Gene expression profiling in human pathogenic dermatophytes in vitro and in vivo

Objectives: Dermatophytes are highly specialized fungi which are the most common agents of superficial mycoses in humans and animals. The particular ability of these microorganisms to invade and multiply within keratinized host structures is presumably linked to their secreted keratinolytic activity, which is therefore a major putative virulence attribute of these fungi. The overall adaptation and transcriptional response of dermatophytes during protein degradation and/or infection is largely unknown. Methods: A Trichophyton rubrum cDNA microarray was developed and used for the transcriptional analysis of T. rubrum and Arthroderma benhamiae cells during growth on protein substrates. Moreover, the gene expression profile in A. benhamiae cells was monitored during infection of guinea pigs. Results: T. rubrum and A. benhamiae cells activate a large set of genes encoding secreted endo- and exoproteases during growth on soy and keratin. In addition, other specifically induced factors with potential implication in protein utilization were identified, e.g. multiple transporters, metabolic enzymes, transcription factors and hypothetical proteins with unknown function. Notably however, the protease gene expression profile in the fungal cells during infection was significantly different from the pattern elicited during in vitro growth on keratin. Conclusions: Our results suggest specific functions of individual proteases during infection, which may not be restricted to the degradation of keratin. This first, broad in vivo transcriptional profiling approach in dermatophytes gives new molecular insights into pathogenicity associated adaptation mechanisms that make these microorganisms the most successful causitive agents of superficial mycoses.

Subject-based steroid profiling and the determination of novel biomarkers for DHT and DHEA misuse in sports.

Doping with natural steroids can be detected by evaluating the urinary concentrations and ratios of several endogenous steroids. Since these biomarkers of steroid doping are known to present large inter-individual variations, monitoring of individual steroid profiles over time allows switching from population-based towards subject-based reference ranges for improved detection. In an Athlete Biological Passport (ABP), biomarkers data are collated throughout the athlete's sporting career and individual thresholds defined adaptively. For now, this approach has been validated on a limited number of markers of steroid doping, such as the testosterone (T) over epitestosterone (E) ratio to detect T misuse in athletes. Additional markers are required for other endogenous steroids like dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA). By combining comprehensive steroid profiles composed of 24 steroid concentrations with Bayesian inference techniques for longitudinal profiling, a selection was made for the detection of DHT and DHEA misuse. The biomarkers found were rated according to relative response, parameter stability, discriminative power, and maximal detection time. This analysis revealed DHT/E, DHT/5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol as best biomarkers for DHT administration and DHEA/E, 16α-hydroxydehydroepiandrosterone/E, 7β-hydroxydehydroepiandrosterone/E and 5β-androstane-3α,17β-diol/5α-androstane-3α,17β-diol for DHEA. The selected biomarkers were found suitable for individual referencing. A drastic overall increase in sensitivity was obtained.The use of multiple markers as formalized in an Athlete Steroidal Passport (ASP) can provide firm evidence of doping with endogenous steroids. Copyright © 2010 John Wiley & Sons, Ltd.

Signaling pathways controlling induced resistance to insect herbivores in Arabidopsis.

Insect attack triggers changes in transcript level in plants that are mediated predominantly by jasmonic acid (JA). The implication of ethylene (ET), salicylic acid (SA), and other signals in this response is less understood and was monitored with a microarray containing insect- and defense-regulated genes. Arabidopsis thaliana mutants coi1-1, ein2-1, and sid2-1 impaired in JA, ET, and SA signaling pathways were challenged with the specialist small cabbage white (Pieris rapae) and the generalist Egyptian cotton worm (Spodoptera littoralis). JA was shown to be a major signal controlling the upregulation of defense genes in response to either insect but was found to suppress changes in transcript level only in response to P. rapae. Larval growth was affected by the JA-dependent defenses, but S. littoralis gained much more weight on coi1-1 than P. rapae. ET and SA mutants had an altered transcript profile after S. littoralis herbivory but not after P. rapae herbivory. In contrast, both insects yielded similar transcript signatures in the abscisic acid (ABA)-biosynthetic mutants aba2-1 and aba3-1, and ABA controlled transcript levels both negatively and positively in insect-attacked plants. In accordance with the transcript signature, S. littoralis larvae performed better on aba2-1 mutants. This study reveals a new role for ABA in defense against insects in Arabidopsis and identifies some components important for plant resistance to herbivory.

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome.

Within the ENCODE Consortium, GENCODE aimed to accurately annotate all protein-coding genes, pseudogenes, and noncoding transcribed loci in the human genome through manual curation and computational methods. Annotated transcript structures were assessed, and less well-supported loci were systematically, experimentally validated. Predicted exon-exon junctions were evaluated by RT-PCR amplification followed by highly multiplexed sequencing readout, a method we called RT-PCR-seq. Seventy-nine percent of all assessed junctions are confirmed by this evaluation procedure, demonstrating the high quality of the GENCODE gene set. RT-PCR-seq was also efficient to screen gene models predicted using the Human Body Map (HBM) RNA-seq data. We validated 73% of these predictions, thus confirming 1168 novel genes, mostly noncoding, which will further complement the GENCODE annotation. Our novel experimental validation pipeline is extremely sensitive, far more than unbiased transcriptome profiling through RNA sequencing, which is becoming the norm. For example, exon-exon junctions unique to GENCODE annotated transcripts are five times more likely to be corroborated with our targeted approach than with extensive large human transcriptome profiling. Data sets such as the HBM and ENCODE RNA-seq data fail sampling of low-expressed transcripts. Our RT-PCR-seq targeted approach also has the advantage of identifying novel exons of known genes, as we discovered unannotated exons in ~11% of assessed introns. We thus estimate that at least 18% of known loci have yet-unannotated exons. Our work demonstrates that the cataloging of all of the genic elements encoded in the human genome will necessitate a coordinated effort between unbiased and targeted approaches, like RNA-seq and RT-PCR-seq.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.

Gene expression profiling reveals consistent differences between clinical samples of human leukaemias and their model cell lines.

Microarray gene expression profiles of fresh clinical samples of chronic myeloid leukaemia in chronic phase, acute promyelocytic leukaemia and acute monocytic leukaemia were compared with profiles from cell lines representing the corresponding types of leukaemia (K562, NB4, HL60). In a hierarchical clustering analysis, all clinical samples clustered separately from the cell lines, regardless of leukaemic subtype. Gene ontology analysis showed that cell lines chiefly overexpressed genes related to macromolecular metabolism, whereas in clinical samples genes related to the immune response were abundantly expressed. These findings must be taken into consideration when conclusions from cell line-based studies are extrapolated to patients.

Detection and chemical profiling of medicine counterfeits by Raman spectroscopy and chemometrics

Raman spectroscopy combined with chemometrics has recently become a widespread technique for the analysis of pharmaceutical solid forms. The application presented in this paper is the investigation of counterfeit medicines. This increasingly serious issue involves networks that are an integral part of industrialized organized crime. Efficient analytical tools are consequently required to fight against it. Quick and reliable authentication means are needed to allow the deployment of measures from the company and the authorities. For this purpose a method in two steps has been implemented here. The first step enables the identification of pharmaceutical tablets and capsules and the detection of their counterfeits. A nonlinear classification method, the Support Vector Machines (SVM), is computed together with a correlation with the database and the detection of Active Pharmaceutical Ingredient (API) peaks in the suspect product. If a counterfeit is detected, the second step allows its chemical profiling among former counterfeits in a forensic intelligence perspective. For this second step a classification based on Principal Component Analysis (PCA) and correlation distance measurements is applied to the Raman spectra of the counterfeits.

Proteomic and transcriptomic profiling of Staphylococcus aureus surface LPXTG-proteins: correlation with agr genotypes and adherence phenotypes.

Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureus physiology should integrate all three approaches.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse. The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Toward a novel forensic intelligence model: systematic profiling of false identity documents

False identity documents represent a serious threat through their production and use in organized crime and by terrorist organizations. The present-day fight against this criminal problem and threats to national security does not appropriately address the organized nature of this criminal activity, treating each fraudulent document on its own during investigation and the judicial process, which causes linkage blindness and restrains the analysis capacity. Given the drawbacks of this case-by-case approach, this article proposes an original model in which false identity documents are used to inform a systematic forensic intelligence process. The process aims to detect links, patterns, and tendencies among false identity documents in order to support strategic and tactical decision making, thus sustaining a proactive intelligence-led approach to fighting identity document fraud and the associated organized criminality. This article formalizes both the model and the process, using practical applications to illustrate its powerful capabilities. This model has a general application and can be transposed to other fields of forensic science facing similar difficulties.

Screening for wound-induced oxylipins in Arabidopsis thaliana by differential HPLC-APCI/MS profiling of crude leaf extracts and subsequent characterisation by capillary-scale NMR

A simple non-targeted differential HPLC-APCI/MS approach has been developed in order to survey metabolome modifications that occur in the leaves of Arabidopsis thaliana following wound-induced stress. The wound-induced accumulation of metabolites, particularly oxylipins, was evaluated by HPLC-MS analysis of crude leaf extracts. A generic, rapid and reproducible pressure liquid extraction procedure was developed for the analysis of restricted leaf samples without the need for specific sample preparation. The presence of various oxylipins was determined by head-to-head comparison of the HPLC-MS data, filtered with a component detection algorithm, and automatically compared with the aid of software searching for small differences in similar HPLC-MS profiles. Repeatability was verified in several specimens belonging to different series. Wound-inducible jasmonates were efficiently highlighted by this non-targeted approach without the need for complex sample preparation as is the case for the 'oxylipin signature' procedure based on GC-MS. Furthermore this HPLC-MS screening technique allowed the isolation of induced compounds for further characterisation by capillary-scale NMR (CapNMR) after HPLC scale-up. In this paper, the screening method is described and applied to illustrate its potential for monitoring polar and non-polar stress-induced constituents as well as its use in combination with CapNMR for the structural assignment of wound-induced compounds of interest