DLPFC, and Experiment 3 (N = 20, a subset of those in Experiment 1) to switch the experimental and control conditions. All participants were healthy male college students. For Experiments 1 and 2, participants were randomly assigned to either the HD-tDCS or the sham stimulation condition. For Experiment 3, participants were assigned to the condition they were not in during Experiment 1. Results showed that HD-tDCS over the left DLPFC increased IGT score, decreased the recency parameter in IGT, and lowered delay discounting rate (k) in the ITC task. We discussed the potential roles of impulse control and time perception in mediating the effect of tDCS stimulation of left DLPFC on decision making. Our results have clinical implications for the treatment of disorders involving poor decision-making, such as addictions.

Catechol-O-methyltransferase (COMT) and brain-derived neurotrophic factor (BDNF) genes have been found to interactively influence working memory (WM) as well as brain activation during WM tasks. However, whether the two genes have interactive effects on resting-state activities of the brain and whether these spontaneous activations correlate with WM are still unknown. This study included behavioral data from WM tasks and genetic data (COMT rs4680 and BDNF Val66Met) from 417 healthy Chinese adults and resting-state fMRI data from 298 of them. Significant interactive effects of BDNF and COMT were found for WM performance as well as for resting-state regional homogeneity (ReHo) in WM-related brain areas, including the left medial frontal gyrus (lMeFG), left superior frontal gyrus (lSFG), right superior and medial frontal gyrus (rSMFG), right medial orbitofrontal gyrus (rMOFG), right middle frontal gyrus (rMFG), precuneus, bilateral superior temporal gyrus, left superior occipital gyrus, right middle occipital gyrus, and right inferior parietal lobule. Simple effects analyses showed that compared to other genotypes, subjects with COMT-VV/BDNF-VV had higher WM and lower ReHo in all five frontal brain areas. The results supported the hypothesis that COMT and BDNF polymorphisms influence WM performance and spontaneous brain activity (i.e., ReHo).

Using functional magnetic resonance imaging, the present study examined whether the comparison distance effect (CDE) and the priming distance effect (PDE) in number processing had the same underlying neural mechanisms. 24 healthy participants completed a number comparison task and a number priming task in the scanner. Imaging data were examined for brain regions selected based on a meta-analysis of previous studies of number processing. Results revealed robust CDE and PDE at both behavioral and neural levels. The CDE had a significant hemodynamic signature in the right parietal cortex but not in the left parietal cortex, although a direct test of this hemispheric laterality did not reach statistical significance. In contrast, the PDE showed significant left-hemisphere laterality with a significant hemodynamic signature in the left parietal cortex but not in the right parietal cortex. These results suggested that the CDE and PDE had different underlying neural mechanisms.

Cooperativeness is an essential behavioral trait evolved to facilitate group living. Social and cognitive mechanisms involved in cooperation (e.g., motivation, reward encoding, action evaluation, and executive functions) are sub-served by the striatal-projected circuits, whose physical existence has been confirmed by animal studies, human postmortem studies, and in vivo human brain studies. The current study investigated the associations between Cooperativeness and fiber connectivities from the striatum to nine subcortical and cortical regions, including the amygdala, hippocampus, medial orbitofrontal cortex, lateral orbitofrontal cortex, ventrolateral prefrontal cortex, dorsolateral prefrontal cortex, posterior cingulate cortex/retrosplenial cortex, dorsal cingulate cortex, and rostral cingulate cortex. Results showed that Cooperativeness was negatively correlated with fiber connectivity for the cognitive control system (from the dorsal caudate to the rostral cingulate cortex and ventrolateral prefrontal cortex), but not with fiber connectivity for the social cognitive system (e.g., connectivity with the medial prefrontal cortex and amygdala). These results partially supported Declerck et al.'s (2013) cognitive neural model of the role of cognitive control and social cognition in cooperation.