Microwave-assisted, rapid synthesis of 2-vinylquinolines and evaluation of their antimalarial activity.

A rapid and efficient synthesis of 2-vinylquinolines via trifluoromethanesulfonamidemediated olefination of 2-methylquinoline and aldehyde under microwave irradiation is reported. Biological evaluation of these scaffolds demonstrates that 2-vinylquinolines 3x - 3z possess excellent antimalarial activities against chloroquine-resistant Dd2 strain of Plasmodium falciparum (IC50 < 100 nM).

Identification and Characterization of Key Charged Residues in the Cofilin Protein Involved in Azole Susceptibility, Apoptosis, and Virulence of Aspergillus fumigatus.

Through some specific amino acid residues, cofilin, a ubiquitous actin depolymerization factor, can significantly affect mitochondrial function related to drug resistance and apoptosis in Saccharomyces cerevisiae; however, this modulation in a major fungal pathogen, Aspergillus fumigatus, was still unclear. Hereby, it was found, first, that mutations on several charged residues in cofilin to alanine, D19A-R21A, E48A, and K36A, increased the formation of reactive oxygen species and induced apoptosis along with typical hallmarks, including mitochondrial membrane potential depolarization, cytochrome c release, upregulation of metacaspases, and DNA cleavage, in A. fumigatus Two of these mutations (D19A-R21A and K36A) increased acetyl coenzyme A and ATP concentrations by triggering fatty acid ß-oxidation. The upregulated acetyl coenzyme A affected the ergosterol biosynthetic pathway, leading to overexpression of cyp51A and -B, while excess ATP fueled ATP-binding cassette transporters. Besides, both of these mutations reduced the susceptibility of A. fumigatus to azole drugs and enhanced the virulence of A. fumigatus in a Galleria mellonella infection model. Taken together, novel and key charged residues in cofilin were identified to be essential modules regulating the mitochondrial function involved in azole susceptibility, apoptosis, and virulence of A. fumigatus.

Elevated MIC Values of Imidazole Drugs against Aspergillus fumigatus Isolates with TR34/L98H/S297T/F495I Mutation.

The use of azole fungicides in agriculture is believed to be one of the main reasons for the emergence of azole resistance in Aspergillus fumigatus Though widely used in agriculture, imidazole fungicides have not been linked to resistance in A. fumigatus This study showed that elevated MIC values of imidazole drugs were observed against A. fumigatus isolates with TR34/L98H/S297T/F495I mutation, but not among isolates with TR34/L98H mutation. Short-tandem-repeat (STR) typing analysis of 580 A. fumigatus isolates from 20 countries suggested that the majority of TR34/L98H/S297T/F495I strains from China were genetically different from the predominant major clade comprising most of the azole-resistant strains and the strains with the same mutation from the Netherlands and Denmark. Alignments of sterol 14α-demethylase sequences suggested that F495I in A. fumigatus was orthologous to F506I in Penicillium digitatum and F489L in Pyrenophora teres, which have been reported to be associated with imidazole resistance. In vitro antifungal susceptibility testing of different recombinants with cyp51A mutations further confirmed the association of the F495I mutation with imidazole resistance. In conclusion, this study suggested that environmental use of imidazole fungicides might confer selection pressure for the emergence of azole resistance in A. fumigatus.

Epidemiology and Molecular Characterizations of Azole Resistance in Clinical and Environmental Aspergillus fumigatus Isolates from China.

Azole resistance in Aspergillus fumigatus has emerged as a worldwide public health problem. We sought here to demonstrate the occurrence and characteristics of azole resistance in A. fumigatus from different parts of China. A total of 317 clinical and 144 environmental A. fumigatus isolates from 12 provinces were collected and subjected to screening for azole resistance. Antifungal susceptibility, cyp51A gene sequencing, and genotyping were carried out for all suspected azole-resistant isolates and a subset of azole-susceptible isolates. As a result, 8 (2.5%) clinical and 2 (1.4%) environmental A. fumigatus isolates were identified as azole resistant. Five azole-resistant strains exhibit the TR34/L98H mutation, whereas four carry the TR34/L98H/S297T/F495I mutation in the cyp51A gene. Genetic typing and phylogenetic analysis showed that there was a worldwide clonal expansion of the TR34/L98H isolates, while the TR34/L98H/S297T/F495I isolates from China harbored a distinct genetic background with resistant isolates from other countries. High polymorphisms existed in the cyp51A gene that produced amino acid changes among azole-susceptible A. fumigatus isolates, with N248K being the most common mutation. These data suggest that the wide distribution of azole-resistant A. fumigatus might be attributed to the environmental resistance mechanisms in China.

Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp.

Metabolic shifts and structural changes in the gut microbiota upon branched-chain amino acid supplementation in middle-aged mice.

The importance of gut microbiota to health has gained extensive attention and is strongly correlated with diet. Dietary supplementation with a branched-chain amino acid-enriched mixture (BCAAem) exerts a variety of beneficial effects in mice and humans. In mice, BCAAem supplementation can promote longevity, but its influence on the gut ecosystem and the underlying mechanism remain unclear. To address this issue, BALB/C mice were fed a BCAAem-supplemented diet and their gut microbiomes were analysed by 16S rDNA sequencing. Quantitative polymerase chain reaction was performed to identify Bifidobacterium spp. in the gut, and gas chromatography-mass spectrometry was conducted for faecal-metabolite detection. The results showed that the structure of the gut microbiota changed, and BCAAem-supplementation in mice slowed the change speed of gut microbiota which is due to age. In addition, the abundance of the Akkermansia and Bifidobacterium increased in BCAAem-supplemented mice, while the ratio of Enterobacteriaceae decreased in BCAAem-supplemented mice. Moreover, 12 different metabolites, representing sugar and lipid metabolism, were altered between the supplemented and control groups. Thus, BCAAem influences the gut microbiota and gut metabolism. In addition, the BCAAem-supplemented group presented lower serum concentrations of lipopolysaccharide-binding protein. The changes are indicative of lower antigen loads in the host gut. These results suggest that dietary supplementation with BCAAem may be considered for improving health and promoting healthy aging.

Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples.

A rapid and sensitive recombinase polymerase amplification (RPA) assay, Bruce-RPA, was developed for detection of Brucella. The assay could detect as few as 3 copies of Brucella per reaction within 20 min. Bruce-RPA represents a candidate point-of-care diagnosis assay for human brucellosis.

A novel New Delhi metallo-ß-lactamase variant, NDM-14, isolated in a Chinese Hospital possesses increased enzymatic activity against carbapenems.

A novel New Delhi metallo-ß-lactamase (NDM) variant, NDM-14, was identified in clinical isolate Acinetobacter lwoffii JN49-1, which was recovered from an intensive care unit patient at a local hospital in China. NDM-14, which differs from other existing enzymes by an amino acid substitution at position 130 (Asp130Gly), possesses enzymatic activity toward carbapenems that is greater than that of NDM-1. Kinetic data indicate that NDM-14 has a higher affinity for imipenem and meropenem.

Emergence and prevalence of non-H2S-producing Salmonella enterica serovar Senftenberg isolates belonging to novel sequence type 1751 in China.

Salmonella enterica serovar Senftenberg is a common nontyphoidal Salmonella serotype which causes human Salmonella infections worldwide. In this study, 182 S. Senftenberg isolates, including 17 atypical non-hydrogen sulfide (H2S)-producing isolates, were detected in China from 2005 to 2011. The microbiological and genetic characteristics of the non-H2S-producing and selected H2S-producing isolates were determined by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis. The phs operons were amplified and sequenced. The 17 non-H2S-producing and 36 H2S-producing isolates belonged to 7 sequence types (STs), including 3 new STs, ST1751, ST1757, and ST1758. Fourteen of the 17 non-H2S-producing isolates belonged to ST1751 and had very similar PFGE patterns. All 17 non-H2S-producing isolates had a nonsense mutation at position 1621 of phsA. H2S-producing and non-H2S-producing S. Senftenberg isolates were isolated from the same stool sample from three patients; isolates from the same patients displayed the same antimicrobial susceptibility, ST, and PFGE pattern but could be discriminated based on CRISPR spacers. Non-H2S-producing S. Senftenberg isolates belonging to ST1751 have been prevalent in Shanghai, China. It is possible that these emerging organisms will disseminate further, because they are difficult to detect. Thus, we should strengthen the surveillance for the spread of this atypical S. Senftenberg variant.

Shigella flexneri T3SS effector IpaH4.5 modulates the host inflammatory response via interaction with NF-κB p65 protein.

Shigella species possess a type III secretion system (T3SS), which is required for human infection and that delivers effector proteins into target host cells. Here, we show that the effector, IpaH4.5 dampens the pro-inflammatory cytokine response. In both the Sereny test and a murine lung infection model, the Shigella ΔipaH4.5 mutant strain caused more severe inflammatory responses and significantly induced higher pro-inflammatory cytokine levels (MIP-2 and TNF-α) in the lung homogenates of wild type-infected mice. Moreover, there was a threefold decrease in bacterial colonization of the mutant compared with the WT and ΔipaH4.5/ipaH4.5-rescued strains. Yeast two-hybrid screening showed that IpaH4.5 specifically interacts with the p65 subunit of NF-κB. Ten truncated versions of IpaH4.5 and p65 spanning different regions were constructed and expressed to further map the IpaH binding sites with p65. The results revealed thatthe p65 region spanning amino acids 1-190 of p65 interacted with the IpaH4.5/1-293 N-terminal region. In vitro, IpaH4.5 displayed ubiquitin ligase activity towards ubiquitin and p65. Furthermore, the transcriptional activity of NF-κB was shown to be inhibited by IpaH4.5 utilizing a dual-luciferase reporter gene detection system containing NF-κB promoter response elements. Thus, we conclude that the IpaH4.5 protein is an E3 ubiquitin ligase capable of directly regulating the host inflammatory response by inhibiting the NF-κB signalling pathway.

T-2 toxin induces developmental toxicity and apoptosis in zebrafish embryos.

T-2 toxin is one of the most important trichothecene mycotoxins occurring in various agriculture products. The developmental toxicity of T-2 toxin and the exact mechanism of action at early life stages are not understood precisely. Zebrafish embryos were exposed to different concentrations of the toxin at 4-6 hours post fertilization (hpf) stage of development, and were observed for different developmental toxic effects at 24, 48, 72, and 144 hpf. Exposure to 0.20 µmol/L or higher concentrations of T-2 toxin significantly increased the mortality and malformation rate such as tail deformities, cardiovascular defects and behavioral changes in early developmental stages of zebrafish. T-2 toxin exposure resulted in significant increases in reactive oxygen species (ROS) production and cell apoptosis, mainly in the tail areas, as revealed by Acridine Orange staining at 24 hpf. In addition, T-2 toxin-induced severe tail deformities could be attenuated by co-exposure to reduced glutathione (GSH). T-2 toxin and GSH co-exposure induced a significant decrease of ROS production in the embryos. The overall results demonstrate that T-2 toxin is able to produce oxidative stress and induce apoptosis, which are involved in the developmental toxicity of T-2 toxin in zebrafish embryos.

Fructose uptake in Bifidobacterium longum NCC2705 is mediated by an ATP-binding cassette transporter.

Recently, a putative ATP-binding cassette (ABC) transport system was identified in Bifidobacterium longum NCC2705 that is highly up-regulated during growth on fructose as the sole carbon source. Cloning and expression of the corresponding ORFs (bl0033-0036) result in efficient fructose uptake by bacteria. Sequence analysis reveals high similarity to typical ABC transport systems and suggests that these genes are organized as an operon. Expression of FruE is induced by fructose, ribose, or xylose and is able to bind these sugars with fructose as the preferred substrate. Our data suggest that BL0033-0036 constitute a high affinity fructose-specific ABC transporter of B. longum NCC2705. We thus suggest to rename the coding genes to fruEKFG and the corresponding proteins to FruE (sugar-binding protein), FruK (ATPase subunit), FruF, and FruG (membrane permeases). Furthermore, protein-protein interactions between the components of the transporter complex were determined by GST pulldown and Western blot analysis. This revealed interactions between the membrane subunits FruF and FruG with FruE, which in vivo is located on the external side of the membrane, and with the cytoplasmatic ATPase FruK. This is in line with the proposed model for bacterial ABC sugar transporters.

Multidrug-resistant atypical variants of Shigella flexneri in China.

We identified 3 atypical Shigella flexneri varieties in China, including 92 strains with multidrug resistance, distinct pulse types, and a novel sequence type. Atypical varieties were prevalent mainly in developed regions, and 1 variant has become the dominant Shigella spp. serotype in China. Improved surveillance will help guide the prevention and control of shigellosis.

Abnormal fecal microbiota community and functions in patients with hepatitis B liver cirrhosis as revealed by a metagenomic approach.

BACKGROUND: Assessment and characterization of human colon microbiota is now a major research area in human diseases, including in patients with hepatitis B liver cirrhosis (HBLC). METHODS: We recruited 120 patients with HBLC and 120 healthy controls. The fecal microbial community and functions in the two groups were analyzed using high-throughput Solexa sequencing of the complete metagenomic DNA and bioinformatics methods. RESULTS: Community and metabolism-wide changes of the fecal microbiota in 20 HBLC patients and 20 healthy controls were observed and compared. A negative correlation was observed between the Child-Turcotte-Pugh scores and Bacteroidetes (P < 0.01), whereas a positive correlation was observed between the scores and Enterobacteriaceae and Veillonella (P < 0.01). Analysis of the additional 200 fecal microbiota samples demonstrated that these intestinal microbial markers might be useful for distinguishing liver cirrhosis microbiota samples from normal ones. The functional diversity was significantly reduced in the fecal microbiota of cirrhotic patients compared with in the controls. At the module or pathway levels, the fecal microbiota of the HBLC patients showed enrichment in the metabolism of glutathione, gluconeogenesis, branched-chain amino acid, nitrogen, and lipid (P < 0.05), whereas there was a decrease in the level of aromatic amino acid, bile acid and cell cycle related metabolism (P < 0.05). CONCLUSIONS: Extensive differences in the microbiota community and metabolic potential were detected in the fecal microbiota of cirrhotic patients. The intestinal microbial community may act as an independent organ to regulate the body's metabolic balance, which may affect the prognosis for HBLC patients.

Changes of predominant species/biovars and sequence types of Brucella isolates, Inner Mongolia, China.

BACKGROUND: Human brucellosis incidence in China was divided into 3 stages, high incidence (1950-1960s), decline (1970-1980s) and re-emergence (1990-2000s). Human brucellosis has been reported in all the 32 provinces, of which Inner Mongolia has the highest prevalence, accounting for over 40% of the cases in China. To investigate the etiology alteration of human brucellosis in Inner Mongolia, the species, biovars and genotypes of 60 Brucella isolates from this province were analyzed. METHODS: Species and biovars of the Brucella strains isolated from outbreaks were determined based on classical identification procedures. Strains were genotyped by multi locus sequence typing (MLST). Sequences of 9 housekeeping genes were obtained and sequence types were defined. The distribution of species, biovars and sequence types (STs) among the three incidence stages were analyzed and compared. RESULTS: The three stages of high incidence, decline and re-emergence were predominated by B. melitensis biovar 2 and 3, B. abortus biovar 3, and B. melitensis biovar 1, respectively, implying changes in the predominant biovars. Genotyping by MLST revealed a total of 14 STs. Nine STs (from ST28 to ST36), accounting for 64.3% of all the STs, were newly defined and different from those observed in other countries. Different STs were distributed among the three stages. ST8 was the most common ST in 1950-1960s and 1990-2000s, while ST2 was the most common in 1970-1980s. CONCLUSIONS: The prevalence of biovars and sequence types of Brucella strains from Inner Mongolia has changed over time in the three stages. Compared with those from other countries, new sequence types of Brucella strains exist in China.

Genome sequences of Brucella melitensis 16M and its two derivatives 16M1w and 16M13w, which evolved in vivo.

Brucella melitensis is an intracellular pathogen that induces chronic infection in humans. Here, we report the genome sequences of 16M and its two derivatives, 16M1w and 16M13w, which were allowed to adapt in vivo for 1 and 13 weeks, respectively. Our findings contribute to the investigation of adaptive mutations and mechanisms of chronic infection by B. melitensis.

Genome sequences of three live attenuated vaccine strains of Brucella species and implications for pathogenesis and differential diagnosis.

Live attenuated vaccines play essential roles in the prevention of brucellosis. Here, we report the draft genome sequences of three vaccine strains, Brucella melitensis M5-10, B. suis S2-30, and B. abortus 104M. Primary genome sequence analysis identified mutations, deletions, and insertions which have implications for attenuation and signatures for differential diagnosis.

Complete genome sequence of Brucella melitensis 133, an isolate of biovar 1 of sequence type 32.

Brucellosis is highly epidemic in China. Of the six classical species, Brucella melitensis and biovar 1 are the most represented species and biovar that cause human brucellosis in China. Here, we report the genome sequence of Brucella melitensis strain 133, a strain of biovar 1 of sequence type 32.

Complete genome sequence of Brucella suis field strain BCB025 of sequence type ST22.

Brucella is a genus of relatively conservative pathogenic bacteria. Brucella suis is the most diversified Brucella species. Strains of B. suis belong to different sequence types. Here, we report the genome sequence of B. suis strain BCB025, one isolate of the sequence type 22 epidemic in China.

Genome sequence of Brucella melitensis strain 128, an isolate of biovar 3 of sequence type 8.

Brucella melitensis is the most common Brucella species causing human brucellosis. B. melitensis is divided into 3 biovars. Here, we report the complete genome sequence of B. melitensis strain 128, a strain of biovar 3 of sequence type 8, which is prevalent in China.