Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species.

BACKGROUND: Fungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs. Candida spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of Candida may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with Tm calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant Candida species. METHODS: Efficiency, coefficient of correlation and the limit of reliable detection were estimated on purified Candida EDTA-whole blood (WB) reference panels seeded with Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler®96, LightCycler® Nano, LightCycler® 2.0. Inter- and intra assay consistencies were also calculated. RESULTS: The limit of reliable detection proved to be 0.2-2 genomic equivalent and the method was reliable on broad concentration ranges (106-10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler®2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ± 0.06 on reference- and % C.V.: 0.14 ± 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinical-panels which is highly acceptable. CONCLUSION: Our assay demonstrates recent advances on Tm calling and HRM analysis for the molecular identification of relevant Candida species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12-24 h).

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions.

Combining standard clinical methods with PCR showed improved diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia.

BACKGROUND: We assessed the diagnostic value of standard clinical methods and combined biomarker testing (galactomannan assay and polymerase chain reaction screening) in a prospective case-control study to detect invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia. METHODS: In this observational study 162 biomarker analyses were performed on samples from 27 febrile neutropenic episodes. Sera were successively screened for galactomannan antigen and for Aspergillus fumigatus specific nucleic acid targets. Furthermore thoracic computed tomography scanning was performed along with bronchoscopy with lavage when clinically indicated. Patients were retrospectively stratified to define a case-group with "proven" or "probable" invasive pulmonary aspergillosis (25.93 %) and a control-group of patients with no evidence for of invasive pulmonary aspergillosis (74.07 %). In 44.44 % of episodes fever ceased in response to antibiotic treatment (group II). Empirical antifungal therapy was administered for episodes with persistent or relapsing fever (group I). 48.15 % of patients died during the study period. Postmortem histology was pursued in 53.85 % of fatalities. RESULTS: Concordant negative galactomannan and computed tomography supported by a polymerase chain reaction assay were shown to have the highest discriminatory power to exclude invasive pulmonary aspergillosis. Bronchoalveolar lavage was performed in 6 cases of invasive pulmonary aspergillosis and in 15 controls. Although bronchoalveolar lavage proved negative in 93 % of controls it did not detect IPA in 86 % of the cases. Remarkably post mortem histology convincingly supported the presence of Aspergillus hyphae in lung tissue from a single case which had consecutive positive polymerase chain reaction assay results but was misdiagnosed by both computed tomography and consistently negative galactomannan assay results. For the galactomannan enzyme-immunoassay the diagnostic odds ratio was 15.33 and for the polymerase chain reaction assay it was 28.67. According to Cohen's kappa our in-house polymerase chain reaction method showed a fair agreement with the galactomannan immunoassay. Combined analysis of the results from the Aspergillus galactomannan enzyme immunoassay together with those generated by our polymerase chain reaction assay led to no misdiagnoses in the control group. CONCLUSION: The data from this pilot-study demonstrate that the consideration of standard clinical methods combined with biomarker testing improves the capacity to make early and more accurate diagnostic decisions.

[Effect of BMP-2 treatment on the morphology and proliferation of human embryonic palatal derived mesenchymal preosteoblast cells]. / BMP-2 hatása a humán embrionális szájpadlásból származó mesenchymalis preosteoblast sejtek morfológiájára és proliferációjára.

In dental implantation missing tooth or teeth are replaced by artificial root. To reduce the time required for the integration newest trends are the enhancement of bone formation around the implant by bioactive molecules, growth factors. Such a molecule is bone morphogenetic protein-2 (BMP-2) accepted by US Food and Drug Administration (FDA). In these kind of applications effect of BMP-2 is tested in vitro on appropriate cell lines. One of these cell lines is the osteoblast like human embrionic palatal mesenchymal cell line (HEPM). In our experiments the effect of BMP-2 homodimer treatment was investigated on the differentiation of HEPM cells to osteoblasts reflected by changes in morphology, and proliferation after a short, 3 days BMP-2 treatment. Results showed that after three days BMP-2 treatment facilitates cell attachment on a concentration dependent manner however changes in cell morphology and proliferation could not be observed. Continuing the BMP-2 treatment inhibitory effect was measured in cell proliferation, which may refer to cell differentiation.

WT1 microdeletion and slowly progressing focal glomerulosclerosis in a patient with male pseudohermaphroditism, childhood leukemia, Wilms tumor and cerebellar angioblastoma.

The Wilms tumor 1 (WT1) gene is currently in focus by pediatric nephrologists as its mutations are associated with nephrotic syndrome, especially as part of complex clinical entities like Denys-Drash or Frasier syndrome. Renal failure may also develop in young WAGR patients, whose condition is attributed to a deletion at chromosomal region 11p13. However, only limited data exist on WT1 microdeletions. A 30-year-old male patient, with a history of genital malformations, a Wilms tumor manifested during the treatment of acute lymphoid leukemia (ALL) at the age of 4, and a cerebellar angioblastoma, was referred with proteinuria and a reduced glomerular filtration rate (GFR). Kidney biopsy revealed FSGS. Although all WT1 exons were amplified with polymerase chain reaction (PCR) and sequenced, none of them showed a mutation. However, an formalin-fixed, paraffin- embedded (FFPE) tissue sample of the patient's childhood Wilms tumor showed WT1- positivity restricted to the renal tumor cells, so the WT1 gene was investigated further. Using quantitative reverse transcription PCR (qRT-PCR), the gene was found to be present in only one copy in the patient's genomic DNA sample, while both copies were detected in both parents. In the patient's sister, the proximal region of WT1 was shown to have an extra copy. Evidence suggests that a heterozygous microdeletion of the gene WT1 is responsible for the patient's disease. It seems reasonable to assume a possible abnormality affecting meiotic crossing over at the WT1 locus in one of the parents.

Circulating microRNA sequencing revealed miRNome patterns in hematology and oncology patients aiding the prognosis of invasive aspergillosis.

Invasive aspergillosis (IA) may occur as a serious complication of hematological malignancy. Delays in antifungal therapy can lead to an invasive disease resulting in high mortality. Currently, there are no well-established blood circulating microRNA biomarkers or laboratory tests which can be used to diagnose IA. Therefore, we aimed to define dysregulated miRNAs in hematology and oncology (HO) patients to identify biomarkers predisposing disease. We performed an in-depth analysis of high-throughput small transcriptome sequencing data obtained from the whole blood samples of our study cohort of 50 participants including 26 high-risk HO patients and 24 controls. By integrating in silico bioinformatic analyses of small noncoding RNA data, 57 miRNAs exhibiting significant expression differences (P < 0.05) were identified between IA-infected patients and non-IA HO patients. Among these, we found 36 differentially expressed miRNAs (DEMs) irrespective of HO malignancy. Of the top ranked DEMs, we found 14 significantly deregulated miRNAs, whose expression levels were successfully quantified by qRT-PCR. MiRNA target prediction revealed the involvement of IA related miRNAs in the biological pathways of tumorigenesis, the cell cycle, the immune response, cell differentiation and apoptosis.

Lack of A-factor production induces the expression of nutrient scavenging and stress-related proteins in Streptomyces griseus.

The small gamma-butyrolactone A-factor is an important autoregulatory signaling molecule for the soil-inhabiting streptomycetes. Starvation is a major trigger for development, and nutrients are provided by degradation of the vegetative mycelium via a process of programmed cell death, reusing proteins, nucleic acids, and cell wall material. The A-factor regulon includes many extracellular hydrolases. Here we show via proteomics analysis that many nutrient-scavenging and stress-related proteins were overexpressed in an A-factor non-producing mutant of Streptomyces griseus B-2682. Transcript analysis showed that this is primarily due to differential transcription of the target genes during early development. The targets include proteins relating to nutrient stress and environmental stress and an orthologue of the Bacillus sporulation control protein Spo0M. The enhanced expression of these proteins underlines the stress that is generated by the absence of A-factor. Wild-type developmental gene expression was restored to the A-factor non-producing mutant by the signaling protein Factor C in line with our earlier observation that Factor C triggers A-factor production.

Nutraceuticals Induced Changes in the Broiler Gastrointestinal Tract Microbiota.

Effects of nutraceuticals on the intestinal microbiota are receiving increased attention; however, there are few studies investigating their effects on broiler meat production. The aim of this study was to implement feeding strategies and carry out a comprehensive trial examining the interplay between natural biologically active compounds such as carotenoids, anthocyanins, fermentable oligosaccharides, and synbiotics and the gastrointestinal tract microbiota. Our feeding program was applied to an intensive production system with a flock of 1,080 Ross 308 broilers. Aging induced significant changes through the feeding experiment. Nutraceuticals were shown to modulate broiler intestinal diversity and differentially enriched Lactobacillus, Enterococcus, Campylobacter, and Streptococcus in the core microbiome during the different stages of broiler rearing. Additionally, they did not remarkably affect animal growth performance; nevertheless, a positive correlation was found between body weight and Corynebacteriales and Pseudomonadales Furthermore, a diet high in carotenoid, fermentable oligosaccharide, and anthocyanin contents affected the number of beneficial genera such as Faecalibacterium, Lactobacillus, Blautia, and Ruminococcus With this comprehensive trial, we revealed that nutraceuticals induced modulations in broiler gastrointestinal tract microbiota. We believe that plant-derived immunostimulants, recycled from plant food waste products, can supplement antibiotic-free broiler meat production.IMPORTANCE In this trial, nutraceuticals were manufactured from waste products of food industry processing of Hungarian red sweet pepper and sour cherry and incorporated into the diet of poultry to investigate their effects on broilers' growth and the broiler gastrointestinal tract microbiota. To avoid the generation of food waste products, we believe that this approach can be developed into a sustainable, green approach that can be implemented in commercial antibiotic-free poultry to provide safe and high-quality meat.

Effects of phytonutrient-supplemented diets on the intestinal microbiota of Cyprinus carpio.

In the aquaculture sector, a strategy for the more efficient use of resources and proper disease control is needed to overcome the challenges of meat production worldwide. Modulation of the gastrointestinal tract microbiota is a promising approach for promoting animal health and preventing infection. This feeding experiment was conducted to discover the phytonutrient-induced changes in the gastrointestinal tract microbiota of common carp (Cyprinus carpio). Acclimatized animals aged 7 months (30 weeks) were divided randomly into five experimental groups to investigate the effects of the applied feed additives. The dietary supplements were manufactured from anthocyanin-containing processing wastes from the food industry, specifically the production of Hungarian sour cherry extract, synbiotics from fermented corn, and fermentable oligosaccharides from Hungarian sweet red pepper seeds and carotenoids from Hungarian sweet red pepper pulps, applied at a dose of 1%. The gut contents of the animals were collected at four time points throughout the 6-week study period. To track the compositional and diversity changes in the microbiota of the carp intestinal tract, V3-V4 16S rRNA gene-based metagenomic sequencing was performed. The growth performance of common carp juveniles was not significantly affected by supplementation of the basal diet with plant extracts. Phytonutrients improve the community diversity, increase the Clostridium and Lactobacillus abundances and decrease the abundances of potentially pathogenic and spoilage bacteria, such as Shewanella, Pseudomonas, Acinetobacter and Aeromonas. The phyla Proteobacteria, Tenericutes and Chlamydiae were positively correlated with the body weight, whereas Spirochaetes and Firmicutes exhibited negatively correlations with the body weight. We hypothesize that the application of phytonutrients in aquaculture settings might be a reasonable green approach for easing the usage of antibiotics.

Free circulating mircoRNAs support the diagnosis of invasive aspergillosis in patients with hematologic malignancies and neutropenia.

Fungal infections represent a worrisome complication in hematologic cancer patients and in the absence of disease specific symptoms, it is important to establish new biological indicators, which can be used during mould-active prophylaxis. Recently, miRNAs have appeared as candidate diagnostic and prognostic markers of several diseases. A pilot clinical study was performed to evaluate the diagnostic utility of 14 microRNAs which can be related to invasive fungal infections. Based on our data miR-142-3p, miR-142-5p, miR-26b-5p and miR-21-5p showed significant overexpression (p < 0.005) due to invasive aspergillosis in hemato-oncology patients with profound neutropenia. A tetramiR assay was designed to monitor peripheral blood specimens. Optimal cut-off was estimated by using the median value (fold change 1.1) of the log10 transformed gene expressions. The biomarker panel was evaluated on two independent sample cohorts implementing different antimicrobial prophylactic strategies. The receiver operating characteristic analysis with area under the curve proved to be 0.97. Three miRNAs (miR-142-5p, miR-142-3p, miR-16-5p) showed significant expression alterations in episodes with sepsis. In summary, the tetramiR assay proved to be a promising diagnostic adjunct with sufficient accuracy and sensitivity to trace invasive aspergillosis in hemato-oncology patients.

Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling.

Here, we developed protocols to improve sensitivity, rigor and comparability of 16S rRNA gene amplification-based next-generation sequencing (NGS) results. A thorough study was performed by evaluating extraction efficiency with respect to the yield, purity, fragmentation of the purified DNA, and sequencing metrics considering the number of quality reads, amplicon sequence variants (ASVs), community structure and biodiversity. We identified batch-effects that significantly bias broiler gastrointestinal tract (GIT) community compositions and made recommendations to improve sensitivity, consistency, and cross-study comparability. We found that the purity of the extracted nucleic acid had a strong effect on the success rate of downstream library preparations. The preparation of stool bacterial suspensions from feces showed a significant positive influence on community biodiversity by enriching Gram-negative bacteria and cataloguing low abundant taxa with greater success than direct processing of fecal material. Applications relying on the automated Roche MagNa Pure 24 magnetic-bead based method provided results with high consistency therefore it seems to be the optimal choice in large-scale studies for investigating broiler GIT microbiota.

Effect of Carotenoids, Oligosaccharides and Anthocyanins on Growth Performance, Immunological Parameters and Intestinal Morphology in Broiler Chickens Challenged with Escherichia coli Lipopolysaccharide.

This study was conducted to investigate the effect of carotenoid, oligosaccharide and anthocyanin supplementation in broiler diets under Escherichia coli lipopolysaccharide (LPS) challenge. Ross 308 chickens were fed 5 diets: basal diet (control diet), diet supplemented with ß-glucan in 0.05% (positive control) and diets with 0.5% carotenoid-, oligosaccharide- or anthocyanin contents. On the 26th days of age, chickens were challenged intraperitoneally 2 mg LPS per kg of body weight. 12 h after injection, birds were euthanized, then spleen and ileum samples were collected. LPS induced increased relative mRNA expression of splenic (p = 0.0445) and ileal (p = 0.0435) interleukin-1ß (IL-1ß), which was lower in the spleen in carotenoid (p = 0.0114), oligosaccharide (p = 0.0497) and anthocyanin (p = 0.0303)-treated chickens compared to LPS-injected control birds. Dietary supplementation of carotenoids also decreased relative gene expression of splenic interleukin-6 (IL-6) (p = 0.0325). In the ileum, ß-glucan supplementation showed lower relative mRNA expression of toll-like receptor 5 (TLR-5) (p = 0.0387) compared to anthocyanin treatment. Gene expression of both splenic and ileal interferon-α (IFN-α), interferon-γ (IFN-γ), toll-like receptor 4 (TLR-4) and toll-like receptor 5 (TLR-5) were not influenced by dietary supplements. In conclusion, carotenoids, oligosaccharides and anthocyanins could partially mitigate the immune stress caused by LPS challenge. All of the compounds impacted longer villus height (p < 0.0001), villus height:crypt depth ratios were higher after ß-glucan (p < 0.0001) and anthocyanin (p = 0.0063) supplementations and thickened mucosa was observed in ß-glucan (p < 0.0001), oligosaccharide (p < 0.0001) and anthocyanin (p = 0.048) treatments. All of these findings could represent a more effective absorption of nutrients.

The Wilms' tumour 1 gene as a factor in non-syndromic hypospadias: evidence and controversy.

Hypospadias is one of the most frequent congenital anomalies of the male external genitalia. Its pathogenesis is due to largely unknown or poorly understood genetic factors and is further complicated by environmental-intrauterine-risk factors. One of the genes currently in focus by molecular biologists and clinicians studying syndromic forms of hypospadias is the Wilms' tumour 1 (WT1) gene. There is controversy over whether WT1 defects are also responsible for isolated hypospadias. In this review, we briefly cover the role of WT1 as a transcription factor and discuss proposed pathogenic pathways leading to hypospadias, outlining possible directions for research. We assess available evidence on the gene's mutations and polymorphisms recently suggested in the background of the disease, and examine the putative role of WT1-associated proteins. We also review relevant aspects of genome-wide association studies carried out so far, and raise some points to consider in future efforts.

Mutation in afsR Leads to A-Factor Deficiency in Streptomyces griseus B2682.

BACKGROUND/AIMS: A-factor, a γ-butyrolactone autoregulator, in Streptomyces griseus is involved in the regulation of differentiation and antibiotic production. Here we studied the S. griseus B2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the afsR gene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. METHODS: Wild afsR was cloned to the pHJL401 shuttle vector and was transformed to the S. griseus AFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. RESULTS: Transformation of AFN by a wild afsR restored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of afsR to the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. CONCLUSIONS: AfsR has an effect on the regulation of A-factor production in S. griseus. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.

Nucleotide Transition 390C-T in the Wilms' Tumor 1 Gene: A Risk Factor of Hypospadias?

INTRODUCTION: The gene Wilms' tumor 1 (WT1) encodes a unique transcription factor. Its defects are known to cause a wide range of complex genitourinary malformations and may contribute to non-syndromic forms of hypospadias. MATERIALS AND METHODS: We performed WT1 mutation analysis and copy number analysis of WT1-interacting protein in 13 Hungarian patients diagnosed with isolated hypospadias. RESULTS: Sequencing of WT1 revealed a high frequency of heterozygosity for transition 390C-T (5 heterozygotes out of 13 patients, including 2 brothers). WT1-interacting protein had a normal copy number in all patients. CONCLUSION: Nucleotide substitution 390C-T may play a role in the pathogenesis of non-syndromic hypospadias. The genotype-phenotype correlation should be confirmed by a larger-scale analysis.

WT1 Expression in Adult Acute Myeloid Leukemia: Assessing its Presence, Magnitude and Temporal Changes as Prognostic Factors.

Expression of the gene Wilms tumor 1 (WT1) has been suggested as a marker of minimal residual disease in acute myeloid leukemia (AML), but literature data are not without controversy. Our aim was to assess the presence, magnitude and temporal changes of WT1 expression as prognostic factors. 60 AML patients were followed until death or the end of the 6-year observation period. Blood samples were taken at diagnosis, post-induction, during remission and in case of a relapse. Using quantitative real-time PCR, we determined WT1 expression from each sample, normalized it against the endogenous control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and classified samples as negative, moderately positive or highly positive. We divided the patients into groups based on detected WT1 expression values, illustrated overall and disease-free survival on Kaplan-Meier curves, and compared differences between each group by the logrank test. Disappearance of WT1-positivity during chemotherapy had a favorable effect on survival. Interestingly, no difference was seen between the survivals of WT1-positive subgroups that expressed moderate or high levels of WT1 mRNA. A 1-log decrease in WT1 expression without becoming negative did not affect prognosis, either. Our results suggest that defining a cut-off value for WT1-positivity, rather than just using logarithmic figures of changes in gene expression, might have prognostic use in post-induction AML patients. We encourage further, larger-scale studies.

Novel and sensitive qPCR assays for the detection and identification of aspergillosis causing species.

Despite concerted efforts, diagnosis of aspergillosis is still a great challenge to clinical microbiology laboratories. Along with the requirement for high sensitivity and specificity, species-specific identification is important. We developed rapid, sensitive and species-specific qPCR assays using the TaqMan technology for the detection and identification of Aspergillus fumigatus and Aspergillus terreus. The assays were designed to target orthologs of the Streptomyces factor C gene that are only found in a few species of filamentous fungi. Fungi acquired this gene through horizontal gene transfer and divergence of the gene allows identification of species. The assays have potential as a molecular diagnosis tool for the early detection of fungal infection caused by Aspergillus fumigatus and Aspergillus terreus, which merits future diagnostic studies. The assays were sensitive enough to detect a few genomic equivalents in blood samples.

WT1 overexpression affecting clinical outcome in non-hodgkin lymphomas and adult acute lymphoblastic leukemia.

The Wilms tumor 1 (WT1) gene has a complex role as a transcriptional regulator, acting as tumor suppressor or oncogene in different malignancies. The prognostic role of its overexpression has been well-studied in leukemias, especially acute myeloid leukemia (AML), but not in lymphomas. For the first time to our knowledge, we present a study demonstrating the correlation of WT1 expression and survival in various non-Hodgkin lymphomas. We also studied the prognostic implications of WT1 overexpression in adult acute lymphoblastic leukemia (ALL). In our sample of 53 patients--25 with diffuse large B-cell lymphoma (DLBCL), 8 with mantle cell lymphoma (MCL), 9 with peripheral T-cell lymphoma (PTCL), 2 with Burkitt's lymphoma, 2 with mucosa-associated lymphoid tissue (MALT) lymphoma, and 7 with B-cell ALL--, we measured WT1 mRNA from blood samples by quantitative RT-PCR, and divided the patients into subgroups based on the level of expression. Kaplan-Meier survival curves were drawn and compared using the logrank test. In the sample of DLBCL patients, the difference in overall and disease-free survival between WT1-positive and negative subgroups was significant (p = 0.0475 and p = 0.0004, respectively), and in a few observed cases, a sudden increase in WT1 expression signified a relapse soon followed by death. Disease-free survival curves in MCL and ALL were similarly suggestive of a potential role played by WT1. In PTCL, though WT1-positivity was detected in 4 out of 9 cases, it did not seem to affect survival. The few cases of MALT and Burkitt's lymphoma all proved to be WT1-negative.

Disruption of SCO5461 gene coding for a mono-ADP-ribosyltransferase enzyme produces a conditional pleiotropic phenotype affecting morphological differentiation and antibiotic production in Streptomyces coelicolor.

The SCO5461 gene of Streptomyces coelicolor A3(2) codes for an ADP-ribosyltransferase enzyme that is predicted to be a transmembrane protein with an extracellular catalytic domain. PCR-targeted disruption of the gene resulted in a mutant that differentiated normally on complex SFM medium; however, morphological differentiation in minimal medium was significantly delayed and this phenotype was even more pronounced on osmotically enhanced minimal medium. The mutant did not sporulate when it was grown on R5 medium, however the normal morphological differentiation was restored when the strain was cultivated beside the wild-type S. coelicolor M145 strain. Comparison of the pattern of ADP-ribosylated proteins showed a difference between the mutant and the wild type, fewer modified proteins were present in the cellular crude extract of the mutant strain. These results support our previous suggestions that protein ADP-ribosylation is involved in the regulation of differentiation and antibiotic production and secretion in Streptomyces.

Identification of ß-lactamases in human and bovine isolates of Staphylococcus aureus strains having borderline resistance to penicillinase-resistant penicillins (PRPs) with proteomic methods.

Methicillin and oxacillin-hydrolyzing enzymes of 6 borderline methicillin-resistant and 1 methicillin-resistant Staphylococcus aureus strains isolated from human clinical samples and 4 borderline methicillin-resistant S. aureus strains isolated from bovine mastitis were investigated. As previous studies suggested the involvement of an additional enzyme besides the penicillinase BlaZ in the determination of borderline resistance, we analyzed the expressed extracellular and membrane-bound ß-lactamases with 2-D gel electrophoresis and mass spectrometry. Our analysis showed that the penicillin-hydrolyzing BlaZ alone was responsible for the hydrolysis of both methicillin and oxacillin. All supernatant and membrane fractions contained the same enzyme with slight sequence variations. The size and pI of the proteins were also variable, probably due to spontaneous hydrolysis and/or posttranslational modifications. Interestingly, we found also cytotoxins and other virulence factors in some nitrocefin-hydrolyzing dots, suggesting that those proteins might have a role in the reduction of local antibiotic concentration.