Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation.

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.

Antibiotic resistant bacteria/genes dissemination in lacustrine sediments highly increased following cultural eutrophication of Lake Geneva (Switzerland).

This study investigates faecal indicator bacteria (FIB), multiple antibiotic resistant (MAR), and antibiotic resistance genes (ARGs), of sediment profiles from different parts of Lake Geneva (Switzerland) over the last decades. MARs consist to expose culturable Escherichia coli (EC) and Enterococcus (ENT) to mixed five antibiotics including Ampicillin, Tetracycline, Amoxicillin, Chloramphenicol and Erythromycin. Culture-independent is performed to assess the distribution of ARGs responsible for, β-lactams (blaTEM; Amoxicillin/Ampicillin), Streptomycin/Spectinomycin (aadA), Tetracycline (tet) Chloramphenicol (cmlA) and Vancomycin (van). Bacterial cultures reveal that in the sediments deposited following eutrophication of Lake Geneva in the 1970s, the percentage of MARs to five antibiotics varied from 0.12% to 4.6% and 0.016% to 11.6% of total culturable EC and ENT, respectively. In these organic-rich bacteria-contaminated sediments, the blaTEM resistant of FIB varied from 22% to 48% and 16% to 37% for EC and ENT respectively, whereas the positive PCR assays responsible for tested ARGs were observed for EC, ENT, and total DNA from all samples. The aadA resistance gene was amplified for all the sediment samples, including those not influenced by WWTP effluent water. Our results demonstrate that bacteria MARs and ARGs highly increased in the sediments contaminated with WWTP effluent following the cultural eutrophication of Lake Geneva. Hence, the human-induced changing limnological conditions highly enhanced the sediment microbial activity, and therein the spreading of antibiotic resistant bacteria and genes in this aquatic environment used to supply drinking water in a highly populated area. Furthermore, the presence of the antibiotic resistance gene aadA in all the studied samples points out a regional dissemination of this emerging contaminant in freshwater sediments since at least the late nineteenth century.

Leukemic cluster growth in culture is an independent risk factor for acute myeloid leukemia and short survival in patients with myelodysplastic syndrome

In patients with myelodysplastic syndrome (MDS) precursor cell cultures (colony-forming unit cells, CFU-C) can provide an insight into the growth potential of malignant myeloid cells. In a retrospective single-center study of 73 untreated MDS patients we assessed whether CFU-C growth patterns were of prognostic value in addition to established criteria. Abnormalities were classified as qualitative (i.e. leukemic cluster growth) or quantitative (i.e. strongly reduced/absent growth). Thirty-nine patients (53%) showed leukemic growth, 26 patients (36%) had strongly reduced/absent colony growth, and 12 patients showed both. In a univariate analysis the presence of leukemic growth was associated with strongly reduced survival (at 10 years 4 vs. 34%, p = 0.004), and a high incidence of transformation to AML (76 vs. 32%, p = 0.01). Multivariate analysis identified leukemic growth as a strong and independent predictor of early death (relative risk 2.12, p = 0.03) and transformation to AML (relative risk 2.63, p = 0.04). Quantitative abnormalities had no significant impact on the disease course. CFU- C assays have significant predictive value in addition to established prognostic factors in MDS. Leukemic growth identifies a subpopulation of MDS patients with poor prognosis.

Leukemic cluster growth in culture is an independent risk factor for acute myeloid leukemia and short survival in patients with myelodysplastic syndrome.

In patients with myelodysplastic syndrome (MDS) precursor cell cultures (colony-forming unit cells, CFU-C) can provide an insight into the growth potential of malignant myeloid cells. In a retrospective single-center study of 73 untreated MDS patients we assessed whether CFU-C growth patterns were of prognostic value in addition to established criteria. Abnormalities were classified as qualitative (i.e. leukemic cluster growth) or quantitative (i.e. strongly reduced/absent growth). Thirty-nine patients (53%) showed leukemic growth, 26 patients (36%) had strongly reduced/absent colony growth, and 12 patients showed both. In a univariate analysis the presence of leukemic growth was associated with strongly reduced survival (at 10 years 4 vs. 34%, p = 0.004), and a high incidence of transformation to AML (76 vs. 32%, p = 0.01). Multivariate analysis identified leukemic growth as a strong and independent predictor of early death (relative risk 2.12, p = 0.03) and transformation to AML (relative risk 2.63, p = 0.04). Quantitative abnormalities had no significant impact on the disease course. CFU-C assays have a significant predictive value in addition to established prognostic factors in MDS. Leukemic growth identifies a subpopulation of MDS patients with poor prognosis.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.

Ectodysplasin regulates hormone-independent mammary ductal morphogenesis via NF-κB.

Ductal growth of the mammary gland occurs in two distinct stages. The first round of branching morphogenesis occurs during embryogenesis, and the second round commences at the onset of puberty. Currently, relatively little is known about the genetic networks that control the initial phases of ductal expansion, which, unlike pubertal development, proceeds independent of hormonal input in female mice. Here we identify NF-κB downstream of the TNF-like ligand ectodysplasin (Eda) as a unique regulator of embryonic and prepubertal ductal morphogenesis. Loss of Eda, or inhibition of NF-κB, led to smaller ductal trees with fewer branches. On the other hand, overexpression of Eda caused a dramatic NF-κB-dependent phenotype in both female and male mice characterized by precocious and highly increased ductal growth and branching that correlated with enhanced cell proliferation. We have identified several putative transcriptional target genes of Eda/NF-κB, including PTHrP, Wnt10a, and Wnt10b, as well as Egf family ligands amphiregulin and epigen. We developed a mammary bud culture system that allowed us to manipulate mammary development ex vivo and found that recombinant PTHrP, Wnt3A, and Egf family ligands stimulate embryonic branching morphogenesis, suggesting that these pathways may cooperatively mediate the effects of Eda.

Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue.

Background: The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. Methods: RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score. Results: Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_ 5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_ 5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Conclusions: Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.

Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis.

BACKGROUND: Histologic grade in breast cancer provides clinically important prognostic information. However, 30%-60% of tumors are classified as histologic grade 2. This grade is associated with an intermediate risk of recurrence and is thus not informative for clinical decision making. We examined whether histologic grade was associated with gene expression profiles of breast cancers and whether such profiles could be used to improve histologic grading. METHODS: We analyzed microarray data from 189 invasive breast carcinomas and from three published gene expression datasets from breast carcinomas. We identified differentially expressed genes in a training set of 64 estrogen receptor (ER)-positive tumor samples by comparing expression profiles between histologic grade 3 tumors and histologic grade 1 tumors and used the expression of these genes to define the gene expression grade index. Data from 597 independent tumors were used to evaluate the association between relapse-free survival and the gene expression grade index in a Kaplan-Meier analysis. All statistical tests were two-sided. RESULTS: We identified 97 genes in our training set that were associated with histologic grade; most of these genes were involved in cell cycle regulation and proliferation. In validation datasets, the gene expression grade index was strongly associated with histologic grade 1 and 3 status; however, among histologic grade 2 tumors, the index spanned the values for histologic grade 1-3 tumors. Among patients with histologic grade 2 tumors, a high gene expression grade index was associated with a higher risk of recurrence than a low gene expression grade index (hazard ratio = 3.61, 95% confidence interval = 2.25 to 5.78; P < .001, log-rank test). CONCLUSIONS: Gene expression grade index appeared to reclassify patients with histologic grade 2 tumors into two groups with high versus low risks of recurrence. This approach may improve the accuracy of tumor grading and thus its prognostic value.

CATMA, a comprehensive genome-scale resource for silencing and transcript profiling of Arabidopsis genes.

BACKGROUND: The Complete Arabidopsis Transcript MicroArray (CATMA) initiative combines the efforts of laboratories in eight European countries 1 to deliver gene-specific sequence tags (GSTs) for the Arabidopsis research community. The CATMA initiative offers the power and flexibility to regularly update the GST collection according to evolving knowledge about the gene repertoire. These GST amplicons can easily be reamplified and shared, subsets can be picked at will to print dedicated arrays, and the GSTs can be cloned and used for other functional studies. This ongoing initiative has already produced approximately 24,000 GSTs that have been made publicly available for spotted microarray printing and RNA interference. RESULTS: GSTs from the CATMA version 2 repertoire (CATMAv2, created in 2002) were mapped onto the gene models from two independent Arabidopsis nuclear genome annotation efforts, TIGR5 and PSB-EuGène, to consolidate a list of genes that were targeted by previously designed CATMA tags. A total of 9,027 gene models were not tagged by any amplified CATMAv2 GST, and 2,533 amplified GSTs were no longer predicted to tag an updated gene model. To validate the efficacy of GST mapping criteria and design rules, the predicted and experimentally observed hybridization characteristics associated to GST features were correlated in transcript profiling datasets obtained with the CATMAv2 microarray, confirming the reliability of this platform. To complete the CATMA repertoire, all 9,027 gene models for which no GST had yet been designed were processed with an adjusted version of the Specific Primer and Amplicon Design Software (SPADS). A total of 5,756 novel GSTs were designed and amplified by PCR from genomic DNA. Together with the pre-existing GST collection, this new addition constitutes the CATMAv3 repertoire. It comprises 30,343 unique amplified sequences that tag 24,202 and 23,009 protein-encoding nuclear gene models in the TAIR6 and EuGène genome annotations, respectively. To cover the remaining untagged genes, we identified 543 additional GSTs using less stringent design criteria and designed 990 sequence tags matching multiple members of gene families (Gene Family Tags or GFTs) to cover any remaining untagged genes. These latter 1,533 features constitute the CATMAv4 addition. CONCLUSION: To update the CATMA GST repertoire, we designed 7,289 additional sequence tags, bringing the total number of tagged TAIR6-annotated Arabidopsis nuclear protein-coding genes to 26,173. This resource is used both for the production of spotted microarrays and the large-scale cloning of hairpin RNA silencing vectors. All information about the resulting updated CATMA repertoire is available through the CATMA database http://www.catma.org.

HIV-1 Subtype Is an Independent Predictor of Reverse Transcriptase Mutation K65R in HIV-1 Patients Treated with Combination Antiretroviral Therapy Including Tenofovir.

Subtype-dependent selection of HIV-1 reverse transcriptase resistance mutation K65R was previously observed in cell culture and small clinical investigations. We compared K65R prevalence across subtypes A, B, C, F, G, and CRF02_AG separately in a cohort of 3,076 patients on combination therapy including tenofovir. K65R selection was significantly higher in HIV-1 subtype C. This could not be explained by clinical and demographic factors in multivariate analysis, suggesting subtype sequence-specific K65R pathways.

The death domain-containing protein Unc5CL is a novel MyD88-independent activator of the pro-inflammatory IRAK signaling cascade.

The family of death domain (DD)-containing proteins are involved in many cellular processes, including apoptosis, inflammation and development. One of these molecules, the adapter protein MyD88, is a key factor in innate and adaptive immunity that integrates signals from the Toll-like receptor/interleukin (IL)-1 receptor (TLR/IL-1R) superfamily by providing an activation platform for IL-1R-associated kinases (IRAKs). Here we show that the DD-containing protein Unc5CL (also known as ZUD) is involved in a novel MyD88-independent mode of IRAK signaling that culminates in the activation of the transcription factor nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase. Unc5CL required IRAK1, IRAK4 and TNF receptor-associated factor 6 but not MyD88 for its ability to activate these pathways. Interestingly, the protein is constitutively autoproteolytically processed, and is anchored by its N-terminus specifically to the apical face of mucosal epithelial cells. Transcriptional profiling identified mainly chemokines, including IL-8, CXCL1 and CCL20 as Unc5CL target genes. Its prominent expression in mucosal tissues, as well as its ability to induce a pro-inflammatory program in cells, suggests that Unc5CL is a factor in epithelial inflammation and immunity as well as a candidate gene involved in mucosal diseases such as inflammatory bowel disease.

Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells.

Engineering of fetal tissue has a high potential for the treatment of acute and chronic wounds of the skin in humans as these cells have high expansion capacity under simple culture conditions and one organ donation can produce Master Cell Banks which can fabricate over 900 million biological bandages (9 x 12cm). In a Phase 1 clinical safety study, cases are presented for the treatment of therapy resistant leg ulcers. All eight patients, representing 13 ulcers, tolerated multiple treatments with fetal biological bandages showing no negative secondary effects and repair processes similar to that seen in 3rd degree burns. Differential gene profiling using Affymetrix gene chips (analyzing 12,500 genes) were accomplished on these banked fetal dermal skin cells compared to banked dermal skin cells of an aged donor in order to point to potential indicators of wound healing. Families of genes involved in cell adhesion and extracellular matrix, cell cycle, cellular signaling, development and immune response show significant differences in regulation between banked fetal and those from banked old skin cells: with approximately 47.0% of genes over-expressed in fetal fibroblasts. It is perhaps these differences which contribute to efficient tissue repair seen in the clinic with fetal cell therapy.

Strong time dependence of the 76-gene prognostic signature for node-negative breast cancer patients in the TRANSBIG multicenter independent validation series.

PURPOSE: Recently, a 76-gene prognostic signature able to predict distant metastases in lymph node-negative (N(-)) breast cancer patients was reported. The aims of this study conducted by TRANSBIG were to independently validate these results and to compare the outcome with clinical risk assessment. EXPERIMENTAL DESIGN: Gene expression profiling of frozen samples from 198 N(-) systemically untreated patients was done at the Bordet Institute, blinded to clinical data and independent of Veridex. Genomic risk was defined by Veridex, blinded to clinical data. Survival analyses, done by an independent statistician, were done with the genomic risk and adjusted for the clinical risk, defined by Adjuvant! Online. RESULTS: The actual 5- and 10-year time to distant metastasis were 98% (88-100%) and 94% (83-98%), respectively, for the good profile group and 76% (68-82%) and 73% (65-79%), respectively, for the poor profile group. The actual 5- and 10-year overall survival were 98% (88-100%) and 87% (73-94%), respectively, for the good profile group and 84% (77-89%) and 72% (63-78%), respectively, for the poor profile group. We observed a strong time dependence of this signature, leading to an adjusted hazard ratio of 13.58 (1.85-99.63) and 8.20 (1.10-60.90) at 5 years and 5.11 (1.57-16.67) and 2.55 (1.07-6.10) at 10 years for time to distant metastasis and overall survival, respectively. CONCLUSION: This independent validation confirmed the performance of the 76-gene signature and adds to the growing evidence that gene expression signatures are of clinical relevance, especially for identifying patients at high risk of early distant metastases.