Enhanced Botrytis cinerea resistance of Arabidopsis plants grown in compost may be explained by increased expression of defense-related genes, as revealed by microarray analysis

Composts are the products obtained after the aerobic degradation of different types of organic matter waste and can be used as substrates or substrate/soil amendments for plant cultivation. There is a small but increasing number of reports that suggest that foliar diseases may be reduced when using compost, rather than standard substrates, as growing medium. The purpose of this study was to examine the gene expression alteration produced by the compost to gain knowledge of the mechanisms involved in compost-induced systemic resistance. A compost from olive marc and olive tree leaves was able to induce resistance against Botrytis cinerea in Arabidopsis, unlike the standard substrate, perlite. Microarray analyses revealed that 178 genes were differently expressed, with a fold change cut-off of 1, of which 155 were up-regulated and 23 were down-regulated in compost-grown, as against perlite-grown plants. A functional enrichment study of up-regulated genes revealed that 38 Gene Ontology terms were significantly enriched. Response to stress, biotic stimulus, other organism, bacterium, fungus, chemical and abiotic stimulus, SA and ABA stimulus, oxidative stress, water, temperature and cold were significantly enriched, as were immune and defense responses, systemic acquired resistance, secondary metabolic process and oxireductase activity. Interestingly, PR1 expression, which was equally enhanced by growing the plants in compost and by B. cinerea inoculation, was further boosted in compost-grown pathogen-inoculated plants. Compost triggered a plant response that shares similarities with both systemic acquired resistance and ABA-dependent/independent abiotic stress responses.

The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Transfer of penicillin resistance between Neisseriae in microcosm

Horizontal gene transfer between commensal and pathogenic Neisseriae is the mechanism proposed to explain how pathogenic species acquire altered portions of the penA gene, which encodes penicillin binding protein 2. These changes resulted in a moderately penicillin-resistant phenotype in the meningococci, whose frequency of isolation in Spain increased at the end of the 1980s. Little has been published about the possibility of this gene transfer in nature or about its simulation in the laboratory. We designed a simple microcosm, formed by solid and liquid media, that partially mimics the upper human respiratory tract. In this microcosm, penicillin-resistant commensal strains and the fully susceptible meningococcus were co-cultivated. The efficiency of gene transfer between the strains depended on the phase of bacterial growth and the conditions of culture. Resistance of penicillin was acquired in different steps irrespective of the source of the DNA. The presence of DNase in the medium had no effect on gene transfer, but it was near zero when nicked DNA was used. Cell-to-cell contact or membrane blebs could explain these results. The analysis of sequences of the transpeptidase domain of PBP2 from transformants, and from donor and recipient strains demonstrated that the emergence of moderately resistant transformants was due to genetic exchange between the co-cultivated strains. Finally, mechanisms other than penA modification could be invoked to explain decreased susceptibility

Potential impact of environmental bacteriophages in spreading antibiotic resistance genes

The idea that bacteriophage transduction plays a role in the horizontal transfer of antibiotic resistance genes is gaining momentum. Such transduction might be vital in horizontal transfer from environmental to human bodyassociated biomes and here we review many lines of evidence supporting this notion. It is well accepted that bacteriophages are the most abundant entities in most environments, where they have been shown to be quite persistent. This fact, together with the ability of many phages to infect bacteria belonging to different taxa, makes them suitable vehicles for gene transfer. Metagenomic studies confirm that substantial percentages of the bacteriophage particles present in most environments contain bacterial genes, including mobile genetic elements and antibiotic resistance genes. When specific genes of resistance to antibiotics are detected by real-time PCR in the bacteriophage populations of different environments, only tenfold lower numbers of these genes are observed, compared with those found in the corresponding bacterial populations. In addition, the antibiotic resistance genes from these bacteriophages are functional and generate resistance to the bacteria when these genes are transfected. Finally, reports about the transduction of antibiotic resistance genes are on the increase.

The role of Serratia marcescens porins in antibiotic resistance

The outer membrane permeability of Serratia marcescens was studied by comparing porin-deficient mutants with their parental strains. Omp1-deficient strains were selected by moxalactam resistance, whereas mutants lacking the Omp2 porin were obtained by experimental infection with the SMP2 phage, whose primary receptor is the Omp2 porin. The role of porins was demonstrated in quinolone accumulation assays, where semi-quantitative differences in accumulation were observed. Permeability coefficients to cephaloridine of Omp1 mutants were determined and compared with those of the parental strain. The clinical isolates S. marcescens HCPR1 and 866 showed 30- to 200-fold reduced permeability coefficients when Omp1 porin was absent

The role of Serratia marcescens porins in antibiotic resistance

The outer membrane permeability of Serratia marcescens was studied by comparing porin-deficient mutants with their parental strains. Omp1-deficient strains were selected by moxalactam resistance, whereas mutants lacking the Omp2 porin were obtained by experimental infection with the SMP2 phage, whose primary receptor is the Omp2 porin. The role of porins was demonstrated in quinolone accumulation assays, where semi-quantitative differences in accumulation were observed. Permeability coefficients to cephaloridine of Omp1 mutants were determined and compared with those of the parental strain. The clinical isolates S. marcescens HCPR1 and 866 showed 30- to 200-fold reduced permeability coefficients when Omp1 porin was absent

The role of Serratia marcescens porins in antibiotic resistance

The outer membrane permeability of Serratia marcescens was studied by comparing porin-deficient mutants with their parental strains. Omp1-deficient strains were selected by moxalactam resistance, whereas mutants lacking the Omp2 porin were obtained by experimental infection with the SMP2 phage, whose primary receptor is the Omp2 porin. The role of porins was demonstrated in quinolone accumulation assays, where semi-quantitative differences in accumulation were observed. Permeability coefficients to cephaloridine of Omp1 mutants were determined and compared with those of the parental strain. The clinical isolates S. marcescens HCPR1 and 866 showed 30- to 200-fold reduced permeability coefficients when Omp1 porin was absent

The role of Serratia marcescens porins in antibiotic resistance

The outer membrane permeability of Serratia marcescens was studied by comparing porin-deficient mutants with their parental strains. Omp1-deficient strains were selected by moxalactam resistance, whereas mutants lacking the Omp2 porin were obtained by experimental infection with the SMP2 phage, whose primary receptor is the Omp2 porin. The role of porins was demonstrated in quinolone accumulation assays, where semi-quantitative differences in accumulation were observed. Permeability coefficients to cephaloridine of Omp1 mutants were determined and compared with those of the parental strain. The clinical isolates S. marcescens HCPR1 and 866 showed 30- to 200-fold reduced permeability coefficients when Omp1 porin was absent

Impact of antibiotic resistance on chemotherapy for pneumococcal infections

Over the past three decades, penicillin-resistant pneumococci have emerged worldwide. In addition, penicillin-resistant strains have also decreased susceptibility to other β-lactams (including cephalosporins) and these strains are often resistant to other antibiotic groups, making the treatment options much more difficult. Nevertheless, the present in vitro definitions of resistance to penicillin and cephalosporins in pneumococci could not be appropriated for all types of pneumococcal infections. Thus, current levels of resistance to penicillin and cephalosporin seem to have little, if any, clinical relevance in nonmeningeal infections (e.g., pneumonia or bacteremia). On the contrary, numerous clinical failures have been reported in patients with pneumococcal meningitis caused by strains with MICs ≥ 0.12 μg/ml, and penicillin should never be used in pneumococcal meningitis except when the strain is known to be fully susceptible to this drug. Today, therapy for pneumococcal meningitis should mainly be selected on the basis of susceptibility to cephalosporins, and most patients may currently be treated with high-dose cefotaxime (±) vancomycin, depending on the levels of resistance in the patient's geographic area. In this review, we present a practical approach, based on current levels of antibiotic resistance, for treating the most prevalent pneumococcal infections. However, it should be emphasized that the most appropriate antibiotic therapy for infections caused by resistant pneumococci remains controversial, and comparative, randomized studies are urgently needed to clarify the best antibiotic therapy for these infections

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.

An update of the mechanisms of resistance to EGFR-tyrosine kinase inhibitors in breast cancer: gefitinib (Iressatrade mark)-induced changes in the expression and nucleo-cytoplasmic trafficking of HER-ligands (Review)

Intrinsic resistance to the epidermal growth factor receptor (EGFR; HER1) tyrosine kinase inhibitor (TKI) gefitinib, and more generally to EGFR TKIs, is a common phenomenon in breast cancer. The availability of molecular criteria for predicting sensitivity to EGFR-TKIs is, therefore, the most relevant issue for their correct use and for planning future research. Though it appears that in non-small-cell lung cancer (NSCLC) response to gefitinib is directly related to the occurrence of specific mutations in the EGFR TK domain, breast cancer patients cannot be selected for treatment with gefitinib on the same basis as such EGFR mutations have beenreported neither in primary breast carcinomas nor in several breast cancer cell lines. Alternatively, there is a generalagreement on the hypothesis that the occurrence of molecular alterations that activate transduction pathways downstreamof EGFR (i.e., MEK1/MEK2 - ERK1/2 MAPK and PI-3'K - AKT growth/survival signaling cascades) significantly affect the response to EGFR TKIs in breast carcinomas. However,there are no studies so far addressing a role of EGF-related ligands as intrinsic breast cancer cell modulators of EGFR TKIefficacy. We recently monitored gene expression profiles andsub-cellular localization of HER-1/-2/-3/-4 related ligands (i.e., EGF, amphiregulin, transforming growth factor-α, ß-cellulin,epiregulin and neuregulins) prior to and after gefitinib treatment in a panel of human breast cancer cell lines. First, gefitinibinduced changes in the endogenous levels of EGF-related ligands correlated with the natural degree of breast cancer cellsensitivity to gefitinib. While breast cancer cells intrinsically resistant to gefitinib (IC50 ≥15 μM) markedly up-regulated(up to 600 times) the expression of genes codifying for HERspecific ligands, a significant down-regulation (up to 106 times)of HER ligand gene transcription was found in breast cancer cells intrinsically sensitive to gefitinib (IC50 ≤1 μM). Second,loss of HER1 function differentially regulated the nuclear trafficking of HER-related ligands. While gefitinib treatment induced an active import and nuclear accumulation of the HER ligand NRG in intrinsically gefitinib-resistant breastcancer cells, an active export and nuclear loss of NRG was observed in intrinsically gefitinib-sensitive breast cancer cells.In summary, through in vitro and pharmacodynamic studies we have learned that, besides mutations in the HER1 gene,oncogenic changes downstream of HER1 are the key players regulating gefitinib efficacy in breast cancer cells. It now appears that pharmacological inhibition of HER1 functionalso leads to striking changes in both the gene expression and the nucleo-cytoplasmic trafficking of HER-specific ligands,and that this response correlates with the intrinsic degree of breast cancer sensitivity to the EGFR TKI gefitinib. Therelevance of this previously unrecognized intracrine feedback to gefitinib warrants further studies as cancer cells could bypassthe antiproliferative effects of HER1-targeted therapeutics without a need for the overexpression and/or activation of other HER family members and/or the activation of HER-driven downstream signaling cascades

Review. Characterization and selection of hexaploid wheats containing resistance to Heterodera avenae or Mayetiola destructor introgressed from Aegilops

Cereal cyst nematode (CCN, Heterodera avenae) and Hessian fly (HF, Mayetiola destructor) are two major pests affecting wheat crops worldwide including important cereal areas of Spain. Aegilops ventricosa and Ae. triuncialis were used as donors in a strategy to introduce resistance genes (RG) for these pests in hexaploid wheat (Triticum aestivum L.). Two 42 chromosomes introgression lines have been derived from Ae. ventricosa: H-93-8 and H-93-33 carrying genes Cre2 and H27 conferring resistance to CCN and HF, respectively. Line TR-3531 with 42 chromosomes has been derived from Ae. triuncialis and carries RGs conferring resistance for CCN (Cre7) and for HF (H30). Alien material has been incorporated in lines H-93 by chromosomal substitution and recombination, while in line TR-3531 homoeologous recombination affecting small DNA fragments has played a major role. It has been demonstrated that Cre2, Cre7, H27 and H30 are major single dominant genes and not allelic of other previously described RGs. Biochemical and molecular-biology studies of the defense mechanism triggered by Cre2 and Cre7 have revealed specific induction of peroxidase and other antioxidant enzymes. In parallel to these basic studies advanced lines carrying resistance genes for CNN and/or HF have been developed. Selection was done using molecular markers for eventually «pyramiding» resistance genes. Several isozyme and RAPD markers have been described and, currently, new markers based on transposable elements and NBS-LRR sequences are being developed. At present, two advanced lines have already been included at the Spanish Catalogue of Commercial Plant Varieties.

Flow resistance equations for mountain rivers

Three models of flow resistance (a Keulegan-type logarithmic law and two models developed for large-scale roughness conditions: the full logarithmic law and a model based on an inflectional velocity profile) were calibrated, validated and compared using an extensive database (N = 1,533) from rivers and flumes, representative of a wide hydraulic and geomorphologic range in the field of gravel-bed and mountain channels. It is preferable to apply the model based on an inflectional velocity profile in the relative submergence (y/d90) interval between 0.5 and 15, while the full logarithmic law is preferable for values below 0.5. For high relative submergence, above 15, either the logarithmic law or the full logarithmic law can be applied. The models fitted to the coarser percentiles are preferable to those fitted to the median diameter, owing to the higher explanatory power achieved by setting a model, the smaller difference in the goodness-of-fit between the different models and the lower influence of the origin of the data (river or flume).

Effect of cattle on Salmonella carriage, diversity and antimicrobial resistance in free-ranging wild boar (Sus scrofa) in northeastern Spain

Salmonella is distributed worldwide and is a pathogen of economic and public health importance. As a multi-host pathogen with a long environmental persistence, it is a suitable model for the study of wildlife-livestock interactions. In this work, we aim to explore the spill-over of Salmonella between free-ranging wild boar and livestock in a protected natural area in NE Spain and the presence of antimicrobial resistance. Salmonella prevalence, serotypes and diversity were compared between wild boars, sympatric cattle and wild boars from cattle-free areas. The effect of age, sex, cattle presence and cattle herd size on Salmonella probability of infection in wild boars was explored by means of Generalized Linear Models and a model selection based on the Akaike’s Information Criterion. Prevalence was higher in wild boars co-habiting with cattle (35.67%, CI 95% 28.19–43.70) than in wild boar from cattle-free areas (17.54%, CI 95% 8.74–29.91). Probability of a wild boar being a Salmonella carrier increased with cattle herd size but decreased with the host age. Serotypes Meleagridis, Anatum and Othmarschen were isolated concurrently from cattle and sympatric wild boars. Apart from serotypes shared with cattle, wild boars appear to have their own serotypes, which are also found in wild boars from cattle-free areas (Enteritidis, Mikawasima, 4:b:- and 35:r:z35). Serotype richness (diversity) was higher in wild boars co-habiting with cattle, but evenness was not altered by the introduction of serotypes from cattle. The finding of a S. Mbandaka strain resistant to sulfamethoxazole, streptomycin and chloramphenicol and a S. Enteritidis strain resistant to ciprofloxacin and nalidixic acid in wild boars is cause for public health concern.

Assessment of insecticide resistance in eggs and neonate larvae of Cydia pomonella (Lepidoptera: Tortricidae)

Spanish Cydia pomonella (L.) field populations have developed resistance to several insecticide groups. Diagnostic concentrations were established as the LC90 calculated on a susceptible strain (S_Spain) for five and seven insecticides and tested on eggs and neonate larvae field populations, respectively. The three most relevant enzymatic detoxification systems (mixed-function oxidases (MFO), glutathione S-tranferases (GST) and esterases (EST)) were studied for neonate larvae. In eggs, 96% of the field populations showed a significantly lower efficacy when compared with the susceptible strain (S_Spain) and the most effective insecticides were fenoxycarb and thiacloprid. In neonate larvae, a significant loss of susceptibility to the insecticides was detected. Flufenoxuron, azinphos-methyl and phosmet showed the lowest efficacy, while lambda-cyhalothrin, alpha-cypermethrin and chlorpyrifos-ethyl showed the highest. Biochemical assays showed that the most important enzymatic system involved in insecticide detoxification was MFO, with highest enzymatic activity ratios (5.1–16.6 for neonates from nine field populations). An enhanced GST and EST activities was detected in one field population, with enzymatic activity ratios of threefold and fivefold for GST and EST, respectively, when compared with the susceptible strain. The insecticide bioassays showed that the LC90 used were effective as diagnostic concentrations. Measures of MFO activity alongside bioassays with insecticide diagnostic concentrations could be used as tools for monitoring insecticide resistance in neonate larvae of C. pomonella.