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1.
Methods Mol Biol ; 2799: 47-54, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727902

RESUMO

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.


Assuntos
Neurônios , Transfecção , Neurônios/metabolismo , Neurônios/citologia , Animais , Transfecção/métodos , Células Cultivadas , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Camundongos , Cultura Primária de Células/métodos , DNA Complementar/genética
2.
Fish Shellfish Immunol ; 149: 109599, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38701990

RESUMO

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS),avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.


Assuntos
Sequência de Aminoácidos , Imunidade Inata , Filogenia , Pinctada , Superóxido Dismutase , Animais , Pinctada/imunologia , Pinctada/genética , Pinctada/enzimologia , Superóxido Dismutase/genética , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Superóxido Dismutase/imunologia , Imunidade Inata/genética , Perfilação da Expressão Gênica/veterinária , Sequência de Bases , Alinhamento de Sequência/veterinária , Escherichia coli , DNA Complementar/genética , Micrococcus luteus/fisiologia , Regulação da Expressão Gênica/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Fish Shellfish Immunol ; 149: 109560, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38615702

RESUMO

The JAK (Janus kinase)-STAT (Signal transducer and activator of transcription) is a well-known functional signaling pathway that plays a key role in several important biological activities such as apoptosis, cell proliferation, differentiation, and immunity. However, limited studies have explored the functions of STAT genes in invertebrates. In the present study, the gene sequences of two STAT genes from the Pacific oyster (Crassostrea gigas), termed CgSTAT-Like-1 (CgSTAT-L1) and CgSTAT-Like-2 (CgSTAT-L2), were obtained using polymerase chain reaction (PCR) amplification and cloning. Multiple sequence comparisons revealed that the sequences of crucial domains of these proteins were conserved, and the similarity with the protein sequence of other molluscan STAT is close to 90 %. The phylogenetic analyses indicated that CgSTAT-L1 and CgSTAT-L2 are novel members of the mollusk STAT family. Quantitative real-time PCR results implied that CgSTAT-L1 and CgSTAT-L2 mRNA expression was found in all tissues, and significantly induced after challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), or poly(I:C). After that, dual-luciferase reporter assays denoted that overexpression of CgSTAT-L1 and CgSTAT-L2 significantly activated the NF-κB signaling, and, interestingly, the overexpressed CgSTAT proteins potentiated LPS-induced NF-κB activation. These results contributed a preliminary analysis of the immune-related function of STAT genes in oysters, laying the foundation for deeper understanding of the function of invertebrate STAT genes.


Assuntos
Sequência de Aminoácidos , Crassostrea , Filogenia , Fatores de Transcrição STAT , Alinhamento de Sequência , Animais , Crassostrea/genética , Crassostrea/imunologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Alinhamento de Sequência/veterinária , Lipopolissacarídeos/farmacologia , Imunidade Inata/genética , Peptidoglicano/farmacologia , Poli I-C/farmacologia , Sequência de Bases , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , DNA Complementar/genética , Clonagem Molecular , Transdução de Sinais
4.
Zoolog Sci ; 41(1): 50-59, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38587517

RESUMO

Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are novel neuropeptides that have been discovered in the hypothalamic infundibulum of chickens. NPGL and NPGM play important roles in lipid metabolism in juvenile chickens. The physiological functions of NPGL and NPGM in sexually mature birds remain unknown. The Japanese quail (Coturnix japonica) seems to be an appropriate model for analyzing NPGL and NPGM during sexual maturity. However, studies on NPGL or NPGM have yet to be reported in the Japanese quail. In the present study, we identified cDNAs encoding precursor proteins of NPGL and NPGM in the quail hypothalamus. In situ hybridization revealed that NPGL mRNA-expressing cells in the hypothalamus were localized in the infundibular nucleus and median eminence, and NPGM mRNA-expressing cells were only found in the mammillary nucleus. Immunohistochemistry revealed that NPGM-like immunoreactive cells were distributed in the mammillary nucleus, whereas NPGL-like immunoreactive cells were not detected in the hypothalamus. Real-time PCR analysis indicated that the expression of NPGL mRNA was higher in the hypothalamus of females than in that of males, and NPGM mRNA expression showed no sex differences. NPGL and NPGM mRNA expression in males was upregulated after 24 h of food deprivation. In females, only NPGM mRNA expression was increased by fasting. These results suggest that the physiological functions of NPGL and NPGM are different in quail, and these factors are involved in sex differences in energy metabolism.


Assuntos
Galinhas , Coturnix , Feminino , Masculino , Animais , Coturnix/genética , Hipotálamo , DNA Complementar , RNA Mensageiro/genética
5.
Zoolog Sci ; 41(2): 141-158, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38587909

RESUMO

The silkworm Bombyx mori exhibits a photoperiodic response (PR) for embryonic diapause induction. This article provides a comprehensive review of literature on the silkworm PR, starting from early works on population to recent studies uncovering the molecular mechanism. Makita Kogure (1933) conducted extensive research on the PR, presenting a pioneering paper on insect photoperiodism. In the 1970s and 80s, artificial diets were developed, and the influence of nutrition on PR was well documented. The photoperiodic photoreceptor has been investigated from organ to molecular level in the silkworm. Culture experiments demonstrated that the photoperiodic induction can be programmed in an isolated brain (Br)-subesophageal ganglion (SG) complex with corpora cardiaca (CC)-corpora allata (CA). The requirement of dietary vitamin A for PR suggests the involvement of opsin pigment in the photoperiodic reception, and a cDNA encoding an opsin (Boceropsin) was cloned from the brain. The effector system concerning the production and secretion of diapause hormone (DH) has also been extensively investigated in the silkworm. DH is produced in a pair of posterior cells of SG, transported to CC by nervi corporis cardiaci, and ultimately released into the hemolymph. Possible involvement of GABAergic and corazonin (Crz) signal pathways was suggested in the control of DH secretion. Knockout (KO) experiments of GABA transporter (GAT) and circadian clock genes demonstrated that GAT plays a crucial role in PR through circadian control. A model outlining the PR mechanism, from maternal photoperiodic light reception to DH secretion, has been proposed.


Assuntos
Bombyx , Diapausa de Inseto , Diapausa , Animais , Bombyx/metabolismo , DNA Complementar , Gânglios , Opsinas/metabolismo
6.
Sci Rep ; 14(1): 9279, 2024 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-38654039

RESUMO

A simple and rapid electrochemical sensing method with high sensitivity and specificity of aptamers was developed for the detection of methylamphetamine (MAMP). A short anti-MAMP thiolated aptamer (Apt) with a methylene blue (MB) probe at 3'-end was immobilized on the surface of a gold electrode (MB-Apt-S/GE). The electrochemical signal appeared when MAMP presenting in the sample solution competed with cDNA for binding with MB-Apt-S. Under optimized conditions, the liner range of this signal-on electrochemical aptasensor for the detection of MAMP achieved from 1.0 to 10.0 nmol/L and 10.0-400 nmol/L. LOD 0.88 nmol/L were obtained. Satisfactory spiked recoveries of saliva and urine were also obtained. In this method, only 5 min were needed to incubate before the square wave voltammetry (SWV) analysis, which was much more rapid than other electrochemical sensors, leading to a bright and broad prospect for the detection of MAMP in biological sample. This method can be used for on-site rapid detection on special occasions, such as drug driving scenes, entertainment venues suspected of drug use, etc.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Metanfetamina , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , Aptâmeros de Nucleotídeos/química , Humanos , Metanfetamina/urina , Metanfetamina/análise , DNA Complementar/genética , Saliva/química , Saliva/metabolismo , Eletrodos , Limite de Detecção , Ouro/química , Azul de Metileno/química
7.
Mol Biol Rep ; 51(1): 547, 2024 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-38642187

RESUMO

BACKGROUND: Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. METHODS AND RESULTS: We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. CONCLUSIONS: The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.


Assuntos
Metschnikowia , Saccharomyces cerevisiae , Vitis , Saccharomyces cerevisiae/metabolismo , Vitis/genética , Vitis/metabolismo , DNA Complementar/metabolismo , Transaminases/genética , Fermentação , RNA/metabolismo
8.
Zhongguo Zhong Yao Za Zhi ; 49(3): 661-670, 2024 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-38621870

RESUMO

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.


Assuntos
Animais Peçonhentos , Anti-Infecciosos , Venenos de Escorpião , Animais , Criança , Humanos , Peptídeos/química , Escorpiões/química , Escorpiões/metabolismo , DNA Complementar , Venenos de Escorpião/farmacologia
9.
Sci Adv ; 10(15): eadk8791, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38608016

RESUMO

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers, enabling their direct integration into CRISPR arrays as 3'-dN-RNAs or 3'-dN-RNA/cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3' proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs (ssDNAs) is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage defense nucleases. Our findings reveal mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.


Assuntos
DNA Polimerase Dirigida por RNA , RNA , DNA Complementar/genética , RNA/genética , DNA Polimerase Dirigida por RNA/genética , DNA/genética , DNA de Cadeia Simples
10.
Methods Mol Biol ; 2757: 289-306, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38668973

RESUMO

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.


Assuntos
Clonagem Molecular , Ctenóforos , DNA Complementar , Biblioteca Gênica , Proteínas Luminescentes , Animais , Ctenóforos/genética , Ctenóforos/metabolismo , Clonagem Molecular/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo
11.
Exp Dermatol ; 33(4): e15072, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38576105

RESUMO

Autosomal recessive congenital ichthyoses (ARCI) is a genetically heterogeneous condition that can be caused by pathogenic variants in at least 12 genes, including ABCA12. ARCI mainly consists of congenital ichthyosiform erythroderma (CIE), lamellar ichthyosis (LI) and harlequin ichthyosis (HI). The objective was to determine previously unreported pathogenic variants in ABCA12 and to update genotype-phenotype correlations for patients with pathogenic ABCA12 variants. Pathogenic variants in ABCA12 were detected using Sanger sequencing or a combination of Sanger sequencing and whole-exome sequencing. To verify the pathogenicity of a previously unreported large deletion and intron variant, cDNA analysis was performed using total RNA extracted from hair roots. Genetic analyses were performed on the patients with CIE, LI, HI and non-congenital ichthyosis with unusual phenotypes (NIUP), and 11 previously unreported ABCA12 variants were identified. Sequencing of cDNA confirmed the aberrant splicing of the variant ABCA12 in the patients with the previously unreported large deletion and intron variant. Our findings expand the phenotype spectrum of ichthyosis patients with ABCA12 pathogenic variants. The present missense variants in ABCA12 are considered to be heterogenous in pathogenicity, and they lead to varying disease severities in patients with ARCI and non-congenital ichthyosis with unusual phenotypes (NIUP).


Assuntos
Eritrodermia Ictiosiforme Congênita , Ictiose Lamelar , Ictiose , Humanos , Ictiose Lamelar/genética , Ictiose Lamelar/patologia , DNA Complementar , Genes Recessivos , Mutação , Ictiose/genética , Eritrodermia Ictiosiforme Congênita/genética , Estudos de Associação Genética , Transportadores de Cassetes de Ligação de ATP/genética
12.
BMC Vet Res ; 20(1): 162, 2024 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-38678249

RESUMO

BACKGROUND: Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro. METHODS: To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells. RESULTS: The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system. CONCLUSION: This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.


Assuntos
Vírus da Cinomose Canina , Plasmídeos , Vírus da Cinomose Canina/genética , Animais , Células Vero , Chlorocebus aethiops , Plasmídeos/genética , Transfecção , Genética Reversa/métodos , DNA Complementar/genética , Cinomose/virologia
13.
Biochem Biophys Res Commun ; 711: 149909, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38615573

RESUMO

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 µg/µL - 0.24 ng/µL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 µL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.


Assuntos
RNA , Humanos , RNA/genética , RNA/análise , Transcrição Reversa , Saliva/metabolismo , Saliva/química , Genética Forense/métodos , Genética Forense/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Padrões de Referência , DNA Complementar/genética , Manchas de Sangue , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas
14.
Int J Mol Sci ; 25(7)2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38612464

RESUMO

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.


Assuntos
Placenta , Sêmen , Masculino , Feminino , Gravidez , Suínos , Animais , Camundongos , DNA Complementar , Espermatozoides , Eutérios , Alanina , Isoantígenos/genética , Fertilização/genética
15.
Microbiol Spectr ; 12(4): e0387223, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38442427

RESUMO

In vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5' end of the viral RNA. A range of cDNA products is generated, most of them indicative for the occurrence of in vitro strand transfer events that involve translocation of cDNA from the 5' end to random positions on the viral RNA. This process results in the formation of cDNAs with large internal deletions. However, near full-length cDNA and cDNA with sequence patterns resembling multiple spliced HIV-1 RNA were also detected. Despite its potential to introduce significant bias in the interpretation of results across various applications, tRNA(Lys-3)-driven reverse transcription has been overlooked thus far. A more in-depth study of this tRNA-driven in vitro reaction may provide new insight into the complex process of in vivo HIV-1 replication.IMPORTANCEThe use of silica-based extraction methods for purifying HIV-1 RNA from viral particles is a common practice, but it involves co-extraction of human tRNA(Lys-3) due to the strong interactions between these molecules. This co-extraction becomes particularly significant when the extracted RNA is used in reverse transcription reactions, as the tRNA(Lys-3) then serves as a primer. Reverse transcription from tRNA(Lys-3) is not confined to cDNA synthesis of the 5' end of the viral RNA but extends across various regions of the viral genome through in vitro strand transfer events. Co-extraction of tRNA(Lys-3) has been overlooked thus far, despite its potential to introduce bias in downstream, reverse transcription-related applications. The observed events in the tRNA(Lys-3)-induced in vitro reverse transcription resemble in vivo replication processes. Therefore, these reactions may offer a unique model to better understand the replication dynamics of HIV-1.


Assuntos
HIV-1 , Transcrição Reversa , Humanos , HIV-1/genética , Artefatos , DNA Complementar/genética , Transcrição Gênica , Sequência de Bases , RNA Viral/genética , RNA de Transferência/genética , Conformação de Ácido Nucleico
16.
Biosci Biotechnol Biochem ; 88(6): 620-629, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38479783

RESUMO

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.


Assuntos
DNA Complementar , Sequenciamento de Nucleotídeos em Larga Escala , Transglutaminases , Humanos , Transglutaminases/genética , Transglutaminases/metabolismo , Especificidade por Substrato , DNA Complementar/genética , Sequência de Aminoácidos , Biblioteca de Peptídeos
17.
Anal Chem ; 96(14): 5471-5477, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38551977

RESUMO

Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/µL DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.


Assuntos
Sistemas CRISPR-Cas , Recombinases , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Nucleotidiltransferases , DNA/genética , DNA de Cadeia Simples , DNA Complementar , Técnicas de Amplificação de Ácido Nucleico
18.
Front Immunol ; 15: 1359169, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38550579

RESUMO

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in ß and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/ß/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the ß and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.


Assuntos
Galinhas , Linfócitos T , Animais , Galinhas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , DNA Complementar , Genoma
19.
Arch Virol ; 169(3): 61, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38441697

RESUMO

The coat protein (CP) of the cucumber mosaic virus (CMV) yellow strain [CMV(Y)], but not the CMV B2 strain [CMV(B2)], serves as an avirulence determinant against the NB-LRR class RCY1 of Arabidopsis thaliana. To investigate the avirulence function, a series of binary vectors were constructed by partially exchanging the CP coding sequence between CMV(Y) and CMV(B2) or introducing nucleotide substitutions. These vectors were transiently expressed in Nicotiana benthamiana leaves transformed with modified RCY1 cDNA. Analysis of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression at leaf sites infiltrated with Agrobacterium indicated that a single amino acid at position 31 of the CP seems to determine the avirulence function.


Assuntos
Arabidopsis , Cucumovirus , Infecções por Citomegalovirus , Humanos , Aminoácidos , Arabidopsis/genética , Cucumovirus/genética , DNA Complementar
20.
Mol Genet Genomic Med ; 12(3): e2353, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38488435

RESUMO

BACKGROUND: Although proteinuria is long recognized as an independent risk factor for progressive chronic kidney diseases, not all forms of proteinuria are detrimental to kidney function, one of which is isolated proteinuria caused by cubilin (CUBN)-specific mutations. CUBN encodes an endocytic receptor, initially found to be responsible for the Imerslund-Gräsbeck syndrome (IGS; OMIM #261100) characterized by a combined phenotype of megaloblastic anemia and proteinuria. METHODS: After analyzing their clinical and pathological characterizations, next-generation sequencing for renal disease genes or whole-exome sequencing (WES) was performed on four patients with non-progressive isolated proteinuria. CUBN biallelic pathogenic variants were identified and further analyzed by cDNA-PCR sequencing, immunohistochemistry, minigene assay, and multiple in silico prediction tools, including 3D protein modeling. RESULTS: Here, we present four patients with isolated proteinuria caused by CUBN C-terminal biallelic pathogenic variants, all of which showed no typical IGS symptoms, such as anemia and vitamin B12 deficiency. Their urine protein levels fluctuated between +~++ and estimated glomerular filtration rate (eGFR) were normal or slightly higher. Mild mesangial hypercellularity was found in three children's renal biopsies. A homozygous splice-site variant of CUBN (c.6821+3 (IVS44) A>G) was proven to result in the exon 44 skipping and premature translation termination by cDNA sequencing and immunohistochemistry. Compound heterozygous mutations were identified among the other three children, including another novel splice-site variant (c.10764+1 (IVS66) G>A) causing the retention of first 4 nucleotides in intron 66 by minigene assay, two unreported missense mutations (c.4907G>A (p.R1636Q); c. 9095 A>G (p.Y3032C)), and two reported missense mutations in China (c.8938G>A (p.D2980N); c. 9287T>C (p.L3096P)), locating behind the vitamin B12-binding domain, affecting CUB11, CUB16, CUB22, CUB23, and CUB27 domains, respectively. CONCLUSION: These results demonstrate that above CUBN mutations may cause non-progressive and isolated proteinuria, expanding the variant spectrum of CUBN and benefiting our understanding of proteinuria and renal function.


Assuntos
Proteinúria , Receptores de Superfície Celular , Criança , Humanos , DNA Complementar , Proteinúria/genética , Proteinúria/patologia , Receptores de Superfície Celular/genética
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