RESUMO
The Minas artisanal cheese from the Serra da Canastra (MAC-CM) microregion is a traditional product due to its production and ripening process. Artisanal chesses manufactured with raw cow's milk and endogenous dairy starters ("also known as pingo") have distinctive flavors and other sensory characteristics because of the unknown microbiota. The aim of this study was to evaluate the microbiota during 30 days of ripening, the physicochemical changes, and their relation in MACs produced in two different microregions located in the Serra da Canastra microregion through culture-dependent and culture-independent methods. The MACs were collected in the cities of Bambuí (MAC-CMB) and Tapiraí (MAC-CMT) in the Canastra microregion (n = 21). Cheeses uniqueness was demonstrated with the multivariate analysis that joined the microbiota and physicochemical characteristics, mainly to the proteolysis process, in which the MAC-CMT showed deeper proteolysis (DI -T0:14.18; T30: 13.95), while the MAC-CMB reached only a primary level (EI -T0:24.23; T30: 31.10). Abiotic factors were responsible for the differences in microbial diversity between the cheese farms. Different microbial groups: the prokaryotes, like Corynebacterium variabile, Lactococcus lactis, and Staphylococcus saprophyticus; and the eukaryotes, like Kluyveromyces lactis and Diutina catenulata dominated ripening over time. The microbial community and proteolysis were responsible for the predominance of volatile groups, with alcohols predominating in MAC-CMB and free fatty acids/acids and esters in MAC-CMT.
Assuntos
Queijo , Microbiologia de Alimentos , Queijo/microbiologia , Queijo/análise , Reação em Cadeia da Polimerase , Microbiota , Eletroforese em Gel de Gradiente Desnaturante , Leite/microbiologia , Leite/química , Animais , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Paladar , Indústria de Laticínios/métodos , Fermentação , ProteóliseRESUMO
Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is a traditional non-culture technique that can provide a fingerprint of the microbial community. In the field of gut microbiota analysis, PCR-DGGE still holds potential for development. In the present study, we utilized an improved nested PCR-DGGE approach targeting the V3 region of 16S ribosomal DNA to investigate the impact of whole grain highland hull-less barley (WHLB), a cereal known for its significant hypocholesterolemic effect, on the gut microbiota profiles of high-fat diet rats. Seventy-two male Sprague-Dawley rats were divided into four groups and fed a normal control diet, a high-fat diet, or a high-fat diet supplemented with a low or high dose of WHLB for 4 or 8 weeks. The results revealed that the dominant bands varied among different dose groups and further changed with different treatment times. The compositions of bacterial communities in feces and cecal content were similar, but the dominant bacterial bands differed. After performing double DGGE, extracting the bands, sequencing the DNA, and aligning the sequences, a total of 19 bands were classified under the Firmicutes and Bacteroidetes phyla, while two bands were identified as unclassified uncultured bacteria. The relative abundance of Lactobacillus gasseri, Uncultured Prevotella sp., and Clostridium sp. increased following the administration of WHLB. Illumina-based sequencing was employed to assess the reliability of DGGE, demonstrating its reliability in analyzing the dominant taxonomic composition, although it may have limitations in accurately detecting the alpha diversity of bacterial species. IMPORTANCE: While next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.
Assuntos
Bactérias , Eletroforese em Gel de Gradiente Desnaturante , Dieta Hiperlipídica , Microbioma Gastrointestinal , Hordeum , RNA Ribossômico 16S , Ratos Sprague-Dawley , Grãos Integrais , Animais , Hordeum/microbiologia , Masculino , Ratos , Microbioma Gastrointestinal/genética , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , RNA Ribossômico 16S/genética , Fezes/microbiologia , DNA Bacteriano/genética , Reação em Cadeia da Polimerase/métodos , Ceco/microbiologiaRESUMO
BACKGROUND: The accurate and expeditious detection of SARS-CoV-2 mutations is critical for monitoring viral evolution, assessing its impact on transmission, virulence, and vaccine efficacy, and formulating public health interventions. In this study, a detection system utilizing micro temperature gradient gel electrophoresis (µTGGE) was developed for the identification of the D614 and G614 variants of the SARS-CoV-2 spike protein. METHODS: The in vitro synthesized D614 and G614 gene fragments of the SARS-CoV-2 spike protein were amplified via polymerase chain reaction and subjected to µTGGE analysis. RESULTS: The migration patterns exhibited by the D614 and G614 variants on the polyacrylamide gel were distinctly dissimilar and readily discernible by µTGGE. In particular, the mid-melting pattern of D614 was shorter than that of G614. CONCLUSIONS: Our results demonstrate the capability of µTGGE for the rapid, precise, and cost-effective detection of SARS-CoV-2 spike protein D614 and G614 variants without the need for sequencing. Therefore, this approach holds considerable potential for use in point-of-care mutation assays for SARS-CoV-2 and other pathogens.
Assuntos
SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Eletroforese em Gel de Gradiente Desnaturante , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Emulsion PCR-DGGE is a molecular biology technique used to amplify and analyze DNA fragments. This technique combines two processes, emulsion PCR and denaturing gradient gel electrophoresis (DGGE), to enhance the specificity and yield of the amplification process and to separate the amplified fragments based on their melting behavior. In the emulsion PCR step, a high-quality DNA template is mixed with the PCR reagents and droplet generator oil to create an oil-in-water emulsion. The emulsion is then subjected to thermal cycling to amplify the target DNA fragments. The amplified fragments are recovered from the droplets and purified to remove any impurities that may interfere with downstream applications. In the DGGE step, the purified amplicon is loaded onto a DGGE apparatus, where the DNA fragments are separated and visualized based on their melting behavior. This method allows for the concurrent amplification and separation of multiple DNA fragments, thereby enhancing the resolution and sensitivity of the analysis. It is widely used in environmental and medical microbiology research, as well as in other fields that require the identification and characterization of microorganisms, such as the study of microbial diversity in soil, water, and other natural environments, as well as in the human gut microbiome and other medical samples.
Assuntos
Pesquisa Biomédica , Humanos , Emulsões , Eletroforese em Gel de Gradiente Desnaturante , Reação em Cadeia da Polimerase , ÁguaRESUMO
In their natural environments, microorganisms usually live in organized communities. Profiling analysis of microbial communities has recently assumed special relevance as it allows a thorough understanding of the diversity of the microbiota, its behavior over time, and the establishment of patterns associated with health and disease. The application of molecular biology approaches holds the advantage of including culture-difficult and as-yet-uncultivated phylotypes in the profiles, providing a more comprehensive picture of the microbial community. This chapter focuses on two particular techniques, namely terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE), both of which have been widely used in environmental studies and have been recently successfully used by the authors in the study of the oral microbial communities associated with conditions of health and disease.
Assuntos
Microbiota , Polimorfismo de Fragmento de Restrição , Eletroforese em Gel de Gradiente Desnaturante , Microbiota/genética , Biologia MolecularRESUMO
Effluents discharged by poultry meat industries are heavily polluted with raw materials, such as fat, blood residues, and proteins. Thus, untreated effluents directly discharged into the environment may constitute a public health threat. This study aims to evaluate the bacterial diversity of three water qualities: industrial poultry wastewater (PWW), tap water (TW), and PWW diluted with TW (50 : 50) (V/V) (TWPWW) by the combination of culture-independent and culture-dependent approaches. The total bacterial DNA was extracted using phenol/chloroform method. The hypervariable 16S rRNA region V3-V5 was amplified by PCR using universal primers. The amplicons were separated by vertical electrophoresis on a polyacrylamide gel of increasing denaturing gradient according to their richness in GC bases. Selected bands were reamplified and sequenced. Pure isolated bacteria from nutrient agar medium were characterized according to their morphological and biochemical characteristics. Genomic DNA from pure strains was extracted by boiling method, and a molecular amplification of the 16S-23S ITS region of the 16S rRNA gene was performed using the universal primers. Selected isolates were identified by sequencing. Results showed a high bacterial load and diversity in PWW in comparison with TW and TWPWW. A collection of 44 strains was obtained, and 25 of them were identified by sequencing. Proteobacteria represented 76% of isolated bacteria Gamma-Proteobacteria was the predominate isolate (68%). Other isolates were Firmicutes (8%), Bacteroidetes (12%), and Actinobacteria (8%). These isolates belong to different genera, namely, Pseudomonas, Acinetobacter, Proteus, Empedobacter, Corynebacterium, Enterobacter, Comamonas, Frondibacter, Leclercia, Staphylococcus, Atlantibacter, Klebsiella, and Microbacterium.
Assuntos
Aves Domésticas , Águas Residuárias , Ágar , Agricultura , Animais , Bactérias , Clorofórmio/análise , Primers do DNA , DNA Bacteriano , Eletroforese em Gel de Gradiente Desnaturante , Fenóis/análise , Filogenia , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Águas Residuárias/análise , ÁguaRESUMO
We investigated the effects of different types of heat treatments on hen's egg white (HEw) and quail egg white (QEw) proteins and their cross-reactivity in young children. Crude extracts of raw and water-boiled HEw and QEw and commercially developed stone-baked HEw were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Patients diagnosed with HEw allergy were enrolled, and pooled sera were tested with each extract in an enzyme-linked immunosorbent assay (ELISA)-inhibition test. A skin prick test (SPT) and oral food challenge (OFC) were also performed. The median age of 12 patients was 2.5 years. SDS-PAGE results revealed strongly stained bands for the ovomucoid of boiled HEw and QEw, while stone-baked HEw displayed remarkable changes for all protein fractions. In the ELISA-inhibition test, pre-incubation of the sera led to a profound decrease, moderate decrease, and minimal decrease in the amount of IgE binding to boiled and raw HEw, boiled and raw QEw, and stone-baked HEw proteins, respectively. SPTs and OFC demonstrated cross-reactivity values of 41.7% (5/12) and 16.7% (2/12) for boiled QEw and stone-baked HEw, respectively. We observed moderate cross-reactivity between QEw and HEw. Boiling had a limited effect on altering egg allergenicity. Commercially developed, stone-baked HEw can be an alternative food for children with HE allergy.
Assuntos
Alérgenos/imunologia , Reações Cruzadas/imunologia , Hipersensibilidade a Ovo/imunologia , Proteínas do Ovo/imunologia , Temperatura Alta , Animais , Galinhas , Pré-Escolar , Culinária , Eletroforese em Gel de Gradiente Desnaturante/métodos , Ovos/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Ovomucina/imunologia , Estudos Prospectivos , Codorniz , Testes CutâneosRESUMO
BACKGROUND: Dogs are the main reservoir hosts of Leishmania infantum; nevertheless, recent investigations indicate a likely role for cats in the epidemiology of Leishmania infection. Feline leishmaniosis (FeL) remains poorly characterised, partly due to the lack of suitable diagnostic tools. This study aimed to compare serum amyloid A (SAA) levels and serum protein electrophoresis (SPE) profiles (specifically, alpha 2 and gamma globulins) in cats naturally exposed to or infected by L. infantum from southern Italy versus those of healthy controls and versus cats with neoplastic or inflammatory conditions from non-endemic areas. METHODS: Serum or plasma samples from four cohorts of cats were analysed for SAA levels and by SPE: (i) G1: healthy controls from Leishmania-non-endemic regions of Switzerland; (ii) G2: cats pre-diagnosed with neoplastic or inflammatory conditions available from the University of Cambridge sample archive; (iii) G3: L. infantum-seropositive, quantitative (q)PCR-negative cats from southern Italy; (iv) G4: L. infantum-seropositive and qPCR-positive cats from southern Italy. SAA data were assessed for normality and homoscedasticity using the Shapiro-Wilk and Levene's tests, respectively; the Kruskall-Wallis test, followed by Dunn's test with Bonferroni correction were subsequently used to compare SAA serum levels between groups. A weighted generalised linear model with a binomial distribution was used to assess statistically significant differences in the numbers of animals displaying elevated gamma globulins and increased alpha 2 globulins between groups. RESULTS: Overall, 68 samples were analysed (G1: n = 16, G2: n = 20, G3: n = 20, G4: n = 12). Cats suffering from neoplastic and inflammatory conditions (G2 ) showed significantly higher SAA levels than healthy controls (G1) (median values [interquartile range]: G1: 0.00 [0.00-0.00] mg/l versus G2: 0.85 [0.00-49.55] mg/l). G2, G3 and G4 cats showed higher percentages of individuals with increased alpha 2 globulins (percentages ± standard error: G1 = 20.0% ± 10.3, G2 = 80.0% ± 8.9, G3 = 70.0% ± 10.2, G4 = 75.0% ± 12.5) and gamma globulins (G1 = 0.0% ± 0, G2 = 65.0% ± 10.7, G3 = 50.0% ± 11.2, G4 = 58.3% ± 14.2) than healthy control cats (G1). For all three markers, no significant difference between cats within G2, G3 and G4 was recorded. CONCLUSIONS: This study indicates that the proportions of animals with elevated levels of alpha 2 and gamma globulins are significantly higher in cats exposed to and infected with L. infantum. Levels of SAA and alpha 2 and gamma globulins may not be used to differentiate between L. infantum infection or exposure, and neoplastic and/or inflammatory conditions.
Assuntos
Doenças do Gato/sangue , Leishmania infantum , Leishmaniose Visceral/veterinária , Proteína Amiloide A Sérica/metabolismo , gama-Globulinas/metabolismo , Animais , Doenças do Gato/virologia , Gatos , Estudos de Coortes , Eletroforese em Gel de Gradiente Desnaturante/veterinária , Leishmaniose Visceral/sangueRESUMO
Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).
Assuntos
Reagentes de Ligações Cruzadas/química , Sondas de DNA/metabolismo , Etildimetilaminopropil Carbodi-Imida/química , RNA/análise , Northern Blotting , Sondas de DNA/química , Eletroforese em Gel de Gradiente Desnaturante , Digoxigenina/química , Eletroforese em Gel de Poliacrilamida Nativa , RNA/químicaRESUMO
Ophiocordyceps sinensis appears as stroma emerging from underground sclerotium enclosed by the skeleton of Thitarodes moth larvae. However, the actual distribution of the fungus in soil still remains unclarified. In this study, 40 soil samples were used for detection of O. sinensis to confirm its distribution in native habitats using denaturing gradient gel electrophoresis, nested internal transcribed spacer (ITS) PCR, and 454 pyrosequencing methods. The soil samples included six types: Os, where both stromata and host moth larvae were found; NL, representing no signs of stromata, but where moth larvae were found; NOs, where neither stroma nor moth larvae were found; BS, with bare soil without the presence of stroma of O. sinensis or moth larvae; AF, from soil surrounding the stroma; and MP, soil particles firmly wrapping the sclerotium of O. sinensis. Of 40 samples tested, 36 showed positive detection of O. sinensis by at least one of the three detection methods, with positive detection in all six sample types at all five sites. The results showed that traces of O. sinensis can be detected in locations with no macroscopically visible evidence of the fungus or its host and at least 100 m away from such locations.
Assuntos
Cordyceps/fisiologia , Microbiologia do Solo , Animais , China , Cordyceps/química , Cordyceps/genética , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Gradiente Desnaturante , Sequenciamento de Nucleotídeos em Larga Escala , Concentração de Íons de Hidrogênio , Larva/microbiologia , Mariposas/microbiologia , Reação em Cadeia da Polimerase , Solo/química , Solo/classificação , Água/análiseRESUMO
BACKGROUND: Elephant populations have greatly reduced mainly due to illegal poaching for their ivory. The trade in elephant products is protected by national laws and CITES agreements to prevent them from further decline. For instance, in Thailand, it is illegal to trade ivory from African elephants; however, the law allows possession of ivory from Asian elephants if permission has been obtained from the authorities. As such, means of enforcement of legislation are needed to classify the legal status of seized ivory products. Many DNA-based techniques have been previously reported for this purpose, although all have a limit of detection not suitable for extremely degraded samples. AIM: We report an assay based on nested PCR followed by DGGE to confirm the legal or illegal status of seized ivory samples where it is assumed that the DNA will be highly degraded. METHOD AND RESULTS: The assay was tested on aged ivory from which the assay was tested for reproducibility, specificity, and, importantly, sensitivity. Blind testing showed 100% identification accuracy. Correct assignment in all 304 samples tested was achieved including confirmation of the legal status of 227 highly degraded, aged ivories, thus underlining the high sensitivity of the assay. CONCLUSION AND RECOMMENDATION: The research output will be beneficial to analyze ivory casework samples in wildlife forensic laboratories.
Assuntos
Degradação Necrótica do DNA , DNA/genética , Eletroforese em Gel de Gradiente Desnaturante , Elefantes/genética , Animais , Conservação dos Recursos Naturais , Crime , Genética Forense/métodos , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
Soils play an important role in the ecosystem of karstic landscapes both as a buffer zone and as a source of acidity to belowground water. Although the microbiota of karstic soils is known to have a great effect on karstification processes, the activity and composition of these communities are largely unknown. This study gives a comparative analysis of soil microbial profiles from different parts of a doline located at Aggtelek, Hungary. The aim was to reveal the relationships between the vegetation type and genetic fingerprints and substrate utilisation (multi-SIR) profiles of the soil microbiota. Soil samples were collected in early and late springs along a transect in a doline covered with different types of vegetation. Genetic fingerprints of bacterial communities were examined by denaturing gradient gel electrophoresis (DGGE) based on the 16S rRNA gene, along with multi-SIR profiles of the microbial communities measured by the MicroResp method using 15 different carbon sources. Genetic fingerprinting indicated that vegetation cover had a strong effect on the composition of soil bacterial communities. Procrustean analysis showed only a weak connection between DGGE and multi-SIR profiles, probably due to the high functional redundancy of the communities. Seasonality had a significant effect on substrate usage, which can be an important factor to consider in future studies.
Assuntos
Microbiota/fisiologia , Parques Recreativos , Microbiologia do Solo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Carbono/metabolismo , Análise por Conglomerados , Eletroforese em Gel de Gradiente Desnaturante , Fenômenos Geológicos , Hungria , RNA Ribossômico 16S/genética , Estações do Ano , Solo/químicaRESUMO
Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
Assuntos
Envelhecimento/metabolismo , Dano ao DNA , Reparo do DNA , Oligonucleotídeos/isolamento & purificação , Poli ADP Ribosilação , Animais , Eletroforese em Gel de Gradiente Desnaturante , Feminino , Células Hep G2 , Humanos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Caracteres SexuaisRESUMO
The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.
Assuntos
Eletroforese em Gel de Gradiente Desnaturante , Corantes Fluorescentes/química , Histidina/análise , Proteínas Recombinantes de Fusão/análise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/análise , Raios Ultravioleta , Histidina/química , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
The intensification of marine aquaculture raises multiple sustainability issues, namely the handling of nutrient-rich effluents that can adversely impact ecosystems. As integrated multi-trophic aquaculture (IMTA) gains momentum, the use of halophyte plants to phytoremediate aquaculture effluents has received growing attention, particularly in aquaponics. It is, therefore, important to obtain a more in-depth knowledge of the microbial communities present in the root systems of these plants, both in their natural environment (sediment) and in aquaponics, in order to understand their nutrient removal potential. The present study used denaturing gradient gel electrophoresis (DGGE) and barcoded pyrosequencing to assess the bacterial community present in the endosphere and rhizosphere of three halophyte plants: Halimione portulacoides, Salicornia ramosissima and Sarcocornia perennis. Species-specific effects were recorded in the profile and diversity of the bacterial communities present in halophyte roots, with significant differences also recorded for the same halophyte species grown in contrasting environments (sediment vs. aquaponics). In aquaponics the most abundant groups belonged to the orders Rhodocyclales, Campylobacterales, Rhodobacterales and Desulfobacterales, while in the natural environment (sediment) the most abundant groups belonged to the orders Rhizobiales, Sphingomonadales and Alteromonadales. An overall enrichment in bacterial taxa involved in nutrient cycling was recorded in the roots of halophytes grown in aquaponics (such as Denitromonas, Mesorhizobium, Colwellia, Dokdonella and Arcobacter), thereby highlighting their potential to reduce the nutrient loads from aquaculture effluents.
Assuntos
Aquicultura/métodos , Raízes de Plantas/microbiologia , Rizosfera , Plantas Tolerantes a Sal/microbiologia , Microbiologia do Solo , Gerenciamento de Resíduos/métodos , Animais , Campylobacterales/metabolismo , Chenopodiaceae/crescimento & desenvolvimento , Chenopodiaceae/microbiologia , Eletroforese em Gel de Gradiente Desnaturante , Peixes , Sequenciamento de Nucleotídeos em Larga Escala , Microbiota/genética , RNA Ribossômico 16S/genética , Plantas Tolerantes a Sal/fisiologiaRESUMO
Anoxic biotrickling filters (BTFs) represent a technology with high H2S elimination capacity and removal efficiencies widely studied for biogas desulfurization. Three changes in the final electron acceptors were made using nitrate and nitrite during an operating period of 520 days. The stability and performance of the anoxic BTF were maintained when a significant perturbation was applied to the system that involved the progressive change of nitrate to nitrite and vice versa. Here the impact of electron acceptor changes on the microbial community was characterized by denaturing gel gradient electrophoresis (DGGE) and next generation sequencing (NGS). Both platforms revealed that the community underwent changes during the perturbations but was resilient because the removal capacity did not significantly change. Proteobacteria and Bacteroidetes were the main Phyla and Sulfurimonas and Thiobacillus the main nitrate-reducing sulfide-oxidizing bacteria (NR-SOB) genera involved in the biodesulfurization process.
Assuntos
Eletroforese em Gel de Gradiente Desnaturante , Elétrons , Filtração , Sequenciamento de Nucleotídeos em Larga Escala , Nitratos/química , Nitritos/química , Epsilonproteobacteria/química , Microbiota , Thiobacillus/químicaRESUMO
Biodeterioration caused by filamentous fungi is often a threat to the architectural heritage (i.e. tombs and historic sites). To specifically understand the deterioration phenomena caused by microorganisms in tombs and how these are shaped due to various environmental factors, the fungal communities in the coffin chamber of the Chinese emperor Yang (BC 569-618) were investigated at different heights using denaturant gradient gel electrophoresis (DGGE) fingerprinting. The associated environmental conditions, such as humidity, temperature, height and illumination, were also assessed. The results showed that a great diversity of fungal species (Cordyceps, Fusarium, Harpochytrium, Emericellopsis, Volutella, Cladosporium, Stachybotrys, Trichoderma, Cochlonema and two unknown fungal species) was present in emperor Yang's coffin chamber. The predominant species were Stachybotrys, Fusarium, Trichoderma and Cochlonema. Redundancy analysis (RDA) indicated that humidity, temperature, height and illumination were the most significantly related factors shaping the fungal communities. Humidity showed the highest degree of variance description (19.2%) than all other environmental factors, followed by illumination (18.3%) and height (12.8%). Furthermore, fungal richness and diversity indices showed a positive correlation with humidity (p < 0.05). These results help in understanding the fungal community in tombs, promoting the mitigation of deterioration phenomena of such building heritage for the present and future.
Assuntos
Fungos/classificação , Fungos/metabolismo , Umidade , Cemitérios , Impressões Digitais de DNA , Eletroforese em Gel de Gradiente Desnaturante , Meio Ambiente , Microbiologia Ambiental , Fungos/isolamento & purificação , Fusarium/genética , Fusarium/isolamento & purificação , Fusarium/metabolismo , Humanos , Micobioma/fisiologia , RNA Ribossômico 18S/genética , Microbiologia do Solo , Stachybotrys/genética , Stachybotrys/isolamento & purificação , Stachybotrys/metabolismo , Temperatura , Trichoderma/genética , Trichoderma/isolamento & purificação , Trichoderma/metabolismoRESUMO
Microorganisms such as thermophilic and psychrotrophic bacteria cause spoilage of milk and milk products [e.g., powdered infant formula (PIF)], mainly because they produce heat-stable extracellular enzymes. However, the dynamic changes in microbial diversity during PIF production are still not well understood. We used denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (HTS) to investigate bacterial community structure and distribution during the major stages of PIF production: raw milk, pasteurization, mixing, evaporation, and spray-drying. Our PCR-DGGE analysis indicated that Lactobacillus and Pseudomonas spp. were the dominant bacteria at the raw milk and pasteurization stages; Lactococcus, Streptococcus, Enterococcus, and Lactobacillus spp. were abundant during mixing, evaporation, and spray-drying. Our HTS analysis showed that Pseudomonas had an abundance of 96.79% at the raw milk stage. Lactobacillus, Streptococcus, Thermus, Acinetobacter, and Bacteroides spp. were most common after pasteurization. The index of bacterial diversity was highest at the evaporation stage, suggesting a high potential risk of microbial contamination. The results from DGGE and HTS were consistent in reflecting changes in dominant flora, but different in reflecting the richness of bacterial communities present during PIF production: HTS revealed a much higher richness of bacterial species than DGGE. Our findings from DGGE and HTS showed that psychrophilic and thermophilic bacteria were the main flora present during PIF production: psychrophilic bacteria were mainly Pseudomonas spp. and thermophilic bacteria were mainly Lactobacillus, Streptococcus, and Bacillus spp. To our knowledge, this is the first study to report dynamic changes in microbial communities during PIF production. Our results provide insight into bacterial communities and identify potential contamination sources that could serve as a guide for reducing microbial risk.
Assuntos
Bactérias/genética , Fórmulas Infantis/microbiologia , Microbiota/genética , Leite/microbiologia , Animais , Análise por Conglomerados , Eletroforese em Gel de Gradiente Desnaturante , Sequenciamento de Nucleotídeos em Larga Escala , Lactobacillus/genética , Pasteurização , Reação em Cadeia da Polimerase , Pós , Análise de Sequência de DNA , Streptococcus/genéticaRESUMO
Joining oligonucleotides together (ligation) is a powerful means of retrieving information from the nanoscale. To recover this information, the linkages created must be compatible with polymerases. However, enzymatic ligation is restrictive and current chemical ligation methods lack flexibility. Herein, a versatile ligation platform based on the formation of urea and squaramide artificial backbones from minimally modified 3'- and 5'-amino oligonucleotides is described. One-pot ligation gives a urea linkage with excellent read-through speed, or a squaramide linkage that is read-through under selective conditions. The squaramide linkage can be broken and reformed on demand, while stable pre-activated precursor oligonucleotides expand the scope of the ligation reaction to reagent-free, mild conditions. The utility of our system is demonstrated by replacing the enzymatically biased RNA-to-DNA reverse transcription step of RT-qPCR with a rapid nucleic-acid-template-dependent DNA chemical ligation system, that allows direct RNA detection.
Assuntos
DNA Polimerase Dirigida por DNA/química , Ácidos Nucleicos/química , Quinina/análogos & derivados , Ureia/química , Eletroforese em Gel de Gradiente Desnaturante , Espectrometria de Massas , Quinina/químicaRESUMO
Monitoring L. helveticus strain dynamics in natural whey starters is of great interest at the industrial level due to the key role that this bacterial population plays in Grana Padano cheese production. In this study, we aimed to develop a PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay based on the slpH locus, in parallel with performing culture-dependent analysis of whey samples using optimized media to maximize the number of isolated strains. We designed new primers targeting the slpH locus to amplify a gene region that would be suitable for PCR-DGGE analysis and discriminating strains. Our results confirmed that the developed PCR-DGGE method was rapid and reliable for monitoring the L. helveticus population in whey starter cultures. All sequences of bands detected in the PCR-DGGE profiles from whey samples showed high similarity to S-layer genes of L. helveticus, and perfectly matched with the slpH locus sequences of dominant strains. Overall, our findings indicated that the target region of the slpH locus was sufficiently heterologous to discriminate L. helveticus strains, and that our PCR-DGGE analysis provided a more accurate picture of the population composition of whey starters compared to culture-dependent techniques that often fail to isolate the most abundant strains.