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1.
Talanta ; 235: 122779, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517637

RESUMO

To ensure the safety of dairy products, especially milk, and consequently protect human health, accurate and simple analytical techniques are highly necessary to determine the low concentration of aflatoxin M1 (AFM1) as an important carcinogen. Herein, a novel, accurate and simple fluorescent aptasensor was designed for selective detection of AFM1 based on bivalent binding aptamer-cDNA (BBA-cDNA) structure. Moreover, MoS2 nanosheets (MoS2 NSs) were used as the fluorescent quencher and FAM-labeled complementary strand of aptamer (FAM-CS) was applied as a fluorescent probe. In this study, we achieved a new result. Unlike previous studies, in this work, the BBA-cDNA structure was not disassembled in the presence of the target. Therefore, as the AFM1 concentration increased, more targets were attached to the BBA-cDNA structure and as a result, the BBA-cDNA structure/AFM1 could not be placed on the surface of MoS2 NSs, leading to the more fluorescent intensity detection. Under optimized conditions, the developed fluorescent analytical method revealed great selectivity toward AFM1 with a limit of detection (LOD) of 0.5 nM and a linear range from 0.7 to 10 nM. This fabricated aptasensor indicated excellent analytical performance for AFM1 detection in milk samples with LOD of 0.1 nM. Overall, the proposed approach could provide an effective basis for small molecule analysis to guarantee food and human safety using appropriate aptamer sequences.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aflatoxina M1/análise , Animais , DNA Complementar , Humanos , Limite de Detecção , Leite/química , Molibdênio
2.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2836-2844, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472301

RESUMO

It has been reported that ODB genes play an important role in homologous recombination-directed DNA repair, suggesting their potential applications in plant breeding. To analyze the expression characteristics of tobacco NtODB gene, the cDNA sequence of NtODB was obtained using in silico cloning technique. The physicochemical properties, signal peptide, and advanced structures of the predicted protein were analyzed using bioinformatics tools. The results showed that the NtODB gene has a 579-bp open reading frame which encodes a protein with 192 amino acid residues. The protein NtODB is predicted to be alkaline and hydrophilic. Real-time quantitative PCR showed that NtODB was constitutively expressed in different tissues. Subcellular localization showed that NtODB was mainly expressed in cell membrane and chloroplast. These results may help us to better understand and elucidate the roles of ODB genes in the homologous recombination-directed DNA repair.


Assuntos
Biologia Computacional , Tabaco , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Simulação por Computador , DNA Complementar , Filogenia , Melhoramento Vegetal , Tabaco/genética
3.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 33(4): 380-386, 2021 Aug 24.
Artigo em Chinês | MEDLINE | ID: mdl-34505445

RESUMO

OBJECTIVE: To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni. METHODS: Total RNA was extracted from S. mansoni, and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni. Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted. RESULTS: The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 106 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively. CONCLUSIONS: A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.


Assuntos
Esparganose , Plerocercoide , Animais , Sequência de Bases , DNA Complementar/genética , Biblioteca Gênica , Humanos
4.
PLoS One ; 16(8): e0244468, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34432798

RESUMO

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity among samples. Mixed allelic frequencies along the 20kb ORF1ab gene in one sample, suggested the presence of a defective viral RNA species subpopulation maintained in mixture with functional RNA in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.


Assuntos
COVID-19/patologia , SARS-CoV-2/genética , Análise de Sequência de DNA/métodos , COVID-19/virologia , DNA Complementar/química , DNA Complementar/metabolismo , Frequência do Gene , Variação Genética , Genoma Viral , Humanos , Fases de Leitura Aberta/genética , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/isolamento & purificação , Carga Viral
5.
J Insect Sci ; 21(4)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34333649

RESUMO

Chitin deacetylases (CDAs) are chitin-degrading enzymes that play a key role in insect molting. In this study, we identified and characterized four full-length cDNAs of CDAs from Sogatella furcifera (Horváth). Developmental expression showed that SfCDA1 and SfCDA2 were expressed at all nymph developmental stages, SfCDA3 and SfCDA4 were mainly expressed in the third-instar to fifth-instar nymph stages, whereas tissue-specific analyses indicated that four CDA genes were mainly high expressed in the integument and head during the fifth-instar nymph. RNA interference (RNAi) results revealed that SfCDA1, SfCDA2, and SfCDA4 are associated with molting defect and high mortality with nymph-adult molting. Furthermore, transcripts of chitin synthase 1 variants (SfCHS1, SfCHS1a, and SfCHS1b) were significantly downregulated and causing significant changes in the expression levels of trehalases (TRE1 and TRE2) in the SfCDA1, SfCDA2, and SfCDA4 dsRNA treatment groups. By contrast, no significant phenotypic characteristics were observed after dsSfCDA3 injection. Taken together, our results suggest that SfCDA1, SfCDA2, and SfCDA4 play a vital role in nymph-adult transition, and these genes could regulate chitin biosynthesis expression levels.


Assuntos
Amidoidrolases/genética , Hemípteros , Animais , Quitina/biossíntese , Quitina/genética , DNA Complementar , Genes de Insetos , Hemípteros/genética , Proteínas de Insetos/genética , Muda/genética , Ninfa/genética , Filogenia , Interferência de RNA , Asas de Animais/crescimento & desenvolvimento
6.
Methods Mol Biol ; 2327: 119-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410643

RESUMO

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.


Assuntos
Genoma Viral , SARS-CoV-2/genética , Saliva/virologia , Sequenciamento Completo do Genoma/métodos , Biologia Computacional , DNA Complementar/genética , Humanos , Sondas Moleculares/genética , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Estreptavidina , Sequenciamento Completo do Genoma/instrumentação
7.
Viruses ; 13(6)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206030

RESUMO

Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4-5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40-47% in 4-5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus-host interaction.


Assuntos
Clonagem Molecular , DNA Complementar , Genoma Viral , Tobamovirus/genética , Sequência de Aminoácidos , Sequência de Bases , Suscetibilidade a Doenças , Genômica/métodos , Fenótipo , Filogenia , Doenças das Plantas/virologia , Análise de Sequência de DNA , Tobamovirus/isolamento & purificação
8.
Vet Microbiol ; 260: 109179, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34271305

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has rapidly spread worldwide. Studies of transmission of the virus carried out in animals have suggested that certain animals may be susceptible to infection with SARS-CoV-2. The aim of the present study was to investigate the infection of SARS-CoV-2 in pets (18 cats and 20 dogs) from owners previously confirmed as COVID-19-positive. Oropharyngeal and rectal swabs were taken and analyzed by real-time RT-PCR assays, while blood samples were taken for antibody detection. Of the total pets analyzed, one cat was found reactive to SARS-CoV-2 by real-time RT-PCR of an oropharyngeal and a rectal swab. This cat presented only sneezing as a clinical sign. Serological analysis confirmed the presence of antibodies in the serum sample from this cat, as well as in the serum from another cat non-reactive to real-time RT-PCR. Complete sequence and phylogenetic analysis allowed determining that the SARS-CoV-2 genome belonged to the B.1.499 lineage. This lineage has been reported in different provinces of Argentina, mainly in the Metropolitan Area of Buenos Aires. This study notifies the first detection of the natural infection and molecular analysis of SARS-CoV-2 in a cat from Argentina whose owner where COVID-19-positive. Although there is currently no evidence that cats can spread COVID-19, results suggest that health authorities should test pets with COVID-19-positive owners.


Assuntos
Doenças do Gato/virologia , Infecções por Coronavirus/veterinária , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Animais , Argentina , Teste de Ácido Nucleico para COVID-19/veterinária , Doenças do Gato/diagnóstico , Gatos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , DNA Complementar/química , Cães , Feminino , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , SARS-CoV-2/classificação
9.
Sensors (Basel) ; 21(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209484

RESUMO

Coronavirus (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as a deadly pandemic. The genomic analysis of SARS-CoV-2 is performed using a reverse transcription-polymerase chain reaction (RT-PCR) technique for identifying viral ribonucleic acid (RNA) in infected patients. However, the RT-PCR diagnostic technique is manually laborious and expensive; therefore, it is not readily accessible in every laboratory. Methodological simplification is crucial to combat the ongoing pandemic by introducing quick, efficient, and affordable diagnostic methods. Here, we report how microcantilever sensors offer promising opportunities for rapid COVID-19 detection. Our first attempt was to capture the single-stranded complementary DNA of SARS-CoV-2 through DNA hybridization. Therefore, the microcantilever surface was immobilized with an oligonucleotide probe and detected using complementary target DNA hybridization by a shift in microcantilever resonance frequency. Our results show that microcantilever sensors can discriminate between complementary and noncomplementary target DNA on a micro to nanoscale. Additionally, the microcantilever sensors' aptitude toward partial complementary DNA determines their potential to identify new variants of coronavirus. Therefore, microcantilever sensing could be a vital tool in the effort to extinguish the spreading COVID-19 pandemic.


Assuntos
COVID-19 , SARS-CoV-2 , DNA Complementar , Humanos , Hibridização de Ácido Nucleico , Pandemias , RNA Viral
10.
Sheng Wu Gong Cheng Xue Bao ; 37(7): 2474-2482, 2021 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-34327912

RESUMO

Spirodela polyrrhiza is a floating plant widely used in biomass utilization and eutrophication phytoremediation. It becomes a common aquatic plant everywhere with the increasingly serious eutrophication. It has been reported that S. polyrrhiza has a good effect on the remediation of eutrophication water. In order to study the absorption and transportation of phosphorus in S. polyrrhiza, we extracted RNA from S. polyrrhiza and then reverse transcribed it into cDNA, which was used as a template to amplify a specific fragment. The full-length sequence of the open reading frame (ORF) was 1 620 bp, encoding 539 amino acids, named SpPHT1;1, and the accession number in GenBank was MN720003. Bioinformatical analysis showed that SpPHT1;1 had no intron. The protein it encoded was a stable, hydrophobic protein with 11 transmembrane domains. SpPHT1;1 structure was similar to that of major facilitator superfamily (MFS) superfamily members. The cluster analysis showed that SpPHT1;1 was closely related to ZMPHT2 in maize and SBPHT1-8 in sorghum. So, it might belong to plant PHT1 family. The expression of SpPHT1;1 in leaf was significantly more than that of root under normal phosphorus condition. Low phosphorus condition could promote gene expression, and the relative expression level of SpPHT1;1 arrived at the peak at 48 h both in root and leaf. High phosphorus condition could inhibit gene expression. These results indicated that SpPHT1;1 expression would be affected by external phosphorus concentration. The results of this study are helpful for further research on the function of phosphate transporter. It also can provide theoretical basis for further development and utilization of S. polyrrhiza.


Assuntos
Araceae , Araceae/genética , Biodegradação Ambiental , Clonagem Molecular , DNA Complementar , Proteínas de Transporte de Fosfato/genética
11.
BMC Genomics ; 22(1): 492, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193038

RESUMO

BACKGROUND: The accumulation of carotenoids in adipose tissue leading to yellow fat is, in sheep, a heritable recessive trait that can be attributed to a nonsense mutation in the beta-carotene oxygenase 2 (BCO2) gene. However, not all sheep breeds suffering from yellow fat have this nonsense mutation, meaning that other functional mechanisms must exist. We investigated one such breed, the Norwegian spælsau. RESULTS: In spælsau we detected an aberration in BCO2 mRNA. Nanopore sequencing of genomic DNA revealed the insertion of a 7.9 kb endogenous Jaagsiekte Sheep Retrovirus (enJSRV) sequence in the first intron of the BCO2 gene. Close examination of its cDNA revealed that the BCO2 genes first exon was spliced together with enJSRV-sequence immediately downstream of a potential -AG splice acceptor site at enJSRV position 415. The hybrid protein product consists of 29 amino acids coded by the BCO2 exon 1, one amino acid coded by the junction sequence, followed by 28 amino acids arbitrary coded for by the enJSRV-sequence, before a translation stop codon is reached. CONCLUSIONS: Considering that the functional BCO2 protein consists of 575 amino acids, it is unlikely that the 58 amino acid BCO2/enJSRV hybrid protein can display any enzymatic function. The existence of this novel BCO2 allele represents an alternative functional mechanism accounting for BCO2 inactivation and is a perfect example of the potential benefits for searching for structural variants using long-read sequencing data.


Assuntos
Retrovirus Jaagsiekte de Ovinos , Tecido Adiposo , Animais , DNA Complementar , Éxons , Retrovirus Jaagsiekte de Ovinos/genética , Ovinos , Carneiro Doméstico/genética
12.
Talanta ; 233: 122505, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215120

RESUMO

Colorimetric sensors are recognized as a promising means for target molecule detection as they provide rapid, cost-effective, and facile sensing visible to the naked eye. Challenges remain though in terms of their detection sensitivity and specificity for short-length target genes. Herein, we demonstrate the successful combination of the catalytic hairpin DNA assembly (CHA) approach with enzyme-linked immunosorbent assay (ELISA)-mimicking techniques for a simple, sensitive, and sequence-specific colorimetric assay to detect short SARS-CoV-2 target cDNA. In the developed CHA-based chemiluminescent assay, a low concentration of target cDNA is continuously recycled to amplify dimeric DNA probes from two biotinylated hairpin DNA until the hairpin DNA is completely consumed. The dimeric DNA probes are effectively immobilized in a neutravidin-coated microplate well and then capture neutravidin-conjugated horseradish peroxidase via biotin-neutravidin interactions, resulting in a sensitive and selective colorless-to-blue color change. The developed sensing system exhibits a high sensitivity with a detection limit of ~1 nM for target cDNA as well as the ability to precisely distinguish a single-base mismatched mutant gene within 2 h. As the proposed system does not require complex protocols or expensive equipment to amplify target cDNA, it has the potential to be utilized as a powerful tool to improve the detection sensitivity of target genes for clinical diagnostics with colorimetric detection.


Assuntos
Técnicas Biossensoriais , COVID-19 , DNA Catalítico , Colorimetria , DNA/genética , DNA Complementar , Humanos , Limite de Detecção , Medições Luminescentes , SARS-CoV-2
13.
Planta ; 254(2): 32, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34287699

RESUMO

MAIN CONCLUSION: A ß-ketoacyl-ACP-synthase II (KAS2) like enzyme and a lysophosphatidic acid acyltransferase (LPAT2) from Consolida ajacis catalyze gondoic acid biosynthesis and incorporation into the sn-2 position of seed TAG in engineered Camelina sativa. Gondoic acid (cis-11 eicosenoic acid, 20:1∆11) is the predominant very-long-chain fatty acid (VLCFA) in camelina (Camelina sativa) seed oil accounting for 12-15% of total triacylglycerol fatty acids. To explore the feasibility of engineering increased levels of this fatty acid in camelina seed, oils from a range of plant species were analyzed to identify those producing 20-Carbon (C20) fatty acids as the only VLCFAs in their seed oil. Seeds of Consolida and Delphinium species (Ranunculaceae) were found to contain moderate levels (0.2% to 25.5%) of C20 fatty acids without accompanying longer chain fatty acids. The C20 fatty acids were abundant in both sn-2 and sn-1/3 positions of seed TAG in Consolida, but were largely absent from the sn-2 position in Delphinium seed TAG. Through generation of a developing seed transcriptome, sequences were identified and cDNAs amplified from Consolida ajacis encoding a ß-ketoacyl-ACP-synthase II like protein (CaKAS2B) that lacked a predicted chloroplast transit peptide, and two homologues of Arabidopsis thaliana lysophosphatidic acid acyltransferase 2 (CaLPAT2a and CaLPAT2b). Expression of CaKAS2B in conventional (WT) camelina and a line previously engineered for high seed oleic acid content (HO) resulted in increased seed VLCFA content. Total VLCFA levels were raised from 24 to 35% and from 7 to 23% in T3 seed from representative transformants in the WT and HO backgrounds, respectively. Gondoic acid was the predominant VLCFA in transformed HO lines with low endogenous cytoplasmic fatty acid elongation activity, suggesting limited capacity of CaKAS2B to elongate beyond C20. Expression in camelina of CaLPAT2b resulted in significantly increased C20-VLCFA esterification at the sn-2 position of seed TAG with VLCFA levels of 33.8% in this position in one transformed line compared to 0.3% at sn-2 in the corresponding control line. Only small changes in total seed VLCFA content were observed in transformed lines implying that increased VLCFA esterification capacity in camelina results in positional redistribution of VLCFAs but does not significantly enhance flux through the fatty acid elongation pathway. The full potential of CaKAS2B and CaLPAT2a for the engineering of high gondoic acid levels in camelina remains to be determined. Seed fatty acid composition of Consolida and Delphinium also provides information that may be of value in the systematics of the Ranunculaceae.


Assuntos
Brassicaceae , Delphinium , Brassicaceae/genética , DNA Complementar/genética , Expressão Ectópica do Gene , Ácidos Graxos , Ácidos Graxos Monoinsaturados , Óleos Vegetais , Plantas Geneticamente Modificadas , Sementes/genética , Triglicerídeos
14.
Langmuir ; 37(29): 8738-8745, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34270267

RESUMO

A kind of blocked aptamer-functionalized molecular beacon (MB) was designed as fluorescence sensors to detect thrombins by binding-induced "turn on" structural transformation. Three MBs named MB(8 + 8), MB(15 + 8), and MB(15 + 6) consisted of two single-stranded oligonucleotides. One long single-stranded oligonucleotide (abbreviated as SS) contained a thrombin aptamer sequence and was modified with a fluorescence group and quenching group on each end side. Another short single-stranded oligonucleotide (written as cDNA) was partially complementary to the long SS. It was interesting to find that the complementary sequence length of cDNA greatly influenced the structure of the MBs. The construction of MB experiments proved that MB(8 + 8) and MB(15 + 8) could form the quenching MBs but MB(15 + 6) could not. MB(8 + 8) was composed of a SS strand paired with a complementary cDNA(8 + 8), which was called one-to-one combination, while MB(15 + 8) was two-to-two combination and MB(15 + 6) was one-to-two combination. When the ratio of SS and cDNA (15 + 8) was 1:1, the quenching efficiency reached maximum. But with the molar ratio of SS and cDNA(8 + 8) increasing, the quenching efficiency increased continuously. Under the optimal conditions that we studied, the detection limit of thrombin by MB(8 + 8) and MB(15 + 8) was 0.19 and 1.2 nM, respectively. In addition, the assay proved to be selective, and the average recovery of thrombin detected by MB(8 + 8) and MB(15 + 8) in diluted serum was 95.4 and 94.5%, respectively.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Complementar , Fluorescência , Trombina
15.
Exp Appl Acarol ; 84(4): 809-823, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34297228

RESUMO

Ticks are hematophagous ectoparasites and cause a major public health threat worldwide. Development of anti-tick vaccines is regarded to be an optimal alternative for tick control. AV422, a unique protein in ticks, is secreted into hosts during blood-feeding, but its roles are not confirmed in Haemaphysalis flava ticks. We retrieved a gene fragment encoding AV422 from a transcriptome dataset of H. flava, and based on it, we reconstructed the full length of AV422 from H. flava (Hf-AV422) by rapid amplification of cDNA ends. Expression profiles of Hf-AV422 in whole ticks and organs of different engorgement levels were determined by qPCR. Then its opening reading frame (ORF) was expressed in Escherichia coli strain BL21 (DE3). The prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) assays were conducted to test anticoagulant activities of the purified recombinant protein (rHf-AV422). The full length of AV422 was 1152 bp. Hf-AV422 showed to be conserved as indicated by multiple sequence alignment. Expression of Hf-AV422 was significantly higher in salivary glands and cuticles than in ovaries. Its expression in whole ticks decreased during engorgement with the highest levels in 1/4 engorged ticks. rHf-AV422 prolonged PT, APTT and TT when incubated with rabbit plasma. Our data demonstrated that Hf-AV422 is a conserved salivary protein with anticoagulant activity. Further studies are needed to test in detail its functional properties to ensure it an adequate antigen candidate for the development of broad-spectrum vaccines against ticks.


Assuntos
Ixodidae , Carrapatos , Animais , DNA Complementar , Ixodidae/genética , Coelhos , Proteínas Recombinantes/genética , Transcriptoma
16.
Methods Mol Biol ; 2323: 99-107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086276

RESUMO

Viroids are small circular, noncoding, highly base-paired RNAs able to infect higher plants. Recently, it has been shown that viroids can be used as very stable scaffolds to produce recombinant RNA in Escherichia coli. Coexpression of an RNA precursor consisting of a viroid monomer, in which the RNA of interest is inserted, flanked by domains of the viroid hammerhead ribozyme, along with a host plant tRNA ligase, the enzyme that catalyzes viroid circularization in infected plants, allows for accumulation of large amounts of the chimeric viroid-RNA of interest in E. coli. Since viroids do not replicate in E. coli, high accumulation most probably results from viroid scaffold stability, resistance to exonucleases due to circularity, and accumulation as a ribonucleoprotein complex with tRNA ligase. Purification of the recombinant RNA from total E. coli RNA is also facilitated by the circular structure of the product.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/metabolismo , RNA Circular/biossíntese , RNA/biossíntese , Viroides , DNA Complementar/genética , Escherichia coli/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plasmídeos/genética , RNA/genética , RNA/isolamento & purificação , RNA Ligase (ATP)/biossíntese , RNA Ligase (ATP)/genética , RNA Catalítico/metabolismo , RNA Circular/genética , RNA Circular/isolamento & purificação , RNA de Plantas/genética , RNA Viral , Solanum melongena/enzimologia , Solanum melongena/genética , Viroides/enzimologia , Viroides/genética
17.
Methods Mol Biol ; 2348: 273-284, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160814

RESUMO

RNA sequencing using nanopore sequencing is a powerful method for transcriptome analysis. The approach is appropriate for comprehensive profiling of the wide range of long noncoding RNAs. Use of nanopore-based sequencing can provide information on novel transcripts, sequence polymorphisms, and splicing variants, and thus has advantages over other gene expression profiling methods such as microarrays. Circulating extracellular long noncoding RNAs are of particular interest because of their potential use as biomarkers. Here, we describe a protocol for cDNA-PCR sequencing of circulating RNA for biomarker discovery in whole blood samples using commercially available kits and nanopore sequencing.


Assuntos
Ácidos Nucleicos Livres/genética , Perfilação da Expressão Gênica , Sequenciamento por Nanoporos/métodos , Transcriptoma , Biomarcadores , Ácidos Nucleicos Livres/isolamento & purificação , DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento por Nanoporos/instrumentação , Reação em Cadeia da Polimerase , Análise de Sequência de RNA
18.
Viruses ; 13(6)2021 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072569

RESUMO

The COVID-19 pandemic, which began in Wuhan (Hubei, China), has been ongoing for about a year and a half. An unprecedented number of people around the world have been infected with SARS-CoV-2, the etiological agent of COVID-19. Despite the fact that the mortality rate for COVID-19 is relatively low, the total number of deaths has currently already reached more than three million and continues to increase due to high incidence. Since the beginning of the pandemic, a large number of sequences have been obtained and many genetic variants have been identified. Some of them bear significant mutations that affect biological properties of the virus. These genetic variants, currently Variants of Concern (VoC), include the so-called United Kingdom variant (20I/501Y), the Brazilian variant (20J/501Y.V3), and the South African variant (20H/501Y.V2). We describe here a novel SARS-CoV-2 variant with distinct spike protein mutations, first obtained at the end of January 2021 in northwest Russia. Therefore, it is necessary to pay attention to the dynamics of its spread among patients with COVID-19, as well as to study in detail its biological properties.


Assuntos
SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Análise Mutacional de DNA , DNA Complementar , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Filogenia , Conformação Proteica , Federação Russa , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química
19.
Nucleic Acids Res ; 49(11): 6420-6436, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096602

RESUMO

The TREX-TAP pathway is vital for mRNA export. For spliced mRNA, the TREX complex is recruited during splicing; however, for intronless mRNA, recruitment is sequence dependent. However, the export of cytoplasmic long noncoding RNA (lncRNA) is poorly characterized. We report the identification of a cytoplasmic accumulation region (CAR-N) in the intronless lncRNA, NKILA. CAR-N removal led to strong nuclear retention of NKILA, and CAR-N insertion promoted the export of cDNA transcripts. In vitro RNP purification via CAR-N, mass spectrometry, and siRNA screening revealed that SRSF1 and SRSF7 were vital to NKILA export, and identified a cluster of SRSF1/7 binding sites within a 55 nucleotide sequence in CAR-N. Significant nuclear enrichment of NKILA was observed for NKILA lacking CAR-N or the cluster of binding sites in knock-in models. Depletion of TREX-TAP pathway components resulted in strong nuclear retention of NKILA. RNA and protein immunoprecipitation verified that SRSF1/7 were bound to NKILA and interacted with UAP56 and ALYREF. Moreover, NKILA lacking CAR-N was unable to inhibit breast cancer cell migration. We concluded that the binding of SRSF1/7 to clustered motifs in CAR-N facilitated TREX recruitment, promoting the export of NKILA, and confirmed the importance of NKILA localization to its function.


Assuntos
Núcleo Celular/metabolismo , RNA Longo não Codificante/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Movimento Celular , Citoplasma/genética , RNA Helicases DEAD-box/metabolismo , DNA Complementar/metabolismo , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Motivos de Nucleotídeos , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo
20.
Nat Protoc ; 16(7): 3672-3694, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34108731

RESUMO

More than 90% of the human genome is transcribed into noncoding RNAs, but their functional characterization has lagged behind. A major bottleneck in the understanding of their functions and mechanisms has been a dearth of systematic methods for identifying interacting protein partners. There now exist several methods, including identification of direct RNA interacting proteins (iDRiP), chromatin isolation by RNA purification (ChIRP), and RNA antisense purification, each previously applied towards identifying a proteome for the prototype noncoding RNA, Xist. iDRiP has recently been modified to successfully identify proteomes for two additional noncoding RNAs of interest, TERRA and U1 RNA. Here we describe the modified protocol in detail, highlighting technical differences that facilitate capture of various noncoding RNAs. The protocol can be applied to short and long RNAs in both cultured cells and tissues, and requires ~1 week from start to finish. Here we also perform a comparative analysis between iDRiP and ChIRP. We obtain partially overlapping profiles, but find that iDRiP yields a greater number of specific proteins and fewer mitochondrial contaminants. With an increasing number of essential long noncoding RNAs being described, robust RNA-centric protein capture methods are critical for the probing of noncoding RNA function and mechanism.


Assuntos
Proteômica/métodos , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Reagentes para Ligações Cruzadas/química , DNA Complementar/genética , Camundongos , Ligação Proteica , Proteoma/metabolismo , Reprodutibilidade dos Testes , Raios Ultravioleta
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