Variations in the Sporulation Efficiency of Pathogenic Freshwater Oomycetes in Relation to the Physico-Chemical Properties of Natural Waters.

Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation.

Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level.

The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a non-destructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.

Nucleotide variations of TP53 exon 4 found in intracranial meningioma and in silico prediction of their significance.

The aim of the present study was to identify TP53 exon 4 mutations in patients with meningioma and to investigate their potential association with specific tumor pathology. Nucleotide alterations were investigated in 48 meningiomas via the direct sequencing of TP53 exon 4 in patient tumor and blood samples using the DNA Sanger method with the BigDyeTerminator v3.1 Cycle Sequencing kit and Applied Biosystems 3730XL apparatus. The results revealed that TP53 exon 4 was frequently altered in meningioma, occurring in 60.4% of the patients investigated. A total of 18 different alterations were detected in the meningioma samples assessed in the current study. The majority of these appeared more than once and some were repeatedly identified in several patients. Changes at codons 72 (c.215G>C) and 62 (c.186delA) were highly prevalent, occurring in 44.8% of patients. Other changes detected via frequency analysis included: Five substitutions on codon 105 (c.315C>T); four insertions on codon 70 (c.209_210insG); three insertions on codon 64 (c.190C>G), 82 (245C>T; 245delC; 243_244insA) and 104 (c.312G>A); and two insertions on codons 108 (c.322G>C), 71 (c.213C>A), 73 (c.217G>A), 91 (c.271T>C) and 100 (c.300G>T). Codons 68 (c.202_203insT), 77 (c.229C>T), 88 (c.263C>G) and 92 (c.276C>A) were altered once. Alterations on codons 82, 91, 108, 104, 105, 70 and 92 were characterized as possibly damaging by PolyPhen-2 and Mutation Taster2 tools. The current study also demonstrated that nucleotide alterations were significantly associated with the loss of p53 expression (P=0.04) and female patients (P=0.049), particularly codon 72. The results present novel data on the mutational spectrum of TP53 in meningeal brain tumors.

Genetic changes of MLH1 and MSH2 genes could explain constant findings on microsatellite instability in intracranial meningioma.

Postreplicative mismatch repair safeguards the stability of our genome. The defects in its functioning will give rise to microsatellite instability. In this study, 50 meningiomas were investigated for microsatellite instability. Two major mismatch repair genes, MLH1 and MSH2, were analyzed using microsatellite markers D1S1611 and BAT26 amplified by polymerase chain reaction and visualized by gel electrophoresis on high-resolution gels. Furthermore, genes DVL3 (D3S1262), AXIN1 (D16S3399), and CDH1 (D16S752) were also investigated for microsatellite instability. Our study revealed constant presence of microsatellite instability in meningioma patients when compared to their autologous blood DNA. Altogether 38% of meningiomas showed microsatellite instability at one microsatellite locus, 16% on two, and 13.3% on three loci. The percent of detected microsatellite instability for MSH2 gene was 14%, and for MLH1, it was 26%, for DVL3 22.9%, for AXIN1 17.8%, and for CDH1 8.3%. Since markers also allowed for the detection of loss of heterozygosity, gross deletions of MLH1 gene were found in 24% of meningiomas. Genetic changes between MLH1 and MSH2 were significantly positively correlated (p = 0.032). We also noted a positive correlation between genetic changes of MSH2 and DVL3 genes (p = 0.034). No significant associations were observed when MLH1 or MSH2 was tested against specific histopathological meningioma subtype or World Health Organization grade. However, genetic changes in DVL3 were strongly associated with anaplastic histology of meningioma (χ2 = 9.14; p = 0.01). Our study contributes to better understanding of the genetic profile of human intracranial meningiomas and suggests that meningiomas harbor defective cellular DNA mismatch repair mechanisms.

AXIN1 Expression and Localization in Meningiomas and Association to Changes of APC and E-cadherin.

BACKGROUND/AIM: Tumor suppressor gene AXIN1 is an inhibitor of Wnt signaling pathway. It down-regulates the pathway's main signaling effector molecule, beta-catenin, in an AXIN-based destruction complex. In the present study we investigated the involvement of AXIN1 in intracranial meningioma. MATERIALS AND METHODS: Loss of heterozygosity and microsatellite instability analyses were performed. The consequences of genetic changes on protein expression levels were studied in the same patients by immunohistochemistry. RESULTS: Allelic deletions of AXIN1 gene were found in 21.1% of meningiomas. Microsatellite instability was also observed in 5.3% of cases. Weak or lack of AXIN1 expression was found in 21.9% of meningiomas. We found strong statistical correlations between cytoplasmic localization of AXIN1 and its weak expression and also between the simultaneous cytoplasmic and nuclear localizations and moderate and strong expression levels (p<0.000). The findings on AXIN1 were compared to concomitant expression of APC, beta-catenin and E-cadherin in the same patients by Chi-Square tests and Pearson's correlations. Analysis revealed that AXIN1 genetic changes were significantly associated to lack of the expression of APC and presence of mutant APC proteins (p<0.018). Moderate and strong cytoplasmic and nuclear AXIN1 expressions were positively correlated to strong expression of E-cadherin (p<0.05). CONCLUSION: Our findings on genetic changes and expression levels of AXIN1 bring novel data on its involvement in meningeal brain tumors and reveal AXIN1's relation to specific Wnt molecules.

Loss of p53 expression is accompanied by upregulation of beta-catenin in meningiomas: a concomitant reciprocal expression.

Crosstalk between Wnt and p53 signalling pathways in cancer has long been suggested. Therefore in this study we have investigated the involvement of these pathways in meningiomas by analysing their main effector molecules, beta-catenin and p53. Cellular expression of p53 and beta-catenin proteins and genetic changes in TP53 were analysed by immunohistochemistry, PCR/RFLP and direct sequencing of TP53 exon 4. All the findings were analysed statistically. Our analysis showed that 47.5% of the 59 meningiomas demonstrated loss of expression of p53 protein. Moderate and strong p53 expression in the nuclei was observed in 8.5% and 6.8% of meningiomas respectively. Gross deletion of TP53 gene was observed in one meningioma, but nucleotide alterations were observed in 35.7% of meningiomas. In contrast, beta-catenin, the main Wnt signalling molecule, was upregulated in 71.2%, while strong expression was observed in 28.8% of meningiomas. The concomitant expressions of p53 and beta-catenin were investigated in the same patients. In the analysed meningiomas, the levels of the two proteins were significantly negatively correlated (P = 0.002). This indicates that meningiomas with lost p53 upregulate beta-catenin and activate Wnt signalling. Besides showing the reciprocal relationship between proteins, we also showed that the expression of p53 was significantly (P = 0.021) associated with higher meningioma grades (II and III), while beta-catenin upregulation was not associated with malignancy grades. Additionally, women exhibited significantly higher values of p53 loss when compared to males (P = 0.005). Our findings provide novel information about p53 involvement in meningeal brain tumours and reveal the complex relationship between Wnt and p53 signalling, they suggest an important role for beta-catenin in these tumours.

Histological groups of human postpubertal testicular germ cell tumours harbour different genetic alterations.

BACKGROUND: Testicular germ cell tumours are the most common malignancies in young males. Molecular biology studies of these tumours are often contradictory. Two histological groups, seminoma and non-seminoma, differ both morphologically and in malignant behaviour. Although a common cytogenetic feature is seen, namely the amplification of the 12p chromosomal region, the development mechanisms of less aggressive seminomas and more aggressive non-seminomas are unknown. MATERIALS AND METHODS: Occurrence of structural genetic alterations was analyzed in 18 seminomas and 22 non-seminomas for genes involved in the malignant tumour phenotype: cadherin 1, Type 1, E-cadherin (Epithelial), CDH1; adenomatous polyposis coli, APC; NME/NM23 nucleoside diphosphate kinase 1, NME1; tumour protein P53, TP53; cyclin-dependent kinase inhibitor 2A, CDKN2A; retinoblastoma 1, RB1; RAD51 recombinase, RAD51; mutS homolog 2, MSH2; MutL homolog 1, MLH1; breast cancer 1, early onset, BRCA1; BCL2-Associated X Protein, BAX; ATP-Binding Cassette, Sub-Family G (WHITE), Member 2, ABCG2. Genetic alterations, loss of heterozygosity and microsatellite instability, were analyzed using restriction fragment or microsatellite repeat length polymorphisms. RESULTS: A difference in genetic alteration occurrence between seminomas and non-seminomas was observed. CONCLUSION: Occurrence of genetic alterations correlates with clinical behaviour of these tumours and may indicate that such alterations could occur early in the development of seminomas and non-seminomas.

Loss of heterozygosity of selected tumor suppressor genes in human testicular germ cell tumors.

Human testicular germ cell tumors (TGCTs) are histologically heterogenous neoplasms with a variable malignant potential. Two main groups of germ cell tumors occur in men: seminomas and nonseminomas. In the present study, a set of four tumor suppressor genes was investigated in testicular cancers. CDH1, APC, p53, and nm23-H1 genes were tested for loss of heterozygosity (LOH). Thirty-eight testicular germ cell tumors (17 seminomas and 21 nonseminomas) were analyzed by PCR using restriction fragment length polymorphism or the dinucleotide/tetranucleotide repeat polymorphism method. An allelic loss of p53 at exon 4 was detected in five nonseminomas, whereas LOH of p53 at intron 6 occurred in one of the seminoma and two of the nonseminoma samples. Allelic losses of the APC gene were present in three seminomas and one nonseminoma, whereas one seminoma and three nonseminomas showed LOH of CDH1. The analysis of allelic losses showed no common structural genetic alterations in tumor tissues, although a different pattern of LOH was observed between the two main histological groups of TGCTs.

Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors.

BACKGROUND: Testicular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs. MATERIALS AND METHODS.: Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method. RESULTS: LOH of the CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component. CONCLUSIONS: The higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior.