Resistance-associated Genes Research Articles

Genome-wide assessment of population structure, linkage disequilibrium and resistant QTLs in Chinese wild grapevine

Chinese wild grapevine is an important germplasm source worldwide; thus, it is necessary to study its genetic diversity, population structure, and linkage disequilibrium (LD). In this study, 81 strains of Chinese wild grapevine were studied and constituted the core collection. These strains were genotyped according to 160 genome-wide SSR (simple sequence repeat) markers. All SSR markers were widely spread across the strains and revealed a total of 3469 alleles. After population structure analysis, all the Chinese wild grapevine strains were grouped into two subgroups that were well matched with the geographical distribution and the results published by our research team. Of all SSR pairs, 8.3% (P<0.01) were in LD. For all inner chromosomal loci pairs, the mean r2 value was 0.0229. The LD decay distance across Chinese wild grapevine was up to 14.13cM, below the critical r2 value of 0.1, following y=−0.0383 ln (x)+0.2016. According to both the linear mixed model and general linear model, the associations of the 160 SSR markers with grape white rot resistance were calculated. Five associated markers were located, four of which had been previously associated with other resistance traits and one of which was new. Chinese wild grapevine association mapping was performed to anchor useful quantitative trait loci (QTLs), and the five markers identified can help us anchor additional resistance-associated genes.

Prevalence and antimicrobial resistance of Salmonella isolates in Moroccan laying hens farms

Increasing emergence of salmonellosis presents a threat to the effective control of foodborne disease in humans. The purpose of this study was to evaluate the prevalence of drug susceptibility and molecular characteristics of non-typhoidal Salmonella (NTS) isolated from laying hens (LH) in 3 Moroccan regions, Rabat-Salé-Zemmour-Zaër (RSZZ), Souss-Massa-Drâa (SMD), and the grand Casablanca (GC). A total of 351 samples were collected from 30 consumer egg laying houses at the end of the egg laying period from April to July 2011. Sixty-four out of these 351 examined samples were contaminated by Salmonella. The Salmonella isolated strains were then serotyped and tested for drug susceptibility and analyzed by polymerase chain reaction (PCR) for the presence of the invasion-associated genes invA and spvC and nalidixic acid resistance-associated qnr gene. The prevalence of NTS infection in LH was estimated to be 73.3%. Seven Salmonella enterica serovars were identified: Enteritidis (37.5%), Kentucky (31.2%), Infantis (10.9%), Typhimurium (6.2%), Thompson (6.2%), Agona (4.6%), and Amsterdam (3.1%). Drug susceptibility testing showed that 65.6% of Salmonella were resistant to at least one antibiotic and 25% were resistant to ciprofloxacin. All isolates were positive for the invasion gene invA and 28% of them were positive for the virulence gene spvC. All nalidixic acid-resistant S. Enteritidis isolates were negative for qnr plasmid genes. Our findings clearly suggest the necessity to establish an NTS monitoring and control program for LH in Morocco.

Exploring the quantitative resistance to Pseudomonas syringae pv. phaseolicola in common bean (Phaseolus vulgaris L.)

Pseudomonas syringae pv. phaseolicola is an important disease that causes halo blight in common bean. The genetic mechanisms underlying quantitative halo blight resistance are poorly understood in this species, as most disease studies have focused on qualitative resistance. The present work examines the genetic basis of quantitative resistance to the nine halo blight races in different organs (primary and trifoliate leaf, stem and pod) of an Andean recombinant inbred line (RIL) progeny. Using a multi-environment quantitative trait locus (QTL) mapping approach, 76 and 101 main-effect and epistatic QTLs were identified, respectively. Most of the epistatic interactions detected were due to loci without detectable QTL additive main effects. Main and epistatic QTLs detected were mainly consistent across the environment conditions. The homologous genomic regions corresponding to 26 of the 76 main-effect detected QTLs were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins and known defence genes. Main-effect QTLs for resistance to races 3, 4 and 5 in leaf, stem and pod were located on chromosome 2 within a 3.01-Mb region, where a cluster of nine NL genes was detected. The NL gene Phvul.002G323300 is located in this region, which can be considered an important putative candidate gene for the non-organ-specific QTL identified here. The present research provides essential information not only for the better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for halo blight resistance in common bean.

Downregulation of ASPP2 improves hepatocellular carcinoma cells survival via promoting BECN1-dependent autophagy initiation.

Autophagy is an important catabolic process, which sustains intracellular homeostasis and lengthens cell survival under stress. Here we identify the ankyrin-repeat-containing, SH3-domain-containing, and proline-rich region-containing protein 2 (ASPP2), a haploinsufficient tumor suppressor, as a molecular regulator of starvation-induced autophagy in hepatocellular carcinoma (HCC). ASPP2 expression is associated with an autophagic response upon nutrient deprivation and downregulation of ASPP2 facilitates autophagic flux, whereas overexpression of ASPP2 blocks this starvation-induced autophagy in HCC cells. Mechanistically, ASPP2 inhibits autophagy through regulating BECN1 transcription and formation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) complex. Firstly, ASPP2 inhibits p65/RelA-induced transcription of BECN1, directly by an ASPP2-p65/RelA-IκBα complex which inhibits phosphorylation of IκBα and the translocation of p65/RelA into the nucleus. Secondly, ASPP2 binds to BECN1, leading to decreased binding of PIK3C3 and UV radiation resistance-associated gene (UVRAG), and increased binding of Rubicon in PIK3C3 complex. Downregulation of ASPP2 enhances the pro-survival and chemoresistant property via autophagy in HCC cells in vitro and in vivo. Decreased ASPP2 expression was associated with increased BECN1 and poor survival in HCC patients. Therefore, ASPP2 is a key regulator of BECN1-dependent autophagy, and decreased ASPP2 may contribute to tumor progression and chemoresistance via promoting autophagy.

Multistep regulation of autophagy by WNK1

The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertension-related disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNA-mediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phosphatidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phosphorylation, and activation of the kinase upstream of ULK1, the AMP-activated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related proline-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or -starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.

Genomic epidemiology of Lineage 4 Mycobacterium tuberculosis subpopulations in New York City and New Jersey, 1999-2009.

BackgroundWhole genome sequencing (WGS) has rapidly become an important research tool in tuberculosis epidemiology and is likely to replace many existing methods in public health microbiology in the near future. WGS-based methods may be particularly useful in areas with less diverse Mycobacterium tuberculosis populations, such as New York City, where conventional genotyping is often uninformative and field epidemiology often difficult. This study applies four candidate strategies for WGS-based identification of emerging M. tuberculosis subpopulations, employing both phylogenomic and population genetics methods.Results M. tuberculosis subpopulations in New York City and New Jersey can be distinguished via phylogenomic reconstruction, evidence of demographic expansion and subpopulation-specific signatures of selection, and by determination of subgroup-defining nucleotide substitutions. These methods identified known historical outbreak clusters and previously unidentified subpopulations within relatively monomorphic M. tuberculosis endemic clone groups. Neutrality statistics based on the site frequency spectrum were less useful for identifying M. tuberculosis subpopulations, likely due to the low levels of informative genetic variation in recently diverged isolate groups. In addition, we observed that isolates from New York City endemic clone groups have acquired multiple non-synonymous SNPs in virulence- and growth-associated pathways, and relatively few mutations in drug resistance-associated genes, suggesting that overall pathoadaptive fitness, rather than the acquisition of drug resistance mutations, has played a central role in the evolutionary history and epidemiology of M. tuberculosis subpopulations in New York City.ConclusionsOur results demonstrate that some but not all WGS-based methods are useful for detection of emerging M. tuberculosis clone groups, and support the use of phylogenomic reconstruction in routine tuberculosis laboratory surveillance, particularly in areas with relatively less diverse M. tuberculosis populations. Our study also supports the use of wider-reaching phylogenomic and population genomic methods in tuberculosis public health practice, which can support tuberculosis control activities by identifying genetic polymorphisms contributing to epidemiological success in local M. tuberculosis populations and possibly explain why certain isolate groups are apparently more successful in specific host populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3298-6) contains supplementary material, which is available to authorized users.

Molecular Detection of Resistance to Azole Components.

Fungal infections have increased significantly in the last few years, and their outcomes are in part complicated by the emergence of antifungal drug-resistant pathogens. Together with Candida species, the mould Aspergillus fumigatus is one of the most prevalent organisms to cause invasive fungal disease. The molecular detection of (tri)azole resistance in both Candida and Aspergillus species may represent a useful means of monitoring the incidence of clinical isolates with antifungal resistance-associated gene alterations. Here, we describe molecular methods that have been developed to allow for accurate detection of azole-resistant isolates among C. glabrata and A. fumigatus fungal species.

Molecular Detection of Resistance to Echinocandins.

In the last years, life-threatening fungal diseases have increased significantly, due to the rising number of human individuals susceptible to fungal infections, which are in part complicated by the emergence of antifungal drug-resistant pathogens. Among yeasts, Candida albicans and Candida glabrata are the most common organisms responsible for invasive fungal diseases. The molecular detection of echinocandin resistance in Candida species may represent a useful means of monitoring the incidence of clinical isolates with antifungal resistance-associated gene alterations. Here, we describe the current methods that enable researchers and/or clinical microbiologists to accurately detect echinocandin-resistant isolates of C. albicans and C. glabrata.

Downregulation of autophagy gene expression in endometria from women with polycystic ovary syndrome

Autophagy, a process of controlled cellular self-digestion, could be involved in cyclic remodeling of the human endometrium. We investigated endometrial mRNA expression of 23 autophagy-related (ATG) genes and transcription factors in healthy controls (n = 12) and anovulatory polycystic ovary syndrome (PCOS) patients (n = 24), as well as in their subgroup (n = 12) before and after metformin treatment. The mRNA levels of transcription factor forkhead box protein O1 (FOXO1) and several molecules involved in autophagosome formation (ATG13, RB1-inducible coiled-coil 1), autophagosome nucleation (ATG14, beclin 1, SH3-domain GRB2-like endophilin B1), autophagosome elongation (ATG3, ATG5, γ-aminobutyric acid receptor-associated protein - GABARAP), and delivery of ubiquitinated proteins to autophagosomes (sequestosome 1), were significantly reduced in anovulatory PCOS compared to healthy endometrium. Free androgen index, but not free estrogen index, insulin levels, or body mass index, negatively correlated with the endometrial expression of ATG3, ATG14, and GABARAP in PCOS patients. Treatment of PCOS patients with metformin (2 g/day for 3 months) significantly increased the endometrial mRNA levels of FOXO1, ATG3, and UV radiation resistance-associated gene. These data suggest that increased androgen availability in PCOS is associated with metformin-sensitive transcriptional downregulation of endometrial autophagy.

Genomic Epidemiology of Lineage 4 Mycobacterium tuberculosis Subpopulations in New York City and New Jersey, 1999–2009

Whole genome sequencing (WGS) has rapidly become an important research tool in tuberculosis epidemiology and is likely to replace many existing methods in public health microbiology in the near future. WGS-based methods may be particularly useful in areas with less diverse Mycobacterium tuberculosis populations, such as New York City, where conventional genotyping is often uninformative and field epidemiology often difficult. This study applies four candidate strategies for WGS-based identification of emerging M. tuberculosis subpopulations, employing both phylogenomic and population genetics methods. M. tuberculosis subpopulations in New York City and New Jersey can be distinguished via phylogenomic reconstruction, evidence of demographic expansion and subpopulation-specific signatures of selection, and by determination of subgroup-defining nucleotide substitutions. These methods identified known historical outbreak clusters and previously unidentified subpopulations within relatively monomorphic M. tuberculosis endemic clone groups. Neutrality statistics based on the site frequency spectrum were less useful for identifying M. tuberculosis subpopulations, likely due to the low levels of informative genetic variation in recently diverged isolate groups. In addition, we observed that isolates from New York City endemic clone groups have acquired multiple non-synonymous SNPs in virulence- and growth-associated pathways, and relatively few mutations in drug resistance-associated genes, suggesting that overall pathoadaptive fitness, rather than the acquisition of drug resistance mutations, has played a central role in the evolutionary history and epidemiology of M. tuberculosis subpopulations in New York City. Our results demonstrate that some but not all WGS-based methods are useful for detection of emerging M. tuberculosis clone groups, and support the use of phylogenomic reconstruction in routine tuberculosis laboratory surveillance, particularly in areas with relatively less diverse M. tuberculosis populations. Our study also supports the use of wider-reaching phylogenomic and population genomic methods in tuberculosis public health practice, which can support tuberculosis control activities by identifying genetic polymorphisms contributing to epidemiological success in local M. tuberculosis populations and possibly explain why certain isolate groups are apparently more successful in specific host populations.

Antimicrobial Susceptibility of Autochthonous Aquatic Vibrio cholerae in Haiti.

We investigated the antimicrobial susceptibility of 50 environmental isolates of Vibrio cholerae non-O1/non-O139 collected in surface waters in Haiti in July 2012, during an active cholera outbreak. A panel of 16 antibiotics was tested on the isolates using the disk diffusion method and PCR detection of seven resistance-associated genes (strA/B, sul1/2, ermA/B, and mefA). All isolates were susceptible to amoxicillin-clavulanic acid, cefotaxime, imipenem, ciprofloxacin, norfloxacin, amikacin, and gentamicin. Nearly a quarter (22.0%) of the isolates were susceptible to all 16 antimicrobials tested and only 8.0% of the isolates (n = 4) were multidrug-resistant. The highest proportions of resistant isolates were observed for sulfonamide (70.0%), amoxicillin (12.0%), and trimethoprim-sulfamethoxazole (10.0%). One strain was resistant to erythromycin and one to doxycycline, two antibiotics used to treat cholera in Haiti. Among the 50 isolates, 78% possessed at least two resistance-associated genes, and the genes sul1, ermA, and strB were detected in all four multidrug-resistant isolates. Our results clearly indicate that the autochthonous population of V. cholerae non-O1/non-O139 found in surface waters in Haiti shows antimicrobial patterns different from that of the outbreak strain. The presence in the Haitian aquatic environment of V. cholerae non-O1/non-O139 with reduced susceptibility or resistance to antibiotics used in human medicine may constitute a mild public health threat.

ZNF32 contributes to the induction of multidrug resistance by regulating TGF-β receptor 2 signaling in lung adenocarcinoma.

Multidrug resistance (MDR) is one of the most important contributors to the high mortality of cancer and remains a major concern. We previously found that zinc finger protein 32 (ZNF32), an important transcription factor associated with cancer in Homo sapiens, protects tumor cells against cell death induced by oxidative stress and other stimuli. We thus hypothesized that ZNF32 might enable the tolerance of cancer cells to anti-tumor drugs because higher ZNF32 expression has been found in cancer tissues and in drug-resistant lung adenocarcinoma (AC) cells. In this study, we found that ZNF32 is upregulated by Sp1 (specificity protein 1) in response to drug treatment and that ZNF32 promotes drug resistance and protects AC cells against cisplatin or gefitinib treatment. ZNF32 overexpression in AC cells conferred resistance to EGFR (epidermal growth factor receptor) inhibitors by enhancing MEK/ERK activation. Moreover, ZNF32 was found to directly bind to the TGF-βR2 (transforming growth factor-beta receptor 2) promoter to promote its expression, and ZNF32-induced resistance was mediated by enhancing TGF-βR2 expression and activating the TGF-βR2/SMAD2 pathway. In both a mouse model and ex vivo cultured patient samples, a high level of ZNF32 expression was closely associated with worse overall survival and cisplatin resistance. ZNF32 appears to be a potential inducer of drug resistance that could increase the expression of the drug resistance-associated gene TGF-βR2 and subsequently facilitate the induction of drug resistance during both conventional chemotherapy and novel target therapy. Thus, ZNF32-associated target therapy is a potential novel adjuvant therapy that might effectively prevent the occurrence of multidrug resistance (MDR) during chemotherapy and improve the survival of patients with AC.

Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria

The rpoB gene codes for the RNA polymerase β subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language.

PtdIns(4,5)P2 signaling regulates ATG14 and autophagy

Autophagy is a regulated self-digestion pathway with fundamental roles in cell homeostasis and diseases. Autophagy is regulated by coordinated actions of a series of autophagy-related (ATG) proteins. The Barkor/ATG14(L)-VPS34 (a class III phosphatidylinositol 3-kinase) complex and its product phosphatidylinositol 3-phosphate [PtdIns(3)P] play key roles in autophagy initiation. ATG14 contains a C-terminal Barkor/ATG14(L) autophagosome-targeting sequence (BATS) domain that senses the curvature of PtdIns(3)P-containing membrane. The BATS domain also strongly binds PtdIns(4,5)P2, but the functional significance has been unclear. Here we show that ATG14 specifically interacts with type Iγ PIP kinase isoform 5 (PIPKIγi5), an enzyme that generates PtdIns(4,5)P2 in mammalian cells. Autophagosomes have associated PIPKIγi5 and PtdIns(4,5)P2 that are colocalized with late endosomes and the endoplasmic reticulum. PtdIns(4,5)P2 generation at these sites requires PIPKIγi5. Loss of PIPKIγi5 results in a loss of ATG14, UV irradiation resistance-associated gene, and Beclin 1 and a block of autophagy. PtdIns(4,5)P2 binding to the ATG14-BATS domain regulates ATG14 interaction with VPS34 and Beclin 1, and thus plays a key role in ATG14 complex assembly and autophagy initiation. This study identifies an unexpected role for PtdIns(4,5)P2 signaling in the regulation of ATG14 complex and autophagy.

Bioinformatics Identification of Drug Resistance-Associated Gene Pairs in Mycobacterium tuberculosis

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Due to the extensive use of anti-tuberculosis drugs and the development of mutations, the emergence and spread of multidrug-resistant tuberculosis is recognized as one of the most dangerous threats to global tuberculosis control. Some single mutations have been identified to be significantly linked with drug resistance. However, the prior research did not take gene-gene interactions into account, and the emergence of transmissible drug resistance is connected with multiple genetic mutations. In this study we use the bioinformatics software GBOOST (The Hong Kong University, Clear Water Bay, Kowloon, Hong Kong, China) to calculate the interactions of Single Nucleotide Polymorphism (SNP) pairs and identify gene pairs associated with drug resistance. A large part of the non-synonymous mutations in the drug target genes that were included in the screened gene pairs were confirmed by previous reports, which lent sound solid credits to the effectiveness of our method. Notably, most of the identified gene pairs containing drug targets also comprise Pro-Pro-Glu (PPE) family proteins, suggesting that PPE family proteins play important roles in the drug resistance of Mtb. Therefore, this study provides deeper insights into the mechanisms underlying anti-tuberculosis drug resistance, and the present method is useful for exploring the drug resistance mechanisms for other microorganisms.

WGS analysis and molecular resistance mechanisms of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae isolates in Europe from 2009 to 2014.

To elucidate the genome-based epidemiology and phylogenomics of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae strains collected in 2009-14 in Europe and clarify the azithromycin resistance mechanisms. Seventy-five azithromycin-resistant (MIC 4 to >256 mg/L) N. gonorrhoeae isolates collected in 17 European countries during 2009-14 were examined using antimicrobial susceptibility testing and WGS. Thirty-six N. gonorrhoeae multi-antigen sequence typing STs and five phylogenomic clades, including 4-22 isolates from several countries per clade, were identified. The azithromycin target mutation A2059G (Escherichia coli numbering) was found in all four alleles of the 23S rRNA gene in all isolates with high-level azithromycin resistance (n = 4; MIC ≥256 mg/L). The C2611T mutation was identified in two to four alleles of the 23S rRNA gene in the remaining 71 isolates. Mutations in mtrR and its promoter were identified in 43 isolates, comprising isolates within the whole azithromycin MIC range. No mutations associated with azithromycin resistance were found in the rplD gene or the rplV gene and none of the macrolide resistance-associated genes [mef(A/E), ere(A), ere(B), erm(A), erm(B), erm(C) and erm(F)] were identified in any isolate. Clonal spread of relatively few N. gonorrhoeae strains accounts for the majority of the azithromycin resistance (MIC >2 mg/L) in Europe. The four isolates with high-level resistance to azithromycin (MIC ≥256 mg/L) were widely separated in the phylogenomic tree and did not belong to any of the main clades. The main azithromycin resistance mechanisms were the A2059G mutation (high-level resistance) and the C2611T mutation (low- and moderate-level resistance) in the 23S rRNA gene.

Adriamycin resistance-associated prohibitin gene inhibits proliferation of human osteosarcoma MG63 cells by interacting with oncogenes and tumor suppressor genes.

The resistance of cancer cells to chemotherapeutic agents is a major obstacle for successful chemotherapy, and the mechanism of chemoresistance remains unclear. The present study developed an adriamycin-resistant human osteosarcoma MG-63 sub-line (MG-63/ADR), and identified differentially expressed proteins that may be associated with adriamycin resistance. Two dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis and a protein identification assay were performed. Western blot analysis was used to examine the prohibitin (PHB) levels in the MG-63/ADR cells. Quantitative polymerase chain reaction was utilized to detect adriamycin resistant-associated genes. Laser-scanning confocal microscope was employed to examine the colocalization of PHB with v-myc avian myelocytomatosis viral oncogene homolog (c-myc), FBJ murine osteosarcoma viral oncogene homolog (c-fos), tumor protein p53 and retinoblastoma 1 (Rb). In addition, the full length of the open reading frame of human PHB was subcloned into a lentiviral vector pLVX-puro. The proliferative rate of MG-63 cells was also investigated. The overall protein expression in MG-63/ADR cells was clearly suppressed. Three notable protein regions, representing high mobility group box 1, Ras homolog gene family, member A, and PHB, were identified to be significantly altered in MG-63/ADR cells when compared with its parental cells. Therefore, PHB modulated the chemoresistance of MG-63/ADR cells by interacting with multiple oncogenes or tumor suppressor genes (c-myc, c-fos, p53 and Rb). In addition, overexpression of PHB decreases the proliferative rate of MG-63 cells. In conclusion, PHB is an adriamycin resistance-associated gene, which may inhibit the proliferation of human osteosarcoma MG-63 cells by interacting with the oncogenes or tumor suppressor genes, c-myc, c-fos, p53 and Rb.

Molecular Characterization and Epidemiologic Study of NDM-1-Producing Extensively Drug-ResistantEscherichia coli

The emergence and dissemination of NDM-1 (New Delhi metallo-β-lactamase-1)-producing Enterobacteriaceae have resulted in a worldwide public health risk. This study described a high incidence and endemic spread of NDM-1-producing extensively drug-resistant Escherichia coli in a teaching hospital in Zhejiang province, China. We recovered six nonduplicated NDM-1-producing E. coli isolates from May 2014 to August 2014 with positive modified Hodge test and EDTA synergistic test. These isolates were highly resistant to β-lactam antimicrobials, aminoglycosides, and quinolones. PCR and DNA sequences analysis showed that all isolates carried the blaNDM-1, blaSHV-11, aac(6')-ib-cr, and qnrB. Several isolates also harbored blaCTX-M-1, blaCTX-M-9, rmtB, and qnrA. Southern blot confirmed that blaNDM-1 was located on the same ∼55 kb plasmid and conjugation experiments further proved the contransferable characteristic of blaNDM-1. The ompC sequences showed various mutations, which was related to multidrug resistance in E. coli. Pulsed-field gel electrophoresis identified four of six isolates that belonged to the same genotype. Multilocus sequence typing assigned them to ST2, except one strain that belonged to ST594. Our study demonstrated that the resistance-associated genes and the loss of the outer membrane proteins could account for high resistance of NDM-1-producing E. coli to multiple antimicrobial drugs. Both horizontal transfer of IncN and transmission of ST2 were responsible for the spread of drug resistance. These findings highlighted an urgent need to limit the further dissemination of NDM-1-producing E. coli in this region.

The Effects and Mechanisms of Periplaneta americana Extract Reversal of Multi-Drug Resistance in BEL-7402/5-FU Cells

The present study reports the reversing effects of extracts from P. americana on multidrug resistance of BEL-7402/5-FU cells, as well as a preliminary investigation on their mechanism of action. A methylthiazolyl tetrazolium (MTT) method was applied to determine the multidrug resistance of BEL-7402/5-FU, while an intracellular drug accumulation assay was used to evaluate the effects of a column chromatography extract (PACC) and defatted extract (PADF) from P. americana on reversing multi-drug resistance. BEL-7402/5-FU reflected high resistance to 5-FU; PACC and PADF could promote drug accumulation in BEL-7402/5-FU cells, among which PADF was more effective than PACC. Moreover, results from the immunocytochemical method showed that PACC and PADF could downregulate the expression of drug resistance-associated proteins (P-gp, MRP, LRP); PACC and PADF had no effects on the expression of multidrug resistance-associated enzymes (GST-π), but PACC could increase the expression of multidrug resistance-associated enzymes (PKC). Results of real-time fluorescence quantitative PCR revealed that PACC and PADF were able to markedly inhibit the expression of multidrug resistance-associated genes (MDR1, LRP and MRP1); PACC presented a significant impact on the gene expression of multidrug resistance-associated enzymes, which increased the gene expression of GST-π and PKC. However, PADF had little impact on the expression of multidrug resistance-associated enzymes. These results demonstrated that PACC and PADF extracted from P. americana could effectively reverse MDR in BEL-7402/5-FU cells, whose mechanism was to inhibit the expression of P-gp, MRP, and LRP, and that PADF was more effective in the reversal of MDR than did PACC. In addition, some of extracts from P. americana altered (sometimes increasing) the expression of multidrug resistance-associated enzymes.

Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, phase-2 and transporter gene expression in mice

Zuotai (mainly β-HgS) and Zhusha (also called as cinnabar, mainly α-HgS) are used in traditional medicines in combination with herbs or even drugs in the treatment of various disorders, while mercury chloride (HgCl2) and methylmercury (MeHg) do not have known medical values but are highly toxic. This study aimed to compare the effects of mercury sulfides with HgCl2 and MeHg on hepatic drug processing gene expression. Mice were orally administrated with Zuotai (β-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHg (3.1mg/kg) for 7days, and the expression of genes related to phase-1 drug metabolism (P450), phase-2 conjugation, and phase-3 (transporters) genes were examined. The mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1–4 family genes and the corresponding nuclear receptors, except for a slight increase in PPARα and Cyp4a10 by HgCl2. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl2 and MeHg, but not by Zuotai and HgS. HgCl2 decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl2 and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes. In conclusion, Zuotai and HgS are different from HgCl2 and MeHg in hepatic drug processing gene expression; suggesting that chemical forms of mercury not only affect their disposition and toxicity, but also affect their effects on the expression of hepatic drug processing genes.