RESUMO
Heavy metals are defined as metals with relatively high density and atomic weight, and their various applications have raised serious concerns about the environmental impacts and potential human health effects. Chromium is an important heavy metal that is involved in biological metabolism, but Cr exposure can induce a severe impact on occupational workers or public health. In this study, we explore the toxic effects of Cr exposure through three exposure routes: dermal contact, inhalation, and ingestion. We propose the underlying toxicity mechanisms of Cr exposure based on transcriptomic data and various bioinformatic tools. Our study provides a comprehensive understanding of the toxicity mechanisms of different Cr exposure routes by diverse bioinformatics analyses.
Assuntos
Cromo , Metais Pesados , Humanos , Cromo/toxicidade , Toxicogenética , Metais Pesados/toxicidade , Biologia Computacional , Perfilação da Expressão GênicaRESUMO
The research of fungi is of great importance in a number of fields, such as environmental and healthcare studies. While there are a large number of optical and molecular methods available for characterization and identification of fungi and their spores, their isolation is still conducted using slow and labor-intensive methods. Here, we develop a microfluidic device for the continuous separation of fungal spores from other eukaryotic cells. The spores were separated through the microfluidic device by expanding pinched flow fractionation (PFF) containing the spores, achieving a spatial separation perpendicular to the flow direction according to the spore size. Further branch flow fractionation (BFF) and co-flow of a Newtonian and viscoelastic fluid were used to enhance the separation performance. Using this microfluidic device, we demonstrated the separation of two different types of fungal spores and further separation of fungal spores from eukaryotic cells with a separation efficiency of above 90%. Compared to the existing conventional methods, our microfluidic flow focusing device requires little manual handling and uses small amounts of samples without any pre-treatment steps of the samples.
Assuntos
Dispositivos Lab-On-A-Chip , Esporos Fúngicos/isolamento & purificação , Alternaria/isolamento & purificação , Aspergillus niger/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Cladosporium/isolamento & purificação , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodosRESUMO
Rapid and sensitive mold detection is becoming increasingly important, especially in indoor environments. Common mold detection methods based on double-mediated electron transfer between an electrode and molds are not highly sensitive and reproducible, although they are rapid and simple. Here, we report a sensitive and reproducible detection method specific to Aspergillus niger ( A. niger), based on a single-mediator system combined with electrochemical-chemical (EC) redox cycling. Intracellular NAD(P)H-oxidizing enzymes in molds can convert electro-inactive hydroxy-nitro(so)arenes into electro-active hydroxy-aminoarenes. Since the membrane and wall of A. niger is well permeable to both a substrate (4-nitro-1-naphthol) and a reduced product (4-amino-1-naphthol) in tris buffer (pH 7.5) solution, the electrochemical signal is increased in the presence of A. niger due to two reactions: (i) enzymatic reduction of the substrate to the reduced product and (ii) electrochemical oxidation of the reduced product to an oxidized product. When a reducing agent (NADH) is present in the solution, the oxidized product is reduced back to the reduced product and then electrochemically reoxidized. This EC redox cycling significantly amplifies the electrochemical signal. Moreover, the background level is low and highly reproducible because the substrate and the reducing agent are electro-inactive at an applied potential of 0.20 V. The calculated detection limit for A. niger in a common double-mediator system consisting of Fe(CN)63- and menadione is â¼2 × 104 colony-forming unit (CFU)/mL, but the detection limit in the single-mediator system combined with EC redox cycling is â¼2 × 103 CFU/mL, indicating that the newly developed single-mediator system is more sensitive. Importantly, the detection method requires only an incubation period of 10 min and does not require a washing step, an electrode modification step, or a specific probe.
Assuntos
Aspergillus niger/isolamento & purificação , Técnicas Eletroquímicas/métodos , Técnicas Microbiológicas/métodos , Aspergillus niger/enzimologia , Humanos , Limite de Detecção , NADPH Oxidases/química , Naftóis/química , Nitrocompostos/química , Oxirredução , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Cadmium (Cd) exposure primarily occurs through inhalation, either by smoking or occupational exposure to contaminated air. Upon inhalation, Cd ultimately reaches the prostate through the bloodstream. In this review, we investigate the carcinogenic potential of Cd in both respiratory organs and the prostate. Specifically, this review examines cellular metabolism, comprehensive toxicity, and carcinogenic mechanisms by exploring gene ontology, biological networks, and adverse outcome pathways. In the respiratory organs, Cd induces lung cancer by altering the expression of IL1B and FGF2, causing DNA damage, reducing cell junction integrity, and promoting apoptosis. In the prostate, Cd induces prostate cancer by modifying the expression of EDN1 and HMOX1, leading to abnormal protein activities and maturation, suppressing tumor suppressors, and inducing apoptosis. Collectively, this review provides a comprehensive understanding of the carcinogenic mechanisms of Cd in two different organs by adopting toxicogenomic approaches. These insights can serve as a foundation for further research on cadmium-induced cancer, contributing to the establishment of future cancer prevention strategies.
RESUMO
This study was conducted to provide basic data for chemical accident response by assessing the health risks of residents living near a chemical accident site due to long-term exposure. The study considered the temporal concentration changes of the leaked chemical (i.e., its behavior in the environment and dilution) until its extinction. A virtual chemical accident was assumed, in which 40 t of formaldehyde was accidentally discharged for 1 h in Ulsan Metropolitan City, Korea. Formaldehyde concentrations over time in each environmental medium after the accident were calculated using a multimedia environmental dynamics model. Exposure subjects divided into four age groups were considered. Carcinogenic risks due to respiration and non-carcinogenic risks due to soil intake were assessed. For all the age groups, the excess cancer risk did not exceed 1.0 × 10-6, indicating that no harmful health impact was caused by inhalation exposure to formaldehyde. The hazard index exceeded 1 for all the age groups, confirming that harmful health impacts were caused by exposure to soil containing the formaldehyde. This study is the first to assess chronic health risks by reflecting long-term residual and temporal concentration changes of a pollutant released in a chemical accident in each environmental medium until its extinction. This work is also significant in that it reflects the exposure characteristics of the toxic chemical.
Assuntos
Vazamento de Resíduos Químicos , Multimídia , Cidades , Exposição Ambiental , Formaldeído/toxicidade , Humanos , República da Coreia , Medição de RiscoRESUMO
To construct a highly sensitive detection system for endocrine disruptors, we have compared the activity of promoters with the ALK1, ICL1, RPS7 and TEF1 for heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of lacZ or hERalpha reporter gene, respectively, and the activity was evaluated by beta-galactosidase assay by lacZ or western blot analysis by hERalpha. The expression analysis revealed that the ALK1 and ICL1 promoter were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly high level of expression in the presence of glucose and glycerol, respectively. Particularly, the TEF1 promoter showed the highest beta-galactosidase activity and a significant signal by western blotting with the anti-estrogen receptor compared with the other promoters. Moreover, the detection system was constructed with promoters were linked to the upstream of expression vector for hERalpha gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hERalpha and CXAU1-2XERE was the most effective system for the E2-dependent induction of the beta-galactosidase activity. This system showed the highest beta-galactosidase activity at 10-6 M E2 and the activity could be detected at even the concentration of 10-10 M E2. As the result, we constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity and reproducibility of the system for characterizing environmental estrogens.
Assuntos
Alcanos/análise , Técnicas Biossensoriais/métodos , Disruptores Endócrinos/análise , Engenharia Genética , Yarrowia/genética , Yarrowia/metabolismo , Alcanos/metabolismo , Disruptores Endócrinos/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Reporter , Humanos , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
The aim of this study was to support management of airborne bacteria in facilities used by pollution-sensitive individuals (in daycares, medical facilities, elder care facilities, and postnatal care centers). A field survey was conducted on 11 facilities from October 2017 to April 2018. Elder care facilities in industrial, urban, and forested areas were excluded. Two indoor, and one outdoor, measuring points were selected per facility. These points were located in areas most often used by the residents. Measurements were taken at random time-points before February 2018 and at specific times in the morning and afternoon thereafter. The relationships among bacterial counts, carbon dioxide concentrations, dust levels, temperature, relative humidity, and ventilation were examined. The pooled average bacterial counts at the daycares, medical facilities, elder care facilities, and postnatal care centers were 540.25 CFU m-3, 245.49 CFU m-3, 149.63 CFU m-3, and 169.65 CFU m-3, respectively. Considering the upper 95% confidence interval, the bacterial counts in many daycares may in fact be >800 CFU m-3, which is the threshold set by the Korean Ministry of the Environment. The pooled average indoor: outdoor bacterial count ratio was 1.13. Indoor airborne bacterial counts were influenced mainly by their sources. This study found no significant correlations among indoor temperature, relative humidity, carbon dioxide concentration, dust levels, and airborne bacterial counts, unlike previous studies. Airborne bacteria management at daycares should be a top priority. The sources of airborne bacteria must also be identified, and a management plan must be developed to control them.
Assuntos
Poluição do Ar em Ambientes Fechados/análise , Bactérias/isolamento & purificação , Logradouros Públicos , Microbiologia do Ar , Monitoramento Ambiental , Humanos , VentilaçãoRESUMO
Currently, low-cost, sensor-based fine dust measurement devices are commercially available in South Korea. This study evaluated the reliability of three such devices-Yi Shan A4, Plantower PMS7003, and Plantower PMS7003-in comparison to long-term consecutive monitoring systems for discharge and prevention facilities regarding fine dust control. The performance of these devices for concentration intervals over time was examined through real-time comparison using a GRIMM (Model: 11-A, dust spectrometer from Grimm Technologies) as a reference; this included a correction factor (C-Factor), calculated by a gravimetric method and an equivalence test. For comparison, the reference and target devices were installed in a chamber with fine dust concentrations of 2 µg/m3, with temperature and humidity maintained at 20 °C and 40%, respectively. The fine particulate matter (PM)2.5 concentrations were classified into five intervals: ≤40 µg/m3, 40â»80 µg/m3, 80â»120 µg/m3, 120â»160 µg/m3, and 200â»230 µg/m3. Statistical analysis was performed using data obtained from national stations for monitoring and controlling fine dust released from facilities under high fine dust loading conditions. The results showed that the measurements of all target devices, which were corrected according to the reference device, provided accurate values at PM2.5 concentrations of ≥40 µg/m3. The statistical analysis results suggest that the evaluated devices are more reliable than the conventional numerical-analysis-based monitoring system.
Assuntos
Poluentes Atmosféricos/análise , Poeira/análise , Monitoramento Ambiental , Custos e Análise de Custo , Monitoramento Ambiental/economia , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Tamanho da Partícula , Reprodutibilidade dos Testes , República da CoreiaRESUMO
Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T>C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A>C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T>C mutation is pathogenic, however, the 13849A>C mutation is of unclear significance. It is likely that the 13849A>C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders.
Assuntos
DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Síndrome MELAS/genética , Proteínas Mitocondriais/genética , Mutação de Sentido Incorreto , Adulto , Povo Asiático , Análise Mutacional de DNA , DNA Mitocondrial/análise , Feminino , Humanos , Coreia (Geográfico) , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo GenéticoRESUMO
In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves.
Assuntos
Carbono-Carbono Liases/metabolismo , Diterpenos/metabolismo , Oryza/enzimologia , Lectinas de Plantas/biossíntese , Sequência de Bases , Células Cultivadas , Primers do DNA , Cinética , RNA Mensageiro/genéticaRESUMO
Surface-enhanced Raman scattering (SERS) of an antifungal reagent, myclobutanil (MCB), was performed on Au and Ag nanoparticles (NPs) to estimate the drug-release behaviors in fungal cells. A density functional theory (DFT) calculation was introduced to predict a favorable binding site of MCB to either the Ag or Au atom. Myclobutanil was presumed to bind more strongly to Au than to Ag in their most stable, optimized geometries of the N4 atom in its 1,2,4-triazole unit binding to the metal atom. Strong intensities were observed in the Ag SERS spectra only at acidic pH values, whereas the most prominent peaks in the Au SERS spectra of MCB matched quite well with those of 1,2,4-triazole regardless of pH conditions. The Raman spectral intensities of the MCB-assembled Ag and Au NPs decreased after treatment with either potato dextrose agar (PDA) or glutathione (GSH). Darkfield microscopy and confocal SERS were performed to analyze the MCB-assembled metal NPs inside Penicillium digitatum fungal cells. The results suggested that MCB was released from the metal NPs in the intracellular GSH in the fungi because we observed only fungal cell peaks.
Assuntos
Antifúngicos/química , Ouro/química , Nanopartículas Metálicas/química , Nitrilas/química , Penicillium/química , Prata/química , Triazóis/química , Adsorção , Análise Espectral Raman/métodosRESUMO
We detected a trace amount of the mycotoxin citrinin using surface-enhanced Raman scattering (SERS) on silver nanoparticle (Ag NP) surfaces. The SERS substrate on hydrophobic Teflon films was also introduced to observe the citrinin peaks. A broad band at â¼1382cm(-1), which was ascribed to the symmetric carboxylate stretching mode, was observed in addition to an antisymmetric carboxylate stretching mode at â¼1568cm(-1) in the Raman spectra. The spectral feature indicated that citrinin would adsorb on Ag NPs via its carboxylate form. Based on density functional theory (DFT) calculations, vibrational mode analysis was performed to compare the Raman spectra of citrinin. DFT calculations also predicted that a bidentate bridge configuration through O15 and O16 atoms in citrinin would be the most stable on three Ag atoms. After treating with Ag NPs, observation of citrinin peaks was attempted in fungal cells of Penicillium citrinum. This work may provide useful insights into the direct observation of the hazardous citrinin mycotoxin using SERS by understanding its adsorption behaviors on Ag surfaces.
Assuntos
Citrinina/análise , Nanopartículas Metálicas/química , Prata/química , Adsorção , Citrinina/química , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Penicillium , Análise Espectral RamanRESUMO
In aquatic invertebrates, particularly marine gastropods, organotin compounds induce irreversible sexual abnormality in females, which is termed imposex, at very low concentrations. Organotin compounds are agonists for nuclear receptors such as RXRs and PPARgamma. However, the imposex phenomenon has not been reported to act as an antagonist on estrogen receptors in other species, including vertebrates and invertebrates. In order to gain insights into the antagonistic activity of organotin compounds on estrogen receptors (ERs), we examined the inhibitive effect of these compounds on estradiol-dependent beta-galactosidase activity using the yeast two-hybrid detection system consisting of a combination of the human estrogen receptor (hERbeta) ligand-binding domain and the co-activator steroid receptor co-activator-1 (SRC1). Tributyltin-hydroxide (TBT-OH) and triphenyltin-chlorine (TPT-Cl) exhibited an inhibitive effect on E2-dependent transcriptional activity, similar to antagonistic chemicals such as 4-hydroxytamoxifen (OHT) or ICI 182,780, at a very low concentration of 10⻹4 M TBT or 10⻹° M TPT, respectively. The yeast growth and transcriptional activity with transcriptional factor GAL4 did not exhibit any effect at the tested concentration of TBT or TPT. Moreover, the yeast two-hybrid system using the interaction between p53 and the T antigen of SV40 large did not describe any effect at the tested concentration of OHT or ICI 182,780. However, the interaction between p53 and T antigen was inhibited at a TBT or TPT concentration of 10â»9 M, respectively. These results indicate that TBT and TPT act as inhibitors of ER-dependent reporter gene transcriptional activation and of the interaction between hERbeta LBD and the co-activator SRC1 in the yeast two-hybrid system. Consequently, our data could partly explain the occurrence of organotin compound-induced imposex on the endocrine system of mammals, including humans.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Compostos Orgânicos de Estanho/farmacologia , Transtornos Ovotesticulares do Desenvolvimento Sexual/etiologia , Receptores de Estrogênio/genética , Ativação Transcricional/efeitos dos fármacos , Compostos de Trialquitina/farmacologia , Disruptores Endócrinos/farmacologia , Estradiol/metabolismo , Feminino , Humanos , Modelos Biológicos , Transtornos Ovotesticulares do Desenvolvimento Sexual/genética , Transtornos Ovotesticulares do Desenvolvimento Sexual/metabolismo , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/antagonistas & inibidores , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA(Leu(UUR))was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA(Lys) of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.
Assuntos
DNA Mitocondrial/genética , Síndrome MELAS/genética , Síndrome MERRF/genética , Adolescente , Adulto , Sequência de Aminoácidos , Povo Asiático/genética , Sequência de Bases , Análise Mutacional de DNA , DNA Mitocondrial/análise , Feminino , Humanos , Síndrome MELAS/diagnóstico , Síndrome MERRF/diagnóstico , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Linhagem , Polimorfismo de Nucleotídeo Único , Homologia de Sequência , Adulto JovemRESUMO
Hydrophobins are small amphipathic proteins that function in a broad range of growth and developmental processes in fungi. They are involved in the formation of aerial structures, the attachment of fungal cells to surfaces, and act in signalling in response to surface cues and pathogenesis. Beauveria bassiana is an important entomopathogenic fungus used as an arthropod biological control agent. To examine the feasibility of using phage display technology to clone cDNAs encoding hydrophobins, biopanning experiments were performed using a variety of affinity resins, including N,N'-diacetylchitobiose-, fucose-, lactose-, maltose- and melibiose-coupled agarose beads. After five rounds of iterative biopanning, cDNAs corresponding to two B. bassiana (class I) hydrophobins were selectively enriched using melibiose- or lactose-coupled agarose beads. Expression analysis revealed that the hyd1 gene was expressed in all samples tested, including aerial conidia, in vitro blastospores, submerged conidia, and cells sporulating on chitin and insect cuticle, with hyd1 expression peaking in growing mycelia. In contrast, the hyd2 gene was not appreciably expressed in any of the single-cell types (aerial conidia, blastospores and submerged conidia), but was constitutively expressed in growing mycelia and when cells were sporulating on chitin and insect cuticle. MS fingerprinting of an approximately 10 kDa protein found in boiling SDS-insoluble, trifluoroacetic acid-soluble extracts from aerial conidia identified the major component of the B. bassiana rodlet layer to be the hyd2 gene product. These results reveal the differential regulation of the isolated hydrophobins and indicate that phage display represents a novel approach to cDNA cloning of hydrophobins.
Assuntos
Beauveria/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Beauveria/química , Beauveria/fisiologia , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/fisiologia , Espectrometria de Massas , Dados de Sequência Molecular , Micélio/genética , Biblioteca de Peptídeos , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Esporos Fúngicos/genéticaRESUMO
We constructed an efficient and reliable yeast two-hybrid detection system to evaluate the estrogenic activity of endocrine disruptors (EDs) (Lee et al., Journal of Biochemistry, 131, 2002). This system employs the interaction between the human estrogen receptor beta (hERbeta) ligand binding domain and the co-activator SRC1. The extent of transcriptional activation by those chemicals correlated with estrogenic activities as measured by other assay systems. We applied this assay system to evaluate anti-estrogenic activities and found that known antagonistic compounds, 4-hydroxytamoxifen (OHT) and ICI 182,780, effectively inhibited reporter gene induction by 17beta-estradiol. We then tested the estrogenic and anti-estrogenic activities of polycyclic aromatic hydrocarbons (PAHs) using this assay system. PAHs only weakly induced the lacZ reporter gene at higher concentrations, but clearly showed an inhibitory effect on reporter gene induction by 10(-9) M 17beta-estradiol. These results suggest that PAHs are potentially anti-estrogenic and that the employed yeast detection system could be applicable to primary screening for effectors on estrogen receptor functions.
Assuntos
Disruptores Endócrinos/farmacologia , Receptor beta de Estrogênio/antagonistas & inibidores , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Microbiologia da Água , Disruptores Endócrinos/metabolismo , Receptor beta de Estrogênio/metabolismo , Coreia (Geográfico)RESUMO
The entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and approximately 2,500 5' end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1,079 contigs, respectively. Almost half (40-45 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2,416 unique sequences, of which 20-30 % were unique to each library, and only approximately 6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental conditions.
Assuntos
Cordyceps/crescimento & desenvolvimento , Cordyceps/patogenicidade , Etiquetas de Sequências Expressas , Biblioteca Gênica , Insetos/microbiologia , Estágios do Ciclo de Vida/fisiologia , Animais , Cordyceps/genética , DNA Fúngico/análise , Bases de Dados Genéticas , Regulação Fúngica da Expressão Gênica , Técnicas In Vitro , Análise de Sequência de DNA , Esporos Fúngicos/fisiologiaRESUMO
In the accompanying paper [Cho, E.-M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006). Microbiology 152, 2843-2854], the analysis of expressed sequence tag (EST) libraries derived from homogeneous single-cell populations of aerial conidia, in vitro blastospores and submerged conidia of the entomopathogenic fungus Beauveria (Cordyceps) bassiana has been reported. Here an extended EST analysis is presented of complex cell mixtures derived from fungal cells sporulating on chitin or grown under culture conditions inducing the production of the B. bassiana secondary metabolite, oosporein. Fungal material used for the construction of the libraries included germinating conidia and blastospores, mycelia, as well as cells in various developmental stages. Approximately 2,500 5' end sequences were determined from random sequencing of clones from each library, and were clustered into 277 contigs with 1,069 singlets, and 306 contigs with 1,064 singlets, for the chitin and oosporein libraries, respectively. Almost half (45-50 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. Approximately 20-25 % of the sequences in each library could be annotated by gene ontology terms. A comparative analysis between the two libraries, as well as the libraries in the accompanying paper, is presented. A set of 4,360 clustered and unique sequences was characterized. The data are indicative of a highly plastic gene expression repertoire being available to B. bassiana for growth during different environmental and developmental conditions, and provides a dataset for gene discovery and genome annotation.