Insights into the Binding Profile of Anti-chlorpyrifos Recombinant Antibodies: From Computational Simulation to Immunoassay Validation.

A novel virtual screening strategy was proposed for the profiling and discovery of active variable regions (VRs) that encode hapten-specific recombinant antibodies (rAbs). Chlorpyrifos, a hazardous organophosphorus pesticide, was selected as the target. First, a VR model-14G4 from anti-chlorpyrifos hybridoma was built via homology modeling. Its binding pattern toward seven organophosphorus analogues was assessed through virtual screening by performing molecular docking. Based on energy scoring, visual examination, and molecular interaction analysis, chlorpyrifos-methyl was also inferred as the high-affinity target for model-14G4 and was then confirmed via a non-competitive surface plasmon resonance (SPR) assay. Subsequently, we attempted to discover hapten-specific VRs by creating a collection of VR models for anonymous testing. Chlorpyrifos and model-14G4 were employed as the known hit and active VRs, respectively. After molecular docking, a novel anti-chlorpyrifos VR (model-1) was identified due to its satisfactory energy scoring and a similar binding pattern to the reference model-14G4. Expressed by HEK293(F) mammalian cells, the newly prepared full-length rAb-model-1 and rAb-14G4 exhibited high sensitivities for detecting chlorpyrifos by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), with IC50 of 3.01 ng/mL and 42.82 ng/mL, respectively. They recognized chlorpyrifos-methyl with a cross-reactivity (CR) of 2.5-17.3%. Moreover, the binding properties of rAb-model-1 for recognizing chlorpyrifos and chlorpyrifos-methyl were confirmed via a non-competitive microscale thermophoresis (MST) method. Thus, the experimental results showed good agreement with computational outputs on antibody profiling. Furthermore, the recognition diversity of rAb-model-1 for chlorpyrifos and chlorpyrifos-methyl was studied via molecular dynamics simulation. Overall, the proposed study provides a versatile and economical strategy for antibody characterization and promotes the in vitro production of rAbs for pesticide monitoring.

[Optimization of extraction process for classic prescription Yihuang Decoction based on Box-Behnken design-response surface methodology, standard relation, and analytic hierarchy process combined with entropy weight method].

Based on the concept of quality by design(QbD), the Box-Behnken design-response surface methodology combined with standard relation(SR) and analytic hierarchy process(AHP)-entropy weight method(EWM) was applied to optimize the extraction process of the classic prescription Yihuang Decoction. The content of geniposidic acid, phellodendrine hydrochloride, and berberine hydrochloride in Yihuang Decoction, the extract yield, and fingerprint similarity were used as the critical quality attributes(CQAs) of the extraction process. The extraction time, water addition, and extraction times were used as the critical process parameters(CPPs). After determining the levels of each factor and level through single-factor experiments, response surface experiments were designed according to the Box-Behnken principle, and the experimental results were analyzed. The SR between each sample and the reference sample under various evaluation indicators of different extraction parameters was calculated. The weights of the five evaluation indicators were determined using AHP-EWM, followed by comprehensive evaluation. A function model between CPPs and CQAs characterized by comprehensive scores was established to predict the optimal extraction process parameters. In the final comprehensive weight coefficients, the yield rate accounted for 43.1%, and the content of berberine hydrochloride, phellodendrine hydrochloride, and geniposidic acid accounted for 35.1%, 6.3%, and 15.5%, respectively. After comprehensive score analysis with SR, the established second-order polynomial model was statistically significant(P<0.01, and the lack of fit was not significant). The predicted optimal extraction conditions for Yihuang Decoction were determined as follows: 8-fold volume of water, extraction time of 1.5 h, and extraction once. The mean comprehensive score of the validation experiment was 85.77, with an RSD of 0.99%, and it met the quality control stan-dards for the reference sample of Yihuang Decoction. The results indicate that the optimized extraction process for Yihuang Decoction is stable and reliable, and the water extract is close in quality attributes to the reference sample. This can serve as a foundation for the research and development of granules in the future. Box-Behnken design-response surface methodology combined with SR and AHP-EWM can provide references for the modern extraction process research of other classic prescriptions.

Emergence of Neisseria gonorrhoeae Clone with Reduced Susceptibility to Sitafloxacin in China: An In Vitro and Genomic Study.

Drug-resistant Neisseria gonorrhoeae poses an urgent threat to public health. Recently, sitafloxacin, a new-generation fluoroquinolone, has shown high in vitro activity against drug-resistant N. gonorrhoeae. However, data on its effectiveness in clinical isolates remains limited. In this study, we collected 507 N. gonorrhoeae isolates from 21 hospitals in Shanghai, China, during 2020 and 2021. Antimicrobial susceptibility testing revealed that sitafloxacin minimum inhibitory concentrations (MICs) exhibited a bimodal distribution, ranging from <0.004 to 2 mg/L. The MIC50 and MIC90 for sitafloxacin were 0.125 mg/L and 0.5 mg/L, respectively, which are 32 and 16 times lower than those for ciprofloxacin (4 mg/L and 8 mg/L, respectively). Sitafloxacin demonstrated high in vitro activity against isolates resistant to either ceftriaxone, azithromycin, or both. Notably, among the isolates with reduced sitafloxacin susceptibility (MIC ≥ MIC90), 83.7% (36/43) were identified as sequence type (ST) 8123. Further phylogenetic analysis showed that ST8123 has evolved into two subclades, designated as subclade-I and subclade-II. A majority of the isolates (80%, 36/45) within subclade-I exhibited reduced susceptibility to sitafloxacin. In contrast, all isolates from subclade-II were found to be susceptible to sitafloxacin. Subsequent genomic investigations revealed that the GyrA-S91F, D95Y, and ParC-S87N mutations, which were exclusively found in ST8123 subclade-I, might be linked to reduced sitafloxacin susceptibility. Our study reveals that sitafloxacin is a promising antibiotic for combating drug-resistant N. gonorrhoeae. However, caution is advised in the clinical application of sitafloxacin for treating N. gonorrhoeae infections due to the emergence of a clone exhibiting reduced susceptibility.

Baicalein-modified chitosan nanofiber membranes with antioxidant and antibacterial activities for chronic wound healing.

Diabetic foot ulcers, burns and many other trauma can lead to the formation of skin wounds, which often remain open for a long period of time, seriously affecting the quality of patient's life. Oxidative stress and infection are the main factors affecting the healing of chronic wounds, so it is important to develop dressings with dual antioxidant and antimicrobial properties for wound management. In this study, functionalized chitosan was synthesized by modifying chitosan with antioxidant baicalein to enhance the antimicrobial and antioxidant activities of chitosan. Then the obtained baicalein-modified chitosan was prepared into nanofibrous membranes by electrospinning. The membrane structures were characterized, and the antioxidant and antibacterial activities were evaluated by in vivo and in vitro experiments. The results showed that the prepared wound dressings had excellent antioxidant and antibacterial activities and significantly accelerated the wound process. This study provided a reference for the development of novel dressing materials to promote wound healing.

Study of the permeation-promoting effect and mechanism of solid microneedles on different properties of drugs.

In transdermal drug delivery systems, the physicochemical properties of the drug affect its percutaneous permeability. However, whether the physicochemical properties of drugs change their transdermal permeability in the presence of pores in the presence of solid microneedles (MNs) has been less studied in this area. In this project, cinnamaldehyde, curcumin, ferulic acid and geniposide were selected as model drugs for the study of their transdermal permeability under the action of MNs, and a combination of classical experiments and visualization means such as scanning electron microscopy and laser confocal was used to investigate the permeation-promoting mechanism of MNs. The results showed that the MNs had significant permeation-promoting effects on different properties of drugs, with the permeation-promoting effects on cinnamaldehyde, curcumin, ferulic acid and geniposide being 6.36, 17.43, 29.54 and 8.91 times, respectively, and the permeation-promoting effects were more pronounced for lipid-soluble and amphiphilic drugs. Using scanning electron microscopy, transmission electron microscopy and other means to confirm that MNs can promote the penetration by acting on the skin to produce pores, and their effect on skin structure is greater than that of drugs. In addition, the existence of pores increases the amount of drug transdermal, which may enhance the diffusion of drug on the skin, and has no effect on lipid exchange and transdermal route. Through the research, it has been found that MNs is equivalent to direct peeling of the stratum corneum (SC), but it is simpler and safer for the patient.

Development of a highly sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the detection of avilamycin in feed.

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed using a highly sensitive and specific monoclonal antibody against avilamycin. The immunising antigen synthesised through the carbonyldiimidazole (CDI) method was used to prepare antibodies. The established ic-ELISA, after optimisation of conditions, possessed a half maximum inhibitory concentration (IC50) value of 7.44 ng mL-1 and the detection limit of 0.21 ng mL-1 in the standard curve. The spiked experiments indicated that the limits of detection were 1.86 µg kg-1 and 2.31 µg kg-1 in swine feed and chicken feed, respectively. In addition, the average recoveries ranged from 74.7 to 105.4% with the coefficient of variation less than 11%. The good correlation (R2 = 0.9818) between the result of ic-ELISA and HPLC demonstrated the reliability of the developed ic-ELISA, which showed that the ic-ELISA developed here achieves the aim of strengthening the monitoring of avilamycin residues and the inspection of import and export related products.

Gut Microbiota Alternation in Disease Progression of Neurosyphilis.

Background: The gut microbiota plays an important role in the development of neurological disorders such as Parkinson's disease and Alzheimer's disease. However, studies on the gut microbiota of patients with neurosyphilis (NS) were rarely reported. Methods: In this study, we collected fecal samples from 62 syphilis patients, including 39 with NS and 23 with non-NS. Among the NS patients, 18 were general paresis (GP). The white blood cell counts, protein concentrations, and Venereal Disease Research Laboratory test positive rates of cerebrospinal fluid from patients in NS or GP group were significantly higher than those from patients in non-NS group. 16S ribosomal RNA sequencing results revealed that the alpha and beta diversities of the gut microbiota were similar between NS and non-NS patients or GP and non-NS patients. Results: Linear discriminant analysis with effect size (LEfSe) analysis showed that some taxa, such as Coprobacter, were increased in both NS group and GP group, compared with non-NS group. Besides, the clade of Akkermansia was also overrepresented in GP Patients. Meanwhile, some taxa such as Clostridia_UCG-014 and SC-I-84 were underrepresented in NS patients. The abundances of class Bacilli and genus Alloprevotella were decreased in GP patients. Among them, the abundances of some taxa such as Coprobacter and Akkermansia have been reported to be associated with other neuropsychiatric disorders. Conclusion: Our findings suggest that the alternation of the gut microbiota in NS patients may contribute to the course of NS, which will deepen our understanding of NS.

A Novel Full-length IgG Recombinant Antibody Highly Specific to Clothianidin and Its Application in Immunochromatographic Assay.

The toxicity of clothianidin to non-target organisms has gradually attracted world-wide attention. It is essential to develop reliable methods for the on-site detection of clothianidin residue. In this study, analogue-based heterologous ic-ELISAs were designed to rapidly screen desirable hybridomas, which could be used for the construction of recombinant antibodies (RAbs) against clothianidin. Based on the antibody variable region genes, two full-length IgG RAbs (1F7-RAb and 5C3-RAb) were produced by the mammalian cell expression system. The performance of the two RAbs was characterized and compared by heterologous ic-ELISAs and non-competitive surface plasmon resonance (SPR) assays. Using heterologous ic-ELISAs, the 1F7-RAb exhibited highly specific and sensitive recognition to clothianidin with an IC50 of 4.62 µg/L, whereas the 5C3-RAb could bind to both clothianidin and dinotefuran. The results of the non-competitive SPR assay further verified that the 1F7-RAb had a higher specificity and affinity to clothianidin than the 5C3-RAb. Finally, a gold immunochromatographic assay based on the novel antibody, 1F7-RAb, was developed for rapid detection of clothianidin with high sensitivity (visual detection limit of 2.5 µg/L), specificity, and good reproducibility, which can be used as an effective supervision tool for clothianidin residue in agricultural and environmental samples.

TFEB- and TFE3-dependent autophagy activation supports cancer proliferation in the absence of centrosomes.

Centrosome amplification is a phenomenon frequently observed in human cancers, so centrosome depletion has been proposed as a therapeutic strategy. However, despite being afflicted with a lack of centrosomes, many cancer cells can still proliferate, implying there are impediments to adopting centrosome depletion as a treatment strategy. Here, we show that TFEB- and TFE3-dependent autophagy activation contributes to acentrosomal cancer proliferation. Our biochemical analyses uncover that both TFEB and TFE3 are novel PLK4 (polo like kinase 4) substrates. Centrosome depletion inactivates PLK4, resulting in TFEB and TFE3 dephosphorylation and subsequent promotion of TFEB and TFE3 nuclear translocation and transcriptional activation of autophagy- and lysosome-related genes. A combination of centrosome depletion and inhibition of the TFEB-TFE3 autophagy-lysosome pathway induced strongly anti-proliferative effects in cancer cells. Thus, our findings point to a new strategy for combating cancer.Abbreviations: AdCre: adenoviral Cre recombinase; AdLuc: adenoviral luciferase; ATG5: autophagy related 5; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DKO: double knockout; GFP: green fluorescent protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LTR: LysoTracker Red; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MITF: melanocyte inducing transcription factor; PLK4: polo like kinase 4; RFP: red fluorescent protein; SASS6: SAS-6 centriolar assembly protein; STIL: STIL centriolar assembly protein; TFEB: transcription factor EB; TFEBΔNLS: TFEB lacking a nuclear localization signal; TFE3: transcription factor binding to IGHM enhancer 3; TP53/p53: tumor protein p53.

Reduced Virulence and Enhanced Host Adaption during Antibiotics Therapy: a Story of a Within-Host Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 Evolution in a Patient with a Serious Scrotal Abscess.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and threatened human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, but how this clone evolves in vivo then adapts to the host and facilitates dissemination remains largely unknown. Here, the genomic dynamics of 4 ST11-CRKP isolates, which were sequentially collected from the urine of a patient with initial serious scrotal abscess and finally recovered without effective medication, were analyzed. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4 to 24 mutations, among which 94% to 100% of them were synonymous or intergenic mutations. During 47 days of antibiotics therapy, CRKP underwent adaptive evolution, including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by a deletion within the ramR ribosomal binding site, which has been described by us previously. On the other hand, mutations associated with two genes, acyltransferase (act) and ompK26, resulted in the attenuation phenotype of ST11-CRKP. act deficiency reduced the capsular polysaccharide (CPS) production, enhanced biofilm formation, weakened capsular protection, and decreased induction of proinflammatory cytokines. Further RNA-Seq analysis revealed that act influenced the expression of ldhA, bglX, mtnK, and metE which likely participate in capsular synthesis and biofilm formation. ompK26 affected the virulence by its overexpression caused by the deletion of the upstream repressor binding site. This study presents a within-host adaption of ST11-CRKP and suggests an important role of CPS in the adaptive evolution of virulence and persistence of CRKP. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated worldwide and can cause life-threatening infections, including pneumonia, bloodstream infections, urinary tract infections, intraabdominal infection, liver abscess, and meningitis. CRKP infection is the leading cause of high mortality in hospitals. The sequence type (ST) 11 CRKP is a dominant clone and accounts for 60% of CRKP infections in China. Recently, the ST11-CRKP with high transmissibility is increasingly identified. Understanding how this clone has evolved is crucial for developing strategies to control its further dissemination. The significance of our research is the identification of the in vivo genomic dynamics of ST11-CRKP and the genetic basis for ST11-CRKP that facilitate persistence and dissemination. Furthermore, our study also highlights the importance of monitoring the within-host evolution of pathogens during the treatment and developing interventions to minimize the potential impact of host adaptation on human health.

Novel immunochromatographic strip assay based on up-conversion nanoparticles for sensitive detection of imidacloprid in agricultural and environmental samples.

Neonicotinoid insecticides are widely used in agriculture for pest control, but the pesticide residues in environmental and agricultural products were a big threat to the health of non-target organisms. In this study, a new immunochromatographic strip test was established for the rapid detection of imidacloprid residue, a neonicotinoid insecticide, based on up-conversion nanoparticles (UCNPs) coupled with the monoclonal antibody against imidacloprid. Under optimal conditions, the half inhibitory concentration (IC50), detection limit, and the linear range of this strip were 8.37 ng/mL, 0.45 ng/mL, and 0.97-250 ng/mL. The strip test could be completed in 30 min. The average recoveries of imidacloprid spiked in water, Chinese cabbages, cucumber, honey, and tea samples were 70.1~101.8%, with coefficient of variations less than 18.9%. The strip was used to test real samples and verified by UPLC-MS/MS method with the good agreement (R2 was 0.9825), indicating this novel strip immunoassay is accurate and reliable.

Molecular analyses of the functional microbial community in composting by PCR-DGGE targeting the genes of the ß-glucosidase.

The study investigated the ß-glucosidase-producing microbial communities and the enzymatic dynamics of CMCase and ß-glucosidase during the process of cattle manure-rice straw composting. In order to analyze the succession of functional community by PCR-denaturing gradient gel electrophoresis (DGGEs), three sets of PCR primers were designed to amplify the family 1 and 3 ß-glucosidase genes from both bacteria and fungi. The results showed in general that the stable functional community composition as well as for the high level enzymatic activities of both cellulase and ß-glucosidase occurred during the last phase (days 14-31) of composting. In the process of composting, that functional groups were determined by the stable bands (GH1-F, GH1-H, GH1-G, GH3E-D and GH3E-E) may significantly contribute to the increase of ß-glucosidase activities in the later phase. Especially, the bands from the family 1 ß-glucosidase genes were appeared before that from the family 3 ß-glucosidase genes from fungi, then the former was substituted for the latter gradually in the cooling phase. We found significant correlations between the ß-glucosidase activity and the communities of the functional bacteria and fungi. The results indicated that different ß-glucosidase-producing microbe release different amounts or activities of ß-glucosidase, and that the composition of microbial communities may play a major role in determining overall ß-glucosidase activity during the composting process.

Entry of Bombyx mori cypovirus 1 into midgut cells in vivo.

In vivo entry of Bombyx mori cypovirus 1 into the silkworm midgut was studied by electron microscopy of ultrathin sections of midguts from silkworm larvae that had been administered virus-contaminated leaves. In 3 h, virions were observed outside and inside midgut cells, including columnar cells, goblet cells and muscle cells. Virions were seen adhering to the plasma membrane of microvilli, embedding in the membrane and settling themselves intact inside the microvilli of the columnar cells. These results suggested that intact virions entered columnar cells by means of direct penetration through the cell membrane. In 12, 24 and 48 h, virogenic stromata and progeny virions were observed in columnar cells, but not in other midgut cells.

Genetic, biochemical, and structural characterization of a new densovirus isolated from a chronically infected Aedes albopictus C6/36 cell line.

We report the isolation, sequencing, biochemical, and structural characterization of a previously undescribed virus in a chronically infected Aedes albopictus C6/36 cell line. This virus is identified as a new densovirus under the Densovirinae subfamily of the Parvoviridae based on its biological and morphologic properties as well as sequence homologies, and is tentatively designated A. albopictus C6/36 cell densovirus (C6/36 DNV). Analysis of the 4094 nt of the C6/36 DNV genome revealed that the plus strand had three large open reading frames (ORFs): a left ORF, a right ORF, and a mid-ORF (within the left ORF), whose potential coding capacities are 91.0, 40.8, and 41.2 kDa, respectively. The left ORF likely encodes the nonstructural protein NS-1, which contains NTP-binding and helicase domains. The right ORF likely encodes structural proteins, VP1 and VP2. Our analyses revealed that C6/36 DNV has a similar genomic organization and shares very high homology in nucleotide sequence and amino acid sequences with Aedes aegypti densovirus (AaeDNV) and A. albopictus densovirus (AalDNV), members of the genus Brevidensovirus of the Densovirinae. Similar to other densoviruses, C6/36 DNV has a different genomic organization and no recognizable sequence homology with viruses in the Parvovirinae. The three-dimensional (3D) reconstruction of the C6/36 DNV at 15.6-A resolution by electron cryomicroscopy (cryoEM) revealed distinctive outer surface features not previously seen in other parvoviruses, indicating structural divergence of densoviruses, in addition to its genomic differences, while the inner surface of the C6/36 DNV capsid exhibits features that are conserved among parvoviruses.