Molecular genetic approaches to the biology of the moss "Physcomitrella patens"

La mousse haplobiontique Physcomitrella patens est utilisée comme système génétique modèle pour l'étude du développement des plantes. Cependant, l'absence d'un protocole efficace de transformation a constitué jusqu'à présent un gros désavantage méthodologique pour le développement futur de ce système expérimental. Les résultats présentés dans le premier chapitre relatent la mise au point d'un protocole de transformation basé sur la technique de transfert direct de gènes dans des protoplastes par précipitation au PEG. Un essai d'expression transitoire de gènes a été mis au point. Ce protocole a été adapté afin de permettre l'introduction in vivo d'anticorps dans des protoplastes. Le protocole modifié permet d'introduire simultanément du DNA et des IgG dans les cellules, et nous avons démontré que ces anticorps peuvent inactiver spécifiquement le produit d'un gène co-introduit (GUS), ainsi que certaines protéines impliquées dans des processus cellulaires (tubuline). Cet essai, baptisé "essai transitoire d'immuno-inactivation in vivo", devrait être directement applicable à d'autres protoplastes végétaux, et permettre l'élaboration de nouvelles stratégies dans l'étude de processus cellulaires. Le second chapitre est consacré aux expériences de transformation de la mousse avec des gènes conférant une résistance à des antibiotiques. Nos résultats démontrent que l'intégration de gènes de résistance dans le génome de P. patens est possible, mais que cet événement est rare. Il s'agit là néanmoins de la première démonstration d'une transformation génétique réussie de cet organisme. L'introduction de gènes de résistance aux antibiotiques dans les protoplastes de P. patens génère à haute fréquence des clones résistants instables. Deux classes de clones instables ont été identifiés. La caractérisation phénotypique, génétique et moléculaire de ces clones suggère fortement que les séquences transformantes sont concaténées pour former des structures de haut poids moléculaire, et que ces structures sont efficacement répliquées et maintenues dans les cellules résistantes en tant qu'éléments génétiques extrachromosomaux. Ce type de transformation nous permet d'envisager des expériences permettant l'identification des séquences génomiques impliquées dans la replication de l'ADN de mousse. Plusieurs lignées transgéniques ont été retransformées avec des plasmides portant des séquences homologues aux séquences intégrées dans le génome, mais conférant une résistance à un autre antibiotique. Les résultats présentés dans le troisième chapitre montrent que les fréquences de transformation intégrative dans les lignées transgéniques sont 10 fois plus élevées que dans la lignée sauvage, et que cette augmentation est associée à une coségrégation des gènes de résistance dans la plupart des clones testés. Ces résultats génétiques indiquent que l'intégration de séquences d'ADN étranger dans le génome de P. patens a lieu en moyenne 10 fois plus fréquemment par recombinaison homologue que par intégration aléatoire. Ce rapport homologue/aléatoire est 10000 fois supérieur aux rapports obtenus avec d'autres plantes, et fournit l'outil indispensable à la réalisation d'expériences de génétique inverse dans cet organisme à haplophase dominante. THESIS SUMMARY The moss Physcomitrella patens is used as a model genetic system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. But further development of this model system was hampered by the lack of a protocol allowing the genetic transformation of this plant. We have developed a transformation protocol based on PEG-mediated direct gene transfer to protoplasts. Our data demonstrate that this procedure leads to the establishment of an efficient transient gene expression assay. A slightly modified protocol has been developed allowing the in vivo introduction of antibodies in moss protoplasts. Both DNA and IgGs can be loaded simultaneously, and specific antibodies can immunodeplete the product of an expression cassette (GUS) as well as proteins involved in cellular processes (tubulins). This assay, named transient in vivo immunodepletion assay, should be applicable to other plant protoplasts, and offers new approaches to study cellular processes. Transformations have been performed with bacterial plasmids carrying antibiotic resistance expression cassette. Our data demonstrate that integrative transformation occurs, but at low frequencies. This is the first demonstration of a successful genetic transformation of mosses. Resistant unstable colonies are recovered at high frequencies following transformation, and two different classes of unstable clones have been identified. Phenotypical, genetic and molecular characterisation of these clones strongly suggests that bacterial plasmids are concatenated to form high molecular arrays which are efficiently replicated and maintained as extrachromosomal elements in the resistant cells. Replicative transformation in P. patens should allow the design of experiments aimed at the identification of genomic sequences involved in moss DNA replication. Transgenic strains have been retransformed with bacterial plasmids carrying sequences homologous to the integrated transloci, but conferring resistance to another antibiotic. Our results demonstrate an order of magnitude increase of integrative transformation frequencies in transgenic strains as compared to wild-type, associated with cosegregation of the resistance genes in most of these double resistant transgenic strains. These observations provide strong genetic evidence that gene targeting occurs about ten times more often than random integration in the genome of P. patens. Such ratio of targeted to random integration is about 10 000 times higher than previous reports of gene targeting in plants, and provides the essential requirement for the development of efficient reverse genetics in the haplodiplobiontic P. patens.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland.

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre(®) YeastOne? test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

Improvement of a testing apparatus for dynamometry: procedures for penetrometry and influence of strain rate to quantify the tensile strength of soil aggregates

Soil penetration resistance (PR) and the tensile strength of aggregates (TS) are commonly used to characterize the physical and structural conditions of agricultural soils. This study aimed to assess the functionality of a dynamometry apparatus by linear speed and position control automation of its mobile base to measure PR and TS. The proposed equipment was used for PR measurement in undisturbed samples of a clayey "Nitossolo Vermelho eutroférrico" (Kandiudalfic Eutrudox) under rubber trees sampled in two positions (within and between rows). These samples were also used to measure the volumetric soil water content and bulk density, and determine the soil resistance to penetration curve (SRPC). The TS was measured in a sandy loam "Latossolo Vermelho distrófico" (LVd) - Typic Haplustox - and in a very clayey "Nitossolo Vermelho distroférrico" (NVdf) - Typic Paleudalf - under different uses: LVd under "annual crops" and "native forest", NVdf under "annual crops" and "eucalyptus plantation" (> 30 years old). To measure TS, different strain rates were applied using two dynamometry testing devices: a reference machine (0.03 mm s-1), which has been widely used in other studies, and the proposed equipment (1.55 mm s-1). The determination coefficient values of the SRPC were high (R² > 0.9), regardless of the sampling position. Mean TS values in LVd and NVdf obtained with the proposed equipment did not differ (p > 0.05) from those of the reference testing apparatus, regardless of land use and soil type. Results indicate that PR and TS can be measured faster and accurately by the proposed procedure.

Antimicrobial resistance of Staphylococcus aureus strains acquired by pig farmers from pigs

Carriage of animal-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) is common among pig farmers. This study was conducted (i) to investigate whether pig farmers are colonized with pig-specific S. aureus genotypes other than CC398 and (ii) to survey antimicrobial resistance of S. aureus isolates from pigs and pig farmers. Forty-eight S. aureus isolates from pig farmers and veterinarians and 130 isolates from pigs collected in Western Switzerland were genotyped by spa typing and amplified fragment length polymorphism (AFLP). Antimicrobial resistance profiles were determined for representative sample of the isolates. The data obtained earlier on healthy S. aureus carriers without exposure to agriculture were used for comparison. The genotype composition of S. aureus isolates from pig farmers and veterinarians was similar to isolates from pigs with predominant AFLP clusters CC398, CC9, and CC49. The resistance to tetracycline and macrolides (clarithromycin) was common among the isolates from farmers and veterinarians (52 and 21%, respectively) and similar to resistance levels in isolates from pigs (39 and 23%, respectively). This was in contrast to isolates from persons without contact with agriculture, where no (0/128) isolates were resistant to tetracycline and 3% of the isolates were resistant to clarithromycin. MRSA CC398 was isolated from pigs (n = 11) and pig farmers (n = 5). These data imply that zoonotic transmission of multidrug-resistant S. aureus from pigs to farmers is frequent, and well-known MRSA transmission merely represents the tip of the iceberg for this phenomenon. We speculate that the relatively low frequency of MRSA isolation is related to lower antimicrobial use in Switzerland compared to, for example, the Netherlands.

Soybean root growth and crop yield in reponse to liming at the beginning of a no-tillage system

Analyzing the soil near crop roots may reveal limitations to growth and yield even in a no-tillage system. The purpose of the present study was to relate the chemical and physical properties of soil under a no-tillage system to soybean root growth and plant yield after five years of use of different types of limestone and forms of application. A clayey Oxisol received application of dolomitic and calcitic limestones and their 1:1 combination in two forms: surface application, maintained on the soil surface; and incorporated, applied on the surface and incorporated mechanically. Soil physical properties (resistance to mechanical penetration, soil bulk density and soil aggregation), soil chemical properties (pH, exchangeable cations, H+Al, and cation exchange capacity) and plant parameters (root growth system, soybean grain yield, and oat dry matter production) were evaluated five years after setting up the experiment. Incorporation of lime neutralized exchangeable Al up to a depth of 20 cm without affecting the soil physical properties. The soybean root system reached depths of 40 cm or more with incorporated limestone, increasing grain yield an average of 31 % in relation to surface application, which limited the effect of lime up to a depth of 5 cm and root growth up to 20 cm. It was concluded that incorporation of limestone at the beginning of a no-tillage system ensures a favorable environment for root growth and soybean yield, while this intervention does not show long-term effects on soil physical properties under no-tillage. This suggests that there is resilience in the physical properties evaluated.

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Sub-inhibitory concentrations of vancomycin prevent quinolone-resistance in a penicillin-resistant isolate of Streptococcus pneumoniae.

BACKGROUND: The continuous spread of penicillin-resistant pneumococci represents a permanent threat in the treatment of pneumococcal infections, especially when strains show additional resistance to quinolones. The main objective of this study was to determine a treatment modality impeding the emergence of quinolone resistance. RESULTS: Exposure of a penicillin-resistant pneumococcus to increasing concentrations of trovafloxacin or ciprofloxacin selected for mutants resistant to these drugs. In the presence of sub-inhibitory concentrations of vancomycin, development of trovafloxacin-resistance and high-level ciprofloxacin-resistance were prevented. CONCLUSIONS: Considering the risk of quinolone-resistance in pneumococci, the observation might be of clinical importance.

Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, ß-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to ß-lactam antibiotics is conferred by ß-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to ß-lactam antibiotics, namely two ß-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.

Distinct Roles of Candida albicans Drug Resistance Transcription Factors TAC1, MRR1, and UPC2 in Virulence.

Azoles are widely used in antifungal therapy in medicine. Resistance to azoles can occur in Candida albicans principally by overexpression of multidrug transporter gene CDR1, CDR2, or MDR1 or by overexpression of ERG11, which encodes the azole target. The expression of these genes is controlled by the transcription factors (TFs) TAC1 (involved in the control of CDR1 and CDR2), MRR1 (involved in the control of MDR1), and UPC2 (involved in the control of ERG11). Several gain-of-function (GOF) mutations are present in hyperactive alleles of these TFs, resulting in the overexpression of target genes. While these mutations are beneficial to C. albicans survival in the presence of the antifungal drugs, their effects could potentially alter the fitness and virulence of C. albicans in the absence of the selective drug pressure. In this work, the effect of GOF mutations on C. albicans virulence was addressed in a systemic model of intravenous infection by mouse survival and kidney fungal burden assays. We engineered a set of strains with identical genetic backgrounds in which hyperactive alleles were reintroduced in one or two copies at their genomic loci. The results obtained showed that neither TAC1 nor MRR1 GOF mutations had a significant effect on C. albicans virulence. In contrast, the presence of two hyperactive UPC2 alleles in C. albicans resulted in a significant decrease in virulence, correlating with diminished kidney colonization compared to that by the wild type. In agreement with the effect on virulence, the decreased fitness of an isolate with UPC2 hyperactive alleles was observed in competition experiments with the wild type in vivo but not in vitro. Interestingly, UPC2 hyperactivity delayed filamentation of C. albicans after phagocytosis by murine macrophages, which may at least partially explain the virulence defects. Combining the UPC2 GOF mutation with another hyperactive TF did not compensate for the negative effect of UPC2 on virulence. In conclusion, among the major TFs involved in azole resistance, only UPC2 had a negative impact on virulence and fitness, which may therefore have consequences for the epidemiology of antifungal resistance.

Prevalence of etravirine mutations and impact on response to treatment in routine clinical care: the Swiss HIV Cohort Study (SHCS).

OBJECTIVES: Etravirine (ETV) is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with reduced cross-resistance to first-generation NNRTIs, which has been primarily studied in randomized clinical trials and not in routine clinical settings. METHODS: ETV resistance-associated mutations (RAMs) were investigated by analysing 6072 genotypic tests. The antiviral activity of ETV was predicted using different interpretation systems: International AIDS Society-USA (IAS-USA), Stanford, Rega and Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS). RESULTS: The prevalence of ETV RAMs was higher in NNRTI-exposed patients [44.9%, 95% confidence interval (CI) 41.0-48.9%] than in treatment-naïve patients (9.6%, 95% CI 8.5-10.7%). ETV RAMs in treatment-naïve patients mainly represent polymorphism, as prevalence estimates in genotypic tests for treatment-naïve patients with documented recent (<1 year) infection, who had acquired HIV before the introduction of NNRTIs, were almost identical (9.8%, 95% CI 3.3-21.4). Discontinuation of NNRTI treatment led to a marked drop in the detection of ETV RAMs, from 51.7% (95% CI 40.8-62.6%) to 34.5% (95% CI 24.6-45.4%, P=0.032). Differences in prevalence among subtypes were found for V90I and V179T (P<0.001). Estimates of restricted virological response to ETV varied among algorithms in patients with exposure to efavirenz (EFV)/nevirapine (NVP), ranging from 3.8% (95% CI 2.5-5.6%) for ANRS to 56.2% (95% CI 52.2-60.1%) for Stanford. The predicted activity of ETV decreased as the sensitivity of potential optimized background regimens decreased. The presence of major IAS-USA mutations (L100I, K101E/H/P and Y181C/I/V) reduced the treatment response at week 24. CONCLUSIONS: Most ETV RAMs in drug-naïve patients are polymorphisms rather than transmitted RAMs. Uncertainty regarding predictions of antiviral activity for ETV in NNRTI-treated patients remains high. The lowest activity was predicted for patients harbouring extensive multidrug-resistant viruses, thus limiting ETV use in those who are most in need.

Mechanisms of CYR61 mediated breast cancer metastasis to the lung

CYR61 (Cysteine-rich angiogenic inducer 61) is a matricellular protein that regulates cell proliferation, adhesion, migration and cell survival through interaction with various types of integrin cell adhesion receptors. At tissue level it is implicated in the regulation of embryonic development, wound healing and angiogenesis. CYR61 has also been involved in cancer progression, however its role appears to be diverse and complex depending on the cancer type and stage. Its contribution to metastasis formation is still unclear. Previous findings reported by our laboratory demonstrated that CYR61 cooperates with avßs integrin to promote invasion and metastasis of cancers growing in a pre-irradiated microenvironment. In this work, we used an orthotopic model of breast cancer to show for the first time that silencing of CYR61 in breast cancer cells suppresses lung metastasis formation. Silencing of MDA-MB-231 reduced both local growth and lung metastasis formation of tumor cells implanted in a pre-irradiated mammary fat pad. CYR61 silencing in tumors growing in non-irradiated mammary fat pads did not impact primary tumor growth but decreased lung metastasis formation. The effect of CYR61 on spontaneous lung metastasis formation during natural cancer progression was further examined by using an experimental model of metastasis. Results from these experiments indicate that CYR61 is critically involved in promoting cancer cells entry into lung parenchyma rather than later steps of colonization. In vitro experiments showed that CYR61 promotes tumor cell spreading, migration and transendothelial migration. CYR61 also supported colony formation under anchorage-independent condition and promotes resistance to anoikis through the involvement of ß1 and ß3 integrin. These results indicate that CYR61 promotes lung metastasis of breast cancer by facilitating extravasation into lung parenchyma through enhanced motility, transendothelial migration and resistance to anoikis. - CYR61 (Cysteine-rich angiogenic inducer 61) est une protéine matricellulaire qui régule la prolifération, l'adhérence, la migration et la survie des cellules par son interaction avec différents types de récepteurs d'adhésion cellulaire de la famille des intégrine. Au niveau des tissus, CYR61 est impliquée dans la régulation du développement embryonnaire, de la cicatrisation et de l'angiogenèse. CYR61 a également été impliquée dans le cancer, mais son rôle semble être divers et complexe en fonction du type du cancer et de son stade. Son rôle dans la formation des métastases n'est pas encore clair. Des résultats antérieurs rapportés par notre laboratoire ont montré que CYR61 coopère avec l'intégrine avß5 pour favoriser l'invasion et la métastase de tumeurs se développant dans un micro-environnement pré-irradié. Dans ce travail, nous avons utilisé un modèle orthotopique de cancer du sein pour démontrer pour la première fois que l'extinction (silencing) du gène CYR61 dans le cancer du sein réduit la formation de métastases pulmonaires. L'extinction de CYR61 dans la lignée cellulaire de cancer du sein humain MDA-MB- 231 réduit à la fois la croissance local ainsi que la formation de métastases pulmonaires à partir de cellules implantés dans les coussinets adipeux mammaires pré-irradié. L'extinction de CYR61 dans des tumeurs grandissant dans les coussinets adipeux mammaires non irradiées n'a pas d'incidence sur la croissance tumorale primaire mais réduit la formation des métastases pulmonaires. Par la suite nous avons examiné l'effet de CYR61 sur la formation de métastases pulmonaires en utilisant un modèle expérimental de métastase. Les résultats de ces expériences indiquent que CYR61 est impliquée de manière cruciale dans les étapes précoces de la formation de métastases, plutôt que dans les étapes tardives de colonisation du poumon. Des expériences in vitro ont montré que CYR61 favorise l'étalement, la migration et la transmigration endothéliale des cellules tumorales. CYR61 favorise également la formation de colonies dans des conditions indépendante de l'ancrage et la résistance à l'anoïkis par l'engagement des intégrines ß1 et ß3. Ces résultats indiquent que CYR61 favorise les métastases pulmonaires du cancer du sein en facilitant l'extravasation dans le parenchyme pulmonaire grâce à la stimulation de la motilità, de la migration transmigration endothéliale et de la résistance à l'anoïkis.

The DNA repair protein ALKBH2 mediates temozolomide resistance in human glioblastoma cells.

INTRODUCTION: Glioblastoma multiforme (GBM; World Health Organization astrocytoma grade IV) is the most frequent and most malignant primary brain tumor in adults. Despite multimodal therapy, all such tumors practically recur during the course of therapy, causing a median survival of only 14.6 months in patients with newly diagnosed GBM. The present study was aimed at examining the expression of the DNA repair protein AlkB homolog 2 (ALKBH2) in human GBM and determining whether it could promote resistance to temozolomide chemotherapy. METHODS: ALKBH2 expression in GBM cell lines and in human GBM was determined by quantitative real-time PCR (qRT-PCR) and gene expression analysis, respectively. Drug sensitivity was assessed in GBM cells overexpressing ALKBH2 and in cells in which ALKBH2 expression was silenced by small-interfering (si)RNA. ALKBH2 expression following activation of the p53 pathway was examined by western blotting and qRT-PCR. RESULTS: ALKBH2 was abundantly expressed in established GBM cell lines and human GBM, and temozolomide exposure increased cellular ALKBH2 expression levels. Overexpression of ALKBH2 in the U87 and U251 GBM cell lines enhanced resistance to the methylating agents temozolomide and methyl methanesulfonate but not to the nonmethylating agent doxorubicin. Conversely, siRNA-mediated knockdown of ALKBH2 increased sensitivity of GBM cells to temozolomide and methyl methanesulfonate but not to doxorubicin or cisplatin. Nongenotoxic activation of the p53 pathway by the selective murine double minute 2 antagonist nutlin-3 caused a significant decrease in cellular ALKBH2 transcription levels. CONCLUSION: Our findings identify ALKBH2 as a novel mediator of temozolomide resistance in human GBM cells. Furthermore, we place ALKBH2 into a new cellular context by showing its regulation by the p53 pathway.

Control of Concrete Deterioration Due to Trace Compounds in Deicers, Phase II, HR-299, 1989

This report presents results of research on ways to reduce the detrimental effects of sulfate-tainted rock salt deicers on portland cement concrete used for highway pavements. Repetitious experiments on the influence of fly ash on the mortar phase of concrete showed significant improvement in resistance to deicing brines is possible. Fifteen to twenty percent by weight of fly ash replacement for portland cement was found to provide optimum improvement. Fly ashes from five sources were evaluated and all were found to be equally beneficial. Preliminary results indicate the type of coarse aggregate also plays an important role in terms of concrete resistance to freeze-thaw in deicing brines. This was particularly true for a porous ferroan dolomite thought to be capable of reaction with the brine. In this case fly ash improved the concrete, but not enough for satisfactory performance. An intermediate response was with a porous limestone where undesirable results were observed without fly ash and adequate performance was realized when 15% fly ash was added. The best combination for making deicer-resistant concrete was found to be with a non-porous limestone. Performance in brines was found to be adequate without fly ash, but better when fly ash was included. Consideration was given to treating existing hardened concrete made with poor aggregate and no fly ash to extend pavement life in the presence of deicers, particularly at joints. Sodium silicate was found to improve freeze-thaw resistance of mortar and is a good candidate for field usage because of its low cost and ease of handling.

KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program.

Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30-40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.

Herbivory in the previous generation primes plants for enhanced insect resistance.

Inducible defenses, which provide enhanced resistance after initial attack, are nearly universal in plants. This defense signaling cascade is mediated by the synthesis, movement, and perception of jasmonic acid and related plant metabolites. To characterize the long-term persistence of plant immunity, we challenged Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) with caterpillar herbivory, application of methyl jasmonate, or mechanical damage during vegetative growth and assessed plant resistance in subsequent generations. Here, we show that induced resistance was associated with transgenerational priming of jasmonic acid-dependent defense responses in both species, caused caterpillars to grow up to 50% smaller than on control plants, and persisted for two generations in Arabidopsis. Arabidopsis mutants that are deficient in jasmonate perception (coronatine insensitive1) or in the biogenesis of small interfering RNA (dicer-like2 dicer-like3 dicer-like4 and nuclear RNA polymerase d2a nuclear RNA polymerase d2b) do not exhibit inherited resistance. The observation of inherited resistance in both the Brassicaceae and Solanaceae suggests that this trait may be more widely distributed in plants. Epigenetic resistance to herbivory thus represents a phenotypically plastic mechanism for enhanced defense across generations.