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.

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.

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.

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.

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.

Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme.

The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its µ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

Evaluation of HAAH/humbug quantitative detection in the diagnosis of hepatocellular carcinoma.

Human aspartyl-(asparaginyl)-ß-hydroxylase (HAAH) is a type 2 transmembrane protein and an α-ketoglutarate-dependent dioxygenase that can stereospecifically catalyze the post-translational hydroxylation reaction of ß-carbon atoms of aspartic acid and asparagine residues present in epidermal growth factor-like domains of certain specific proteins. Humbug is a truncated isoform of aspartyl (asparaginyl) ß-hydroxylase that lacks the catalytic domain. A series of reports demonstrated that overexpression of HAAH/humbug was identified in hepatocellular carcinoma (HCC) and various tumor tissues. However, the prognostic value of HAAH/humbug expression in HCC remains unclear. The purpose of this study was to investigate the expression of the HAAH/humbug gene at the mRNA and protein levels in HCC and to assess the overexpression of HAAH/humbug as a diagnostic and prognostic marker in HCC. HAAH/humbug mRNA levels were measured in 120 HCCs and 40 paired non-tumor liver tissues by molecular beacon (MB) quantitative RT-PCR. Immunohistochemical staining was used to detect the protein level of the HAAH/humbug in the same specimens. ROC analysis was performed based on the expression levels of the HAAH/humbug gene in 120 cases of HCC tissues and 40 cases of adjacent non-tumor liver tissues. The results showed that 117 (97.5%) of the 120 frozen sections of patients with HCCs had HAAH/humbug-positive immunoreactivity, whereas the 40 adjacent non-tumor liver tissues exhibited no staining. Higher levels of HAAH/humbug mRNA were found in 114 (95%) of the 120 HCC tissues relative to the adjacent cancer­free tissue. ROC curve analysis exhibited that the sensitivity was 90.1%, specificity was 97.6% and ROC AUC was 0.986. The specific value of HAAH/ß-actin abundance used as a cut­off point was 0.315, while the gene copy number (7.35 copies/µl) was used a as cut­off point, with sensitivity being 99.2%, specificity 96.7% and the ROC AUC used 0.990. No statistically significant difference was observed for these two factors. HAAH/humbug expression levels were upregulated in almost all the HCC tissues when compared to the adjacent cancer-free tissue, irrespective of the cut­off point used. Results of the present study suggested that HAAH/humbug is a potential diagnostic and prognostic biomarker for the treatment of HCC.

Redox modification of nuclear actin by MICAL-2 regulates SRF signaling.

The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. However, pathways that regulate nuclear actin levels are poorly understood. Here, we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription.

N-Glycolyl GM3 ganglioside immunoexpression in oral mucosal melanomas of Chinese.

OBJECTIVE: The aim of this study was to determine the expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in oral mucosal melanomas. MATERIALS AND METHODS: To assess the presence of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) mRNA, an RT-PCR assay was performed in melanoma cell line (ME), an oral mucosal ME, and two fresh oral mucosal melanoma tissues. Expression of NeuGcGM3 ganglioside was evaluated by immunohistochemistry in 44 primary oral mucosal melanomas and 10 oral melanotic nevi. Also, the expression of NeuGcGM3 was examined in ME by immunocytochemistry. RESULTS: We first checked the expression of CMAH in ME and two fresh oral mucosal melanoma tissues. Presence of NeuGcGM3 ganglioside was evident in 37 of 44 cases (84.1%), showing a diffuse cytoplasmic and membranous staining. Patients with primary occurrence showed high levels of NeuGcGM3 ganglioside compared to patients with secondary occurrence. On the other hand, negative immunoreaction was evidenced in oral melanotic nevi. ME also presented the expression of NeuGcGM3 by immunocytochemistry. CONCLUSIONS: In this work, we for the first time evaluated the expression of 14F7 MAb immunorecognition in oral mucosal melanomas. Our results were in agreement with the assumption that NeuGcGM3 ganglioside may be considered as target for passive and active immunotherapy in oral mucosal melanomas expressing this molecule and indicate less risk of recurrence and a better prognosis. Moreover, ME provides a platform for more studies on the specific function of NeuGcGM3 in oral mucosal melanomas.

Overexpression of the HIF hydroxylases PHD1, PHD2, PHD3 and FIH are individually and collectively unfavorable prognosticators for NSCLC survival.

INTRODUCTION: Hypoxia induced factors (HIFs) are at the heart of the adaptive mechanisms cancer cells must implement for survival. HIFs are regulated by four hydroxylases; Prolyl hydroxylase (PHD)-1,-2,-3 and factor inhibiting HIF (FIH). We aimed to investigate the prognostic impact of these oxygen sensors in NSCLC. METHODS: Tumor tissue samples from 335 resected stages I to IIIA NSCLC patients was obtained and tissue microarrays (TMAs) were constructed. Hydroxylase expression was evaluated by immunohistochemistry. PRINCIPAL FINDINGS: There was scorable expression for all HIF hydroxylases in tumor cells, but not in stroma. In univariate analyses, high tumor cell expression of all the HIF hydroxylases were unfavorable prognosticators for disease-specific survival (DSS); PHD1 (P = 0.023), PHD2 (P = 0.013), PHD3 (P = 0.018) and FIH (P = 0.033). In the multivariate analyses we found high tumor cell expression of PHD2 (HR = 2.03, CI 95% 1.20-3.42, P = 0.008) and PHD1 (HR = 1.45, CI 95% 1.01-2.10, P = 0.047) to be significant independent prognosticators for DSS. Besides, there was an additive prognostic effect by the increasing number of highly expressed HIF hydroxylases. Provided none high expression HIF hydroxylases, the 5-year survival was 80% vs. 23% if all four were highly expressed (HR = 6.48, CI 95% 2.23-18.8, P = 0.001). CONCLUSIONS: HIF hydroxylases are, in general, poor prognosticators for NSCLC survival. PHD1 and PHD2 are independent negative prognostic factors in NSCLC. Moreover, there is an additive poor prognostic impact by an increasing number of highly expressed HIF hydroxylases.

[The distribution and expression pro-files of Aspartyl/Asparaginyl beta-hydroxylase (ASPH) in some tumorous cell lines and tissues].

AIM: To investigate the distribution and expression profiles of Aspartyl/Asparaginyl beta-hydroxylase (ASPH) in some tumorous cell lines and some tissues. To evaluate the distribution and expression profile of Aspartyl/Asparaginyl beta-hydroxylase, ASPH in some tumor cell lines and tumor tissues. METHODS: The expression of ASPH in tumor cell lines on transcriptional level transcriptical level was tested by using RT-PCR, and on translational level was tested by cellular immunofluorescence and Western blot. The histologic sections from tumorous and normal tissue were tested immunohistochemistry. RESULTS: The expression of ASPH could be detected at both the transcriptional level and the translational level in the tumorous cell lines and it distributed in the cell membrane and the cytoplasmic domain. CONCLUSION: ASPH can be wildly used as a potential prognostic marker in neoplasms detection.

Susceptibilidad de Aedes aegypti a DDT, deltametrina y lambdacialotrina en Colombia / Susceptibility of Aedes aegypti to DDT, deltamethrin, and lambda-cyhalothrin in Colombia

OBJETIVOS: Evaluar el estado de susceptibilidad a insecticidas piretroides deltametrina y lambdacialotrina y al organoclorado DDT, e identificar los mecanismos bioquímicos asociados con resistencia en 13 poblaciones naturales de Aedes aegypti recolectadas en localidades de Colombia donde el dengue es un grave problema de salud pública. MÉTODOS: Se recolectaron y criaron en condiciones controladas formas inmaduras de diferentes criaderos naturales del vector para cada localidad. Con la generación F2 se realizaron bioensayos utilizando las metodologías OMS 1981 (papeles impregnados) y CDC 1998 (botellas impregnadas). En las poblaciones con mortalidades compatibles con disminución de la susceptibilidad, se midieron los niveles de esterasas no específicas (ENE), oxidasas de función mixta (OFM) y acetilcolinesterasa modificada (ACEM) mediante pruebas colorimétricas. RESULTADOS: Todas las poblaciones del mosquito evaluadas evidenciaron resistencia al organoclorado DDT. En cuanto a los piretroides, se encontró resistencia generalizada a lambdacialotrina pero no a deltametrina. Los mecanismos bioquímicos de resistencia evaluados permitieron encontrar 7 de 11 poblaciones con ENE elevadas y una población con OFM incrementadas. CONCLUSIONES: Se descarta la resistencia cruzada de tipo fisiológico entre el DDT y lambdacialotrina en las poblaciones de A. aegypti evaluadas. La resistencia fisiológica a lambdacialotrina parece asociarse con el incremento de las ENE. El comportamiento diferencial en los niveles de susceptibilidad y los valores enzimáticos entre poblaciones se asociaron con la variabilidad genética y presión de selección química a nivel local.

OBJECTIVES: To assess the susceptibility status of 13 natural populations of Aedes aegypti (collected from sites in Colombia where dengue is a serious public health problem) to the pyrethroids, deltamethrin and lambda-cyhalothrin, and to the organochlorine, DDT, and to identify any biochemical mechanisms associated with resistance. METHODS: Immature forms of the vector were collected from natural breeding spots at each site and then raised under controlled conditions. Using the F2 generation, bioassays were performed using the World Health Organization's 1981 methodology (impregnated paper) and United States Centers for Disease Control and Prevention's 1998 methodology (impregnated bottles). In populations where mortality rates were consistent with decreased susceptibility, levels of nonspecific esterases (NSE), mixed-function oxidases (MFO), and acetylcholinesterase (AChE) were measured using colorimetric tests. RESULTS: All of the mosquito populations that were tested showed resistance to the organochlorine DDT. In the case of the pyrethroids, widespread resistance to lambda-cyhalothrin was found, but not to deltamethrin. Assessing the biochemical resistance mechanisms showed that 7 of the 11 populations had elevated NSE, and one population, increased MFO. CONCLUSIONS: Physiological cross-resistance between DDT and lambda-cyhalothrin in the A. aegypti populations tested was dismissed. Physiological resistance to lambda-cyhalothrin appears to be associated with increased NSE. The differences in susceptibility levels and enzyme values among the populations were associated with genetic variations and chemicals in use locally.

Rapid single-nucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2.

BACKGROUND: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.

Bioluminescent assays for ADMET.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.