Characterization of antibiotic resistance genes and mobile genetic elements in Escherichia coli isolated from captive black bears.

The objective of this study was to analyze the antimicrobial resistance (AMR) characteristics produced by antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and gene cassettes in Escherichia coli isolated from the feces of captive black bears. Antimicrobial susceptibility testing was performed by using the disk diffusion method, and both MGEs and integron gene cassettes were detected by polymerase chain reaction. Our results showed that 43.7% (62/142) of the isolates were multidrug resistant strains and 97.9% (139/142) of the isolates were resistant to at least one antibiotic. The highest AMR phenotype was observed for tetracycline (79.6%, 113/142), followed by ampicillin (50.0%, 71/142), trimethoprim-sulfamethoxazole (43.7%, 62/142) and cefotaxime (35.9%, 51/142). However, all isolates were susceptible to tobramycin. tetA had the highest occurrence in 6 ARGs in 142 E. coli isolates (76.8%, 109/142). Ten mobile genetic elements were observed and IS26 was dominant (88.0%, 125/142). ISECP1 was positively associated with five ß-lactam antibiotics. ISCR3/14, IS1133 and intI3 were not detected. Seventy-five E. coli isolates (65 intI1-positive isolates, 2 intI2-positive isolates and 8 intI1 + intI2-positive isolates) carried integrons. Five gene cassettes (dfrA1, aadA2, dfrA17-aadA5, aadA2-dfrA12 and dfrA1-aadA1) were identified in the intI1-positive isolates and 2 gene cassettes (dfrA1-catB2-sat2-aadA1 and dfrA1-catB2-sat1-aadA1) were observed in the intI2-positive isolates. Monitoring of ARGs, MGEs and gene cassettes is important to understand the prevalence of AMR, which may help to introduce measures to prevent and control of AMR in E. coli for captive black bears.

Characteristics of MDR E. coli strains isolated from Pet Dogs with clinic diarrhea: A pool of antibiotic resistance genes and virulence-associated genes.

The increasing number of multi-drug resistant (MDR) bacteria in companion animals poses a threat to both pet treatment and public health. To investigate the characteristics of MDR Escherichia coli (E. coli) from dogs, we detected the antimicrobial resistance (AMR) of 135 E. coli isolates from diarrheal pet dogs by disc diffusion method (K-B method), and screened antibiotic resistance genes (ARGs), virulence-associated genes (VAGs), and population structure (phylogenetic groups and MLST) by polymerase chain reaction (PCR) for 74 MDR strains, then further analyzed the association between AMRs and ARGs or VAGs. Our results showed that 135 isolates exhibited high resistance to AMP (71.11%, 96/135), TET (62.22%, 84/135), and SXT (59.26%, 80/135). Additionally, 54.81% (74/135) of the isolates were identified as MDR E. coli. In 74 MDR strains, a total of 12 ARGs in 6 categories and 14 VAGs in 4 categories were observed, of which tetA (95.95%, 71/74) and fimC (100%, 74/74) were the most prevalent. Further analysis of associations between ARGs and AMRs or VAGs in MDR strains revealed 23 significant positive associated pairs were observed between ARGs and AMRs, while only 5 associated pairs were observed between ARGs and VAGs (3 positive associated pairs and 2 negative associated pairs). Results of population structure analysis showed that B2 and D groups were the prevalent phylogroups (90.54%, 67/74), and 74 MDR strains belonged to 42 STs (6 clonal complexes and 23 singletons), of which ST10 was the dominant lineage. Our findings indicated that MDR E. coli from pet dogs carry a high diversity of ARGs and VAGs, and were mostly belong to B2/D groups and ST10. Measures should be taken to prevent the transmission of MDR E. coli between companion animals and humans, as the fecal shedding of MDR E. coli from pet dogs may pose a threat to humans.

Mechanochemical Iron-Catalyzed Nitrene Transfer Reactions: Direct Synthesis of N-Acyl Sulfonimidamides from Sulfinamides and Dioxazolones.

A mechanochemical synthesis of sulfonimidamides by iron(II)-catalyzed exogenous ligand-free N-acyl nitrene transfer to sulfinamides is reported. The one-step method tolerates a wide range of sulfinamides with various substituents under solvent-free ambient conditions. Compared to its solution-phase counterpart, this mechanochemical approach shows better conversion and chemoselectivity. Mechanistic investigations by ESI-MS revealed the generation of crucial nitrene iron intermediates.

Distribution and associations for antimicrobial resistance and antibiotic resistance genes of Escherichia coli from musk deer (Moschus berezovskii) in Sichuan, China.

This study aimed to investigate the antimicrobial resistance (AMR), antibiotic resistance genes (ARGs) and integrons in 157 Escherichia coli (E. coli) strains isolated from feces of captive musk deer from 2 farms (Dujiang Yan and Barkam) in Sichuan province. Result showed that 91.72% (144/157) strains were resistant to at least one antimicrobial and 24.20% (38/157) strains were multi-drug resistant (MDR). The antibiotics that most E. coli strains were resistant to was sulfamethoxazole (85.99%), followed by ampicillin (26.11%) and tetracycline (24.84%). We further detected 13 ARGs in the 157 E. coli strains, of which blaTEM had the highest occurrence (91.72%), followed by aac(3')-Iid (60.51%) and blaCTX-M (16.56%). Doxycycline, chloramphenicol, and ceftriaxone resistance were strongly correlated with the presence of tetB, floR and blaCTX-M, respectively. The strongest positive association among AMR phenotypes was ampicillin/cefuroxime sodium (OR, 828.000). The strongest positive association among 16 pairs of ARGs was sul1/floR (OR, 21.667). Nine pairs positive associations were observed between AMR phenotypes and corresponding resistance genes and the strongest association was observed for CHL/floR (OR, 301.167). Investigation of integrons revealed intl1 and intl2 genes were detected in 10.19% (16/157) and 1.27% (2/157) E. coli strains, respectively. Only one type of gene cassettes (drA17-aadA5) was detected in class 1 integron positive strains. Our data implied musk deer is a reservoir of ARGs and positive associations were common observed among E. coli strains carrying AMRs and ARGs.

Discovery of a potent and highly selective inhibitor of ataxia telangiectasia mutated and Rad3-Related (ATR) kinase: Structural activity relationship and antitumor activity both in vitro and in vivo.

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is an important regulator of the DNA damage response (DDR), especially in response to replication stress (RS). Tumor cells with ataxia-telangiectasia mutated (ATM) kinase loss of function or DDR defects that promote replicative stress are often more reliant on ATR for survival, highlighting ATR as a good antitumor target under the principle of synthetic lethality. Herein we report the discovery of a potent and highly selective ATR inhibitor, SKLB-197, which was obtained through structural optimization and structure-activity relationship (SAR) studies towards a hit compound (Cpd-1). SKLB-197 showed an IC50 value of 0.013 µM against ATR but very weak or no activity against other 402 protein kinases. It displayed potent antitumor activity against ATM-deficent tumors both in vitro and in vivo. In addition, this compound exhibited good pharmacokinetic properties. Overall, SKLB-197 could be a promising lead compound for drug discovery targeting ATR and deserves further in-depth studies.

Discovery of a novel and potent inhibitor with differential species-specific effects against NLRP3 and AIM2 inflammasome-dependent pyroptosis.

The NLRP3 inflammasome, which regulated a proinflammatory programmed cell death form termed pyroptosis, is involved in the pathological process of various human diseases, such as multiple sclerosis, type 2 diabetes, and gout. Thus, compounds inhibiting activation of the NLRP3 inflammasome can be promising treatments for these diseases. In this study, we conducted a phenotypic screening against NLRP3-dependent pyroptosis and discovered the hit compound 1, which showed moderate antipyroptotic activity. Chemistry efforts to improve potency of 1 resulted in a novel compound 59 (J114), which exhibited a half-maximal inhibitory concentration (IC50) of 0.077 ± 0.008 µM against cell pyroptosis. Interestingly, unlike all pyroptosis inhibitors currently reported, the activity of J114 showed significant differences in human- and mouse-derived cells. The IC50 of J114-mediated inhibition of IL-1ß secretion by human THP-1 macrophages was 0.098 µM, which was nearly 150-fold and 500-fold more potent than that of J774A.1 (14.62 µM) and bone marrow-derived macrophages (BMDMs) (48.98 µM), respectively. Further studies showed that J114 displayed remarkable inhibitory activity against NLRP3- and AIM2-but not NLRC4-dependent activation of caspase-1 and the release of IL-1ß in human THP-1 macrophages. Mechanistically, J114 disturbed the interaction of NLRP3 or AIM2 with the adaptor protein ASC and inhibited ASC oligomerization. Overall, our study identified a unique molecule that inhibits NLRP3 and AIM2 inflammasome activation and has species differences, which is worthy of further research to understand the differential regulation of the NLRP3 and AIM2 inflammasomes in humans and mice.

Structural Optimization and Structure-Activity Relationship Studies of 6,6-Dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one Derivatives as A New Class of Potent Inhibitors of Pan-Trk and Their Drug-Resistant Mutants.

Tropomyosin receptor kinases (TrkA, TrkB, and TrkC) are attractive therapeutic targets for multiple cancers. Two first-generation small-molecule Trks inhibitors, larotrectinib and entrectinib, have just been approved to use clinically. However, the drug-resistance mutations of Trks have already emerged, which calls for new-generation Trks inhibitors. Herein, we report the structural optimization and structure-activity relationship studies of 6,6-dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one derivatives as a new class of pan-Trk inhibitors. The prioritized compound 11g exhibited low nanomolar IC50 values against TrkA, TrkB, and TrkC and various drug-resistant mutants. It also showed good kinase selectivity. 11g displayed excellent in vitro antitumor activity and strongly suppressed Trk-mediated signaling pathways in intact cells. In in vivo studies, compound 11g exhibited good antitumor activity in BaF3-TEL-TrkA and BaF3-TEL-TrkCG623R allograft mouse models without exhibiting apparent toxicity. Collectively, 11g could be a promising lead compound for drug discovery targeting Trks and deserves further investigation.

Interactions of Muscovy duck reovirus, gut microbiota, and host innate immunity: Transcriptome and gut microbiota analysis.

It has been shown that Muscovy duck reovirus (MDRV) infection causes severe intestinal barrier damage and intestinal mucosal immune suppression. The health and balance of gut microbes is essential for the progression of intestinal infectious diseases. To investigate the interaction of MDRV, intestinal bacteria with host intestinal innate immunity, an MDRV contact-infection model was established in this study. High-throughput sequencing technology was used to sequence 16S rDNA and transcripts in ileal samples from experimental Muscovy ducklings. Our results suggest that intestinal opportunistic pathogens such as Streptococcus and Corynebacterium proliferated massively in MDRV-infected Muscovy ducklings. The body initiates antiviral and antibacterial immunity and actively fights the infection of gut microbes. The synthesis of peptidoglycan, lipopolysaccharide, and flagellin by intestinal bacteria activates the Toll-like receptor signaling pathway resulting in increased secretion of IFN-ß, IL-1ß, and IL-8. The RIG-I-like receptor signaling pathway is an important signaling pathway for the interaction between MDRV and the host. At the same time, we also observed that multiple genes in the JAK-STAT signaling pathway were significantly different. These genes are important targets for studying the immunosuppression caused by MDRV. In conclusion, we analyzed the interaction of MDRV, intestinal flora and host immune system during MDRV infection, which provides a basis for the further study on the mechanism of intestinal immunosuppression caused by MDRV.

Discovery and structure-activity relationship studies of 1-aryl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione derivatives as potent dual inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and trytophan 2,3-dioxygenase (TDO).

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO), which mediate kynurenine pathway of tryptophan degradation, have emerged as potential new targets in immunotherapy for treatment of cancer because of their critical role in immunosuppression in the tumor microenvironment. In this investigation, we report the structural optimization and structure-activity relationship studies of 1-phenyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione derivatives as a new class of IDO1/TDO dual inhibitors. Among all the obtained dual inhibitors, 1-(3-chloro-4-fluorophenyl)-6-fluoro-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (38) displayed the most potent IDO1 and TDO inhibitory activities with IC50 (half-maximal inhibitory concentration) values of 5 nM for IDO1 and 4 nM for TDO. It turned out that compound 38 was not a PAINS compound. Compound 38 could efficiently inhibit the biofunction of IDO1 and TDO in intact cells. In LL2 (Lewis lung cancer) and Hepa1-6 (hepatic carcinoma) allograft mouse models, this compound also showed considerable in vivo anti-tumor activity and no obvious toxicity was observed. Therefore, 38 could be a good lead compound for cancer immunotherapy and deserving further investigation.

High prevalence of multi-drug resistances and diversity of mobile genetic elements in Escherichia coli isolates from captive giant pandas.

The purpose of this study was to characterize the antimicrobial resistance produced by mobile genetic elements and integron gene cassettes in Escherichia coli isolated from the feces of captive giant pandas. We performed a standard disk diffusion antimicrobial susceptibility test with 84 E. coli isolates and further evaluated the mobile genetic elements and integron gene cassettes. The antimicrobial susceptibility test demonstrated that 43.37% (36/84) of the isolates showed multiple drug resistances. The E. coli isolates mainly showed resistance to aztreonam (86.90%, 73/84) and amoxicillin/clavulanic acid (80.95%, 68/84). The most frequently observed resistance patterns were ampicillin/amoxicillin-clavulanic acid (13.10%, n = 11), and doxycycline/amoxicillin-clavulanic acid (4.76%, n = 4). Further analyses detected 11 mobile genetic elements, of which merA (54/84, 64.30%) had the highest frequency. All isolates were negative for intI3, traA, tnpU, traF, tnp513, tnsA, ISkpn7, ISpa7, ISkpn6, and ISCR1. We further analyzed antimicrobial resistance-related integrons among 30 E. coli isolates (the 27 intI1-positive isolates and the 3 intI2-positive isolates); six gene cassette profiles (dfrA17+aadA5, aadA2, dfrA12+aadA2, dfrA1+aadA1, dfrA1, and aadA1) were identified in the 27 intI1-positive isolates, but not in the three intI2-positive ones. Our study sheds light on the prevalence of multiple drug resistances and the diversity of mobile genetic elements in E. coli isolates, and highlights the necessity to monitor antibiotic resistance in more E. coli strains from captive giant pandas.

Pd-Catalyzed Dearomative Asymmetric Allylic Alkylation of Naphthols with Alkoxyallenes.

A Pd-catalyzed linear selective intermolecular asymmetric dearomative allylic alkylation reaction of naphthols with alkoxyallenes under mild reaction conditions is reported. The transformation is successfully promoted by Pd2(dba)3 and the chiral Trost ligand and provides a general atom-efficient protocol to obtain various ß-naphthalenones bearing an all carbon quaternary stereogenic center in good yields and chemo- and stereoselectivities.

Palladium-Catalyzed Allylic Substitution Reaction of Benzothiazolylacetamide with Allylic Alcohols in Water.

An efficient tetrakis(triphenylphosphine)palladium- and Brønsted acid catalyzed allylic substitution reaction of benzothiazolylacetamide with allylic alcohols in water has been developed, and the corresponding allylated products were afforded in good to excellent (up to 99%) yields with high regioselectivities. This straightforward protocol exhibits good functional group tolerance and scalability.

Indium-Mediated Synthesis of Benzylic Hydroperoxides.

An indium(0)-metal-mediated efficient synthesis of benzylic hydroperoxides is described. The reaction proceeds efficiently with a broad range of benzyl bromides under aerobic conditions at room temperature to afford benzyl hydroperoxides in good to excellent yields. In addition, the tandem hydroperoxidation-Michael addition of ( E)-1-(bromomethyl)-2-(2-nitrovinyl)benzene was also demonstrated.

Crown ether microfunctionalized carbon nanotubes for dispersive micro-solid-phase extraction of sudan dyes and their metabolites.

In this study, dispersive micro solid phase extraction (DMSPE) combined with ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry method was established to extract and determine sudan dyes and their metabolites in real samples. The crown ether microfunctionalized multi-walled carbon nanotubes (MWCNTs) was applied as the sorbent in DMSPE procedure. Several experimental parameters that can effect the extraction performance of the DMSPE method were investigated separately by a univariate method. The validation data showed that the limits of detection were in the range of 0.084-13.13 µg/kg, the mean recoveries were ranged from 80.15 to 103.58% for six samples. Compared with other published methods, the proposed method was more effective, more time-saving and more eco-friendly. Finally, the developed method was successfully applied to enrich and detect sudan dyes and their metabolites in real samples.

Synthesis of Triphenylenes Starting from 2-Iodobiphenyls and Iodobenzenes via Palladium-Catalyzed Dual C-H Activation and Double C-C Bond Formation.

A novel and facile approach for the synthesis of triphenylenes has been developed via palladium-catalyzed coupling of 2-iodobiphenyls and iodobenzenes. The reaction involves dual palladium-catalyzed C-H activations and double palladium-catalyzed C-C bond formations. A range of unsymmetrically functionalized triphenylenes can be synthesized with the reaction. The approach features readily available starting materials, high atom- and step-economy, and access to various unsymmetrically functionalized triphenylenes.

Synthesis of 2-substituted tetraphenylenes via transition-metal-catalyzed derivatization of tetraphenylene.

A new strategy for the synthesis of 2-substituted tetraphenylenes through a transition-metal-catalyzed derivatization has been developed. Three types of functionalities, including OAc, X (Cl, Br, I) and carbonyl, were introduced onto tetraphenylene, which allows the easy access to a variety of monosubstituted tetraphenylenes. These reactions could accelerate research on the properties and application of tetraphenylene derivatives.

[The Isolation and Identification of Infectious Bronchitis Virus PTFY Strain in Muscovy Ducks].

In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks.

Palladium-catalyzed C-H ethoxycarbonyldifluoromethylation of electron-rich heteroarenes.

The first Pd-catalyzed C-H ethoxycarbonyldifluoromethylation with BrCF2CO2Et has been developed. The use of a bidentate phosphine ligand (Xantphos) is critical for the reaction to occur. A variety of electron-rich heteroarenes, including indoles, furans, thiophenes, and pyrroles, can be ethoxycarbonyldifluoromethylated in moderate to excellent yields. The reactions take place at the C-H bonds adjacent to the heteroatoms with high regioselectivity. This method provides a new protocol for the introduction of difuoroalkyl groups into electron-rich heteroarenes.

Silver-catalyzed C-H trifluoromethylation of arenes using trifluoroacetic acid as the trifluoromethylating reagent.

Direct trifluoromethylation of arenes using TFA as the trifluoromethylating reagent was achieved with Ag as the catalyst. This reaction not only provides a new protocol for aryl C-H trifluoromethylation, but the generation of CF3· from TFA may prove useful in other contexts and could potentially be extended to other trifluoromethylation reactions.