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1.
Proc Natl Acad Sci U S A ; 120(47): e2311565120, 2023 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-37956285

RESUMO

Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aß), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aß, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aß, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.


Assuntos
Doença de Alzheimer , Aptâmeros de Nucleotídeos , Grafite , Doença de Parkinson , Humanos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Doença de Parkinson/diagnóstico , Biomarcadores , Proteínas tau
2.
Cereb Cortex ; 34(5)2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38771241

RESUMO

The functional brain connectome is highly dynamic over time. However, how brain connectome dynamics evolves during the third trimester of pregnancy and is associated with later cognitive growth remains unknown. Here, we use resting-state functional Magnetic Resonance Imaging (MRI) data from 39 newborns aged 32 to 42 postmenstrual weeks to investigate the maturation process of connectome dynamics and its role in predicting neurocognitive outcomes at 2 years of age. Neonatal brain dynamics is assessed using a multilayer network model. Network dynamics decreases globally but increases in both modularity and diversity with development. Regionally, module switching decreases with development primarily in the lateral precentral gyrus, medial temporal lobe, and subcortical areas, with a higher growth rate in primary regions than in association regions. Support vector regression reveals that neonatal connectome dynamics is predictive of individual cognitive and language abilities at 2  years of age. Our findings highlight network-level neural substrates underlying early cognitive development.


Assuntos
Encéfalo , Cognição , Conectoma , Imageamento por Ressonância Magnética , Humanos , Conectoma/métodos , Feminino , Masculino , Imageamento por Ressonância Magnética/métodos , Cognição/fisiologia , Recém-Nascido , Encéfalo/crescimento & desenvolvimento , Encéfalo/diagnóstico por imagem , Encéfalo/fisiologia , Pré-Escolar , Desenvolvimento da Linguagem , Desenvolvimento Infantil/fisiologia
3.
Anal Chem ; 96(21): 8641-8647, 2024 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-38716697

RESUMO

Pathogenic bacterial infections, even at extremely low concentrations, pose significant threats to human health. However, the challenge persists in achieving high-sensitivity bacterial detection, particularly in complex samples. Herein, we present a novel sandwich-type electrochemical sensor utilizing bacteria-imprinted polymer (BIP) coupled with vancomycin-conjugated MnO2 nanozyme (Van@BSA-MnO2) for the ultrasensitive detection of pathogenic bacteria, exemplified by Staphylococcus aureus (S. aureus). The BIP, in situ prepared on the electrode surface, acts as a highly specific capture probe by replicating the surface features of S. aureus. Vancomycin (Van), known for its affinity to bacterial cell walls, is conjugated with a Bovine serum albumin (BSA)-templated MnO2 nanozyme through EDC/NHS chemistry. The resulting Van@BSA-MnO2 complex, serving as a detection probe, provides an efficient catalytic platform for signal amplification. Upon binding with the captured S. aureus, the Van@BSA-MnO2 complex catalyzes a substrate reaction, generating a current signal proportional to the target bacterial concentration. The sensor displays remarkable sensitivity, capable of detecting a single bacterial cell in a phosphate buffer solution. Even in complex milk matrices, it maintains outstanding performance, identifying S. aureus at concentrations as low as 10 CFU mL-1 without requiring intricate sample pretreatment. Moreover, the sensor demonstrates excellent selectivity, particularly in distinguishing target S. aureus from interfering bacteria of the same genus at concentrations 100-fold higher. This innovative method, employing entirely synthetic materials, provides a versatile and low-cost detection platform for Gram-positive bacteria. In comparison to existing nanozyme-based bacterial sensors with biological recognition materials, our assay offers distinct advantages, including enhanced sensitivity, ease of preparation, and cost-effectiveness, thereby holding significant promise for applications in food safety and environmental monitoring.


Assuntos
Compostos de Manganês , Óxidos , Polímeros , Staphylococcus aureus , Vancomicina , Staphylococcus aureus/isolamento & purificação , Compostos de Manganês/química , Óxidos/química , Vancomicina/química , Polímeros/química , Soroalbumina Bovina/química , Técnicas Eletroquímicas/métodos , Análise de Célula Única , Antibacterianos/química , Antibacterianos/farmacologia , Animais , Limite de Detecção , Polímeros Molecularmente Impressos/química , Humanos
4.
Anal Chem ; 2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39136665

RESUMO

Respiratory pathogens pose significant challenges to public health, demanding efficient diagnostic methods. This study presents an integrated microfluidic chip for the simultaneous detection of multiple respiratory pathogens. The chip integrates magnetic bead-based nucleic acid extraction and purification, acoustic streaming-driven mixing, liquid equalization, and multiplex PCR amplification with in situ fluorescence detection. Nucleic acid extraction takes only 12 min, yielding results comparable to commercial kits. Efficient mixing of magnetic beads is achieved through a combination of designed micropillars and bubble-trapping array structures. The micropillars maintain the aqueous phase in the mixing chamber, while the bubble-trapping arrays enable stable formation of bubbles, serving as a micromixer under the acoustic field. To prevent cross-contamination, an oil-encapsulated water droplet system is incorporated throughout nucleic acid extraction and PCR amplification. This assay displays remarkable multiplex analysis capability on a single chip, enabling the simultaneous detection of 12 common respiratory pathogens with a low detection limit of 10 copies/µL. Moreover, this method demonstrates excellent practical applicability in clinical nasal samples. Compared to many microfluidic chip-based molecular biology methods, the assay exhibits comparable or superior multipathogen analysis capability, sensitivity, and speed, completing the sample-to-answer process in approximately 70 min. This integrated microfluidic device offers a promising multiplex molecular diagnosis platform for on-site simultaneous detection of multiple pathogens.

5.
BMC Plant Biol ; 24(1): 964, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39402446

RESUMO

The southern root-knot nematode, Meloidogyne incognita, is a highly serious plant parasitic nematode species that causes significant economic losses in various crops, including cucumber (Cucumis sativus L.). Currently, there are no commercial cultivars available with resistance to M. incognita in cucumber. However, the African horned melon (Cucumis metuliferus Naud.), a semi-wild relative of cucumber, has shown high resistance to M. incognita. In this study, we constructed an ultrahigh-density genetic linkage bin-map using low-coverage sequences from an F2 population generated through the cross between C. metuliferus inbred lines CM3 and CM27. Finally, we identified a QTL (quantitative trait locus, QTL3.1) with a LOD (logarithm of the odds) score of 3.84, explaining 8.4% of the resistance variation. Subsequently, by combining the results of qPCR (quantitative PCR) and VIGS (virus-induced gene silencing), we identified two genes, EVM0025394 and EVM0006042, that are potentially involved in the resistance to M. incognita in CM3. The identification of QTLs and candidate genes in this study serve as a basis for further functional analysis and lay the groundwork for harnessing this resistance trait.


Assuntos
Resistência à Doença , Doenças das Plantas , Locos de Características Quantitativas , Tylenchoidea , Tylenchoidea/fisiologia , Animais , Doenças das Plantas/parasitologia , Doenças das Plantas/genética , Resistência à Doença/genética , Cucumis/genética , Cucumis/parasitologia , Genes de Plantas , Mapeamento Cromossômico , Ligação Genética
6.
Immunol Invest ; 53(7): 1102-1112, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39206848

RESUMO

INTRODUCTION: Brucellosis is an important zoonosis worldwide, affecting humans and animals. There are no specific medicines available to treat brucellosis. Astragalus polysaccharide (APS) is derived from Astragalus membranaceus and exhibits impressive bioactivity, including anti-aging, anti-tumor, and immunomodulatory functions. METHODS: Mice were intraperitoneally inoculated with Brucella melitensis M5 and then treated with APS intraperitoneally injection daily for 7 d. RESULTS: Compared to the M5-infected group, the lower bacteria loads in the APS-treated groups were proved, especially at the acute stage of infection. APS treatment relieved splenomegaly, excess expressions of several pro-inflammatory cytokines (including CXCL1, IFN-γ, IL-1ß, IL-2, IL-12p70, and TNF-α). The raised level of IL-4 was observed in APS-treated mice. APS contributed to raising the ratio of M1 macrophage and reducing the ratio of M2 macrophage in the blood. DISCUSSION: The present study provides some evidence on the potential application of APS in controlling and treating brucellosis and should be further explored.


Assuntos
Brucella melitensis , Brucelose , Citocinas , Macrófagos , Camundongos Endogâmicos BALB C , Polissacarídeos , Animais , Brucelose/imunologia , Brucelose/tratamento farmacológico , Brucelose/prevenção & controle , Polissacarídeos/farmacologia , Brucella melitensis/imunologia , Camundongos , Citocinas/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Astrágalo/química , Modelos Animais de Doenças , Feminino , Humanos
7.
Int J Mol Sci ; 25(8)2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38673861

RESUMO

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.


Assuntos
Arabidopsis , Betaína , Peptídeo Sintases , Tylenchoidea , Betaína/metabolismo , Animais , Tylenchoidea/metabolismo , Tylenchoidea/genética , Arabidopsis/parasitologia , Arabidopsis/metabolismo , Arabidopsis/genética , Peptídeo Sintases/metabolismo , Peptídeo Sintases/genética , Interações Hospedeiro-Parasita , Doenças das Plantas/parasitologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Nematoides/metabolismo , Nematoides/genética
8.
Plant Biotechnol J ; 21(7): 1496-1509, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37074757

RESUMO

Root-knot nematodes (RKNs) are infamous plant pathogens in tomato production, causing considerable losses in agriculture worldwide. Mi-1 is the only commercially available RKN-resistance gene; however, the resistance is inactivated when the soil temperature is over 28 °C. Mi-9 in wild tomato (Solanum arcanum LA2157) has stable resistance to RKNs under high temperature but has not been cloned and applied. In this study, a chromosome-scale genome assembly of S. arcanum LA2157 was constructed through Nanopore and Hi-C sequencing. Based on molecular markers of Mi-9 and comparative genomic analysis, the localization region and candidate Mi-9 genes cluster consisting of seven nucleotide-binding sites and leucine-rich repeat (NBS-LRR) genes were located. Transcriptional expression profiles confirmed that five of the seven candidate genes were expressed in root tissue. Moreover, virus-induced gene silencing of the Sarc_034200 gene resulted in increased susceptibility of S. arcanum LA2157 to Meloidogyne incognita, and genetic transformation of the Sarc_034200 gene in susceptible Solanum pimpinellifolium conferred significant resistance to M. incognita at 25 °C and 30 °C and showed hypersensitive responses at nematode infection sites. This suggested that Sarc_034200 is the Mi-9 gene. In summary, we cloned, confirmed and applied the heat-stable RKN-resistance gene Mi-9, which is of great significance to tomato breeding for nematode resistance.


Assuntos
Solanum lycopersicum , Solanum , Tylenchoidea , Animais , Solanum/genética , Temperatura Alta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Melhoramento Vegetal , Solanum lycopersicum/genética , Cromossomos/metabolismo , Raízes de Plantas/genética , Doenças das Plantas/genética
9.
New Phytol ; 240(6): 2468-2483, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37823217

RESUMO

Meloidogyne enterolobii is an emerging root-knot nematode species that overcomes most of the nematode resistance genes in crops. Nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we show the MeMSP1 effector is secreted into giant cells and promotes M. enterolobii parasitism. Using co-immunoprecipitation and bimolecular fluorescent complementation assays, we identified glutathione-S-transferase phi GSTFs as host targets of the MeMSP1 effector. This protein family plays important roles in plant responses to abiotic and biotic stresses. We demonstrate that MeMSP1 interacts with all Arabidopsis GSTF. Moreover, we confirmed that the N-terminal region of AtGSTF9 is critical for its interaction, and atgstf9 mutant lines are more susceptible to root-knot nematode infection. Combined transcriptome and metabolome analyses showed that MeMSP1 affects the metabolic pathways of Arabidopsis thaliana, resulting in the accumulation of amino acids, nucleic acids, and their metabolites, and organic acids and the downregulation of flavonoids. Our study has shed light on a novel effector mechanism that targets plant metabolism, reducing the production of plant defence-related compounds while favouring the accumulation of metabolites beneficial to the nematode, and thereby promoting parasitism.


Assuntos
Arabidopsis , Tylenchoidea , Animais , Arabidopsis/genética , Interações Hospedeiro-Parasita , Tylenchoidea/fisiologia , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Doenças das Plantas/genética
10.
Analyst ; 148(14): 3239-3246, 2023 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-37341575

RESUMO

Microfluidic impedance cytometry is emerging as a label-free, low-cost and portable solution for cell analysis. Impedance-based cell or particle characterization is provided by microfluidic and electronic devices. We report the design and characterization of a miniaturized flow cytometer based on a 3-dimensional (3D) hydrodynamic focusing mechanism. A sheath adaptively concentrated the sample laterally and vertically at the bottom of the microchannel, reducing the variance of particle translocation height and increasing the signal-to-noise ratio of the particle impedance pulse. Through simulation and confocal microscopy experiments, it has been verified that an increase in the ratio of sheath to sample decreased the cross-sectional area of the concentrated stream, which can be reduced to 26.50% of the pre-focusing value. The appropriate sheath flow settings increased the impedance pulse amplitude for different particles, and the coefficient of variation reduced by at least 35.85%, contributing to a more accurate representation of the particle impedance characteristic distribution. The system displayed the difference of HepG2 cell impedance before and after drug treatment, which is consistent with the results of flow cytometry, providing a convenient and inexpensive solution for monitoring cell status.


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Técnicas Analíticas Microfluídicas/métodos , Citometria de Fluxo/métodos , Impedância Elétrica , Hidrodinâmica
11.
Analyst ; 148(12): 2758-2766, 2023 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-37194305

RESUMO

This paper introduces an enclosed microfluidic chip that integrates sample preparation and the chamber-based digital polymerase chain reaction (cdPCR). The sample preparation of the chip includes nucleic acid extraction and purification based on magnetic beads, which adsorb nucleic acids by moving around the reaction chambers to complete the reactions including lysis, washing, and elution. The cdPCR area of the chip consists of tens of thousands of regularly arranged microchambers. After the sample preparation processes are completed, the purified nucleic acid can be directly introduced into the microchambers for amplification and detection on the chip. The nucleic acid extraction performance and digital quantification performance of the system were examined using synthetic SARS-CoV-2 plasmid templates at concentrations ranging from 101-105 copies per µL. Further on, a simulated clinical sample was used to test the system, and the integrated chip was able to accurately detect SARS-CoV-2 virus particle samples doped with interference (saliva) with a detection limit of 10 copies per µL. This integrated system could provide a promising tool for point-of-care testing of pathogenic infections.


Assuntos
Microfluídica , Microfluídica/métodos , Reação em Cadeia da Polimerase , Ácidos Nucleicos/análise , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação
12.
Analyst ; 148(9): 1939-1947, 2023 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-36916483

RESUMO

Diagnosis of cancer by biomarkers plays an important role in human health and life. However, current laboratory techniques for detecting cancer biomarkers still require laborious and time-consuming operation by skilled operators and associated laboratory instruments. This work presents a colorimetric biosensor for the rapid and sensitive detection of carcinoembryonic antigen (CEA) based on an automated immunomagnetic separation platform and a droplet array microfluidic chip with the aid of an image analysis system. Immunomagnetic nanoparticles (MNPs) were used to capture CEA in the samples. CEA-detecting antibodies and horseradish peroxidase (HRP) were modified on polystyrene microspheres (PS), catalysing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) as signal outputs. Color reaction data were analyzed to establish a CEA concentration standard curve. The movement of MNPs between droplets in the microfluidic chip is achieved using an automatically programmable magnetic control system. This colorimetric biosensor has been used for the simultaneous detection of six CEA samples ranging from 100 pg mL-1 to 100 ng mL-1 with a detection limit of 14.347 pg mL-1 in 10 min, following the linear equation: y = -4.773 ln(x) + 156.26 with a correlation of R2 = 0.9924, and the entire workflow can be completed within 80 minutes. The microfluidic immunosensor designed in this paper has the advantages of low cost, automation, low sample consumption, high throughput, and promising applications in biochemistry.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Humanos , Antígeno Carcinoembrionário/análise , Separação Imunomagnética/métodos , Microfluídica , Imunoensaio/métodos , Técnicas Biossensoriais/métodos , Anticorpos Monoclonais , Limite de Detecção , Ouro
13.
J Appl Microbiol ; 134(2)2023 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-36611228

RESUMO

AIMS: Root-knot nematodes (RKNs) are plant pathogens that cause huge economic losses worldwide. The biological management of RKNs may be a sustainable alternative to chemical control methods. Here, the biocontrol potential of Methylorubrum rhodesianum M520 against the RKN Meloidogyne incognita was investigated to theoretically support its application as a biocontrol agent in field production. METHODS AND RESULTS: In-vitro assays showed 91.9% mortality of M. incognita second-stage juveniles in the presence of strain M520 and that the hatching rate of M. incognita eggs was 21.7% lower than that of eggs treated with sterile water. In pot experiments, the M520 treatment caused 70.8% reduction in root-knots and increased plant shoot length and stem and root fresh weights, compared to control plant values. In split-root experiments, cucumber roots treated with M520 showed 25.6% decrease in root gall number, compared to that in control roots. CONCLUSION: M520 has multiple mechanisms against RKNs and might be used as a biocontrol agent against M. incognita in cucumber, laying a foundation for further studying M520 biocontrol against RKNs.


Assuntos
Cucumis sativus , Methylobacteriaceae , Tylenchida , Tylenchoidea , Animais , Raízes de Plantas
14.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674475

RESUMO

Fusarium oxysporum f. sp. phaseoli, the causal agent of cowpea fusarium wilt, is a serious threat to cowpea production in China. In this study, a sample of cowpea fusarium wilt was identified as Fusarium oxysporum f. sp. phaseoli using the methods of morphological characters and molecular detection. We further reported the first genome assembly for Fusarium oxysporum f. sp. phaseoli, with 53.7 Mb genome sequence comprising 14,694 genes. Comparative genomic analysis among five Fusarium oxysporum genomes showed that four accessory chromosomes in the five Fusarium oxysporum display similar characteristics, with low sequence similarity (55.35%, vs. overall average of 81.76%), low gene density (2.18 genes/10 kb vs. 3.02 genes/Mb) and highly transposable element density (TEs) (15.01/100 kb vs. 4.89/100 kb), indicating that variable accessory chromosomes are the main source of Fusarium oxysporum evolution. We identified a total of 100 Fusarium oxysporum f. sp. phaseoli-specific effectors in the genome and found 13 specific effector genes located in large insertion or deletion regions, suggesting that insertion or deletion events can cause the emergence of species-specific effectors in Fusarium oxysporum. Our genome assembly of Fusarium oxysporum f. sp. phaseoli provides a valuable resource for the study of cowpea fusarium wilt, and the comparative genomic study of Fusarium oxysporum could contribute to the knowledge of genome and effector-associated pathogenicity evolution in Fusarium oxysporum study.


Assuntos
Fusarium , Fusarium/genética , Doenças das Plantas , Genoma Fúngico
15.
Plant J ; 107(1): 136-148, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33866620

RESUMO

Cucumis metuliferus (African horned cucumber), a wild relative of Cucumis sativus (cucumber) and Cucumis melo (melon), displays high-level resistance to several important plant pathogens (e.g., root-knot nematodes and several viruses). Here, we report a chromosome-level genome assembly for C. metuliferus, with a 316 Mb genome sequence comprising 29 039 genes. Phylogenetic analysis of related species in family Cucurbitaceae indicated that the divergence time between C. metuliferus and melon was 17.8 million years ago. Comparisons between the C. metuliferus and melon genomes revealed large structural variations (inversions and translocations >1 Mb) in eight chromosomes of these two species. Gene family comparison showed that C. metuliferus has the largest number of resistance-related nucleotide-binding site leucine-rich repeat (NBS-LRR) genes in Cucurbitaceae. The loss of NBS-LRR loci caused by large insertions or deletions (indels) and pseudogenization caused by small indels explained the loss of NBS-LRR genes in Cucurbitaceae. Population structure analysis suggested that C. metuliferus originated in Zimbabwe, then spread to other southern African regions where it likely underwent similar domestic selection as melon. This C. metuliferus reference sequence will accelerate the understanding of the molecular evolution of resistance-related genes and enhance cucurbit crop improvement efforts.


Assuntos
Cucumis/genética , Genes de Plantas , Genoma de Planta , Filogenia , África , Cromossomos de Plantas , Cucumis melo/genética , Evolução Molecular , Variação Genética , Genética Populacional , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Seleção Genética , Zimbábue
16.
Anal Biochem ; 656: 114877, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36055398

RESUMO

The lack of reliable and practical method for detecting rare hot mutation of epidermal growth factor receptor (EGFR) in circulating tumor DNA (ctDNA) for lung cancer has remained a challenge for general clinical application due to excess wild type DNA in clinical samples. In this study, we developed a droplet digital PCR (ddPCR) platform, integrating a PDMS chip and double-layer glass reservoir. The duplex T-junction droplet generators in PDMS chip can produce about one million uniform droplets of 4.187 pL within ∼10 min, which were then stored in the glass reservoir. The double-layer glass reservoir can protect droplets from evaporation and breaking, solving the problem of instability during thermal-cycling. The quantitative capabilities of the ddPCR chip were evaluated by testing EGFR exon gene 21, with a good linear correlation in the wide range of 101 to 106 copies/µL (R2 = 0.9998). We then demonstrated that the proposed ddPCR device can recognize rare EGFR L858R mutation under a background of 106 copies/µL wild-type DNA at a sensitivity of 0.0001%. Finally, we demonstrated this ddPCR platform could identify low amount of EGFR L858R mutation in ctDNA and CTCs of patients with lung cancer.


Assuntos
DNA Tumoral Circulante , Neoplasias Pulmonares , DNA Tumoral Circulante/genética , Receptores ErbB/genética , Genes erbB-1 , Humanos , Neoplasias Pulmonares/diagnóstico , Mutação , Reação em Cadeia da Polimerase/métodos
17.
Analyst ; 147(8): 1722-1729, 2022 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-35343521

RESUMO

Quantification of tumor cell heterogeneity is critical for clinical diagnostic and therapeutic applications, including evaluation of the cancerous stage of tumors. In this work, we presented a novel method to effectively distinguish the grade of bladder cancer at a single-cell level in both cell line and clinical cell samples. This was achieved by taking advantage of microdroplets and microelectrodes, which can encapsulate and then trap single cells for measuring their impedance in a label-free and non-invasive manner. These findings suggested that this impedance analysis device based on droplet microfluidics is promising in the fields of clinical and point-of-care diagnostics.


Assuntos
Técnicas Analíticas Microfluídicas , Neoplasias da Bexiga Urinária , Linhagem Celular , Impedância Elétrica , Humanos , Microeletrodos , Microfluídica/métodos , Neoplasias da Bexiga Urinária/diagnóstico
18.
New Phytol ; 232(5): 2124-2137, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34449897

RESUMO

Root-knot nematodes, Meloidogyne spp., secrete effectors to modulate plant immune responses and establish a parasitic relationship with host plants. However, the functions and plant targets of C-type lectin (CTL)-like effectors of Meloidogyne incognita remain unknown. Here, we characterized a CTL-like effector of M. incognita, MiCTL1a, and identified its target and role in nematode parasitism. In situ hybridization demonstrated the expression of MiCTL1 in the subventral glands; and in planta, immunolocalization showed its secretion during M. incognita parasitism. Virus-induced gene silencing of the MiCTL1 reduced the infection ability of M. incognita in Nicotiana benthamiana. The ectopic expression in Arabidopsis not only increased susceptibility to M. incognita but also promoted root growth. Yeast two-hybrid and co-immunoprecipitation assays revealed that MiCTL1a interacts with Arabidopsis catalases, which play essential roles in hydrogen peroxide homeostasis. Knockout or overexpression of catalases showed either increased or reduced susceptibility to M. incognita, respectively. Moreover, MiCTL1a not only reduced catalase activity in vitro and in planta but also modulated stress-related gene expressions in Arabidopsis. Our data suggest that MiCTL1a interacts with plant catalases and interferes with catalase activity, allowing M. incognita to establish a parasitic relationship with its host by fine-tuning responses mediated by reactive oxygen species.


Assuntos
Tylenchoidea , Animais , Catalase , Proteínas de Helminto , Lectinas Tipo C , Doenças das Plantas
19.
Analyst ; 146(17): 5380-5388, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34338259

RESUMO

A microfluidic chip has been integrated with a capacitive biosensor based on mass-producible three-dimensional (3D) interdigital electrode arrays. To achieve the monitoring of biosensor preparation and cardiac- and periodontitis-related biomarkers, all the processes were detected in a continuously on-site way. Fabrication steps for the microfluidic chip-bonded 3D interdigital capacitor biosensor include gold thiol modification, the activation of EDC/sulfo-NHS, and the bioconjugation of antibodies. Fluorescent characterization and X-ray photoelectron spectroscopy analysis were applied to assess the successful immobilization of the C-reactive protein (CRP) antibody. The experimental results indicate the good specificity and high sensitivity of the microfluidic integrated 3D capacitive biosensor. The limit of detection of the 3D capacitive biosensor for CRP label-free detection was about 1 pg mL-1. This 3D capacitive biosensor with integrated microfluidics is mass-producible and has achieved the on-site continuous detection of cardiac- and periodontitis-related biomarkers with high performance.


Assuntos
Técnicas Biossensoriais , Microfluídica , Proteína C-Reativa , Eletrodos , Ouro
20.
Plant Dis ; 105(8): 2061-2070, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33599517

RESUMO

Root-knot nematodes (Meloidogyne spp.) are soilborne pathogens that infect vegetable crops and cause major economic losses worldwide annually. Therefore, there is an urgent need for novel nematicides or biological control agents to reduce the damage caused by root-knot nematodes. In this study, we tested efficacy of the Bacillus cereus strain Bc-cm103, isolated from the rhizoplane of Cucumis metuliferus, against Meloidogyne incognita. Strain Bc-cm103 fermentation broth caused 100% mortality of the nematode second-stage juveniles within 12 h and decreased the egg hatching rate by 40.06% within 72 h compared with sterile water. Confocal laser-scanning microscopy revealed that strain Bc-cm103 formed a biofilm on cucumber (C. sativus) roots, which protected the roots from the infection of M. incognita. Additionally, strain Bc-cm103 activated the defense-responsive genes PR1, PR2, LOX1, and CTR1 in cucumber. Furthermore, strain Bc-cm103 significantly reduced the appearance of root galls in pot, split-root, and field tests. These results indicated that B. cereus strain Bc-cm103 had a strong suppressive effect on M. incognita and therefore could be used as a potential biocontrol agent against this pathogen.


Assuntos
Solanum lycopersicum , Tylenchoidea , Animais , Antinematódeos , Bacillus cereus , Agentes de Controle Biológico
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