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Objectives: To study the ability of theaflavin-3,3'-digallate (TF3)/ethanol solution to crosslink demineralized dentin collagen, resist collagenase digestion, and explore the potential mechanism. Methods: Fully demineralized dentin blocks were prepared using human third molars that were caries-free. Then, these blocks were randomly allocated into 14 separate groups (n = 6), namely, control, ethanol, 5% glutaraldehyde (GA), 12.5, 25, 50, and 100 mg/ml TF3/ethanol solution groups. Each group was further divided into two subgroups based on crosslinking time: 30 and 60 s. The efficacy and mechanism of TF3's interaction with dentin type I collagen were predicted through molecular docking. The cross-linking, anti-enzymatic degradation, and biomechanical properties were studied by weight loss, hydroxyproline release, scanning/transmission electron microscopy (SEM/TEM), in situ zymography, surface hardness, thermogravimetric analysis, and swelling ratio. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy were utilized to explore its mechanisms. Statistical analysis was performed using one and two-way analysis of variance and Tukey's test. Results: TF3/ethanol solution could effectively crosslink demineralized dentin collagen and improve its resistance to collagenase digestion and biomechanical properties (p < 0.05), showing concentration and time dependence. The effect of 25 and 50 mg/ml TF3/ethanol solution was similar to that of 5% GA, whereas the 100 mg/mL TF3/ethanol solution exhibited better performance (p < 0.05). TF3 and dentin type I collagen are mainly cross-linked by hydrogen bonds, and there may be covalent and hydrophobic interactions. Conclusion: TF3 has the capability to efficiently cross-link demineralized dentin collagen, enhancing its resistance to collagenase enzymatic hydrolysis and biomechanical properties within clinically acceptable timeframes (30 s/60 s). Additionally, it exhibits promise in enhancing the longevity of dentin adhesion.
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The cell nucleus serves as the pivotal command center of living cells, and delivering therapeutic agents directly into the nucleus can result in highly efficient anti-tumor eradication of cancer cells. However, nucleus-targeting drug delivery is very difficult due to the presence of numerous biological barriers. Here, three antitumor drugs (DNase I, ICG: indocyanine green, and THP: pirarubicin) were sequentially triggered protein self-assembly to produce a nucleus-targeting and programmed responsive multi-drugs delivery system (DIT). DIT consisted of uniform spherical particles with a size of 282 ± 7.7 nm. The acidic microenvironment of tumors and near-infrared light could successively trigger DIT for the programmed release of three drugs, enabling targeted delivery to the tumor. THP served as a nucleus-guiding molecule and a chemotherapy drug. Through THP-guided DIT, DNase I was successfully delivered to the nucleus of tumor cells and killed them by degrading their DNA. Tumor acidic microenvironment had the ability to induce DIT, leading to the aggregation of sufficient ICG in the tumor tissues. This provided an opportunity for the photothermal therapy of ICG. Hence, three drugs were cleverly combined using a simple method to achieve multi-drugs targeted delivery and highly effective combined anticancer therapy.
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Antineoplásicos , Núcleo Celular , Desoxirribonucleasa I , Doxorrubicina , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Animales , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Núcleo Celular/metabolismo , Desoxirribonucleasa I/metabolismo , Doxorrubicina/farmacología , Doxorrubicina/química , Doxorrubicina/administración & dosificación , Doxorrubicina/análogos & derivados , Portadores de Fármacos/química , Verde de Indocianina/química , Microambiente Tumoral/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
The variable amplification efficiency of each thermal cycle of qPCR obeys the Poisson distribution, and the qPCR system dynamically changes, so there must be a detection error in its quantitative analysis. Here, more than 20 cycles of the linear amplification of qPCR can be produced as the BSA hydrogel is introduced to achieve the controlled release of Taq DNA polymerase. There is a significant negative correlation between the slope of linear amplification and Ct values (r = -0.9455), and it is well evident that the slope can reflect the amplification efficiency and a linear positive correlation exists between them. Through the change in the concentration of primers in the qPCR system, an exponential equation between Ct values and the slopes can be fitted (R2 = 0.9995). The slopes and Ct values of each qPCR system can be corrected by using this equation to guarantee that there will be significant consistency in their amplification efficiency because the degree of linear fitting (R2) between Ct values and the logarithm of their corresponding concentration of the DNA template increased significantly. By this time, the accurate amplification efficiency can be calculated in a known multiple of two initial concentrations of DNA templates. With the aid of the relationship between the known primer concentration and the fluorescence intensity at the end of PCR (End RFU), the initial concentrations of DNA templates can be reversely calculated in the absence of standard curves.
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ADN , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ADN/genéticaRESUMEN
Accurately identifying multidrug-resistant (MDR) bacteria from clinical samples has long been a challenge. Herein, we report a simple and programmable dual-mode aptasensor called DAPT to reliably detect MDR bacteria. The DAPT method comprises two elements, namely the mode of dynamic light scattering (Mode-DLS) for ultrasensitive detection and the mode of fluorescence (Mode-Flu) for reliable quantification as a potent complement. Benefiting from the states of aptamer-modified gold nanoparticles (AptGNPs) sensitively changing from dispersion to aggregation, the proposed Mode-DLS achieved the rapid, specific, and ultrasensitive detection of methicillin-resistant Staphylococcus aureus (MRSA) at the limit of detection (LOD) of 4.63 CFU mL-1 in a proof-of-concept experiment. Simultaneously, the Mode-Flu ensured the accuracy of the detection, especially at a high concentration of bacteria. Moreover, the feasibility and universality of the DAPT platform was validated with four other superbugs by simply reprogramming the corresponding sequence. Overall, the proposed DAPT method based on a dual-mode aptasensor can provide a universal platform for the rapid and ultrasensitive detection of pathogenic bacteria due to its superior programmability.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Nanopartículas del Metal , Staphylococcus aureus Resistente a Meticilina , Oro , Inhibidores de Agregación Plaquetaria , Límite de Detección , Técnicas Biosensibles/métodosRESUMEN
Programmed response, carrier-free, and multimodal therapy drug delivery systems (DDS) are promising solutions to multidirectional cytotoxic effects, inefficient antitumor, and severe side effects for cancer therapy. Here, three widely used clinical drugs, interferon α1b (IFNα1b), indocyanine green (ICG), and doxorubicin (DOX), were prepared into carrier-free DDS IFNα1b-ICG-DOX (IID) by a simple one-step method without additional any reagents. IID can achieve smart and programmed DDS by combining low pH and near-infrared (NIR) light stimuli-responsive controlled release. In pH = 7.4 environments, our IID is about 380 nm in size with negative charge rounded particles; while they enter into the acid environment (pH < 7), hydrogen ions (H+) trigger DOX release, their size becomes larger and the surface charge turns positive. These larger particles are rapidly disintegrated after exposure to NIR light and then the remaining DOX, IFNα1b, and ICG are released. In vivo, the IID with larger size and positive charge resulting from low pH is is easy to accumulate in tumor tissue. Tumors can be exposed to NIR light when needed to control the release of these three drugs. Hence, DOX, ICG, and IFNα1b can be enriched in the tumor to the high efficiency of combined chemotherapy, photothermal therapy, and immunotherapy.
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Antineoplásicos , Neoplasias , Humanos , Doxorrubicina , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Terapia Combinada , Neoplasias/tratamiento farmacológico , Fototerapia , Línea Celular Tumoral , Verde de Indocianina , Liberación de FármacosRESUMEN
The detection of pH change in the gastrointestinal tract (GIT) is invasive and the intestinal pH detection is even harder. Here, we develop an AuI integrated contrast agent (Au NCs@DA) for noninvasive GIT imaging to detect pH change. This agent is composed of gold nanoclusters (Au NCs) and diatrizoic acid (DA) and is extremely sensitive to the acid-base response. Au NCs@DA about 400 nm in size can be stable in acid solution and make the fluorescence intensity of Au NCs to drop significantly. After entering a neutral environment, Au NCs@DA can rapidly form sediment, and then its CT value and fluorescence increase. Alkaline pH can trigger the release of DA from Au NCs@DA to make its fluorescence intensity to recover. As entering GIT, Au NCs@DA can successively outline their anatomy for optical/CT double-modal imaging under gastrointestinal motility. The variations of the fluorescence and CT images triggered by different pH are also recorded to analyze the pH change of GIT. Furthermore, the clearance rate of stable Au NCs@DA in acid pH increases, which also can assist to evaluate pH value. Therefore, Au NCs@DA can definitely be an excellent candidate for the noninvasive detection of pH change in GIT through optical/CT double-modal imaging.
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Medios de Contraste , Nanopartículas del Metal , Tracto Gastrointestinal/diagnóstico por imagen , Oro , Concentración de Iones de Hidrógeno , Tomografía Computarizada por Rayos XRESUMEN
The development of nanomedicine has recently achieved several breakthroughs in the field of cancer treatment; however, biocompatibility and targeted penetration of these nanomaterials remain as limitations, which lead to serious side effects and significantly narrow the scope of their application. The self-assembly of intermediate filaments with arginine-glycine-aspartate (RGD) peptide (RGD-IFP) was triggered by the hydrophobic cationic molecule 7-amino actinomycin D (7-AAD) to synthesize a bifunctional nanoparticle that could serve as a fluorescent imaging probe to visualize tumor treatment. The designed RGD-IFP peptide possessed the ability to encapsulate 7-AAD molecules through the formation of hydrogen bonds and hydrophobic interactions by a one-step method. This fluorescent nanoprobe with RGD peptide could be targeted for delivery into tumor cells and released in acidic environments such as endosomes/lysosomes, ultimately inducing cytotoxicity by arresting tumor cell cycling with inserted DNA. It is noteworthy that the RGD-IFP/7-AAD nanoprobe tail-vein injection approach demonstrated not only high tumor-targeted imaging potential, but also potent antitumor therapeutic effects in vivo. The proposed strategy may be used in peptide-driven bifunctional nanoparticles for precise imaging and cancer therapy.
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Four nucleotides (A, U, C and G) constitute 64 codons at free combination but 64 codons are unequally assigned to 21 items (20 amino acids plus one stop). About 500 amino acids are known but only 20 are selected to make up the proteins. However, the relationships between amino acid and codon and between 20 amino acids have been unclear. In this paper, we studied the relationships between 20 amino acids in 33 species and found there were three constraints between 20 amino acids, such as the relatively stable mean carbon and hydrogen (C : H) ratios (0.50), similarity interactions between the constituent ratios of amino acids, and the frequency of amino acids according with Poisson distribution under certain conditions. We demonstrated that the unequal distribution of 64 codons and the choice of amino acids in molecular evolution would be constrained to remain stable C : H ratios. The constituent ratios and frequency of 20 amino acids in a species or a protein are two determinants of protein sequence evolution, so this finding showed the constraints between 20 amino acids played an important role in protein sequence evolution.
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An outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began in December 2019. Accurate, rapid, convenient, and relatively inexpensive diagnostic methods for SARS-CoV-2 infection are important for public health and optimal clinical care. The current gold standard for diagnosing SARS-CoV-2 infection is reverse transcription-polymerase chain reaction (RT-PCR). However, RTPCR assays are designed for use in well-equipped laboratories with sophisticated laboratory infrastructure and highly trained technicians, and are unsuitable for use in under-equipped laboratories and in the field. In this study, we report the development of an accurate, rapid, and easy-to-implement isothermal and nonenzymatic signal amplification system (a catalytic hairpin assembly (CHA) reaction) coupled with a lateral flow immunoassay (LFIA) strip-based detection method that can detect SARSCoV-2 in oropharyngeal swab samples. Our method avoids RNA isolation, PCR amplification, and elaborate result analysis, which typically takes 6-8 h. The entire CHA-LFIA detection method, from nasopharyngeal sampling to obtaining test results, takes less than 90 min. Such methods are simple and require no expensive equipment, only a simple thermostatically controlled water bath and a fluorescence reader device. We validated our method using synthetic oligonucleotides and clinical samples from 15 patients with SARS-CoV-2 infection and 15 healthy individuals. Our detection method provides a fast, simple, and sensitive (with a limit of detection (LoD) of 2000 copies/mL) alternative to the SARS-CoV-2 RT-PCR assay, with 100 % positive and negative predictive agreements.
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The emergence of drug-resistant bacteria emphasizes the urgent need for novel antibiotics. The antimicrobial peptide TS shows extensive antibacterial activity in vitro and in vivo, especially in gram-negative bacteria; however, its antibacterial mechanism is unclear. Here, we find that TS without hemolytic activity disrupts the integrity of the outer bacterial cell membrane by displacing divalent cations and competitively binding lipopolysaccharides. In addition, the antimicrobial peptide TS can inhibit and kill E. coli by disintegrating the bacteria from within by interacting with bacterial DNA. Thus, antimicrobial peptide TS's multiple antibacterial mechanisms may not easily induce bacterial resistance, suggesting use as an antibacterial drug to be for combating bacterial infections in the future.
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Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/síntesis química , Proteínas Citotóxicas Formadoras de Poros/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/patología , Hemólisis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas Citotóxicas Formadoras de Poros/químicaRESUMEN
Rationale: Drug combination therapy for cancer treatment exerts a more potent antitumor effect. The targeted delivery and release of multiple drugs in a patient's body thus presents a more effective treatment approach, warranting further research. Methods: Two antitumor drugs (ICG: indocyanine green and THP: pirarubicin) were successfully screened to sequentially trigger self-assembling peptides (P60) to produce bacteria-sized particles (500-1000 nm, P60-ICG-THP). First, after mixing equal amount of P60 and ICG, trace amount of water (the mass ratio between P60 and water: 100:1) was used to trigger their assembly into P60-ICG. Subsequently, the assembly of P60-ICG and THP was further triggered by ultrasound treatment to produce P60-ICG-THP. Results: P60-ICG-THP constituted a cluster of several nanoparticles (50-100 nm) and possessed a negative charge. Owing to its size and charge characteristics, P60-ICG-THP could remain outside the cell membrane, avoiding the phagocytic clearance of blood and normal tissue cells in vivo. However, after localizing in the tumor, the size and charge switches of P60-ICG-THP, rapidly triggered by the low pH of the tumor microenvironment, caused P60-ICG-THP to segregate into two parts: (i) positively charged nanoparticles with a size of approximately 50 nm, and (ii) negatively charged particles of an uneven size. The former, mainly carrying THP (chemotherapeutic agent), could immediately cross the cell membrane and deliver pirarubicin into the nucleus of tumor cells. The latter, carrying ICG (used for photothermal therapy), could also enter the cell via the endocytosis pathway or accumulate in tumor blood vessels to selectively block the supply of nutrients and oxygen (cancer starvation). Both these particles could avoid the rapid excretion of ICG in the liver and were conducive to accumulation in the tumor tissue for photothermal therapy. Conclusion: Our drug delivery system not only achieves the precise subcellular delivery of two anticancer drugs due to their size and charge switches in the tumor site, but also provides a new strategy to combine chemotherapy, photothermal therapy, and cancer starvation therapy for the development of a highly efficient antitumor therapeutic regimen.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Neoplasias/terapia , Fotoquimioterapia/métodos , Terapia Fototérmica/métodos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Núcleo Celular/metabolismo , Doxorrubicina/administración & dosificación , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Liberación de Fármacos , Humanos , Concentración de Iones de Hidrógeno , Verde de Indocianina/administración & dosificación , Verde de Indocianina/farmacocinética , Nanopartículas/química , Neoplasias/patología , Tamaño de la Partícula , Péptidos/administración & dosificación , Péptidos/farmacocinética , Distribución Tisular , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recently, the long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was reported to be involved in the pathogenesis of several cancers, including human colorectal cancer (CRC). However, the molecular basis for cancer initiation, development, and progression remains unclear. In this study, we observe that upregulated PVT1 is associated with poor prognosis and bad clinicopathological features of CRC patients. In vitro means of PVT1 loss in a CRC cell line inhibit cell proliferation, migration, and invasion. Furthermore, dual-luciferase reporter and RNA pull-down assays indicated that PVT1 binds to miR-16-5p, which has been shown to play strong tumor suppressive roles in CRC. Targeted loss of miR-16-5p partially rescues the suppressive effect induced by PVT1 knockdown. Vascular endothelial growth factor A (VEGFA), a direct downstream target of miR-16-5p, was suppressed by PVT1 knockdown in CRC cells. Overexpression of VEGFA is known to modulate the AKT signaling cascade by activating vascular endothelial growth factor receptor 1 (VEGFR1). We, therefore, show that PVT1 loss combined with miR-16-5p overexpression reduces tumor volume maximally when propagated within a mouse xenograft model. We conclude that the PVT1-miR-16-5p/VEGFA/VEGFR1/AKT axis directly coordinates the response in CRC pathogenesis and suggest PVT1 as a novel target for potential CRC therapy.
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Bimetallic nanoparticles act as a multifunctional platform because their properties are dependent on the composition, size, and shape, so their synthetic approaches and technological applications have fascinated many researchers. However, the rigorous reaction conditions and the hazardous chemicals are required during the chemical synthesizing processes. In this study, we develop a biosynthesis method of the bimetallic Au-Ag nanoparticles at room temperature without stabilizers or surfactants. In the solution containing Escherichia coli and Au ions, Au nanoparticles are first obtained upon increasing the pH. After Ag ions join, the core-shell Au-Ag nanoparticles are orderly produced. Transmission electron microscopy (TEM), UV-vis, Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction analysis (XRD), and X-ray photoelectron spectroscopy (XPS) are performed to confirm the structure and composition of biosynthetic Au-Ag nanoparticles. Furthermore, we have demonstrated that our bimetallic Au-Ag nanoparticles have greater application prospects in the ultrafast colorimetric detection of H2O2, photothermal therapy, and antibiotic therapy in comparison with single Au or Ag nanoparticles. Our bimetallic Au-Ag NPs could achieve the rapid and colorimetric detection of H2O2 without 3,3',5,5'-tetramethylbenzidine (TMB) and peroxidase. Moreover, Au-Ag NPs could enhance antibacterial ability but not increase their cytotoxicity, which provides a guarantee for the clinical applications of silver.
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Nanopartículas del Metal , Plata , Escherichia coli , Oro , Peróxido de HidrógenoRESUMEN
Stimuli-responsive drug delivery systems based on tumor microenvironment conditions show tremendous promise to enhance tumor-targeted delivery and drug release. Herein, a multifunctional peptide (P51) was developed for programmed delivery of the hydrophobic chemotherapeutic agent pirarubicin. P51 was prepared with a ligand-specific targeting for the cancer biomarker Arg-Gly-Asp (RGD), and three tumor microenvironment-sensitive release triggers, acid environment, reducing agent, and a specific enzyme. The peptides Cys-s-s-Cys (disulfide linkage) and Pro-Val-Gly-Leu-Ile-Gly correspond to the cleavage sites of a reducing agent (DTT) and an enzyme (MMP-2). The peptides act as a junction between Ser-Glu-Glu-Asp-Pro (a negatively charged sequence) and a 41-residue peptide containing an α-helix that has the capacity to encapsulate pirarubicin via electrostatic and hydrophobic interactions. These interactions can be disrupted by the acidic tumor microenvironment. Self-assembly of P51 and pirarubicin (P51-THP NPs) results into stable spherical nanoparticles in a single step. We have demonstrated that the acid environment, DTT, and MMP-2 stimulate the release of pirarubicin from P51-THP NPs and, more importantly, the efficiency of drug release is markedly increased when all three release triggers are present. In addition, more effective tumor targeting, antitumor effect, and reduced systemic toxicity of P51-THP NPs have been confirmed by in vitro and in vivo results.
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Antineoplásicos/administración & dosificación , Doxorrubicina/análogos & derivados , Sistemas de Liberación de Medicamentos , Péptidos/química , Animales , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Liberación de Fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Nanopartículas , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Heavy metals not only pollute the environment but also are health and environmental hazard. Bacteria constitute inexpensive and eco-friendly material to eliminate and recycle heavy metals via biomineralization and biosorption. However, the effect of metal biomineralization in bacterial biofilms on the ecological balance of bacteria and infectious diseases is unclear. This study aimed to explore the interaction between a eukaryotic cell line HEK293T and mineralized Escherichia coli, using a model of gold biomineralization on E. coli biofilms (E. coli-Au). In our present model, bacterial activity was not disrupted and bacterial adhesion and invasion were enhanced. E. coli-Au invaded the cytoplasm and nuclei of HEK293T cells and damaged them via intracellular growth and multiplication. The present findings indicate that metal biomineralization in bacterial biofilms for leaching of heavy metal ions is hazardous to eukaryotic cells and even human health.
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The aim of this study was to analyze the relationship between the percent of euploid embryo and the tolerance of embryo biopsy in preimplantation genetic screening (PGS).PubMed and trial registers were searched for clinical studies that patients were randomized to the PGS group or the control group from 1995 to October 2017. The patients of advanced maternal age, repeated implantation failure, and good prognosis with or without PGS in randomized controlled trials (RCTs) were collected.Original data from 9 RCT studies comparing in-vitro fertilization with and without PGS including 1642 patients were obtained and they were divided into 3 subgroups according to the percent of euploid embryo. PGS significantly increased live birth babies per embryo transferred (risk ratio: 2.98, 95% confidence interval: 1.54-5.75) in ≤30% of euploid embryo subgroups and but in other 2 groups, PGS has no effect. Significant negative correlation was found between the percent of euploid embryo and the tolerance of embryo biopsy in PGS (râ=â0.80, Pâ=â0.010)The tolerance of embryo biopsy in PGS was associated negatively with the percent of euploid embryo. There was a beneficial effect when PGS was used in the patients with the lowest percent of euploid embryo.
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Aneuploidia , Transferencia de Embrión , Diagnóstico Preimplantación , Biopsia , Femenino , Humanos , Embarazo , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Antimicrobial peptides killed bacteria through intercalating into the bacterial membrane. Their antimicrobial efficiencies varied in bacterial species and were affected by ion strength in the culture medium. A recombinant IGP protein consisted of an antimicrobial peptide, Ib-AMP4 fused with the Green Fluorescent Protein was expressed from E. coli cells and was found to maintain the antimicrobial activity. We demonstrated the interaction between the lipid membranes with IGP by quartz crystal microbalance with dissipation and tried to elucidate the effect of calcium ions by lipopolysaccharide monolayer surface isotherm assays. Ten most frequent clinic isolates were subjected to IGP incubation in buffers containing different calcium ion concentrations. The yielded fluorescent intensities ranging from several thousand to several million, differed greatly between species allowing big coefficient of variances that rendered this method a superior reproducibility and resolution. The classification and data treatment were performed by pattern identification with linear discriminant analysis. Seventy-nine isolates of the 10 most frequent clinic species were classified in the blind test with accuracy >70% by a single measurement and with a 100% accuracy by combined measurements for each species. In conclusion, the concept is based on a solid fact that antimicrobial proteins inhibit bacterial growth at a constant minimal inhibitory concentration through intercalating into the biomembrane. The developed method has a good resolution and high-faulty tolerance rate in discriminating bacteria.
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Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Bacterias/crecimiento & desarrollo , Proteínas Fluorescentes Verdes/química , Tecnicas de Microbalanza del Cristal de Cuarzo , Proteínas Recombinantes de Fusión/química , Péptidos Catiónicos Antimicrobianos/genética , Bacterias/aislamiento & purificación , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Cell-free DNA (cfDNA) in blood, which stems from the fetus of pregnant women and tumor in cancer patients, has gained attention in molecular diagnosis. However, cfDNA is less stable, and its amount in the serum is extremely low; these are critical barriers for the utilization of this resource. In this study, a DNA-modified polyacrylamide hydrogel (DNA-Gel) was prepared, and a specialized device was designed to simultaneously catch, purify, concentrate, and detect targeted cfDNA by electrophoresis. We demonstrated that 20-1000 bp ssDNA and dsDNA could be caught and released by the DNA-Gel-based device with high specificity and sensitivity. Upon increasing the number of cycles and electrophoresis time, higher DNA purity and density were achieved, and the separation of serum proteins, untargeted cfDNA, and other charged molecules was promoted. As low as 10 pg µL-1 of DNA could be detected using the DNA-Gel after four cycles of concentration. We also detected 1 fg µL-1 of DNA in the serum with 16 cycles of concentration, followed by 25 PCR cycles. We also designed a device to obtain DNA from the DNA-Gel. We found that the DNA loss rate was around 50%, and A260/A280 was close to 1.7. Thus, we have designed a cost-effective and highly economical device to purify DNA at low concentrations with high specificity and selectivity.
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Background: Tumor vessels can potentially serve as diagnostic, prognostic and therapeutic targets for solid tumors. Fluorescent dyes are commonly used as biological indicators, while photobleaching seriously hinders their application. In this study, we aim to generate a fluorescent silica nanoparticles (FSiNPs) theranostic system marked by the mouse endgolin (mEND) aptamer, YQ26. Methods: A highly specific YQ26 was selected by using gene-modified cell line-based SELEX technique. FSiNPs were prepared via the reverse microemulsion method. The YQ26-FSiNPs theranostic system was developed by combining YQ26 with the FSiNPs for in vivo tumor imaging, treatment and monitoring. Results: Both in vitro experiments (i.e. cellular and tumor tissue targeting assays) and in vivo animal studies (i.e. in vivo imaging and antitumor efficacy of YQ26-FSiNPs) clearly demonstrated that YQ26-FSiNPs could achieve prominently high targeting efficiency and therapeutic effects via aptamer YQ26-mediated binding to endoglin (END) molecule. Conclusion: This simple, sensitive, and specific YQ26-FSiNPs theranostic system has a great potential for clinical tumor targeting imaging and treatment.
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Aptámeros de Nucleótidos/farmacocinética , Endoglina/metabolismo , Nanopartículas/metabolismo , Neovascularización Patológica/terapia , Nanomedicina Teranóstica/métodos , Animales , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/uso terapéutico , Línea Celular Tumoral , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/uso terapéutico , Neovascularización Patológica/diagnóstico por imagen , Unión Proteica , Técnica SELEX de Producción de Aptámeros , Dióxido de Silicio/química , Distribución TisularRESUMEN
Low fusion efficiency and nominal activity of fusion cells (FCs) restrict the clinical application of dendritic cell (DC)/tumor fusion cells. Collagen I (Col I) is an interstitial collagen with a closely-knit structure used to repair damaged cell membranes. This study evaluated whether Col I could improve the fusion efficiency of polyethylene glycol (PEG)-induction and enhance the immunogenicity of fusion vaccine. DC/B16 melanoma and controlled DC/H22 hepatoma cell fusions were induced by PEG with or without Col I. Col I/PEG treatment increased the levels of DC surface molecules and the secretion of lactate, pro- and anti-inflammatory cytokines in fusion cells. Col I/PEG-treated FCs enhanced T-cell proliferation and cytotoxic T lymphocyte activity. The Col I-prepared fusion vaccine obviously suppressed tumor growth and prolonged mice survival time. Thus Col I treatment could significantly improve the efficiency of PEG-induced DC/tumor fusion and enhance the anticancer activity of the fusion vaccine. This novel fusion strategy might promote the clinical application of DC/tumor fusion immunotherapy.