Reference gene catalog and metagenome-assembled genomes from the gut microbiome reveal the microbial composition, antibiotic resistome, and adaptability of a lignocellulose diet in the giant panda.

The giant panda, a strict herbivore that feeds on bamboo, still retains a typical carnivorous digestive system. Reference catalogs of microbial genes and genomes are lacking, largely limiting the antibiotic resistome and functional exploration of the giant panda gut microbiome. Here, we integrated 177 fecal metagenomes of captive and wild giant pandas to construct a giant panda integrated gene catalog (GPIGC) comprised of approximately 4.5 million non-redundant genes and reconstruct 393 metagenome-assembled genomes (MAGs). Taxonomic and functional characterization of genes revealed that the captivity of the giant panda significantly changed the core microbial composition and the distribution of microbial genes. Higher abundance and prevalence of antibiotic resistance genes (ARGs) were detected in the guts of captive giant pandas, and ARG distribution was influenced by geography, for both captive and wild individuals. Escherichia, as the prevalent genus in the guts of captive giant pandas, was the main carrier of ARGs, meaning there is a high risk of ARG transmission by Escherichia. We also found that multiple mcr gene variants, conferring plasmid-mediated mobile colistin resistance, were widespread in the guts of captive and wild giant pandas. There were low proportions of carbohydrate-active enzyme (CAZyme) genes in GPIGC and MAGs compared with several omnivorous and herbivorous mammals. Many members of Clostridium MAGs were significantly enriched in the guts of adult, old and wild giant pandas. The genomes of isolates and MAGs of Clostridiaceae harbored key genes or enzymes in complete pathways for degrading lignocellulose and producing short-chain fatty acids (SCFAs), indicating the potential of these bacteria to utilize the low-nutrient bamboo diet. Overall, our data presented an exhaustive reference gene catalog and MAGs in giant panda gut and provided a comprehensive understanding of the antibiotic resistome and microbial adaptability for a high-lignocellulose diet.

First Steps toward the Giant Panda Metabolome Database: Untargeted Metabolomics of Feces, Urine, Serum, and Saliva by 1H NMR.

Differences in the concentration of metabolites in the biofluids of animals closely reflect their physiological diversities. In order to set the basis for a metabolomic atlas for giant panda (Ailuropoda melanoleuca), we characterized the metabolome of healthy giant panda feces (23), urine (16), serum (6), and saliva (4) samples by means of 1H NMR. A total of 107 metabolites and a core metabolome of 12 metabolites was quantified across the four biological matrices. Through univariate analysis followed by robust principal component analysis, we were able to describe how the molecular profile observed in giant panda urine and feces was affected by gender and age. Among the molecules modified by age in feces, fucose plays a peculiar role because it is related to the digestion of bamboo's hemicellulose, which is considered as the main source of energy for giant panda. A metagenomic investigation directed toward this molecule showed that its concentration was indeed positively related to the two-component system pathway and negatively related to the amino sugar and nucleotide sugar metabolism pathway. Such work is meant to provide a robust framework for further -omics research studies on giant panda to accelerate our understanding of the interaction of giant panda with its natural environment.

Preparation and characterization of magnetic molecular imprinted polymers with ionic liquid for the extraction of carbaryl in food.

Magnetic molecular imprinted polymers with ionic liquid used as an auxiliary solvent (IL@MMIPs) for the recognition of the methyl carbamate pesticide carbaryl (CBR) in foodstuff have been synthesized. The properties and application of IL@MMIPs were determined. The kinetic and isotherm adsorption processes were found to follow the pseudo-second-order and the Scatchard models, respectively. The selective experiment showed that the IL@MMIPs exhibited good selectivity to CBR compared to magnetic nonimprinted polymers with IL (IL@MNIPs). By using the IL@MMIPs as an adsorbent for the enrichment of CBR in food samples, the limit of detection (LOD, S/N = 3) and the limit of quantitation (LOQ, S/N = 10) of this method were 3 µg kg-1 and 10 µg kg-1, respectively. Compared with the traditional method, the IL@MMIP method has better recoveries (83.23-99.83%), precision (1.12-2.09%), and stabilization (intraday, 1.08-2.81%; interday, 2.26-3.30%). IL@MMIPs are an ideal adsorbent that could be applied to conveniently detect CBR in complex food, and the proposed method can be considered as a selective and sensitive alternative to traditional methods with affordable cost, avoiding the complex pretreatment procedure. Graphical abstract .

A phosphorescent probe for cephalexin consisting of mesoporous thioglycolic acid-modified Mn:ZnS quantum dots coated with a molecularly imprinted polymer.

The authors have synthesized a phosphorescent probe of type SiO2-QD-MIP, where QD stands for Mn:ZnS quantum dots and MIP is a polymer coating that was molecularly imprinted with cephalexin. The nanoprobe with high specificity was prepared via sol-gel polymerization using thioglycolic acid (TGA)-modified QDs as luminescent materials, cephalexin as the template, 3-aminopropyltriethoxysilane as the functional monomer, and tetraethoxysilane as the crosslinking agent. The SiO2-QD-MIPs were characterized by X-ray powder diffraction, transmission electron microscopy, scanning electron microscopy, and Fourier transform infrared spectrometry. The orange emission of the probe, with excitation/emission maxima at 295/590 nm, decreases linearly in the 2.5-50 µg·L-1 cephalexin concentration range with a limit of detection (LOD) of 0.81 µg·L-1. The nanoprobe was successfully applied to the determination of cephalexin in (spiked) raw milk and milk powder. The recoveries ranged from 91.7 to 103.7%.

YKL071W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of glycolaldehyde and furfural derived from lignocellulose.

Aldehydes generated as by-products during the pretreatment of lignocellulose are the key inhibitors to Saccharomyces cerevisiae, which is considered as the most promising microorganism for industrial production of biofuel, xylitol as well as other special chemicals from lignocellulose. S. cerevisiae has the inherent ability to in situ detoxify aldehydes to corresponding alcohols by multiple aldehyde reductases. Herein, we report that an uncharacterized open reading frame YKL071W from S. cerevisiae encodes a novel "classical" short-chain dehydrogenase/reductase (SDR) protein with NADH-dependent enzymatic activities for reduction of furfural (FF), glycolaldehyde (GA), formaldehyde (FA), and benzaldehyde (BZA). This enzyme showed much better specific activities for reduction of GA and FF than FA and BZA, and displayed much higher Km and Kcat/Km but lower Vmax and Kcat for reduction of GA than FF. For this enzyme, the optimum pH was 5.5 and 6.0 for reduction of GA and FF, and the optimum temperature was 30 °C for reduction of GA and FF. Both pH and temperature affected stability of this enzyme in a similar trend for reduction of GA and FF. Cu2+, Zn2+, Ni2+, and Fe3+ had severe inhibition effects on enzyme activities of Ykl071wp for reduction of GA and FF. Transcription of YKL071W in S. cerevisiae was significantly upregulated under GA and FF stress conditions, and its transcription is most probably regulated by transcription factor genes of YAP1, CAD1, PDR3, and STB5. This research provides guidelines to identify more uncharacterized genes with reductase activities for detoxification of aldehydes derived from lignocellulose in S. cerevisiae.

Preparation of biochar-based surface molecularly imprinted polymers and evaluation of their selective adsorption and removal of carbaryl from rice and corn.

The residue of carbaryl in food is a threat to human health. In this study, activated soybean shell biochar (A-SBC) was used as a carrier, methacrylic acid (MAA) was used as a functional monomer, and carbaryl was used as a template molecule to synthesize the activated biochar surface molecularly imprinted polymer (A-SBC@MIP). The synthesized A-SBC@MIP was characterized by SEM, FT-IR, XRD and XPS techniques, and then applied as adsorbent for carbaryl removal. The adsorption capacity of A-SBC@MIP for carbaryl was 8.6 mg‧g-1 and the imprinting factor was 1.49 at the optimum ionic strength and pH. The kinetic and isothermal data indicated that it had fast mass transfer rate and high binding capacity(Qmax=47.9 mg‧g-1). A-SBC@MIP showed good regenerative properties and the adsorption of carbaryl was excellent in its structural analogues. A solid-phase extraction (SPE) column composed of A-SBC@MIP was developed for the detection of rice and corn under optimized conditions, with recoveries of 93-101% for the spiked carbaryl. The limit of detection (LOD) of the method was 3.6 µg‧kg-1 with good linearity (R2=0.994) in the range of 0.01-5.00 mg‧L-1. The results show that the developed MIPs-SPE can enrich carbaryl from food samples as a specific and cost-effective method.

[Isolation, identification and cellulase production of a cellulolytic bacterium from intestines of giant panda].

OBJECTIVE: To isolate and identify the cellulose-producing bacterium from fresh feces of healthy giant pandas, and characterize its cellulase production. METHODS: A strain with high activity of cellulase was isolated and purified by carboxymethyl cellulose-Na (CMC-Na) medium. The morphological, physiological and biochemical characteristics and 16S rDNA gene were analyzed to identify the taxonomic position of the strain. Meanwhile, its cellulase producing conditions and degradation of several cellulose substrates were studied. RESULTS: A cellulose-producing strain P2 was obtained. Strain P2 is a gram-positive and aerobic bacterium. Its growth temperature ranges from 20 to 50 degrees C (optimum at 37 degrees C) and pH 6.0 to 9.0 (optimum at 7.0), and NaCl concentration at 0%-15% (optimum at 2.0%). It took 24 h to reach the peak of cellulase production. Phylogenetic analysis based on 16S rDNA sequence showed that strain P2 is most closely related to the Bacillus amyloliquefaciens NBRC15535 with the similarity of 99. 66% . Strain P2 posses different abilities to degrade four different cellulose substrates including filter paper, absorbent cotton, straw, and bamboo fiber. During the degradation, it shows different enzymatic activity curves with endoglucanases, exoglucanases, beta-glucosidases and cellulase. CONCLUSION: An aerobic cellulolytic bacterium was isolated from feces of giant pandas for the first time, and was identified as Bacillus amyloliquefaciens. Due to its ability to degrade the cellulose materials with different structures, strain P2 could be used in the further study on the digestion mechanism of bamboo of giant panda.

A magnetic phosphorescence molecularly imprinted polymers probe based on manganese-doped ZnS quantum dots for rapid detection of trace norfloxacin residual in food.

This paper reports the development of a novel probe based on magnetic room-temperature phosphorescence quantum dots with molecularly imprinted polymers (MQD-MIPs) for the rapid detection of trace norfloxacin (NFX) residual in complex food matrix. The highly selective probe was constructed by surface molecular imprinting technology using magnetic materials (Fe3O4 nanoparticles) as core, Mn-doped ZnS quantum dots (Mn-ZnS QDs) as phosphorescent materials, NFX as template, 3-aminopropyltriethoxysilane as functional monomer, and tetraethoxysilane as crosslinking agent. The as-obtained MQD-MIPs were characterized in detail by transmission electron microscopy, scanning electron microscopy, X-ray powder diffraction, Fourier transform infrared spectrometry, and vibrating sample magnetometer. A magnetic strength of 37.64 emu g-1 was recorded. Also, the probe displayed excellent room temperature phosphorescence properties with excitation/emission peaks at 300/590 nm. Under the optimized conditions, the detection time was less than 40 min, phosphorescence intensity varied linearly with concentration from 1 to 90 µg·L-1, and detection limit reached as low as 0.80 µg·L-1. Furthermore, the MQD-MIPs-based probe successfully detected norfloxacin residues in spiked fish and milk samples with recoveries of 90.92-111.53% and RSD <7%, outperforming the standard control method-HPLC-FLD (recoveries of 85.89-118.28%).

Preparation and application of magnetic molecular imprinted polymers for extraction of cephalexin from pork and milk samples.

A highly selective and effective method was successfully developed using magnetic molecular imprinted polymers (MMIPs) as solid-phase extraction (SPE) coupled with high performance liquid chromatography-ultraviolet detector (HPLC-UV) to rapidly determine cephalexin (CFX) in complex animal-derived food. MMIPs were creatively synthesized via suspension polymerization using Fe3O4 magnetic nanoparticles as supporter, CFX as template, acrylamide (AM) as functional monomer, and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The MMIPs were characterized using X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), scanning electron microscopy (SEM), and vibrating sample magnetometry (VSM). The binding process fitted well with pseudo-second-order model with good selectivity. Scatchard plot analysis suggested that MMIPs have two types of binding sites with the Qmax of 24.18 mg g-1 and 40.25 mg g-1, respectively. And Langmuir model proved that the recognition sites were uniformly distributed in a monolayer on the surface of MMIPs. The methodological assessment showed good applicability of MMIPs with excellent recovery (85.5%-94.0%), precision (1.2%-2.4%), and stability (intra-day 1.3%-3.6%; inter-day 2.6%-4.3%) in determining CFX content. In addition, the linearity of the calibration curve was good in the range of 0.02-5.00 mg L-1, with a sensitive detection limit of 5.00 µg kg-1. The results above suggest that the obtained MMIPs exert good performance for separation of CFX in animal-derived food, and the proposed method is suitable for the reliable determination of CFX in complex samples.