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Mycoplasma synoviae (MS) is an essential pathogenic mycoplasma in poultry worldwide, posing a serious threat to the poultry industry's health. Timely detection is imperative for early diagnosis, prevention, and control of MS infection. Current laboratory methods for MS detection are generally complicated, time-consuming, and require sophisticated equipment. Therefore, a simple and rapid method is urgently needed. This study developed a novel real-time fluorescence-based recombinase-aided amplification (RF-RAA) technique for detecting MS nucleic acids, enabling target gene amplification within 20 min at 39°C. The RF-RAA outcomes are interpretable in 2 modalities: real-time fluorescence monitoring employing a temperature-controlled fluorescence detector or direct visual inspection facilitated by a portable blue light transilluminator. This method exhibits robust specificity, demonstrating no cross-reactivity with various common poultry pathogens, and achieves high sensitivity, detecting as low as 10 copies/µL for the standard plasmid. Seventy-one clinical samples of chicken throat swabs were detected by RF-RAA and real-time fluorescence quantitative polymerase chain reaction (qPCR) methods. The diagnostic coincidence rates of qPCR with RF-RAA (fluorescence monitoring) and RF-RAA (visual observation) were determined to be 100% and 97.2% (69/71), respectively. In conclusion, the RF-RAA method developed in this study provides a rapid and visually observable approach for MS detection, offering a novel technique to diagnosing MS infection, especially in resource-limited settings.
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Neutral rhodol-based red emitters are shown to efficiently localize in mitochondria, as demonstrated by confocal microscopy and co-localization studies. A simple model is proposed to explain the localization mechanism of neutral molecules. The model takes into account the strong coupling between the molecular dipole moment and the electric field of the inner mitochondrial membrane.
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Colorantes Fluorescentes , Mitocondrias , Mitocondrias/metabolismo , Mitocondrias/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Humanos , Microscopía Confocal , Xantonas/química , Estructura Molecular , Células HeLaRESUMEN
In recent years, cationic polymer vectors have been viewed as a promising method for delivering nucleic acids. With the advancement of synthetic polymer chemistry, we can control chemical structures and properties to enhance the efficacy of gene delivery. Herein, a facile, cost-effective, and scalable method was developed to synthesize PEGylated PDMAEMA polymers (PEO-PDMAEMA-PEO), where PEGylation could enable prolonged polyplexes circulation time in the blood stream. Two polymers of different molecular weights were synthesized, and polymer/eGFP polyplexes were prepared and characterized. The correlation between polymers' molecular weight and physicochemical properties (size and zeta potential) of polyplexes was investigated. Lipofectamine 2000, a commercial non-viral transfection reagent, was used as a standard control. PEO-PDMAEMA-PEO with higher molecular weight exhibited slightly better transfection efficiency than Lipofectamine 2000, and the cytotoxicity study proved that it could function as a safe gene vector. We believe that PEO-PDMAEMA-PEO could serve as a model to investigate more potential in the gene delivery area.
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Técnicas de Transferencia de Gen , Nylons , Polietilenglicoles , Transfección , Polietilenglicoles/química , Humanos , Nylons/química , Nylons/síntesis química , Transfección/métodos , Metacrilatos/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Lípidos/química , Supervivencia Celular/efectos de los fármacosRESUMEN
Photodynamic therapy (PDT) is an effective and U.S. Food and Drug Administration (FDA) approved treatment for cancer and other diseases. Photosensitizer is one of the three key components that harvest the energy of light at a certain wavelength. Compared to the conventional fluorophores used as photosensitizers, boron dipyrromethene (BODIPY) derivatives have grown fast in recent years due to their low dark toxicity, versatile tunable sites, and easiness of being paired with other treatments. In this paper, two pH-sensitive BODIPY-based photosensitizers (BDC and BDBrC) were synthesized by adding carbazole moieties onto the BODIPY cores (BD and BDBr) through condensation reactions. BDBrC has two Br atoms at the BODIPY core that promote singlet oxygen generation and further red-shift the absorption maximum peak. Both compounds showed sensitivity toward pH change and generated more singlet oxygen under acidic conditions. The cellular uptake and cell imaging experiments showed that BDBrC can selectively target the lysosome organelle. The further dark cell viability and light cytotoxicity indicate the light triggered PDT treatment can be accomplished with BDBrC.
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Far-red BODIPY-based oxime esters for photo-uncaging were designed to release molecules of interest with carboxylic acids. The low power red LED light breaks the N-O oxime ester bond and frees the caged molecules. We studied the mechanism and kinetics of the uncaging procedure using a 1H NMR spectrometer. Moreover, the drug delivery strategy to release valproic acid (VPA) on demand was tested in vitro using this far-red BODIPY photo-uncaging strategy to induce apoptosis in tumor cells.
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Ésteres , Oximas , Ésteres/química , Oximas/química , Luz , Compuestos de Boro/químicaRESUMEN
Photobase generators (PBGs) are compounds that utilize light-sensitive chemical-protecting groups to offer spatiotemporal control of releasing organic bases upon targeted light irradiation. PBGs can be implemented as an external control to initiate anionic polymerizations such as thiol-ene Michael addition reactions. However, there are limitations for common PBGs, including a short absorption wavelength and weak base release that lead to poor efficiency in photopolymerization. Therefore, there is a great need for visible-light-triggered PBGs that are capable of releasing strong bases efficiently. Here, we report two novel BODIPY-based visible-light-sensitive PBGs for light-induced activation of the thiol-ene Michael "click" reaction and polymerization. These PBGs were designed by connecting the BODIPY-based light-sensitive protecting group with tetramethylguanidine (TMG), a strong base. Moreover, we exploited the heavy atom effect to increase the efficiency of releasing TMG and the polymerization rate. These BODIPY-based PBGs exhibit extraordinary activity toward thiol-ene Michael addition-based polymerization, and they can be used in surface coating and polymer network formation of different thiol and vinyl monomers.
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Endoplasmic reticulum (ER) stress and unfolded protein response are cells' survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a pro-PrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPI-anchored PrP to pro-PrP via a conserved ATF6-miRNA449c-5p-PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 3'-UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6-miR449c-5p-PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Estrés del Retículo Endoplásmico , Glicosilfosfatidilinositoles , Neoplasias Pancreáticas , Proteínas Priónicas , Animales , Humanos , Ratones , Factor de Transcripción Activador 6/genética , Adenocarcinoma/patología , Glicosilfosfatidilinositoles/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Neoplasias PancreáticasRESUMEN
The capability to image subtle mechanical motion at cellular and sub-cellular scales can be used to study how extracellular particles interact with cultured cells and, more generally, how cells interact with their environment. However, current technologies need to provide sufficient spatial resolution, temporal resolution, and motion sensitivity to image cellular and sub-cellular motion in the en face plane. To address this unmet need, we investigate a full-field Doppler phase microscopy (FF-DPM) technology based on an innovative optical computation strategy that enables depth-resolved imaging and phase quantification. In this study, we validated the motion tracking (displacements and velocities) capability of FF-DPM by imaging samples actuated by a piezo transducer (PZT). We demonstrated FF-DPM imaging of magnetic particles under different conditions with different motion characteristics. Our results show that free particles (suspended in a cell culture medium) had a significantly larger magnitude of motion than particles adhered to a cell. The key innovation of this study is the use of an optical computation strategy to perform depth-resolved phase quantification and Doppler measurement. The FF-DPM will have a significant impact, as it provides a unique capability to quantitatively measure subtle motion for models based on cultured cells.
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In this study, we investigated a complex optically computed phase microscopy (complex-OCPM) technology. Based on a low coherence interferometer and an innovative optical computation approach, the complex-OCPM imaging system achieves depth resolved quantitative phase measurement. Particularly, the complex-OCPM imaging system directly measures the complex amplitude of the optical field emerging from the sample, extracts the phase as the argument of a complex signal, and achieves a high spatial resolution in phase imaging. We evaluated the performance of complex-OCPM imaging using resolution targets and live cells. Our results show that the complex-OCPM system achieves quantitative phase imaging with sub-cellular resolution on label-free cells.
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Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases that has brought significant economic losses to the swine industry worldwide. Rapid and accurate PRRS virus (PRRSV) detection is one of the key factors for PRRS prevention and control. This study developed a real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) method for type 2 PRRSV (PRRSV-2) detection. The RF-RT-RAA assay could be performed at 42 °C for 20 min with the optimal primers and a probe. RF-RT-RAA results could be monitored using real-time fluorescence read-out or visually observed with the naked eye using a portable blue light transilluminator. The method had a strong specificity; no cross-reaction was identified with the detected common swine viruses. Moreover, the technique yielded high sensitivity with the lowest detection limit of 101 copies/µL and exhibited good repeatability and reproductively with the coefficients of variation (CV) less than 10%. Eighty-seven clinical samples were tested using RF-RT-RAA and a commercial PRRSV-2 RT-qPCR detection kit. The coincidence rate was 100% between RF-RT-RAA (real-time fluorescence read-out) and RT-qPCR, and 97.7% between RF-RT-RAA (visually observed) and RT-qPCR. The RF-RT-RAA assay provides a new method for rapid and visual detection of PRRSV-2.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Porcinos , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Transcripción Reversa , Recombinasas , Síndrome Respiratorio y de la Reproducción Porcina/diagnóstico , HidrolasasRESUMEN
Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/µL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.
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Mycoplasma synoviae , Recombinasas , Animales , Pollos , Escherichia coli , Mycoplasma synoviae/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Sensibilidad y EspecificidadRESUMEN
Hypochlorous acid (HClO) is produced by white blood cells to defend against injury and bacteria. However, as one of the reactive oxygen species, high intracellular HClO concentration could lead to chronic diseases that affect the cardiovascular and nervous systems. To monitor HClO concentrations in bio-samples, the fluorescent probe is preferred to have: a) absorbability in the far-red window with reduced light-toxicity and improved tissue penetration depth, b) ratiometric feature for accurate analysis. In this study, we reported a far-red ratiometric HClO fluorescence probe based on BODIPY chromophore and aldoxime sensing group. Not only the color change of the probe solution can be detected by naked eyes, but also the emission ratios (I645/I670) showed a significant increase upon the introduction of HClO. More importantly, the feasibility of HClO monitoring in bio-samples was demonstrated in vitro using a confocal microscope.
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A new PEGylated macroiniferter was prepared based on the polycondensation reaction of polyethylene oxide (PEO), methylene diphenyl diisocyanate (MDI), and 1,1,2,2-tetraphenyl-1,2-ethanediol (TPED). The macroiniferter consists of PEO end groups and readily reacts with acrylamides (such as N-isopropylacrylamide, NIPAM) and forms ABA block copolymers (PEO-PNIPAM-PEO). This approach of making amphiphilic ABA block copolymers is robust, versatile, and useful, particularly for the development of polymers for biomedical applications. The resulting amphiphilic PEO-PNIPAM-PEO block copolymers are also temperature sensitive, and their phase transition temperatures are close to human body temperature and therefore they have been applied as drug carriers for cancer treatment. Two PEO-PNIPAM-PEO polymers with different molecular weights were prepared and selected to make temperature-sensitive micelles. As a result of the biocompatibility of these micelles, cell viability tests proved that these micelles have low toxicity toward cancer cells. The resultant polymer micelles were then used as drug carriers to deliver the hydrophobic anticancer drug doxorubicin (DOX), and the results showed that they exhibit significantly higher cumulative drug release efficiency at higher temperatures. Moreover, after loading DOX into the micelles, cellular uptake experiments showed easy uptake and cell viability tests showed that DOX-loaded micelles possess a better therapeutic effect than free DOX at the same dose.
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Supercapacitors (SCs) are short-term energy storage elements that find many applications, e.g., electronic charging devices and suppressors of power fluctuations in grids that are interfaced with sustainable sources. The capacitance of an ordinary capacitor increases when dispersing metallic colloids in its dielectric. A similar strategy for SCs means deployment of nano-scale metal colloids (in our case, Au nanoparticles, or AuNPs) at the very narrow interface between an electrolyte and a porous electrode (here, active carbon film, AC, on a grafoil current collector). Unlike previous studies, here we placed AuNPs at a small distance from the electrode. This was achieved by coating the AuNPs with a negatively charged ligand that also enables strong adhesion to the electrode. A very large specific capacitance amplification was demonstrated: for example, C-V data at a scan rate of 20 mV s-1 indicated a specific capacitance amplification of more than 10 when 30 µg of AuNPs was incorporated with 200 mg of active carbon while using a 1 M Na2SO4 electrolyte and a 5% cellulose acetate butyrate binder. Upon replacing the 1 M Na2SO4 electrolyte with 1 M KOH, and keeping the same set of electrodes, the amplification factor decreased but remained large, â¼3, as determined using C-V traces at the same scan rate. This proves that the AuNPs adhered well to the AC electrodes. Simulations indicated the importance of keeping the AuNPs in close proximity to the electrodes, but not in direct contact with them, in order to maintain a substantial amplified polarization effect. Unlike semiconductor embedded electrodes, optical effects were found to be minimal.
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Chemoresistance is one of the major challenges for cancer treatment, more recently ascribed to defective mitochondrial outer membrane permeabilization (MOMP), significantly diminishing chemotherapeutic agent-induced apoptosis. A boron-dipyrromethene (BODIPY) chromophore-based triarylsulfonium photoacid generator (BD-PAG) was used to target mitochondria with the aim to regulate mitochondrial pH and further depolarize the mitochondrial membrane. Cell viability assays demonstrated the relative biocompatibility of BD-PAG in the dark while live cell imaging suggested high accumulation in mitochondria. Specific assays indicated that BD-PAG is capable of regulating mitochondrial pH with significant effects on mitochondrial membrane depolarization. Therapeutic tests using chlorambucil in combination with BD-PAG revealed a new strategy in chemoresistance suppression.
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Antineoplásicos , Mitocondrias , Neoplasias , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Neoplasias/tratamiento farmacológicoRESUMEN
INTRODUCTION: Marek's disease virus (MDV) can cause malignant T-cell lymphomas and immunosuppression in chickens. Macrophage migration inhibitory factor (MIF) not only plays a critical role in inhibiting T-cell responses, but also contributes to multiple aspects of tumour progression. The aim of this study was to reveal the potential role of MIF in the pathogenesis of MDV infection. MATERIAL AND METHODS: MIF gene expression levels were measured by using real-time PCR. Expression was assayed at different times in chicken embryo fibroblast (CEF) cells and tissue samples of SPF chickens infected with different MDV strains and fold change was calculated by the 2-â³â³CT method. RESULTS: The expression of MIF was significantly downregulated (p < 0.05 and FC > 2) in CEF cells infected with the very virulent MDV RB1B strain at 48 h post infection (hpi) and in the skin and spleen at 14 days post infection (dpi). The reduction of MIF expression was also found in CEF cells infected by reticuloendotheliosis virus (REV), avian leukosis virus subgroup J (ALV-J), and MDV vaccine strain CVI988 or in HD11 cells stimulated with TLR2, 3, 4, and 7 ligands. Interestingly, MIF expression decreased continuously from 7 to 28 dpi in the thymus after RB1B virus infection while it increased after CVI988 virus infection. Upregulated expression of MIF was found in CEF infected with RB1B at 96 hpi and in the spleen and skin at 21 and 28 dpi. CONCLUSION: The present study revealed the different expression pattern of MIF in response to MDV infection and indicated that MIF level may be associated with MDV pathogenesis.
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To develop an attenuated vaccine candidate against K88ac enterotoxigenic Escherichia coli (ETEC), a novel Escherichia coli (E. coli) K88ac LT(S63K)ΔSTb with LT(S63K) mutation and ST1 deletion was generated using site mutagenesis and λ-Red homologous recombination based on wild paternal ETEC strain C83902. E. coli K88ac LT(S63K)ΔSTb showed very similar fimbriae expression and growth kinetics to the wild strain C83902, but it was significantly attenuated according to the results of a rabbit ligated ileal loop assay and mouse infection study. Oral inoculation with E. coli K88ac LT(S63K)ΔSTb stimulated the mucosa immune response and induced the secretion of IgA to K88ac in the intestines in mice. A challenge experiment revealed that the attenuated strain provided efficient protection against C83902 in the following 7 days and at the 24th day post-inoculation, suggesting that the attenuated isolate could act as an ecological protectant and vaccine in preventing K88ac ETEC.