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Pangenome inference is an indispensable step in bacterial genomics, yet its scalability poses a challenge due to the rapid growth of genomic collections. This paper presents PanTA, a software package designed for constructing pangenomes of large bacterial datasets, showing unprecedented efficiency levels multiple times higher than existing tools. PanTA introduces a novel mechanism to construct the pangenome progressively without rebuilding the accumulated collection from scratch. The progressive mode is shown to consume orders of magnitude less computational resources than existing solutions in managing growing datasets. The software is open source and is publicly available at https://github.com/amromics/panta and at 10.6084/m9.figshare.23724705 .
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Genoma Bacteriano , Software , Genômica/métodos , Bactérias/genética , FilogeniaRESUMO
Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications.
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Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Células HEK293 , Proteômica/métodos , Fluxo de Trabalho , Ultracentrifugação/métodos , MicroRNAs/metabolismoRESUMO
Background: Biomarker testing has gradually become standard of care in precision oncology to help physicians select optimal treatment for patients. Compared to single-gene or small gene panel testing, comprehensive genomic profiling (CGP) has emerged as a more time- and tissue-efficient method. This study demonstrated in-depth analytical validation of K-4CARE, a CGP assay that integrates circulating tumor DNA (ctDNA) tracking for residual cancer surveillance. Methods: The assay utilized a panel of 473 cancer-relevant genes with a total length of 1.7 Mb. Reference standards were used to evaluate limit of detection (LOD), concordance, sensitivity, specificity and precision of the assay to detect single nucleotide variants (SNVs), small insertion/deletions (Indels), gene amplification and fusion, microsatellite instability (MSI) and tumor mutational burden (TMB). The assay was then benchmarked against orthogonal methods using 155 clinical samples from 10 cancer types. In selected cancers, top tumor-derived somatic mutations, as ranked by our proprietary algorithm, were used to detect ctDNA in the plasma. Results: For detection of somatic SNVs and Indels, gene fusion and amplification, the assay had sensitivity of >99%, 94% and >99% respectively, and specificity of >99%. Detection of germline variants also achieved sensitivity and specificity of >99%. For TMB measurement, the correlation coefficient between whole-exome sequencing and our targeted panel was 97%. MSI analysis when benchmarked against polymerase chain reaction method showed sensitivity of 94% and specificity of >99%. The concordance between our assay and the TruSight Oncology 500 assay for detection of somatic variants, TMB and MSI measurement was 100%, 89%, and 98% respectively. When CGP-informed mutations were used to personalize ctDNA tracking, the detection rate of ctDNA in liquid biopsy was 79%, and clinical utility in cancer surveillance was demonstrated in 2 case studies. Conclusion: K-4CARE™ assay provides comprehensive and reliable genomic information that fulfills all guideline-based biomarker testing for both targeted therapy and immunotherapy. Integration of ctDNA tracking helps clinicians to further monitor treatment response and ultimately provide well-rounded care to cancer patients.
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BACKGROUND: Small extracellular vesicles from adipose-derived stem cells (ASC-sEVs) have gained remarkable attention for their regenerative and protective properties against skin aging. However, the use of ASC-sEVs to further encapsulate certain natural anti-aging compounds for synergistic effects has not been actively explored. For large-scale production in skincare industry, it is also crucial to standardize cost-effective methods to produce highly pure ASC-sEVs. METHODS: Human ASCs were expanded in serum-free media with different compositions to first optimize the sEV production. ASC-sEVs from different batches were then purified using tangential flow filtration and sucrose cushion ultracentrifugation, followed by extensive characterization for identity and content profiling including proteomics, lipidomics and miRNA sequencing. ASC-sEVs were further loaded with nicotinamide riboside (NR) and resveratrol by sonication-incubation method. The therapeutic effect of ASC-sEVs and loaded ASC-sEVs was tested on human keratinocyte cell line HaCaT exposed to UVB by measuring reactive oxygen species (ROS). The loaded ASC-sEVs were later applied on the hand skin of three volunteers once a day for 8 weeks and skin analysis was performed every 2 weeks. RESULTS: Our standardized workflow produced ASC-sEVs with high yield, high purity and with stable characteristics and consistent biocargo among different batches. The most abundant subpopulations in ASC-sEVs were CD63+ (â¼30%) and CD81+ -CD63+ (â¼35%). Purified ASC-sEVs could be loaded with NR and resveratrol at the optimized loading efficiency of â¼20%. In UVB-exposed HaCaT cells, loaded ASC-sEVs could reduce ROS by 38.3%, higher than the sEVs (13.3%) or compounds (18.5%) individually. In human trial, application of loaded ASC-sEVs after 8 weeks substantially improved skin texture, increased skin hydration and elasticity by 104% and reduced mean pore volume by 51%. CONCLUSIONS: This study demonstrated a robust protocol to produce ASC-sEVs and exogenously load them with natural compounds. The loaded ASC-sEVs exhibited synergistic effects of both sEVs and anti-aging compounds in photoaging protection and skin rejuvenation.
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Envelhecimento da Pele , Humanos , Espécies Reativas de Oxigênio , Rejuvenescimento , Resveratrol , Células-TroncoRESUMO
The aim of this work is to fabricate a sensitive and novel enzymeless electrochemical sensor for the simultaneous determination of parathion and paraoxon using the Nd-UiO-66@MWCNT nanocomposite. For this purpose, Neodymium (Nd) was introduced into a Universitetet i Oslo (UiO-66) structure to construct Nd-UiO-66 and then, adding multi-walled carbon nanotubes to the Nd-UiO-66 to increase the electrocatalytic activity and surface area of the obtained composite. The Nd-UiO-66@MWCNT has numerous advantages like excellent conductivity, tunable texture, and large surface area and can be used as a distinctive structure for the construction of modified glassy carbon electrode (GCE) to enhance the charge-transfer and the efficiency of electrochemical sensors. This modified electrode showed sensitive and selective determination of paraoxon and parathion over the linear ranges of 0.7-100 and 1-120 nM, with detection limits of 0.04 and 0.07 nM, respectively. The proposed Nd-UiO-66@MWCNT/GCE sensor in this study can be applied in environmental and toxicological laboratories and field tests to detect parathion and paraoxon levels.
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Estruturas Metalorgânicas , Nanotubos de Carbono , Paration , Técnicas Eletroquímicas , Eletrodos , Neodímio , Paraoxon , Ácidos FtálicosRESUMO
Microsphere biolasers have attracted a great deal of interest due to their potential for biosensing and cell tracking. Here we demonstrate a novel, to the best of our knowledge, microfluidic-based fabrication of nearly monodisperse dye-doped protein microsphere biolasers with a tunable size from 150 to 50 µm. In particular, for an 85 µm-bead, about 70% of the fabricated microspheres have the same size of 85 µm. Under optical pumping, the fabricated microspheres emit whispering gallery mode lasing emission with a lasing threshold of ${{7}}\;\unicode{x00B5} {\rm{J}}\;{{\rm{mm}}^{- 2}}$ and quality ($\!Q$) factor up to 3000. Interestingly, microspheres with the same size exhibit a similar lasing threshold and spectrum. The result indicates a high reproducibility of microsphere biolasers by the microfluidic-based fabrication technique. This Letter provides an effective method for mass production of high-$Q$ factor microsphere biolasers which is a significant step toward real biosensing and medical applications.
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Técnicas Biossensoriais/instrumentação , Lasers , Medições Luminescentes/instrumentação , MicroesferasRESUMO
Alport Syndrome (AS) is a genetic disorder characterized by impaired kidney function. The development of a noninvasive tool for early diagnosis and monitoring of renal function during disease progression is of clinical importance. Hyperpolarized 13C MRI is an emerging technique that enables non-invasive, real-time measurement of in vivo metabolism. This study aimed to investigate the feasibility of using this technique for assessing changes in renal metabolism in the mouse model of AS. Mice with AS demonstrated a significant reduction in the level of lactate from 4- to 7-week-old, while the levels of lactate were unchanged in the control mice over time. This reduction in lactate production in the AS group accompanied a significant increase of PEPCK expression levels, indicating that the disease progression in AS triggered the gluconeogenic pathway and might have resulted in a decreased lactate pool size and a subsequent reduction in pyruvate-to-lactate conversion. Additional metabolic imaging parameters, including the level of lactate and pyruvate, were found to be different between the AS and control groups. These preliminary results suggest that hyperpolarized 13C MRI might provide a potential noninvasive tool for the characterization of disease progression in AS.
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In magnetic resonance imaging (MRI), the scan time for acquiring an image is relatively long, resulting in patient uncomfortable and error artifacts. Fortunately, the compressed sensing (CS) and parallel magnetic resonance imaging (pMRI) can reduce the scan time of the MRI without significantly compromising the quality of the images. It has been found that the combination of pMRI and CS can better improve the image reconstruction, which will accelerate the speed of MRI acquisition because the number of measurements is much smaller than that by pMRI. In this paper, we propose combining a combined CS method and pMRI for better accelerating the MRI acquisition. In the combined CS method, the under-sampled data of the K-space is performed by taking both regular sampling and traditional random under-sampling approaches. MRI image reconstruction is then performed by using nonlinear conjugate gradient optimization. The performance of the proposed method is simulated and evaluated using the reconstruction error measure, the universal image quality Q-index, and the peak signal-to-noise ratio (PSNR). The numerical simulations confirmed that, the average error, the Q index, and the PSNR ratio of the appointed scheme are remarkably improved up to 59, 63, and 39% respectively as compared to the traditional scheme. For the first time, instead of using highly computational approaches, a simple and efficient combination of CS and pMRI is proposed for the better MRI reconstruction. These findings are very meaningful for reducing the imaging time of MRI systems.
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Algoritmos , Processamento de Imagem Assistida por Computador , Aceleração , Humanos , Imageamento por Ressonância Magnética , Razão Sinal-RuídoRESUMO
In this work, single-phase nanostructured NdFe1-xCoxO3 (x = 0, 0.1, 0.2, and 0.3) perovskite materials were obtained by annealing stoichiochemistry mixtures of their component hydroxides at 750 °C for 60 min. The partial substitution of Fe by Co in the NdFeO3 crystal lattice leads to significant changes in the structural characteristics, and as a consequence, also alters both the magnetic and optical properties of the resulting perovskites. The low optical band gap (Eg = 2.06 ÷ 1.46 eV) and high coercivity (Hc = 136.76 ÷ 416.06 Oe) give Co-doped NdFeO3 nanoparticles a huge advantage for application in both photocatalysis and hard magnetic devices.
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A colorimetric paper-based sensor is proposed for the rapid monitoring of six major organophosphate and carbamate pesticides. The assay was constructed by dropping gold and silver nanoparticles on the hydrophilic zones of a paper substrate. The nanoparticles were modified by L-arginine, quercetin, and polyglutamic acid. The mechanism of sensing is based on the interaction between the pesticide and the nanoparticles. The color of nanoparticles changed during the interactions. A digital camera recorded these changes. The assay provided a unique response for each studied pesticide. This method can determine six individual pesticides including carbaryl, paraoxon, parathion, malathion, diazinon, and chlorpyrifos. The limit of detection for these pesticides were 29.0, 22.0, 32.0, 17.0, 45.0, and 36.0 ng mL-1, respectively. The assay was applied to simultaneously determine the six studied pesticides in a mixture using the partial least square method (PLS). The root mean square errors of prediction were 11, 8.7, 9.2, 10, 12, and 11 for carbaryl, paraoxon, parathion, malathion, diazinon, and chlorpyrifos, respectively. The paper-based device can differentiate two types of studied pesticide (organophosphate and carbamate) as well as two types of organophosphate structures (oxon and thion). Furthermore, this sensor showed high selectivity to the pesticides in the presence of other potential species (e.g., metal ions, anions, amino acids, sugar, and vitamins). This assay is capable of determining the pesticide compounds in tap water, apple juice, and rice samples.Graphical abstract.
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Colorimetria/instrumentação , Malus/química , Oryza/química , Praguicidas/química , Carbamatos/química , Colorimetria/métodos , Contaminação de Alimentos , Sucos de Frutas e Vegetais/análise , Ouro , Nanopartículas Metálicas , Organofosfatos/química , Papel , Prata , Água/química , Poluentes Químicos da ÁguaRESUMO
This study introduces an applicable colorimetric sensor array for the detection of pesticides in the vapor phase. The array consisted of six metal nanoparticles spotted on the piece of filter paper. 3D-origami pattern was used for the fabrication of a paper-based sensor to decrease the effect of the nanoparticles leaching after exposure to analytes. Exposure to pesticide aerosols caused changes in the color of the array due to the aggregation of nanoparticles. These changes provided selective responses to thion pesticides such as malathion, parathion, chlorpyrifos, and diazinon. The sensing assay could also differentiate between aliphatic and aromatic thions and discriminate amine-containing compounds from the other studied analytes. These finding results are clearly confirmed by both visual detection and multivariate statistical methods. The proposed sensor was successfully developed for the quantitative measurement of pesticide aerosols at a very low concentration. The limit of detection of this method determined for malathion, parathion, chlorpyrifos and diazinon were 58.0, 103.0, 81.0 and 117.0, respectively. Moreover, the array could be employed to simultaneously analyze four studied pesticides. The statistcal results confirmed that the method has high performance for concurrent detection of thions as a major air pollutant without the interference of other species.
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Compressive sampling (CS) has been commonly employed in the field of magnetic resonance imaging (MRI) to accurately reconstruct sparse and compressive signals. In a MR image, a large amount of encoded information focuses on the origin of the k-space. For the 2D Cartesian K-space MRI, under-sampling the frequency-encoding (kx) dimension does not affect to the acquisition time, thus, only the phase-encoding (ky) dimension can be exploited. In the traditional random under-sampling approach, it acquired Gaussian random measurements along the phaseencoding (ky) in the k-space. In this paper, we proposed a hybrid under-sampling approach; the number of measurements in (ky) is divided into two portions: 70% of the measurements are for random under-sampling and 30% are for definite under-sampling near the origin of the k-space. The numerical simulation consequences pointed out that, in the lower region of the under-sampling ratio r, both the average error and the universal image quality index of the appointed scheme are drastically improved up to 55 and 77% respectively as compared to the traditional scheme. For the first time, instead of using highly computational complexity of many advanced reconstruction techniques, a simple and efficient CS method based simulation is proposed for MRI reconstruction improvement. These findings are very useful for designing new MRI data acquisition approaches for reducing the imaging time of current MRI systems.
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Compressão de Dados , Imageamento por Ressonância Magnética , Algoritmos , Simulação por Computador , Processamento de Imagem Assistida por Computador , Distribuição NormalRESUMO
This work presents an effective strategy for the well-oriented immobilization of antibodies in which boronic acid is directly attached to the surface and with no need of the long and flexible spacer. A magnetic graphene nanoribbon-boronic-acid-based immunosensor was developed and tested for the impedimetric detection of lymphoma cancer cells, a blood cancer biomarker. Magnetic graphene nanoribbons (MGNRs) were modified with boronic acid (BA) to create a supporting matrix that is utilized by immobilizing anti-CD20 antibodies with good orientation. The prepared biosensing layer (MGNR/BA/Ab) with well-oriented antibodies was premixed into whole blood samples to interact with lymphoma cancer cell receptors. In the presence of target cell receptors, an immunocomplex was formed between anti-CD20 antibodies and lymphoma cancer cell receptors. Then, the biosensing layer was magnetically collected on a screen-printed carbon electrode (SPCE) and placed in a homemade electrochemical cell configuration to measure impedimetric signals. The fabrication steps of the immunosensor were characterized by various techniques, such as resonance light scattering, fluorescence, electrochemical impedance spectroscopy, and cyclic voltammetry. The assay is highly sensitive: the calculated limit of detection of lymphoma cancer cells was as low as 38 cells/mL, and the detection was linear from 100 to 1â¯000â¯000 cells/mL. The specificity of the immunosensor is also very high, and there is no interference effect with several potential interferents, such as the breast cancer (MCF-7), human embryonic kidney (HEK293), and leukemia (HL-60 and KCL-22) cell lines. The performance of the immunosensor for lymphoma cancer cells in clinical blood samples is consistent with that of commercial flow cytometric assays.
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Anticorpos Imobilizados/imunologia , Biomarcadores Tumorais/sangue , Separação Celular/métodos , Imunoensaio/métodos , Linfoma/sangue , Nanotubos de Carbono/química , Anticorpos Imobilizados/química , Biomarcadores Tumorais/imunologia , Ácidos Borônicos/química , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Células HEK293 , Humanos , Limite de Detecção , Linfoma/patologia , Fenômenos Magnéticos , Rituximab/química , Rituximab/imunologiaRESUMO
Cell migration has been recognized as one hallmark of malignant tumor progression. By integrating the method of electrical cell-substrate impedance sensing (ECIS) with the Boyden chamber design, the state-of-the-art techniques provide kinetic information about cell migration and invasion processes in three-dimensional (3D) extracellular matrixes. However, the information related to the initial stage of cell migration with single-cell resolution, which plays a unique role in the metastasis-invasion cascade of cancer, is not yet available. In this paper, we present a microfluidic device integrated with ECIS for investigating single cancer cell migration in 3D matrixes. Using microfluidics techniques without the requirement of physical connections to off-chip pneumatics, the proposed sensor chip can efficiently capture single cells on microelectrode arrays for sequential on-chip 2D or 3D cell culture and impedance measurement. An on-chip single-cell migration assay was successfully demonstrated within several minutes. Migration of single metastatic MDA-MB-231 cells in their initial stage can be monitored in real time; it shows a rapid change in impedance magnitude of approximately 10 Ω/s, whereas no prominent impedance change is observed for less-metastasis MCF-7 cells. The proposed sensor chip, allowing for a rapid and selective detection of the migratory properties of cancer cells at the single-cell level, could be applied as a new tool for cancer research.