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Highly sensitive detection of low-frequency EGFR-L858R mutation is particularly important in guiding targeted therapy of nonsmall-cell lung carcinoma (NSCLC). To this end, a ligase chain reaction (LCR)-based electrochemical biosensor (e-LCR) with an inverted sandwich-type architecture was provided by combining a cooperation of lambda exonuclease-RecJf exonuclease (λ-RecJf exo). In this work, by designing a knife-like DNA substrate (an overhang ssDNA part referred to the "knife arm") and introducing the λ-RecJf exo, the unreacted DNA probes in the LCR were specially degraded while only the ligated products were preserved, after which the ligated knife-like DNA products were hybridized with capture probes on the gold electrode surface through the "knife arms", forming the inverted sandwich-type DNA structure and bringing the methylene blue-label close to the electrode surface to engender the electrical signal. Finally, the sensitivity of the e-LCR could be improved by 3 orders of magnitude with the help of the λ-RecJf exo, and due to the mutation recognizing in the ligation site of the employed ligase, this method could detect EGFR-L858R mutation down to 0.01%, along with a linear range of 1 fM-10 pM and a limit detection of 0.8 fM. Further, the developed method could distinguish between L858R positive and negative mutations in cultured cell samples, tumor tissue samples, and plasma samples, whose accuracy was verified by the droplet digital PCR, holding a huge potential in liquid biopsy for precisely guiding individualized-treatment of NSCLC patients with advantages of high sensitivity, low cost, and adaptability to point-of-care testing.
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BACKGROUND: Currently, there is no research available on the prognosis, potential effect and therapeutic value of USP31 in clear cell renal cell carcinoma (ccRCC). To address this gap, the present study aimed to shed light on its potential roles and possible mechanisms in ccRCC. METHODS: R software was utilized to conduct bioinformatics analyses with the data derived from The Cancer Genome Atlas (i.e. KIRC) and Gene Expression Omnibus datasets. The expression of USP31 in ccRCC was validated by a PCR. The independent prognostic ability of USP31 was evaluated by Cox regression analysis. We conducted gene set enrichment analysis (GSEA) to explore the potential USP31-related pathways. We also discussed the relationships between USP31 and immunity, by predicting its possible upstream transcription factors (TFs) by ChEA3. RESULTS: In ccRCC, USP31 demonstrated a high level of expression and this increased expression was correlated with a poor prognosis (p < 0.05). Through univariate and multivariate Cox regression analysis, USP31 was identified as an independent prognostic factor for ccRCC (p < 0.05). Furthermore, eight USP31-related pathways were identified by GSEA (p < 0.05). Moreover, USP31 was found to be associated with microsatellite instability, tumor microenvironment, a variety of immune cells and immune checkpoints and immune infiltration (p < 0.05). Additionally, Patients with high USP31 expression in ccRCC were shown to have better curative effects after immunotherapy (p < 0.05). Finally, we found that AR, USF1, MXI1 and CLOCK could be the potential upstream TFs of USP31. CONCLUSIONS: USP31 could serve as a potential biomarker for predicting both prognosis and immune responses, revealing its potential mechanisms of TF-USP31 mRNA networks in ccRCC.
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Carcinoma de Células Renales , Carcinoma , Neoplasias Renales , Humanos , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/terapia , Biomarcadores , Neoplasias Renales/genética , Neoplasias Renales/terapia , Inmunidad , ARN , Microambiente Tumoral/genética , Proteasas Ubiquitina-EspecíficasRESUMEN
BACKGROUND/AIM: To investigate the independent relationships of visual impairment (VI) and Subjective cognitive complaints (SCC) with physical function impairment (PFI) and the interaction effect between VI and SCC on PFI in American older adults. METHODS: The data of this cross-sectional study was obtained from the 2005-2008 National Health and Examination Survey (NHANES) conducted in the United States. The VI criterion included both subjective self-reported eyesight conditions and objective visual acuity test results. The self-reported questionnaires were utilized to determine PFI and SCC. According to the survey design of NHANS, original data were weighted to produce nationally representative estimates. Both the unweighted original data and weighted estimates underwent analysis. Crude and adjusted logistic models were employed to assess the pairwise associations among VI, SCC, and PFI. To assess the interactive effect, measures such as the relative excess risk due to interaction (RERI), attributable proportion due to interaction (AP), and synergy index (S) were calculated. RESULTS: A total of 2,710 subjects (weighted n = 38,966,687) aged 60 years or older were included. Compared with subjects without subjective visual impairment (SVI), those with SVI had a significant positive association with PFI [weighted OR (95%CI): 3.11 (2.25, 4.31)]. After multi-variable adjusting, the relationship remained significant [weighted OR (95%CI): 1.90 (1.32, 2.72)]. Similarly, those with objective visual impairment (OVI) were positively associated with the risk of PFI in the crude model [weighted OR (95%CI): 2.35 (1.53, 3.61)] and adjusted model [weighted OR (95%CI): 1.84 (1.07, 3.17)]. Moreover, we found the association of SCC with an increased risk of FPI [crude weighted OR (95%CI): 5.02 (3.40, 7.40); adjusted weighted OR (95%CI): 3.29 (2.01, 5.38)]. Ultimately, the additive interaction showed there was a significant positive interaction term between SVI and SCC on PFI, while OVI and SCC did not. CONCLUSION: Both VI and SCC were significantly associated with PFI in elder adults. Besides, there was a significant synergistic interaction between SVI and SCC on PFI, which indicated the improvement of SVI and SCC may be beneficial for the prevention of PFI. For the elderly, especially those with multiple disabilities, comprehensive and targeted approaches are imperative to foster their overall well-being and health.
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Baja Visión , Anciano , Humanos , Estados Unidos/epidemiología , Encuestas Nutricionales , Estudios Transversales , Ejercicio Físico , CogniciónRESUMEN
5-Methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT) is a novel psychoactive substance exhibiting a tryptamine structure. Despite its increasing prevalence, the environmental impact of 5-MeO-MiPT remains unexplored. Our prior investigation revealed that 5-MeO-MiPT induced inhibited spontaneous movement and prompted anxiety-like behavior in adult zebrafish-a validated toxicological model. To elucidate this phenomenon and establish a correlation between metabolomics and behavioral changes induced by 5-MeO-MiPT, zebrafish were administered varying drug concentrations. Zebrafishes were subjected to injections of different 5-MeO-MiPT concentrations. Subsequent metabolomic analysis of endogenous metabolites affected by the drug unveiled substantial variations in metabolic levels between the control group and the drug-injected cohorts. A total of 22 distinct metabolites emerged as potential biomarkers. Further scrutiny identified seven pathways significantly influenced by 5-MeO-MiPT. A focused exploration into amino acid metabolism, lipid metabolism, and energy metabolism unveiled that the metabolic repercussions of 5-MeO-MiPT on zebrafish resulted in observable brain damage. Notably, the study identified a consequential disruption in the liver-brain pathway. The comprehensive metabolomic approach employed herein effectively discerned the impact of 5-MeO-MiPT on zebrafish metabolism. This approach also shed light on the mechanism underpinning the anxiety-like behavior observed in zebrafish post-drug injection. Specifically, our findings indicate that 5-MeO-MiPT induces brain damage, particularly within the liver-brain pathway.
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5-Metoxitriptamina/análogos & derivados , Triptaminas , Pez Cebra , Animales , Pez Cebra/metabolismo , Triptaminas/toxicidad , Triptaminas/metabolismo , Metabolómica/métodos , Hígado/metabolismoRESUMEN
As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (µM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.
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Técnicas Biosensibles , ARN , Química Clic/métodos , Técnicas Biosensibles/métodos , ADN/química , Sondas de ADN/genética , Sondas de ADN/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas Electroquímicas/métodos , Límite de Detección , Hibridación de Ácido NucleicoRESUMEN
Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized "negative" signal of wobble-base pair-mediated amplification was fully utilized as a "positive" one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue (IMB) and current of ferrocene (IFc) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: IMB/IFc > 2; GG homozygote: IMB/IFc < 1; GA heterozygote: 1 < IMB/IFc < 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.
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Técnicas Biosensibles , Reacción en Cadena de la Ligasa , Humanos , Alelos , Genotipo , Citocromo P-450 CYP2C19 , Técnicas Electroquímicas , Oro , Límite de DetecciónRESUMEN
Speciation is a central topic in evolutionary biology. However, how genomic divergence originates and accumulates in the face of gene flow during ecological adaptation remains poorly understood. Closely related species that have adapted to distinct environments but inhabit some overlapping ranges provide an ideal system to evaluate this issue. Here, we combine population genomics and species distribution models (SDMs) to examine genomic divergences between two sister plant species, Medicago ruthenica and M. archiducis-nicolai, that occur in northern China and the northeast Qinghai-Tibet Plateau, respectively, with overlapping distributions in the border of the two regions. M. ruthenica and M. archiducis-nicolai are well-delimited based on population genomic data, although hybrids exist in sympatric sampling locations. Coalescent simulations and SDMs suggest that the two species diverged from each other in the Quaternary but have been in continuous contact with gene flow occurring between the two species since then. We also discovered positive selection signatures associated with genes both outside and within genomic islands in both species that are probably involved in adaptations to arid and high-altitude environments. Our findings provide insights into how natural selection and climatic changes in the Quaternary initiated and maintained interspecific divergence of these two sister species.
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Evolución Biológica , Medicago , Tibet , China , Genómica , FilogeniaRESUMEN
OBJECTIVES: To construct and validate a prediction model based on full-sequence MRI for preoperatively evaluating the invasion depth of bladder cancer. METHODS: A total of 445 patients with bladder cancer were divided into a seven-to-three training set and test set for each group. The radiomic features of lesions were extracted automatically from the preoperative MRI images. Two feature selection methods were performed and compared, the key of which are the Least Absolute Shrinkage and Selection Operator (LASSO) and the Max Relevance Min Redundancy (mRMR). The classifier of the prediction model was selected from six advanced machine-learning techniques. The receiver operating characteristic (ROC) curves and the area under the curve (AUC) were applied to assess the efficiency of the models. RESULTS: The models with the best performance for pathological invasion prediction and muscular invasion prediction consisted of LASSO as the feature selection method and random forest as the classifier. In the training set, the AUC of the pathological invasion model and muscular invasion model were 0.808 and 0.828. Furthermore, with the mRMR as the feature selection method, the external invasion model based on random forest achieved excellent discrimination (AUC, 0.857). CONCLUSIONS: The full-sequence models demonstrated excellent accuracy for preoperatively predicting the bladder cancer invasion status. CLINICAL RELEVANCE STATEMENT: This study introduces a full-sequence MRI model for preoperative prediction of the depth of bladder cancer infiltration, which could help clinicians to recognise pathological features associated with tumour infiltration prior to invasive procedures. KEY POINTS: ⢠Full-sequence MRI prediction model performed better than Vesicle Imaging-Reporting and Data System (VI-RADS) for preoperatively evaluating the invasion status of bladder cancer. ⢠Machine learning methods can extract information from T1-weighted image (T1WI) sequences and benefit bladder cancer invasion prediction.
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Imagen por Resonancia Magnética , Neoplasias de la Vejiga Urinaria , Humanos , Estudios Retrospectivos , Imagen por Resonancia Magnética/métodos , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Neoplasias de la Vejiga Urinaria/cirugía , Curva ROC , Aprendizaje AutomáticoRESUMEN
Oral administration is the preferred route for drug administration that leads to better therapy compliance. The intestine plays a key role in the absorption and metabolism of oral drugs, therefore, new intestinal models are being continuously proposed, which contribute to the study of intestinal physiology, drug screening, drug side effects, and drug-drug interactions.Advances in pharmaceutical processes have produced more drug formulations, causing challenges for intestinal models. To adapt to the rapid evolution of pharmaceuticals, more intestinal models have been created. However, because of the complexity of the intestine, few models can take all aspects of the intestine into account, and some functions must be sacrificed to investigate other areas. Therefore, investigators need to choose appropriate models according to the experimental stage and other requirements to obtain the desired results.To help researchers achieve this goal, this review summarised the advantages and disadvantages of current commonly used intestinal models and discusses possible future directions, providing a better understanding of intestinal models.
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Mucosa Intestinal , Intestinos , Preparaciones Farmacéuticas/metabolismo , Mucosa Intestinal/metabolismo , Administración Oral , Permeabilidad , Simulación por Computador , Absorción Intestinal , Modelos BiológicosRESUMEN
Subway vehicle roofs must be inspected when entering and exiting the depot to ensure safe subway vehicle operations. This paper presents an improved method for detecting foreign objects on subway vehicle roofs based on the YOLOv7 algorithm. First, we capture images of foreign objects using a line-scan camera at the depot entrance and exit, creating a dataset of foreign roof objects. Subsequently, we address the shortcomings of the YOLOv7 algorithm by introducing the Ghost module, an improved weighted bidirectional feature pyramid network (WBiFPN), and the Wise intersection over union (WIoU) bounding-box regression loss function. These enhancements are incorporated to build the subway vehicle roof foreign object detection model based on the improved YOLOv7, which we refer to as YOLOv7-GBW. The experimental results demonstrate the practicality and usability of the proposed method. The analysis of the experimental results indicates that the YOLOv7-GBW algorithm achieves a detection accuracy of 90.29% at a speed of 54.3 frames per second (fps) with a parameter count of 15.51 million. The improved YOLOv7 model outperforms mainstream detection algorithms in terms of detection accuracy, speed, and parameter count. This finding confirms that the proposed method meets the requirements for detecting foreign objects on subway vehicle roofs.
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Many plants have the capability to accumulate anthocyanins for coloration, and anthocyanins are advantageous to human health. In the case of hulless barley (Hordeum vulgare L. var. nudum), investigation into the mechanism of anthocyanin formation is limited to the level of protein-coding genes (PCGs). Here, we conducted a comprehensive bioinformatics analysis to identify a total of 9414 long noncoding RNAs (lncRNAs) in the seed coats of purple and white hulless barley along a developmental gradient. Transcriptome-wide profiles of lncRNAs documented several properties, including GC content fluctuation, uneven length, a diverse range of exon numbers, and a wide variety of transcript classifications. We found that certain lncRNAs in hulless barley possess detectable sequence conservation with Hordeum vulgare and other monocots. Furthermore, both differentially expressed lncRNAs (DElncRNAs) and PCGs (DEPCGs) were concentrated in the later seed development stages. On the one hand, DElncRNAs could potentially cis-regulate DEPCGs associated with multiple metabolic pathways, including flavonoid and anthocyanin biosynthesis in the late milk and soft dough stages. On the other hand, there was an opportunity for trans-regulated lncRNAs in the color-forming module to affect seed coat color by upregulating PCGs in the anthocyanin pathway. In addition, the interweaving of hulless barley lncRNAs and diverse TFs may function in seed coat coloration. Notably, we depicted a dynamic portrait of the anthocyanin synthesis pathway containing hulless barley lncRNAs. Therefore, this work provides valuable gene resources and more insights into the molecular mechanisms underlying anthocyanin accumulation in hulless barley from the perspective of lncRNAs, which facilitate the development of molecular design breeding in crops.
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Hordeum , ARN Largo no Codificante , Antocianinas/genética , Antocianinas/metabolismo , Hordeum/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Semillas/genética , Tibet , TranscriptomaRESUMEN
Thanks to its preparatory ease, close affinity, and low cost, the aptasensor can serve as a promising substitute for antibody-dependent biosensors. However, the available aptasensors are mostly subject to a single-mode readout and the interference of unbound aptamers in solution and non-target-induced transition events. Herein, we proposed a multimodal aptasensor for multimode detection of ochratoxin A (OTA) with cross-validation using the 3'-6-carboxyfluorescein (FAM)-enhanced exonuclease I (Exo I) tool and magnetic microbead carrier. Specifically, the 3'-FAM-labeled aptamer/biotinylated-cDNA hybrids were immobilized onto streptavidin-magnetic microbeads via streptavidin-biotin interaction. With the presence of OTA, an antiparallel G-quadruplex conformation was formed, protecting the 3'-FAM labels from Exo I digestion, and then anti-FAM-horseradish peroxidase (HRP) was bound via specific antigen-antibody affinity; for the aptamers without the protection of OTA, the distal ssDNA was hydrolyzed from 3' â 5', releasing 3'-FAM labels to the solution. Therefore, the OTA was detected by analyzing the "signal-off" fluorescence of the supernatant and two "signal-on" signals in electrochemistry and colorimetry through the detection of the coating magnetic microbeads in HRP's substrate. The results showed that the 3'-FAM labels increased the activity of Exo I, producing a low background due to a more thorough digestion of unbound aptamers. The proposed multimodal aptasensor successfully detected the OTA in actual samples. This work first provides a novel strategy for the development of aptasensors with Exo I and 3'-FAM labels, broadening the application of aptamer in the multimode detection of small molecules.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Ocratoxinas , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Exodesoxirribonucleasas , Límite de Detección , Fenómenos Magnéticos , Microesferas , Ocratoxinas/análisis , Estreptavidina/químicaRESUMEN
Bacterial non-enzymatic Mn(II) oxidation involving reactive oxygen species (ROS) (i.e., indirect oxidation), initially discovered from a marine alpha-proteobacterium, is believed to be of importance in controlling biogeochemical cycles. For soil-borne bacteria, however, evidence of indirect Mn(II) oxidation remains unclear. In this study, the indirect Mn(II) oxidation was evidenced in a soil-borne bacterium, Providencia sp. LLDRA6. First, with and without 50 mM of Mn(II) exposure for LLDRA6, 300 differentially expressed genes were found to be linked to Mn(II) exposure via transcriptome sequencing. Among them, an operon, responsible for phenylacetic acid catabolism, was sharply upregulated in transcription, drawing us a special attention, since its transcriptional upregulation has recently shown to be important for withstanding ROS. Next, a fluorometric probe, 2',7'-Dichlorofluorescin diacetate (DCFDA), was used to qualitatively detect ROS from cells, showing a distinct increase in fluorescence intensities of ROS during Mn(II) exposure. Furthermore, concentrations of superoxide and hydrogen peroxide from cells were detected, respectively, with and without Mn(II) exposure, exhibiting that when Mn(II) oxidation occurred, superoxide concentration significantly increased but hydrogen peroxide concentration significantly decreased. Particularly, superoxide produced by LLDRA6 was proven to be the oxidant for Mn(II) in the formation of Mn oxides. Finally, we predicted links between phenylacetic acid metabolism pathway and ROS during Mn(II) exposure, proposing that the excessive ROS, generated in response to Mn(II) exposure, transcriptionally activate phenylacetic acid catabolism presumably by increasing concentrations of highly reactive oxepins.
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Óxidos , Superóxidos , Bacterias , Peróxido de Hidrógeno , Manganeso , Oxidación-Reducción , Providencia , SueloRESUMEN
BACKGROUND: Epigenetic abnormalities in acute myeloid leukaemia provide us with a target for novel therapeutic strategies. The aim of the study was to verify the epigenetic regulatory mechanism of E-cadherin gene silencing induced by long non-coding RNA MALAT-1 in AML. METHODS: Expression of MALAT-1, E-cadherin, EZH2, SUZ12 and EED genes in AML patients was detected by RT-qPCR. After MALAT-1 silencing in AML cell lines, levels of the E-cadherin, EZH2, SUZ12, EED, DNMT1, DNMT3A and DNMT3B genes and encoded proteins were detected by RT-qPCR and Western blotting. The level of CpG island methylation and trimethylation modification of histone H3K27 in the promoter region of E-cadherin was detected by pyrosequencing and ChIP-qPCR. RIP-qPCR was used to detect the interaction between MALAT-1 and proteins. RESULTS: MALAT-1, EZH2 and EED gene expression was markedly increased in AML patients with E-cadherin down-regulation. A positive correlation between EZH2 or SUZ12 and MALAT-1 expression was observed. After MALAT-1 silencing, the expression of E-cadherin was up-regulated, whereas the expression of EZH2, SUZ12, DNMT1, DNMT3A and DNMT3B was down-regulated. Results of Western blotting were consistent with those of RT-qPCR. Methylation levels of E-cadherin in AML patients were higher than that in normal controls, which appeared to increase with age. Methylation of the CpG island and H3K27 trimethylation of E-cadherin were decreased after MALAT-1 silencing. RIP-qPCR suggested that MALAT-1 might be enriched by EZH2 and SUZ12. CONCLUSION: Our findings verified that MALAT-1 might lead to the transcriptional silencing of E-cadherin gene through the trimethylation of H3K27 mediated by recruiting EZH2 and SUZ12.
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Antígenos CD/metabolismo , Cadherinas/metabolismo , Leucemia Mieloide Aguda , ARN Largo no Codificante/metabolismo , Cadherinas/genética , Epigénesis Genética/genética , Histonas/genética , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , ARN Largo no Codificante/genéticaRESUMEN
Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H2O2-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10-15 to1.0 × 10-10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.
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Técnicas Biosensibles , Sondas de ADN/química , Técnicas Electroquímicas , Reacción en Cadena de la Ligasa , ARN/genética , HumanosRESUMEN
BACKGROUND: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC. RESULTS: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences. CONCLUSIONS: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.
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Reordenamiento Génico/genética , Genoma de Plastidios/genética , Medicago/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Genes de Plantas/genética , Melilotus/genética , Filogenia , Plastidios/genéticaRESUMEN
For periprosthetic joint infection (PJI) patients, an early and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) in joint synovial fluid is of great significance for receiving timely treatment and avoiding side effects. In clinical practice, the methods for detecting MRSA include the culture-based method and the PCR-based mecA gene detection method with fluorescent readout. However, the culture-based method requires up to 3-7 days for incubation and elaborative screening. The PCR-based molecular diagnosis, due to its high sensitivity, improves the detection time but sacrifices cost and gives false-positive results. Herein, a ligation chain reaction (LCR)-based electrochemical biosensor was developed to detect the mecA of MRSA with the advantages of rapidity, accuracy and low cost. In this system, an integrated dsDNA labeled with thiol and biotin at both terminals is generated only in the presence of the target DNA after LCR, followed by immobilization of the integrated dsDNAs on the bovine serum albumin (BSA)-coated gold electrode, and then the streptavidin horseradish peroxidase (SA-HRPs) is specifically bound to the biotin labels via biotin-streptavidin interaction, generating the catalytic amperometric readout. Impressively, the developed method achieved the detection of rare mecA in the joint synovial fluid of PJI patients (417-666 copies as quantified by qPCR). The proposed electrochemistry-based method is highly convenient for the point-of-care testing and was comparable with PCR in sensitivity, but superior in selectivity (single-base differentiation) and cost (nanomolar DNA probe consumption and simple device), demonstrating its huge potential in clinical applications for MRSA diagnosis.
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Técnicas Biosensibles , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Proteínas Bacterianas/genética , ADN , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Proteínas de Unión a las Penicilinas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones Estafilocócicas/diagnóstico , Líquido SinovialRESUMEN
Effective and simultaneous monitoring of the abnormal expression of certain microRNAs (miRNAs), especially for miRNA-21 and miRNA-155, can indicate drug resistance in lung cancer. In this work, T7 exonuclease (T7 Exo)-assisted target recycling amplification coupled with the extensive fluorescence quenching of graphene oxide (GO) was designed for the simultaneous detection of miRNA-21 and miRNA-155 using FAM- and ROX-labeled single-strand DNA probes. Through this method, the variable emission intensities of FAM and ROX caused by the introduction of miRNA-21 and miRNA-155, respectively, were obtained with high sensitivity. The method exhibited excellent analytical performance for simultaneous detection of miRNA-21 and miRNA-155 without cross-interference. The linear range was from 0.005 nM to 5 nM over three orders of magnitude, with detection limits as low as 3.2 pM and 4.5 pM for miRNA-21 and miRNA-155, respectively. Furthermore, the recovery (92.49-103.67%) and relative standard deviation (RSD < 4.8%) of the standard addition test of miRNA-21 and miRNA-155 in human plasma suggested the potential for drug resistance warning in clinical practice via this simple strategy. A homogeneous T7 Exo-assisted signal amplification combined with GO quenching platform was developed for accurate, sensitive and simultaneous analysis of miRNA-21 and miRNA-155 for drug resistance warning in lung cancer. This simple method exhibited a wide linear range and low LODs for miR-21 and miR-155.
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Técnicas Biosensibles , Exodesoxirribonucleasas/metabolismo , Neoplasias Pulmonares/sangre , MicroARNs/análisis , Sondas de ADN/química , Polarización de Fluorescencia , Grafito/química , Humanos , Límite de Detección , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Plasma/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia/métodosRESUMEN
In medical diagnostic studies, the Youden index is a summary measure widely used in the evaluation of the diagnostic accuracy of a medical test. When covariates are not considered, the diagnostic accuracy of the test can be biased or misleading. By incorporating information from covariates using linear regression models, we propose generalized confidence intervals for the covariate-adjusted Youden index and its optimal cut-off point. Furthermore, under heteroscedastic regression models, we propose various confidence intervals for the covariate-adjusted Youden index and its optimal cut-off point. Extensive simulation studies are conducted to evaluate the finite sample performance of various confidence intervals for the Youden index and its optimal cut-off point in the presence of covariates. To illustrate the application of our recommended methods, we apply the methods to a dataset on postprandial blood glucose measurements.
Asunto(s)
Pruebas Diagnósticas de Rutina , Biomarcadores , Simulación por Computador , Intervalos de Confianza , Humanos , Curva ROCRESUMEN
An innovative, rapid and stable method for simultaneous determination of three tetracycline (oxytetracycline, tetracycline and doxycycline) and two fluoroquinolone (ciprofloxacin and enrofloxacin) residues in poultry eggs by ultra-high performance liquid chromatography-fluorescence detection (UPLC-FLD) was established and optimized. The samples were homogenized and extracted with acetonitrile/ultrapure water (90:10, v/v) and then purified by solid-phase extraction (SPE). LC separation was achieved on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm), and the mobile phase was composed of acetonitrile and a 0.1 mol/L malonic acid solution containing 50 mmol/L magnesium chloride (the pH was adjusted to 5.5 with ammonia). When the five target drugs were spiked at the limit of quantification, 0.5 times the maximum residue limit (MRL), 1.0 MRL and 2.0 MRL, the recoveries were above 83.5% and the precision ranged from 1.99% to 6.24%. These figures of merit complied with the parameter validation regulations of the EU and U.S. FDA. The limits of detection and quantifications of the targets were 0.1-13.4 µg/kg and 0.3-40.1 µg/kg, respectively. The proposed method was easily extended to quantitative analyses of target drug residues in 85 egg samples, thus demonstrating its reliability and applicability.