Ratiometric fluorescence sensing of formaldehyde in food samples based on bifunctional MOF.

A new fluorescence strategy was described for ratiometric sensing of formaldehyde (FA) with bifunctional MOF, which acted as a fluorescence reporter as well as biomimetic peroxidase. With the assistance of H2O2, NH2-MIL-101 (Fe) catalyzes the oxidation of non-luminescent substrate o-phenylenediamine (OPD) to produce fluorescent product (oxOPD) with the maximum emission at 570 nm. Besides, intrinsic fluorescence of MOF (λem = 445 nm) was quenched by oxOPD through inner filter effect (IFE). However, FA and OPD reacted to generate Schiff bases, which competitively consumed OPD inhibiting the generation of oxOPD. Under the excitation wavelength of 375 nm, a ratiometric strategy was designed to detect FA with the fluorescence intensity ratio at 445 nm and 570 nm (F445/F570) as readout signal. This strategy exhibited a wide linear range (0.1-50 µM) and low detection limit of 0.03 µM. This method was confirmed for FA detection in food samples. In addition to establishing a new method to detect FA, this work will open new applications of MOF in food safety.

Structural and functional definition of a vulnerable site on the hemagglutinin of highly pathogenic avian influenza A virus H5N1.

Most neutralizing antibodies against highly pathogenic avian influenza A virus H5N1 recognize the receptor-binding site (RBS) on the globular head domain and the stem of H5N1 hemagglutinin (HA). Through comprehensive analysis of multiple human protective antibodies, we previously identified four vulnerable sites (VS1-VS4) on the globular head domain. Among them, the VS1, occupying the opposite side of the RBS on the same HA, was defined by the epitope of antibody 65C6. In this study, we report the crystal structures of two additional human H5N1 antibodies isolated from H5N1-infected individuals, 3C11 and AVFluIgG01, bound to the head at 2.33- and 2.30-Å resolution, respectively. These two new antibody epitopes have large overlap with and extend beyond the original VS1. Site-directed mutagenesis experiments identified eight pivotal residues (Ser-126b, Lys-165, Arg-166, Ser-167, Tyr-168, Asn-169, Thr-171, and Asn-172) critical for 65C6-, 3C11-, and AVFluIgG01-binding and neutralization activities. These residues formed a unique "Y"-shaped surface on H5N1 globular head and are highly conserved among H5N1 viruses. Our results further support the existence of a vulnerable site distinct from the RBS and the stem region of H5N1 HA, and future design of immunogens should take this particular site into consideration.

Complementary recognition of the receptor-binding site of highly pathogenic H5N1 influenza viruses by two human neutralizing antibodies.

The highly pathogenic avian influenza virus H5N1 is a major threat to global public health and therefore a high-priority target of current vaccine development. The receptor-binding site (RBS) on the globular head of hemagglutinin (HA) in the viral envelope is one of the major target sites for antibody recognition against H5N1 and other influenza viruses. Here, we report the identification and characterization of a pair of human RBS-specific antibodies, designated FLD21.140 and AVFluIgG03, that are mutually complementary in their neutralizing activities against a diverse panel of H5N1 viruses. Crystallographic analysis and site-directed mutagenesis revealed that the two antibodies share a similar RBS-binding mode, and their individual specificities are governed by residues at positions 133a, 144, and 145. Specifically, FLD21.140 preferred Leu-133a/Lys-144/Ser-145, whereas AVFluIgG03 favored Ser-133a/Thr-144/Pro-145 residue triplets, both of which perfectly matched the most prevalent residues in viruses from epidemic-originating regions. Of note, according to an analysis of 3758 H5 HA sequences available in the Influenza Virus Database at the National Center for Biotechnology Information, the residues Leu-133a/Ser-133a and Ser-145/Pro-145 constituted more than 87.6 and 99.3% of all residues at these two positions, respectively. Taken together, our results provide a structural understanding for the neutralizing complementarity of these two antibodies and improve our understanding of the RBS-specific antibody response against H5N1 infection in humans.

Colorimetric determination of the activities of tyrosinase and catalase via substrate-triggered decomposition of MnO2 nanosheets.

The authors describe novel colorimetric assays for tyrosinase (TYR) and catalase (CAT) based on the substrate-triggered decomposition of MnO2 nanosheets (NSs). The MnO2 NSs can act as oxidase mimics that catalyze the oxidation of the substrate tetramethylbenzidine (TMB) to form a blue dye with an absorption maximum at 652 nm. The oxidase-mimicking activity of the MnO2 NSs is inhibited by dopamine (DA)/hydrogen peroxide (H2O2) due to their decomposition of the MnO2 NSs. TYR catalyzes the oxidation of DA while CAT can decompose H2O2 into water and oxygen. Therefore, the oxidase-mimicking activity of MnO2 NSs is restored in the presence of both enzymes and their substrates. Based on the competitive consumption of substrates between enzymes and MnO2 NSs, a colorimetric method for determination of enzyme activity and its substrate is developed. The detection limits for TYR and CAT are 6 mU·mL-1 and 33 mU·mL-1, respectively. Graphical abstractA colorimetric method for monitoring enzyme activity and its substrate is described. It is based on the substrate-inhibited oxidase-mimicking activity of MnO2 nanosheets.

Molecular beacon-templated silver nanoclusters as a fluorescent probe for determination of bleomycin via DNA scission.

The authors describe a molecular beacon-based fluorescent probe for the determination of the cancer drug bleomycin (BLM). The probe was tagged with DNA-templated silver nanoclusters (DNA-AgNCs) and guanine-rich sequences (GRSs) at two terminals serving as signal reporter with a loop. In the absence of the BLM-iron(II) complex [BLM-Fe(II)], the probe has a hairpin shape and displays strong fluorescence because the AgNCs are close to the GRSs. In the presence of the BLM-Fe(II) complex, it will selectively cleave the probe at the 5'-GC-3' scission site of the loop. This displaces the AgNCs away from the GRSs and causes a decrease in fluorescence, best measured at excitation/emission wavelengths of 565/623 nm. This effect enables BLM to be detected with a detection limit as low as 33 pM, which was 1-3 orders of magnitude more sensitive than most of the previous reports. The probe was applied for the determination of BLM in spiked human serum samples, and excellent performance was achieved. In our perception, the method described here represents a promising tool for highly sensitive and specific analysis of BLM during cancer treatment. Graphical abstract Schematic of a highly sensitive fluorometric assay forbleomycin. It is based on molecular beacon-templated silver nanoclusters and DNA scission.

Ratiometric fluorescence assay for sulfide ions with fluorescent MOF-based nanozyme.

A novel ratiometric fluorescence strategy for sulfide ions (S2-) analysis has been developed using metal-organic framework (MOF)-based nanozyme. NH2-Cu-MOF displays blue fluorescence (λem = 435 nm) originating from 2-amino-1,4-benzenedicarboxylic acid ligand. Besides, it possesses oxidase-like activity due to Cu2+ node, which can trigger chromogenic reaction. o-Phenylenediamine (OPD), as a common enzyme substrate, can be oxidized by NH2-Cu-MOF to form luminescent products (oxOPD) (λem = 570 nm). Inner filter effect occurs between oxOPD and MOF. Upon exposure to S2-, oxidase-like activity of MOF is depressed significantly because of the generation of CuS. On one hand, the amount of free Cu2+ decreases, affecting the yielding of oxOPD. On the other hand, CuNPs with larger size are obtained during the oxidation-reduction reaction between Cu2+ and OPD, which show weaker autocatalytic ability for OPD oxidation. These result in the decrease and increase of intensities at 570 and 435 nm, respectively. This method exhibits sensitive and selective responses towards S2- with LOD of 0.1 µM. Furthermore, such ratiometric strategy has been applied to detect S2- in food samples.

Cascade amplification strategy combined with analyte-triggered fluorescence switching of dual-quenching system for highly sensitive detection of isoniazide.

A sensitive fluorescence sensing platform consisting of manganese dioxide nanosheets (MnO2) and gold nanoparticles (AuNPs) as dual nanoquenchers has been constructed to detect isoniazid combined with analyte-triggered cascade reactions. The fluorescence of 2,3-diaminophenazine (DAP) is quenched simultaneously by MnO2 and AuNPs via inner filter effect. MnO2 is decomposed by isoniazid to generate Mn2+, which makes AuNPs aggregated. The quenching abilities of both the decomposed MnO2 and aggregated AuNPs are inhibited, causing remarkable fluorescence recovery. The usage of dual nanoquenchers enhances the quenching efficiency and reduces the fluorescence background. Moreover, the isoniazid-triggered cascade reaction further amplifies the readout signal. Thus, this strategy exhibits higher sensitivity towards the detection of isoniazid. Compared with MnO2-based fluorescence assay, this strategy possesses lower limit of detection. This strategy has been successfully used to detect isoniazid in pharmaceutical preparations, which is of great significance for drug analysis.

Enhanced detection of ascorbic acid with cascaded fluorescence recovery of a dual-nanoquencher system.

An innovative strategy with target-triggered cascade fluorescence recovery of a dual-nanoquencher system was developed to detect ascorbic acid (AA). Herein, manganese dioxide (MnO2) nanosheets and gold nanoparticles (AuNPs) were used as nanoquenchers simultaneously. Owing to their synergistic effects, the fluorescence of 2,3-diaminophenazine (DAP) was decreased efficiently, thus minimizing the background fluorescence. The introduction of AA triggered the decomposition of MnO2 into Mn2+, which induced the aggregation of AuNPs. Both the decomposed MnO2 and aggregated AuNPs possess weak quenching abilities towards DAP. Such a cascade amplification strategy enhanced the detection sensitivity for AA with a LOD as low as 6.7 nM, which was two orders of magnitude lower than that of MnO2-based fluorescence assay. Furthermore, this amplification strategy was successfully applied to detect AA in food samples.

Effect of Clostridium butyricum Supplementation on in vitro Rumen Fermentation and Microbiota With High Grain Substrate Varying With Media pH Levels.

Clostridium butyricum (C. butyricum) can survive at low pH, and it has been widely used as an alternative to antibiotics for the improvement of feed efficiency and animal health in monogastrics. A recent study suggested that the improved ruminal fermentation with supplementing C. butyricum is may be associated with increasing the abundance of rumen microbiota in Holstein heifers, as ruminal pH plays a key role in rumen microbiota and the probiotics are often active in a dose-dependent manner. The objective of this study was to determine the effects of increasing the doses of C. butyricum on gas production (GP) kinetics, dry matter disappearance (DMD), fermentation characteristics, and rumen microbiota using a high grain substrate in batch culture varying with media pH levels. The doses of C. butyricum were supplemented at 0 (control), 0.5 × 106, 1 × 106, and 2 × 106 CFU/bottle, respectively, at either media pH 6.0 or pH 6.6. The fermentation microbiota at 0 and 1 × 106 CFU/bottle were determined using the 16S rRNA high throughput sequencing technology. Overall, the GP, DMD, total volatile fatty acid (VFA) concentration, and the ratio of acetate:propionate were higher (P <0.01) at media pH 6.6 than at pH 6.0. However, there was interaction between pH × dose of C. butyricum for rate constant of GP (P = 0.01), average GP rate (P = 0.07), and volume of GP (P = 0.06); with the increase in C. butyricum supplementation, the GP kinetics were not changed at media pH 6.0, but the volume (P = 0.02), rate of GP (P = 0.01), and average GP rate (P = 0.01) were quadratically changed at media pH 6.6. The DMD was not affected by increasing the supplementation of C. butyricum. The molar proportions of propionate (P <0.09), butyrate (P <0.06), and NH3-N concentration (P = 0.02) were quadratically changed with increasing supplementation of C. butyricum regardless of media pH levels. The interactions between media pH level and dose of C. butyricum supplementation were noticed for alpha diversity indexes of Shannon (P = 0.02) and Evenness (P = 0.04). The alpha diversity indexes increased (P <0.05) except for Chao1 with supplementation of C. butyricum. The unweighted uniFrac analysis showed that the group of control at media pH 6.0 and control at media pH 6.6, and supplementation of C. butyricum and control at media pH 6.0 clustered separately from each other. At the phylum level, relative abundance (RA) of Bacteroidota was lower (P <0.01) and Firmicutes was higher (P <0.01) at media pH 6.6 than pH 6.0. Moreover, RA of Proteobacteria decreased (P <0.05) with supplemented C. butyricum at either media pH 6.6 or pH 6.0. At media pH 6.6, RA of Rikenellaceae_RC9_gut_group and Prevotella were decreased, and CAG-352 was increased (at genus level) compared to pH 6.0. Supplementation of C. butyricum decreased RA of Rikenellaceae_RC9_gut_group and increased CAG-352 at media pH 6.0. It could hence be concluded that manipulating media pH level and supplementation of C. butyricum effectively modulated in vitro rumen fermentation characteristics and microbiota but in a dose depending manner of C. butyricum addition.

A potent and protective human neutralizing antibody targeting a novel vulnerable site of Epstein-Barr virus.

Epstein-Barr virus (EBV) is associated with a range of epithelial and B cell malignancies as well as autoimmune disorders, for which there are still no specific treatments or effective vaccines. Here, we isolate EBV gH/gL-specific antibodies from an EBV-infected individual. One antibody, 1D8, efficiently neutralizes EBV infection of two major target cell types, B cells and epithelial cells. In humanized mice, 1D8 provides protection against a high-dose EBV challenge by substantially reducing viral loads and associated tumor burden. Crystal structure analysis reveals that 1D8 binds to a key vulnerable interface between the D-I/D-II domains of the viral gH/gL protein, especially the D-II of the gH, thereby interfering with the gH/gL-mediated membrane fusion and binding to target cells. Overall, we identify a potent and protective neutralizing antibody capable of reducing the EBV load. The novel vulnerable site represents an attractive target that is potentially important for antibody and vaccine intervention against EBV infection.

Potent neutralizing monoclonal antibodies against Ebola virus infection.

Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection.

Transport, fate, and stimulating impact of silver nanoparticles on the removal of Cd(II) by Phanerochaete chrysosporium in aqueous solutions.

Despite the knowledge about increasing discharge of silver nanoparticles (AgNPs) into wastewater and its potential toxicity to microorganisms, the interaction of AgNPs with heavy metals in the biological removal process remains poorly understood. This study focused on the effect of AgNPs (hydrodynamic diameter about 24.3±0.37 nm) on the removal of cadmium (Cd(II)) by using a model white rot fungus species, Phanerochaete chrysosporium. Results showed that the biological removal capacity of Cd(II) increased with the concentration of AgNPs increasing from 0.1 mg/L to 1 mg/L. The maximum removal capacity (4.67 mg/g) was located at 1 mg/L AgNPs, and then decreased with further increasing AgNPs concentration, suggesting that an appropriate concentration of AgNPs has a stimulating effect on the removal of Cd(II) by P. chrysosporium instead of an inhibitory effect. Results of Ag(+) and total Ag concentrations in the solutions together with those of SEM and XRD demonstrated that added AgNPs had undergone oxidative dissolution and transported from the solution to the surface of fungal mycelia (up to 94%). FTIR spectra confirmed that amino, carboxyl, hydroxyl, and other reducing functional groups were involved in Cd(II) removal, AgNPs transportation, and the reduction of Ag(+) to AgNPs.

Physiological fluxes and antioxidative enzymes activities of immobilized Phanerochaete chrysosporium loaded with TiO2 nanoparticles after exposure to toxic pollutants in solution.

Immobilized Phanerochaete chrysosporium loaded with TiO2 nanoparticles (PTNs) are novel high-value bioremediation materials for adsorbing cadmium and for degrading 2,4-dichlorophenol (2,4-DCP). The real-time changes in H(+) and O2 fluxes were measured using the noninvasive microtest technique (NMT). The H(+) influx increased after the addition of 2,4-DCP, and shifted to efflux following the addition of Cd(2+). The O2 flux decreased after the addition of both 2,4-DCP and Cd(2+). A larger Cd(2+) flux was immediately observed after exposure to 0.5mM Cd(2+) (-351.25 pmol cm(-2) s(-1)) than to 0.1 mM Cd(2+) (-107.47 pmol cm(-2) s(-1)). The removal of Cd(2+) by the PTNs increased more after treatment with the 0.5 mM exposure solution (27.6 mg g(-1)) than with the 0.1 mM exposure solution (3.49 mg g(-1)). The enzyme activities were analyzed to review the antioxidative defense system of PTNs in a solution containing various concentrations of Cd(2+). The activities of the coenzyme nicotinamide adenine dinucleotide (NADH) oxidase as well as the enzyme catalase (CAT) plateaued at 6.5 U g(-1) FW and 9.7 U g(-1) FW, respectively, after exposure to 0.25 mM Cd(2+). The activity of superoxide dismutase (SOD) increased gradually in solutions containing 0.1-0.6 mM Cd(2+), and eventually reached a maximum (68.86 U g(-1) FW). These results illustrate how the antioxidative defense system and the physiological fluxes of PTNs respond to the stress caused by toxic pollutants.

Polyvinyl alcohol-immobilized Phanerochaete chrysosporium and its application in the bioremediation of composite-polluted wastewater.

A novel biosorbent, polyvinyl alcohol (PVA)-immobilized Phanerochaete chrysosporium, was applied to the bioremediation of composite-polluted wastewater, containing both cadmium and 2,4-dichlorophenol (2,4-DCP). The optimum removal efficiency achieved was 78% for Cd(II) and 95.4% for 2,4-DCP at initial concentrations of 20 mg/L Cd(II) and 40 mg/L 2,4-DCP. PPBs had significantly enhanced the resistance of P. chrysosporium to 2,4-DCP, leading to the degradation rates of 2,4-DCP beyond 90% with varying initial 2,4-DCP concentrations. This research demonstrated that 2,4-DCP and secreted proteins might be used as carbon and nitrogen sources by PVA-immobilized P. chrysosporium beads (PPBs) for Cd(II) removal. Fourier transform infrared spectroscopy analysis showed that hydroxyl and carboxyl groups on the surface of PPBs were dominant in Cd(II) binding. The mechanism underlying the degradation of 2,4-DCP into fumaric acid and 1-hexanol was investigated. The adsorption-desorption studies indicated that PPBs kept up to 98.9% of desorption efficiency over three cycles.

Comprehensive analysis of antibody recognition in convalescent humans from highly pathogenic avian influenza H5N1 infection.

Understanding the mechanism of protective antibody recognition against highly pathogenic avian influenza A virus H5N1 in humans is critical for the development of effective therapies and vaccines. Here we report the crystal structure of three H5-specific human monoclonal antibodies bound to the globular head of hemagglutinin (HA) with distinct epitope specificities, neutralization potencies and breadth. A structural and functional analysis of these epitopes combined with those reported elsewhere identifies four major vulnerable sites on the globular head of H5N1 HA. Chimeric and vulnerable site-specific mutant pseudoviruses are generated to delineate broad neutralization specificities of convalescent sera from two individuals who recovered from the infection with H5N1 virus. Our results show that the four vulnerable sites on the globular head rather than the stem region are the major neutralizing targets, suggesting that during natural H5N1 infection neutralizing antibodies against the globular head work in concert to provide protective antibody-mediated immunity.

Hydrogen sulfide alleviates 2,4-dichlorophenol toxicity and promotes its degradation in Phanerochaete chrysosporium.

In this study, the H2S donor, sodium hydrosulfide (NaHS) was used to pretreat Phanerochaete chrysosporium in order to improve its ability to degrade 2,4-dichlorophenol (2,4-DCP). When pretreated with 100µM NaHS, P. chrysosporium was able to degrade 2,4-DCP completely in 24h, whereas the degradation efficiency of the untreated control was only 57%. The 2,4-DCP-induced oxidative stress was alleviated by NaHS, and the percentage of surviving cells increased by 32%. H2S or HS(-), rather than other compounds derived from NaHS, were responsible for promoting 2,4-DCP degradation by P. chrysosporium. The results of this study suggest that H2S treatment is a potential strategy to alleviate environmental stress and improve the efficiency of the biological removal of pollutants from wastewater.