Tracing TET1 expression in prostate cancer: discovery of malignant cells with a distinct oncogenic signature.

BACKGROUND: Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is involved in DNA demethylation and transcriptional regulation, plays a key role in the maintenance of stem cell pluripotency, and is dysregulated in malignant cells. The identification of cancer stem cells (CSCs) driving tumor growth and metastasis is the primary objective of biomarker discovery in aggressive prostate cancer (PCa). In this context, we analyzed TET1 expression in PCa. METHODS: A large-scale immunohistochemical analysis of TET1 was performed in normal prostate (NOR) and PCa using conventional slides (50 PCa specimens) and tissue microarrays (669 NOR and 1371 PCa tissue cores from 371 PCa specimens). Western blotting, RT-qPCR, and 450 K methylation array analyses were performed on PCa cell lines. Genome-wide correlation, gene regulatory network, and functional genomics studies were performed using publicly available data sources and bioinformatics tools. RESULTS: In NOR, TET1 was exclusively expressed in normal cytokeratin 903 (CK903)-positive basal cells. In PCa, TET1 was frequently detected in alpha-methylacyl-CoA racemase (AMACR)-positive tumor cell clusters and was detectable at all tumor stages and Gleason scores. Pearson's correlation analyses of PCa revealed 626 TET1-coactivated genes (r > 0.5) primarily encoding chromatin remodeling and mitotic factors. Moreover, signaling pathways regulating antiviral processes (62 zinc finger, ZNF, antiviral proteins) and the pluripotency of stem cells were activated. A significant proportion of detected genes exhibited TET1-correlated promoter hypomethylation. There were 161 genes encoding transcription factors (TFs), of which 133 were ZNF-TFs with promoter binding sites in TET1 and in the vast majority of TET1-coactivated genes. CONCLUSIONS: TET1-expressing cells are an integral part of PCa and may represent CSCs with oncogenic potential.

Preliminary results of neutron and X-ray diffraction data collection on a lytic polysaccharide monooxygenase under reduced and acidic conditions.

Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper-oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.

A single quantum dot-based fluorescence resonance energy transfer biosensor for antibody-free detection of ten-eleven translocation 1.

We developed a single quantum dot-based fluorescence resonance energy transfer biosensor for antibody-free detection of ten-eleven translocation 1 (TET1). This biosensor can sensitively detect TET1 in a homogeneous manner without the involvement of any specific antibodies, and it can be used for accurate measurement of TET1 activity in human neuroblastoma cells and the screening of TET1 inhibitors.

Prognostic role of high TET1 expression in patients with solid tumors: A meta-analysis.

BACKGROUND: Recently, increased expression of TET1 has been shown to inhibit tumor development in many studies. Therefore, a meta-analysis was conducted to assess the prognostic role of TET1 in solid tumors. METHODS: PubMed, Embase, and the Web of Science (last updated on June 13, 2019) were searched and 16 eligible studies involving 3100 patients were eventually taken forward into the meta-analysis. RESULTS: Pooled results indicated that higher TET1 expression in cancer tissues was associated with improved overall survival (OS) [hazard ratio (HR) = 0.736, 95% confidence interval (95% CI) = 0.542-0.998, P = .049]. In the subgroup analysis, higher TET1 expression in respiratory tumors (HR = 0.778, 95% CI = 0.639-0.946, P = .012) and breast cancer in Asian patients (HR = 0.326, 95% CI = 0.199-0.533, P < .001) were significantly associated with better OS. In addition, the association between high TET1 expression and prolonged OS was also statistically significant in the following subgroups; data source from samples (HR = 0.561, 95% CI = 0.384-0.819, P = .003), reported in text (HR = 0.539, 95% CI = 0.312-0.931, P = .027), TET1 protein (HR = 0.635, 95% CI = 0.409-0.984, P = .042), Asians (HR = 0.563, 95% CI = 0.376-0.844, P = .005). CONCLUSION: This meta-analysis displays that high expression levels of TET1 in tissues is significantly associated with better survival in patients with solid tumors. This finding can be used as evidence to the tone that TET1 may be a useful target for the treatment of patients with solid tumors in the future.

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata / Determinación de concentraciones letales y niveles de enzimas metabólicas de insecticidas en Triatoma dimidiata

Abstract: Objective: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. Materials and methods: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. Results: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and β-eterase levels. Conclusion: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

Resumen: Objetivo: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. Material y métodos: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. Resultados: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y β-eterasas. Conclusión: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata.

OBJECTIVE: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. MATERIALS AND METHODS: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. RESULTS: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and ß-eterase levels. CONCLUSIONS: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

OBJETIVO: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. MATERIAL Y MÉTODOS: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. RESULTADOS: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y ß-eterasas. CONCLUSIONES: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Ciliary and cytoskeletal functions of an ancient monooxygenase essential for bioactive amidated peptide synthesis.

Many secreted peptides used for cell-cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies.

TP53 inactivation and expression of methylation-associated proteins in gastric adenocarcinoma with enteroblastic differentiation.

Gastric adenocarcinoma with enteroblastic differentiation (GAED) is a rare variant of aggressive adenocarcinoma. We demonstrated previously that GAED is genetically characterized by frequent TP53 mutation. In this study, we aimed to further clarify the mechanism of inactivation of TP53 in GAED in the light of promoter methylation of TP53, and expression of methylation-associated proteins such as Ten-eleven translocation (TET) 1 and 5-hydroxymethylcytosine (5-hmc) in addition to ATM mutations. We analyzed 51 cases of GAED. The ATM mutation was detected in only one case. Promoter methylation of TP53 was detected in 18% and frequency of loss of heterozygosity (LOH) at TP53 locus was 37.2%. Reduced TET1 expression was found in 29 cases (56.9%) and was significantly associated with advanced stage (p = 0.01), lymph node metastasis (p = 0.04), and macroscopic type (p = 0.01). Reduced 5-hmc expression was found in 28 cases (54.9%) and was significantly associated with advanced stage (p = 0.01), gender (p = 0.01), tumor location (p = 0.03), tumor size (p = 0.01), and lymph node metastasis (p = 0.01). Among 9 cases with TP53 promoter methylation, reduced expression of TET1 was observed in 6 cases, and reduced expression of 5-hmc was observed in 5 cases. Reduced expression of both TET1 and 5-hmc was significantly associated with adverse clinical outcomes. In summary, promoter methylation of TP53 is partly involved in loss of p53 expression. Aberrant methylation by reduced TET1 and 5-hmc may be involved in the development of aggressive GAED.

Effect of common processing of soybeans on the enzymatic activity and detectability of the protein, Dicamba Mono-Oxygenase (DMO), introduced into dicamba-tolerant MON 87708.

Assessing the safety of genetically engineered crops includes evaluating the risk (hazard and exposure) of consuming their newly expressed proteins. The dicamba monooxygenase (DMO) protein, introduced into soybeans to confer tolerance (DT) to dicamba herbicide, was previously characterized and identified to pose no food or feed safety hazards. Most agricultural commodities (e.g., soybeans, maize) enter the food supply after processing methods that can include exposure to high temperatures, harsh solvents or pH extremes that can adversely impact the structure and function of proteins. To understand the likelihood of exposure to DMO in foods from DT soy, enzymatically active and/or immunodetectable forms of DMO were measured in pilot-scale productions of two soy foods (soymilk and tofu), and eight processed fractions (full fat flour, inactivated full fat flour, defatted flour, toasted meal, protein isolate, protein concentrate, crude lecithin, and refined, bleached and deodorized oil). Western blot analysis detected DMO in tofu and in five of the eight processed fractions. DMO activity was not detected in either soymilk or tofu, nor in six of the eight processed fractions. Therefore, many commercial soy processing methods can denature and/or degrade introduced proteins, like DMO. Although the DMO protein has shown no evidence of hazard, this study demonstrates that processing further reduces any food or feed risk by limiting dietary exposure to intact DMO protein.

Insecticide resistance status, frequency of L1014F Kdr and G119S Ace-1 mutations, and expression of detoxification enzymes in Anopheles gambiae (s.l.) in two regions of northern Benin in preparation for indoor residual spraying.

BACKGROUND: This study aims to provide baseline data on the resistance status to insecticides, the frequency of mechanisms involved and the impact of the association with the synergist piperonyl butoxide (PBO) on resistant Anopheles gambiae (s.l.) populations in two regions of northern Benin, prior to an indoor residual spraying campaign and introduction of next generation long-lasting insecticidal nets (LLINs) incorporating PBO. METHODS: Adult Anopheles gambiae (s.l.) originating from larvae collected in two study regions (Alibori within the Kandi-Gogounou-Segbana districts and Donga within the Djougou-Copargo-Ouake districts) were tested with impregnated papers (bendiocarb 0.1%, pirimiphos-methyl 0.25%, permethrin 0.75% and deltamethrin 0.05%). The synergist PBO was used to check for the involvement of detoxification enzymes in pyrethroid resistant populations. Molecular analyses were performed for the identification of species within the Anopheles gambiae (s.l.) complex and kdr L1014F and G119S Ace-1 mutations. Biochemical assays assessed the activity of detoxification enzymes. RESULTS: Anopheles gambiae (s.l.) was resistant to pyrethroids, with a mortality range of 25-83% with deltamethrin and 6-55% with permethrin. A significant increase in mortality was observed after pre-exposure to PBO for both deltamethrin (63-99%) and permethrin (56-99%). With bendiocarb, An. gambiae (s.l.) were susceptible in Kandi (99% mortality), with possible resistance (92-95%) recorded in Djougou, Copargo, Gogounou, Ouake and Segbana. All study populations were fully susceptible to pirimiphos-methyl. The frequencies of resistant mutations varied according to species and sites: 0.67-0.88 for L1014F kdr and 0-0.06 for G119S Ace-1. Three study locations (Djougou, Gogounou and Kandi) showed high oxidase activity and four sites (Djougou, Ouake, Copargo and Kandi) showed elevated esterase activity. CONCLUSIONS: This study confirms resistance to pyrethroids and suggests emerging bendiocarb resistance in An. gambiae (s.l.) populations in northern Benin. However, recovery of susceptibility to pyrethroids after PBO exposure, and susceptibility to organophosphates in the An. gambiae (s.l.) populations indicate that next generation LLINs incorporating PBO synergist combined with an indoor residual spraying (IRS) campaign with organophosphate insecticides may be regarded as alternative control tools.

Recent advances in research on aspartate ß-hydroxylase (ASPH) in pancreatic cancer: A brief update.

Pancreatic cancer (PC) is a highly aggressive tumor, often difficult to diagnose and treat. Aspartate ß-hydroxylase (ASPH) is a type II transmembrane protein and the member of α-ketoglutarate-dependent dioxygenase family, found to be overexpressed in different cancer types, including PC. ASPH appears to be involved in the regulation of proliferation, invasion and metastasis of PC cells through multiple signaling pathways, suggesting its role as a tumor biomarker and therapeutic target. In this review, we briefly summarize the possible mechanisms of action of ASPH in PC and recent progress in the therapeutic approaches targeting ASPH.

Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) originate from enterochromaffin cells scattered in the intestinal mucosa of the ileum and jejunum. Loss of one copy of chromosome 18 is the most frequent observed aberration in primary tumors and metastases. The aim of this study was to investigate possible involvement of 5-hydroxymethylcytosine (5hmC), TET1 and TET2 in SI-NETs. METHODS: The analysis was conducted using 40 primary tumors and corresponding 47 metastases. The level of 5hmC, TET1 and TET2 was analyzed by DNA immune-dot blot assay and immunohistochemistry. Other methods included a colony forming assay, western blotting analysis, and quantitative bisulfite pyrosequencing analysis. The effect of the exportin-1 nuclear transport machinery inhibitors on cell proliferation and apoptosis was also explored using two SI-NET cell lines. RESULTS: Variable levels of 5hmC and a mosaic staining appearance with a mixture of positive and negative cell nuclei, regardless of cell number and staining strength, was observed overall both in primary tumors and metastases. Similarly aberrant staining pattern was observed for TET1 and TET2. In a number of tumors (15/32) mosaic pattern together with areas of negative staining was also observed for TET1. Abolished expression of TET1 in the tumors did not seem to involve hypermethylation of the TET1 promoter region. Overexpression of TET1 in a colony forming assay supported a function as cell growth regulator. In contrast to 5hmC and TET1, TET2 was also observed in the cytoplasm of all the analyzed SI-NETs regardless of nuclear localization. Treatment of CNDT2.5 and KRJ-I cells with the exportin-1 (XPO1/CRM1) inhibitor, leptomycin B, induced reduction in the cytoplasm and nuclear retention of TET2. Aberrant partitioning of TET2 from the nucleus to the cytoplasm seemed therefore to involve the exportin-1 nuclear transport machinery. Reduced cell proliferation and induction of apoptosis were observed after treatment of CNDT2.5 and KRJ-I cells with leptomycin B or KPT-330 (selinexor). CONCLUSIONS: SI-NETs are epigenetically dysregulated at the level of 5-hydroxymethylcytosine/ TET1/TET2. We suggest that KPT-330/selinexor or future developments should be considered and evaluated for single treatment of patients with SI-NET disease and also in combinations with somatostatin analogues, peptide receptor radiotherapy, or everolimus.

Correlation between high expression levels of jumonji domain-containing 4 and short survival in cases of colon adenocarcinoma.

In this study, we investigated the expression of jumonji domain-containing 4 (JMJD4) in colon adenocarcinoma (CA) look for evidences for future studies on clinical diagnostic and prognostic value. The immunohistochemical (IHC) reactivity of JMJD4 was assessed in human tissue microarrays using monoclonal antibodies. An initial investigation revealed that the expression of JMJD4 protein was significantly higher in tumor tissue of the colon and liver than in normal tissue. Upon further investigation, we observed significant positivity of JMJD4 between 59 paired samples from CA tissue and adjacent normal tissue. JMJD4 protein expression in CA differed significantly according to the histological grade and M-class (distant metastasis). We also determined that the mRNA or protein expression of JMJD4 was significantly associated with poor survival in patients with CA. Finally, univariate and multivariate analysis revealed that JMJD4 expression could be a prognostic indicator for patients with CA and may provide a new target for the development of novel therapies for the treatment of CA.

Cardiomyocytes have mosaic patterns of protein expression.

Skeletal myocytes have well-established fast and slow twitch fibers with unique gene and protein specific expression patterns. By immunohistochemical staining, these show a mosaic pattern across myocytes. We hypothesized cardiac myocytes may behave similarly where some proteins are differentially expressed between mature cardiomyocytes. We utilized the tool HPASubC on over 52,000 cardiac images of the Human Protein Atlas to identify differential protein expression patterns by immunohistochemistry across the cardiomyocytes. We matched identified proteins to open chromatin and gene expression data. We identified 143 putative proteins with mosaic patterns of expression across the cardiomyocytes. We validated four of these proteins (MYL3, MYL4, PAM, and MYOM1) and demonstrated unique atrial or ventricular patterns of expression for each. Acetylation of histone H3K27 at the promoters of these four genes were consistent with the atrial/ventricular expression patterns. Despite the generally accepted homogeneity of cardiomyocytes, a small subset of proteins varies between cardiomyocytes in a mosaic pattern. This fundamental process has been previously uncharacterized. These changes may inform on different functional and disease-related activities of proteins in individual cardiomyocytes.

Evidence for multiple-insecticide resistance in urban Aedes albopictus populations in southern China.

BACKGROUND: Aedes albopictus (Skuse) is an invasive mosquito that has become an important vector of chikungunya, dengue and Zika viruses. In the absence of specific antiviral therapy or a vaccine, vector management is the sole method available for reducing Aedes-induced disease morbidity. Determining the resistance status of Ae. albopictus to insecticides and exploring the resistance mechanisms is essential for future vector control planning. METHODS: Aedes albopictus larvae and pupae were sampled from six sites (two sites each from urban, suburban and rural) in Guangzhou. The resistance bioassays were conducted against Bacillus thuringiensis israelensis (Bti): deltamethrin, propoxur and malathion for larvae; and deltamethrin, DDT, propoxur and malathion for adults. P450 monooxygenase (P450s), glutathione S-transferase (GSTs) and carboxylesterase (COEs) activities of adult mosquitoes were measured. Mutations at the knockdown resistance (kdr) gene were analyzed, and the association between kdr mutations and phenotypic resistance was tested. RESULTS: Adult bioassays revealed varied susceptibility against DDT, deltamethrin and propoxur in the six Ae. albopictus populations. Significantly lower mortality rates were found in urban populations than suburban and rural populations. Urban mosquito populations showed resistance against DDT, deltamethrin and propoxur, while one rural population was resistant to DDT. All populations tested were susceptible to malathion. Larval bioassays results indicated that all populations of Ae. albopictus were sensitive to the larvicide Bti and malathion. Resistance to deltamethrin and propoxur was common in larval populations. The F1534S and F1534 L mutations were found to be significantly associated with deltamethrin resistance. Biochemical assays indicated elevated detoxification enzyme activities in the field mosquito populations. CONCLUSIONS: Aedes albopictus populations in Guangzhou, especially in urban areas, have developed resistance to the commonly used insecticides, primarily DDT and deltamethrin. This finding calls for resistance management and developing counter measures to mitigate the spread of resistance.

A Degradome-Based Polymerase Chain Reaction to Resolve the Potential of Environmental Samples for 2,4-Dichlorophenol Biodegradation.

A clean way to overcome environmental pollution is biodegradation. In this perspective, at the intersection of biodegradation and metagenomics, the degradome is defined as the totality of genes related to the biodegradation of a certain compound. It includes the genetic elements from both culturable and uncultured microorganisms. The possibility of assessing the biodegradation potential of an environmental samples, using a degradome-based polymerase chain reaction, was explored. 2,4-Dichlorophenol (2,4-DCP) was chosen as a model and the use of tfdB gene as a biodegradation marker was confirmed by bioinformatics study of TfdB protein. Five primer pairs were designed for the detection of different tfdB gene families. A total of 16 environmental samples were collected from Egyptian agricultural soils and wastewaters and tested for the presence of 2,4-DCP. The biodegradation capacity of 2,4-DCP was determined, for all isolated consortia, to reach up to 350 mg/l. Metagenomic DNA was extracted directly from the soil samples while successive 2,4-DCP-degrading microbial communities were enriched, with increasing concentrations of 2,4-DCP, then their DNA was extracted. The extracted DNA was tested for the distribution of the tfdB gene using a degradome-based polymerase chain reaction. tfdB-1 and tfdB-2 were detected in 5 and 9 samples, respectively. However, the co-existence of both genes was detected only in five samples. All tfdB positive samples were capable of 2,4-DCP degradation. The developed approach of assessing the potential of different environments for degrading 2,4-DCP was successfully measured in terms of accuracy (81.25%) and specificity (100%).

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.

Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.

The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption.

Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. To establish this new assay method, a chitin-active LPMO from Bacillus atrophaeus (BatLPMO10) was cloned as a model enzyme. An expression system using B. subtilis as the host cell yielded a simple purification process without complicated periplasmic fractionation, as well as improved productivity by 3.7-fold higher than that of Escherichia coli BL21(DE3). At the optimum pH determined using a newly developed assay, BatLPMO10 showed the highest activity in terms of promoting chitin degradation by a chitinase. In addition, the assay method indicated that BatLPMO10 was inhibited by sodium ions, and BatLPMO10 and a chitinase mutually enhanced each other's activities upon degrading chitin as the substrate. In conclusion, this hydrolase-free ascorbate assay allows quantitative analysis of BatLPMO10 without a coupled enzyme.