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Background Locoweeds are widely distributed all over the world. Goats and other herbivores often get poisoning if they eat a large number of locoweeds by mistake. Swainsonine (SW), the main toxin in locoweeds, can inhibit lysosomes α-Mannosidase (LAM) competitively in animal cells. The insufficient activity of lysosomes α-Mannosidase can lead to abnormal metabolism of animals, forming alpha mannosidosis. However，the details of interaction between SW and LAM are not clear yet. Methods In this study, molecular docking was used to predict the interaction points between SW and LAM. The effect of putative points was investigated by constructing mutated LAM (LAMM) and analyzing its biochemical characteristics. Results The results showed that the Trp at the 28th position and Tyr at the 599th position of the LAM were the candidates of interaction points. Both tryptophan at position 28 of A peptide residue and tyrosine at position 599 of D peptide residue of goat LAM were mutated into glycine, and goat lysosomes were obtained α- The mutant sequence of mannosidase; The recombinant yeast GS115 / pPIC9K LAMM was successfully constructed; After SW induction, the sensitivity of goat LAMM to SW decreased significantly, and the enzyme activity decreased about 3 times compared with wild-type LAM; The optimum temperature of LAMM decreased from 55 °C to 50 °C, and the optimum pH value increased from 4.5 to 5.0; All the trivalent metal ions (Fe3+, Al3+, Cr3+) had significant activation (P < 0.05), and EDTA had inhibitory effect on goat LAM before and after mutation. Conclusions These results indicated that the sensitivity of goat LAMM to SW was significantly reduced, but other characteristics changed little, which was consistent with the molecular docking results of goat LAMM. This study provides help for the study of interaction mechanism between SW and goat LAM, and also provides a new idea for the development of animal locoweed disease control technology.

Persistent environmental pollutants are a growing problem around the world. The effective control of the pollutants is of great significance for human health. Some microbes, especially Arthrobacter, can degrade pollutants into nontoxic substances in various ways. Here, we review the biological properties of Arthrobacter adapting to a variety of environmental stresses, including starvation, hypertonic and hypotonic condition, oxidative stress, heavy metal stress, and low-temperature stress. Furthermore, we categorized the Arthrobacter species that can degrade triazines, organophosphorus, alkaloids, benzene, and its derivatives. Metabolic pathways behind the various biodegradation processes are further discussed. This review will be a helpful reference for comprehensive utilization of Arthrobacter species to tackle environmental pollutants.

Background Extended-spectrum β-lactamases (ESBLs)-producing Escherichia coli (E. coli) isolates in environment water become progressively a potential threat to public health, while the detailed information about the ESBL-producing E. coli isolates in the rivers and lakes in Northwest China is scarce. In the present study, it was aimed to characterize the ESBL-producing E. coli isolated from the surface waters in Northwest China. Results A total of 2686 E. coli isolates were obtained from eleven rivers and lakes in Northwest China to screen for ESBL producers. Seventy-six (2.8%) isolates were classified as ESBL producers, and phylogenic groups D and A accounted for 59.2% of the ESBL producers. CTX-Ms were the predominant ESBLs genotype, and they were represented by seven bla CTX-M subtypes. bla CTX-M-14 was the most prevalent specific CTX-M gene, followed by bla CTX-M-9, bla CTX-M-123, bla CTX-M-15, bla CTX-M-27, bla CTX-M-1 and bla CTX-M-65. Moreover, 54 of the 76 ESBL producers carried at least one plasmid-mediated quinolone resistance (PMQR) gene, and aac(6′)-Ib-cr was predominant. The overall occurrence of virulence factors ranged from 1.3% (eae) to 48.7% (traT). Thirty-seven sequence types (STs) were confirmed among the 76 ESBL producers, and the predominant was ST10, which was represented by 10 isolates; importantly, clone B2-ST131, associated with severe infections in humans and animals, was detected three times. Conclusion The prevalence of ESBL-producing E. coli from the rivers and lakes in Northwest China was low (2.8%), and the extraintestinal pathogenic E. coli (ExPEC) pathotype was the most commonly detected on the basis of the virulence factor profiles. 76.3% of ESBL producers harbored more than one β-lactamase gene, and bla CTX-M-14 was the predominant genotype. Notably, one ST131 isolate from Gaogan Canal simultaneously harbored bla CTX-M-9, bla CTX-M-15, bla CTX-M-123, bla KPC-2, bla NDM-1, bla OXA-2 as well as the PMQR genes qnrA, qnrS and aac(6′)-Ib-cr.

Objectives: The aim of the present study was to explore the prevalence and molecular characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli collected from pig farms in Northwest China. Methods: Between May 2015 and June 2017, a total of 456 E. coli isolates were collected from fecal samples of healthy and diarrheal pigs in Northwest China to screen the ESBL producers. The β-lactamases, plasmid-mediated quinolone resistance (PMQR) genes and virulence genes among ESBL producers were corroborated by PCR and sequencing. Finally, ESBL producers were further grouped according to phylogenetic background and genetic relatedness. Results: Forty-four (9.6%) out of the 456 E. coli isolates were identified as ESBL-producing isolates. All ESBL producers exhibited multidrug resistance (MDR) phenotype, and more than 90% of the ESBL producers were resistant to amoxicillin, amoxicillin-clavulanic acid, oxytetracycline, enrofloxacin and sulfamethoxazole/trimethoprim. All ESBL producers harbored at least one type of β-lactamase, with blaCTX−M, blaTEM, blaSHV, blaOXA−48, and blaKPC−2 being detected in forty, thirty, seven, four, two and one isolates, respectively. Sequencing revealed the most common blaCTX−M subtype was blaCTX−M−14 (n = 24), followed by blaCTX−M−15 (n = 14), blaCTX−M−64 (n = 11), blaCTX−M−9 (n = 10) and blaCTX−M−123 (n = 9). qnrS (n = 23) was the predominant PMQR gene, and all PMQR genes were detected in co-existence with β-lactamase genes. estA (n = 18) and F4 (n = 18) were the most prevalent enterotoxin and fimbrial adhesin, respectively, and 27 different virotypes were found with respect to the association of enterotoxins and fimbrial adhesins. Twenty-four different sequence types (STs) were identified among 44 ESBL producers, and clones ST405, ST10 and ST648 were strongly present in more than one-third (34.1%) of ESBL producers. Conclusion: All ESBL-producing E. coli isolates exhibited MDR phenotype, and showed high prevalence of β-lactamase and PMQR genes. Especially, one isolate harbored ESBL genes blaTEM, blaSHV, blaCTX−M−9, blaCTX−M−14, blaCTX−M−64, and carbapenemase gene blaOXA−48 and blaKPC−2, as well as PMQR genes qnrS, qnrB, qnrD, qepA and aac(6')-Ib-cr.

Background Fibrinogen-like protein 2 (FGL2), a new member of the fibrinogen-like family, has recently been identified as a novel immunosuppressive molecule. Aim The purpose of this work was to investigate intestinal and peripheral expression of FGL2 in patients with inflammatory bowel disease (IBD), mainly ulcerative colitis (UC) and Crohn’s disease (CD). Methods FGL2 expression in mucosal biopsies from three groups (UC group (n = 61), CD group (n = 54), and controls group (n = 35)) was detected by immunohistochemistry. Concentrations of FGL2 in plasma from 50 UC patients, 45 CD patients, and 30 controls were analyzed by enzyme-linked immunosorbent assay. Western blot of FGL2 protein and real-time fluorescent quantitative PCR of FGL2 mRNA expression by peripheral mononuclear cells was performed. Correlations of FGL2 expression with disease type, activity, and location, and with measured laboratory data, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), were examined. Results Intestinal and peripheral FGL2 protein data showed that FGL2 expression was significantly up-regulated in both UC and CD patients compared with controls (P

Swainsonine is an indolizidine alkaloid that has been found in locoweeds and some fungi. Our previous study demonstrated that Arthrobacter sp. HW08 or its crude enzyme extract could degrade swainsonie efficiently. However, the mechanism of swainsonine degradation in bacteria remains unclear. In this study, we used label-free quantitative proteomics method based on liquid chromatography-electrospray ionization-tandem mass spectrometry to dissect the mechanism of swainsonine biodegradation by Arthrobacter sp. HW08. The results showed that 129 differentially expressed proteins were relevant to swainsonine degradation. These differentially expressed proteins were mostly related to the biological process of metabolism and the molecular function of catalytic activity. Among the 129 differentially expressed proteins, putative sugar phosphate isomerase/epimerase A1R5X7, Acetyl-CoA acetyltransferase A0JZ95, and nicotinamide adenine dinucleotide phosphate (NADP)-dependent alcohol dehydrogenase A1R6C3 were found to contribute to the swainsonine degradation. Notably, NADP-dependent alcohol dehyrodgenase A1R6C3 appeared to play a major role in degrading swainsonine, but not as much as Arthrobacter sp. HW08 did. Collectively, our findings here provide insights to understand the mechanism of swainsonine degradation in bacteria.

Plant cytochrome P450 is a key enzyme responsible for the herbicide resistance but the molecular basis of the mechanism is unclear. To understand this, four typical plant P450s and a widely resistant herbicide chlortoluron were analysed by carrying out homology modelling, molecular docking, molecular dynamics simulations and binding free energy analysis. Our results demonstrate that: (i) the putative hydrophobic residues located in the F-helix and polar residues in I-helix are critical in the herbicide resistance; (ii) the binding mode analysis and binding free energy calculation indicate that the distance between catalytic site of chlortoluron and heme of P450, as well as the binding affinity are key elements affecting the resistance for plants. In conclusion, this work provides a new insight into the interactions of plant P450s with herbicide from a molecular level, offering valuable information for the future design of novel effective herbicides which also escape from the P450 metabolism.

Swainsonine (SW) is an indolizidine triol plant alkaloid isolated from the species Astragalus, colloquially termed locoweed. When chronically ingested by livestock and wildlife, symptoms include severe neuronal disturbance. Toxicity to the central and peripheral nervous system is caused by inhibition of lysosomal α-mannosidase (AMA) and accumulation of intracellular oligosaccharide. Consequently, SW has been used as a model substance in investigations of lysosomal storage diseases. Involvement of the basal ganglia has been postulated due to the neuronal symptoms of affected animals. Therefore, primary midbrain cultures from embryonic mice containing dopaminergic neurons were utilized in this study. Neural cells were exposed to SW (0.01-100 μM) for 72 h. AMA activity was 50 % inhibited at 1 μM SW. Cytotoxic changes in cultures were observed above 25 μM SW by increases in lactate dehydrogenase activity and nitric oxide content. Neurotoxicity to dopaminergic cells was visualized by tyrosine hydroxylase immunohistochemistry. Structural degeneration scored as dendritic shortening and shrinkage of cell bodies was dose-dependent and resulted in nerve loss above 25 μM. SW exposure caused progression from reversible to irreversible cytotoxicity. Partial regeneration of AMA-activity in culture was observed on removal of SW. The antioxidative vitamins ascorbic acid and tocopherol (both 100 μM) partially reversed the toxic effect on dopaminergic cells and ascorbic acid decreased AMA inhibition. Thus, neuronal midbrain cell cultures can demonstrate the neurotoxic action of SW and cytoprotective strategies may be tested at a single nerve cell level.

In order to obtain structural features of 3-arylpyrimidin-2,4-diones emerged as promising inhibitors of insect γ-aminobutyric acid (GABA) receptor, a set of ligand-/receptor-based 3D-QSAR models for 60 derivatives are generated using Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA). The statistically optimal CoMSIA model is produced with highest q(2) of 0.62, r(2) (ncv) of 0.97, and r(2) (pred) of 0.95. A minor/bulky electronegative hydrophilic polar substituent at the 1-/6-postion of the uracil ring, and bulky substituents at the 3'-, 4'- and 5'-positions of the benzene ring are beneficial for the enhanced potency of the inhibitors as revealed by the obtained 3D-contour maps. Furthermore, homology modeling, molecular dynamics (MD) simulation and molecular docking are also carried out to gain a better understanding of the probable binding modes of these inhibitors, and the results show that residues Ala-183(C), Thr-187(B), Thr-187(D) and Thr-187(E) in the second transmembrane domains of GABA receptor are responsible for the H-bonding interactions with the inhibitor. The good correlation between docking observations and 3D-QSAR analyses further proves the model reasonability in probing the structural features and the binding mode of 3-arylpyrimidin-2,4-dione derivatives within the housefly GABA receptor.

In order to isolate and characterize 2,2,6,6-Tetramethyl-4-piperidone (TMPD)- degrading bacteria and to select the optimal degrading conditions, different bacterial types which can degrade TMPD were isolated from soil samples. Three strains (LF-1, LF-2 and LF-3) showed strong biodegradation ability by UV-visible spectrophotometer analysis. Morphological and biochemical analyses, as well as 16S rRNA gene sequence comparisons of three strains were further carried out. They were identified as Klebsiella oxytoca, Staphylococcus pasteur and Bacillus flexus, respectively. K. oxytoca, S. pasteur and B. flexus had degrading capabilities for TMPD in mineral salt medium of 80.9, 69.8 and 64.9%, respectively, during 24 days of incubation. Maximum degrading rates were obtained by an initial concentration of the TMPD of 200 mg/L, an incubation temperature of 30°C, and constant aeration (180 rev/min). In conclusion, K. oxytoca, S. pasteur and B. flexus show their potentials in TMPD detoxifying and in protecting the TMPD contaminated environment. Key words: 2,2,6,6-Tetramethyl-4-piperidone (TMPD), biodegradation, Klebsiella oxytoca, Staphylococcus pasteur, Bacillus flexus.

Locoweeds including the toxic species of Astragalus spp and Oxytropis spp. are widely distributed in the western region of China and result in a chronic neurological disease known as locoism in animals. To determine the presence of swainsonine-producing fungal endophyte of major locoweed species in China, endophytes were isolated from 8 locoweed species that including A. variabilis, A. strictus, O. glacialis, O. kansuensis, O. ochrocepala, O. sericopetala, O. glabra and O. latibracteata. Seven species of locoweed were confirmed contain substantial amounts of swainsonine and infect swainsonine-producing fungal endophyte. These endophytes were classified as Undifilim oxytropis according to the fungal morphology and phylogenetic analysis based on sufficient ITS sequences. PCR-RFLP analysis of IGS region showed that the interspecific or intraspecific variations were present among the endophytes from different locoweed species.

Golgi α-mannosidase II (GMII), a key glycosyl hydrolase in the N-linked glycosylation pathway, has been demonstrated to be closely associated with the genesis and development of cancer. In this study, we cloned cDNA-encoding Capra hircus GMII (chGMII) and expressed it in Pichia pastoris expression system. The chGMII cDNA contains an open reading frame of 3432 bp encoding a polypeptide of 1144 amino acids. The deduced molecular mass and pI of chGMII was 130.5 kDa and 8.04, respectively. The gene expression profile analysis showed GMII was the highest expressed gene in the spleen. The recombinant chGMII showed maximum activity at pH 5.4 and 42 °C and was activated by Fe(2+), Zn(2+), Ca(2+), and Mn(2+) and strongly inhibited by Co(2+), Cu(2+), and EDTA. By homology modeling and molecular docking, we obtained the predicted 3D structure of chGMII and the probable binding modes of chGMII-GnMan5Gn, chGMII-SW. A small cavity containing Tyr355 and zinc ion fixed by residues Asp290, His176, Asp178, and His570 was identified as the active center of chGMII. These results not only provide a clue for clarifying the catalytic mechanism of chGMII but also lay a theoretical foundation for subsequent investigations in the field of anticancer therapy for mammals.

The endophytic fungus XJ-AC03, which was isolated from the healthy roots of Aconitum leucostomum, produced aconitine when grown in potato dextrose agar (PDA) medium. The presence of aconitine was confirmed by the chromatographic and spectroscopic analyses. The yield of aconitine was recorded as 236.4 μg/g by high performance liquid chromatography (HPLC). The mass spectrometry was shown to be identical to authentic aconitine. Further analysis with nuclear magnetic resonance (NMR) spectroscopy to show the chemical structure of the fungal aconitine indicated that the fungal aconitine produced an NMR spectrum identical to that of authentic aconitine. Strain XJ-AC03 was identified as Cladosporium cladosporioides by its characteristic culture morphology and ITS rDNA sequence analysis.

Nicotinic acetylcholine receptor (nAChR) is a target for insect-selective neonicotinoid insecticides (NNs), exemplified by imidacloprid (IMI). In the present study, 78 IMI derivatives reported as inhibitors of Drosophila melanogaster nAChR (Dm-nAChR) and Musca domestica nAChR (Md-nAChR) were used for three-dimensional quantitative structure-activity relationship (3D-QSAR) studies. Two optimal models with good predictive power were obtained: Q(2) = 0.64, R(2)(pred) = 0.72 for Dm-nAChR, and Q(2) = 0.63, R(2)(pred) = 0.62 for Md-nAChR. In addition, homology modeling, molecular dynamic (MD) simulation, and molecular docking also showed that amino acids located within loops A, C, D and E play key roles in the interaction of Dm-/Md-nAChR with NNs. This is highly consistent with the results of graphical analysis of 3D-QSAR contour plots. Mutation analysis also implicates the Y/S mutation within loop B as being associated closely with NN resistance in Drosophila and Musca. The results obtained lead to a better understanding not only of interactions between these antagonists and Dm-/Md-nAChR, but also of the essential features that should be considered when designing novel inhibitors with desired activities.