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1.
Acta Neuropathol Commun ; 12(1): 76, 2024 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-38755736

RESUMEN

Activated microglia play an important role in driving photoreceptor degeneration-associated neuroinflammation in the retina. Controlling pro-inflammatory activation of microglia holds promise for mitigating the progression of photoreceptor degeneration. Our previous study has demonstrated that pre-light damage treatment of hyperoside, a naturally occurring flavonol glycoside with antioxidant and anti-inflammatory activities, prevents photooxidative stress-induced photoreceptor degeneration and neuroinflammatory responses in the retina. However, the direct impact of hyperoside on microglia-mediated neuroinflammation during photoreceptor degeneration remains unknown. Upon verifying the anti-inflammatory effects of hyperoside in LPS-stimulated BV-2 cells, our results here further demonstrated that post-light damage hyperoside treatment mitigated the loss of photoreceptors and attenuated the functional decline of the retina. Meanwhile, post-light damage hyperoside treatment lowered neuroinflammatory responses and dampened microglial activation in the illuminated retinas. With respect to microglial activation, hyperoside mitigated the pro-inflammatory responses in DNA-stimulated BV-2 cells and lowered DNA-stimulated production of 2'3'-cGAMP in BV-2 cells. Moreover, hyperoside was shown to directly interact with cGAS and suppress the enzymatic activity of cGAS in a cell-free system. In conclusion, the current study suggests for the first time that the DNA sensor cGAS is a direct target of hyperoside. Hyperoside is effective at mitigating DNA-stimulated cGAS-mediated pro-inflammatory activation of microglia, which likely contributes to the therapeutic effects of hyperoside at curtailing neuroinflammation and alleviating neuroinflammation-instigated photoreceptor degeneration.


Asunto(s)
Microglía , Nucleotidiltransferasas , Quercetina , Degeneración Retiniana , Animales , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Quercetina/farmacología , Quercetina/análogos & derivados , Degeneración Retiniana/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/prevención & control , Ratones , Nucleotidiltransferasas/metabolismo , Ratones Endogámicos C57BL , ADN/metabolismo , Línea Celular , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/metabolismo , Masculino
2.
J Nanobiotechnology ; 22(1): 274, 2024 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-38773614

RESUMEN

Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.


Asunto(s)
Técnicas Biosensibles , Vesículas Extracelulares , Límite de Detección , Estructuras Metalorgánicas , MicroARNs , Papel , Estructuras Metalorgánicas/química , Vesículas Extracelulares/química , Humanos , Técnicas Biosensibles/métodos , ADN Catalítico/química , Grafito/química , Oro/química , ADN/química , Nanopartículas del Metal/química , Hibridación de Ácido Nucleico , Técnicas Electroquímicas/métodos , Electrodos , Circonio/química
3.
Elife ; 122024 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-38767330

RESUMEN

A protein's genetic architecture - the set of causal rules by which its sequence produces its functions - also determines its possible evolutionary trajectories. Prior research has proposed that the genetic architecture of proteins is very complex, with pervasive epistatic interactions that constrain evolution and make function difficult to predict from sequence. Most of this work has analyzed only the direct paths between two proteins of interest - excluding the vast majority of possible genotypes and evolutionary trajectories - and has considered only a single protein function, leaving unaddressed the genetic architecture of functional specificity and its impact on the evolution of new functions. Here, we develop a new method based on ordinal logistic regression to directly characterize the global genetic determinants of multiple protein functions from 20-state combinatorial deep mutational scanning (DMS) experiments. We use it to dissect the genetic architecture and evolution of a transcription factor's specificity for DNA, using data from a combinatorial DMS of an ancient steroid hormone receptor's capacity to activate transcription from two biologically relevant DNA elements. We show that the genetic architecture of DNA recognition consists of a dense set of main and pairwise effects that involve virtually every possible amino acid state in the protein-DNA interface, but higher-order epistasis plays only a tiny role. Pairwise interactions enlarge the set of functional sequences and are the primary determinants of specificity for different DNA elements. They also massively expand the number of opportunities for single-residue mutations to switch specificity from one DNA target to another. By bringing variants with different functions close together in sequence space, pairwise epistasis therefore facilitates rather than constrains the evolution of new functions.


Asunto(s)
Epistasis Genética , Evolución Molecular , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , ADN/genética , ADN/metabolismo , Mutación , Unión Proteica
4.
Adv Immunol ; 161: 109-126, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38763699

RESUMEN

Besides the canonical B-form, DNA also adopts alternative non-B form conformations which are highly conserved in all domains of life. While extensive research over decades has centered on the genomic functions of B-form DNA, understanding how non-B-form conformations influence functional genomic states remains a fundamental and open question. Recent studies have ascribed alternative DNA conformations such as G-quadruplexes and R-loops as important functional features in eukaryotic genomes. This review delves into the biological importance of alternative DNA structures, with a specific focus on hematopoiesis and adaptive immunity. We discuss the emerging roles of G-quadruplex and R-loop structures, the two most well-studied alternative DNA conformations, in the hematopoietic compartment and present evidence for their functional roles in normal cellular physiology and associated pathologies.


Asunto(s)
Inmunidad Adaptativa , G-Cuádruplex , Hematopoyesis , Humanos , Hematopoyesis/genética , Animales , ADN/inmunología , Conformación de Ácido Nucleico
5.
J Nanobiotechnology ; 22(1): 230, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38720322

RESUMEN

Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO2) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A50 or T20 DNA single strands to obtain MnO2/OVA/A50 and MnO2/OVA/T20, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO2/OVA/A50-T20 particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO2/OVA/A50 and MnO2/OVA/T20, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.


Asunto(s)
Vacunas contra el Cáncer , Ganglios Linfáticos , Compuestos de Manganeso , Ratones Endogámicos C57BL , Nanopartículas , Ovalbúmina , Óxidos , Animales , Vacunas contra el Cáncer/inmunología , Ganglios Linfáticos/inmunología , Ratones , Ovalbúmina/inmunología , Ovalbúmina/química , Óxidos/química , Nanopartículas/química , Compuestos de Manganeso/química , Inmunidad Celular , Femenino , Línea Celular Tumoral , ADN/química , ADN/inmunología , Inmunoterapia/métodos , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Tamaño de la Partícula , Antígenos de Neoplasias/inmunología
6.
Biotechnol J ; 19(5): e2300734, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38719571

RESUMEN

Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.


Asunto(s)
ADN , Ganglios Espinales , Factor de Crecimiento Nervioso , Regeneración Nerviosa , Animales , Ratas , Células PC12 , ADN/química , Ganglios Espinales/citología , Factor de Crecimiento Nervioso/química , Factor de Crecimiento Nervioso/farmacología , Nanoestructuras/química , Neuronas , Nervio Ciático , Andamios del Tejido/química , Ratas Sprague-Dawley
7.
Sci Rep ; 14(1): 10826, 2024 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-38734799

RESUMEN

Sequencing the DNA nucleobases is essential in the diagnosis and treatment of many diseases related to human genes. In this article, the encapsulation of DNA nucleobases with some of the important synthesized chiral (7, 6), (8, 6), and (10, 8) carbon nanotubes were investigated. The structures were modeled by applying density functional theory based on tight binding method (DFTB) by considering semi-empirical basis sets. Encapsulating DNA nucleobases on the inside of CNTs caused changes in the electronic properties of the selected chiral CNTs. The results confirmed that van der Waals (vdW) interactions, π-orbitals interactions, non-bonded electron pairs, and the presence of high electronegative atoms are the key factors for these changes. The result of electronic parameters showed that among the CNTs, CNT (8, 6) is a suitable choice in sequencing guanine (G) and cytosine (C) DNA nucleobases. However, they are not able to sequence adenine (A) and thymine (T). According to the band gap energy engineering approach and absorption energy, the presence of G and C DNA nucleobases decreased the band gap energy of CNTs. Hence selected CNTs suggested as biosensor substrates for sequencing G and C DNA nucleobases.


Asunto(s)
ADN , Guanina , Nanotubos de Carbono , Nanotubos de Carbono/química , ADN/química , Guanina/química , Teoría Funcional de la Densidad , Adenina/química , Citosina/química , Timina/química , Análisis de Secuencia de ADN/métodos , Electrones , Modelos Moleculares , Humanos
8.
PLoS One ; 19(5): e0298857, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38696375

RESUMEN

High-cost DNA extraction procedures pose significant challenges for budget-constrained laboratories. To address this, we introduce OpenCell, an economical, open-source, 3-in-1 laboratory device that combines the functionalities of a bead homogenizer, a microcentrifuge, and a vortex mixer. OpenCell utilizes modular attachments that magnetically connect to a central rotating brushless motor. This motor couples to an epicyclic gearing mechanism, enabling efficient bead homogenization, vortex mixing, and centrifugation within one compact unit. OpenCell's design incorporates multiple redundant safety features, ensuring both the device's and operator's safety. Additional features such as RPM measurement, programmable timers, battery operation, and optional speed control make OpenCell a reliable and reproducible laboratory instrument. In our study, OpenCell successfully isolated DNA from Spinacia oleracea (spinach), with an average yield of 2.3 µg and an A260/A280 ratio of 1.77, demonstrating its effectiveness for downstream applications such as Polymerase Chain Reaction (PCR) amplification. With its compact size (20 cm x 28 cm x 6.7 cm) and lightweight design (0.8 kg), comparable to the size and weight of a laptop, OpenCell is portable, making it an attractive component of a 'lab-in-a-backpack' for resource-constrained environments in low-and-middle-income countries and synthetic biology in remote field stations. Leveraging the accessibility of 3D printing and off-the-shelf components, OpenCell can be manufactured and assembled at a low unit cost of less than $50, providing an affordable alternative to expensive laboratory equipment costing over $4000. OpenCell aims to overcome the barriers to entry in synthetic biology research and contribute to the growing collection of frugal and open hardware.


Asunto(s)
ADN , ADN/aislamiento & purificación , Diseño de Equipo , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/economía , Reacción en Cadena de la Polimerasa/instrumentación , ADN de Plantas/aislamiento & purificación , ADN de Plantas/genética
9.
Proc Natl Acad Sci U S A ; 121(19): e2322934121, 2024 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-38701119

RESUMEN

EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that EPHA2 and EPHA4 expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (Ki: 4.0 nM) and EPHA4 (Ki: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC50: 461 nM) and EPHA4 (IC50: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (Ki: 0.13 nM) and EPHA4 (Ki: 0.38 nM) with excellent cell-based potency EPHA2 (IC50: 8.0 nM) and EPHA4 (IC50: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1ß-mediated expression of prostaglandin synthase 2 (PTGS2). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.


Asunto(s)
Inhibidores de Proteínas Quinasas , Humanos , Femenino , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Endometriosis/tratamiento farmacológico , Endometriosis/metabolismo , Endometriosis/patología , ADN/metabolismo , Receptores de la Familia Eph/metabolismo , Receptores de la Familia Eph/antagonistas & inhibidores , Receptor EphA2/metabolismo , Receptor EphA2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Movimiento Celular/efectos de los fármacos
10.
Anal Chem ; 96(19): 7747-7755, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38691774

RESUMEN

Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTDAND-AND) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTDOR-AND) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTDAND-AND and MTDOR-AND, while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.


Asunto(s)
ADN , Molécula de Adhesión Celular Epitelial , MicroARNs , Humanos , Molécula de Adhesión Celular Epitelial/metabolismo , ADN/química , MicroARNs/análisis , MicroARNs/metabolismo , Mucina-1/metabolismo , Mucina-1/análisis , Computadores Moleculares , Células MCF-7 , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/análisis , Membrana Celular/metabolismo , Membrana Celular/química , Células Hep G2
11.
Anal Chem ; 96(19): 7679-7686, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38698534

RESUMEN

Despite the success of surface-enhanced Raman spectroscopy (SERS) for detecting DNA immobilized on plasmonic metal surfaces, its quantitative response is limited by the rapid falloff of enhancement with distance from the metal surface and variations in sensitivity that depend on orientation and proximity to plasmonic "hot spots". In this work, we assess an alternative approach for enhancing detection by immobilizing DNA on the interior surfaces of porous silica particles. These substrates provide over a 1000-fold greater surface area for detection compared to a planar support. The porous silica substrate is a purely dielectric material with randomly oriented internal surfaces, where scattering is independent of proximity and orientation of oligonucleotides relative to the silica surface. We characterize the quantitative response of Raman scattering from DNA in porous silica particles with sequences used in previous SERS investigations of DNA for comparison. The results show that Raman scattering of DNA in porous silica is independent of distance of nucleotides from the silica surface, allowing detection of longer DNA strands with constant sensitivity. The surface area enhancement within particles is reproducible (<4% particle-to-particle variation) owing to the uniform internal pore structure and surface chemistry of the silica support. DNA immobilization with a bis-thiosuccinimide linker provides a Raman-active internal standard for quantitative interpretation of Raman scattering results. Despite the high (30 mM) concentrations of immobilized DNA within porous silica particles, they can be used to measure nanomolar binding affinities of target molecules to DNA by equilibrating a very small number of particles with a sufficiently large volume of low-concentration solution of target molecules.


Asunto(s)
ADN , Dióxido de Silicio , Espectrometría Raman , Propiedades de Superficie , Dióxido de Silicio/química , Espectrometría Raman/métodos , Porosidad , ADN/química , ADN/análisis
12.
Langmuir ; 40(19): 10157-10170, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38700902

RESUMEN

I-Motif (iM) DNA structures represent among the most significant noncanonical nucleic acid configurations. iM-forming DNA sequences are found in an array of vital genomic locations and are particularly frequent in the promoter islands of various oncogenes. Thus, iM DNA is a crucial candidate for anticancer medicines; therefore, binding interactions between iM DNA and small molecular ligands, such as flavonoids, are critically important. Extensive sets of spectroscopic strategies and thermodynamic analysis were utilized in the present investigation to find out the favorable interaction of quercetin (Que), a dietary flavonoid that has various health-promoting characteristics, including anticancer properties, with noncanonical iM DNA structure. Spectroscopic studies and thermal analysis revealed that Que interacts preferentially with HRAS1 iM DNA compared with VEGF, BCL2 iM, and duplex DNA. Que, therefore, emerged as a suitable natural-product-oriented antagonist for targeting HRAS1 iM DNA. The innovative spectroscopic as well as mechanical features of Que and its specific affinity for HRAS1 iM may be useful for therapeutic applications and provide crucial insights for the design of compounds with remarkable medicinal properties.


Asunto(s)
ADN , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas p21(ras) , Quercetina , Quercetina/química , Quercetina/farmacología , Quercetina/metabolismo , ADN/química , ADN/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Termodinámica , Humanos , Motivos de Nucleótidos , Sitios de Unión
13.
ACS Nano ; 18(19): 12401-12411, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38701333

RESUMEN

Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.


Asunto(s)
ADN , Polimorfismo de Nucleótido Simple , Polimorfismo de Nucleótido Simple/genética , Humanos , ADN/genética , ADN/química , Neoplasias Colorrectales/genética , Reacción en Cadena de la Polimerasa , Colorantes Fluorescentes/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/química
14.
Mikrochim Acta ; 191(6): 298, 2024 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-38709403

RESUMEN

As a real-time fluid biopsy method, the detection of circulating tumor cells (CTCs) provides important information for the early diagnosis, precise treatment, and prognosis of cancer. However, the low density of CTCs in the peripheral blood hampers their capture and detection with high sensitivity and selectivity using currently available methods. Hence, we designed a sandwich-type electrochemical aptasensor that utilizes holothurian-shaped AuPd nanoparticles (AuPd HSs), tetrahedral DNA nanostructures (TDNs), and CuPdPt nanowire networks (NWs) interwoven with a graphdiyne (GDY) sheet for ultrasensitive non-destructive detection of MCF-7 breast cancer cells. CuPdPt NW-GDY effectively enhanced the electron transfer rate and coupled with the loaded TDNs. The TDNs could capture MCF-7 cells with precision and firmness, and the resulting composite complex was combined with AuPd HSs to form a sandwich-type structure. This novel aptasensor showed a linear range between 10 and 106 cells mL-1 and an ultralow detection limit of 7 cells mL-1. The specificity, stability, and repeatability of the measurements were successfully verified. Moreover, we used benzonase nuclease to achieve non-destructive recovery of cells for further clinical studies. According to the results, our aptasensor was more sensitive measuring the number of CTCs than other approaches because of the employment of TDNs, CuPdPt NW-GDY, and AuPd HSs. We designed a reliable sensor system for the detection of CTCs in the peripheral blood, which could serve as a new approach for cancer diagnosis at an early stage.


Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN , Técnicas Electroquímicas , Oro , Límite de Detección , Nanopartículas del Metal , Células Neoplásicas Circulantes , Paladio , Células Neoplásicas Circulantes/patología , Humanos , Células MCF-7 , Nanopartículas del Metal/química , Técnicas Electroquímicas/métodos , Aptámeros de Nucleótidos/química , Oro/química , ADN/química , Técnicas Biosensibles/métodos , Paladio/química
15.
Hereditas ; 161(1): 16, 2024 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-38711122
16.
PeerJ ; 12: e17071, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38711623

RESUMEN

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn't allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization.


Asunto(s)
Tejido Adiposo , ADN , ARN , Animales , ARN/aislamiento & purificación , ARN/genética , Porcinos , ADN/aislamiento & purificación , ADN/genética , Tejido Adiposo/metabolismo
17.
Amino Acids ; 56(1): 34, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38691208

RESUMEN

Breast cancer is the most common cancer among women worldwide, and marine creatures are the most abundant reservoir of anticancer medicines. Tachyplesin peptides have shown antibacterial capabilities, but their potential to inhibit cancer growth and trigger cancer cell death has not been investigated. A synthetic tachyplesin nucleotide sequence was generated and inserted into the pcDNA3.1( +) Mammalian Expression Vector. PCR analysis and enzyme digesting procedures were used to evaluate the vectors' accuracy. The transfection efficiency of MCF-7 and MCF10-A cells was 57% and 65%, respectively. The proliferation of MCF-7 cancer cells was markedly suppressed. Administration of plasmid DNA (pDNA) combined with tachyplesin to mice with tumors did not cause any discernible morbidity or mortality throughout treatment. The final body weight curves revealed a significant reduction in weight among mice treated with pDNA/tachyplesin and tachyplesin at a dose of 100 µg/ml (18.4 ± 0.24 gr, P < 0.05; 11.4 ± 0.24 gr P < 0.01) compared to the control group treated with PBS (22 ± 0.31 gr). Animals treated with pDNA/tachyplesin and tachyplesin exhibited a higher percentage of CD4 + Foxp3 + Tregs, CD8 + Foxp3 + Tregs, and CD4 + and CD8 + T cell populations expressing CTLA-4 in their lymph nodes and spleen compared to the PBS group. The groups that received pDNA/tachyplesin exhibited a substantial upregulation in the expression levels of caspase-3, caspase-8, BAX, PI3K, STAT3, and JAK genes. The results offer new possibilities for treating cancer by targeting malignancies using pDNA/tachyplesin and activating the mTOR and NFκB signaling pathways.


Asunto(s)
Péptidos Catiónicos Antimicrobianos , Apoptosis , Proteínas de Unión al ADN , Péptidos Cíclicos , Plásmidos , Animales , Apoptosis/efectos de los fármacos , Humanos , Ratones , Femenino , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Cíclicos/farmacología , Células MCF-7 , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Neoplasias de la Mama/inmunología , ADN , Ratones Endogámicos BALB C
18.
Cryo Letters ; 45(3): 177-184, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38709189

RESUMEN

BACKGROUND: Ovarian tissue cryopreservation for fertility preservation carries a risk of malignant cell re-seeding. Artificial ovary is a promising method to solve such a problem. However, ovary decellularization protocols are limited. Hence, further studies are necessary to get better ovarian decellularization techniques for the construction of artificial ovary scaffolds. OBJECTIVE: To establish an innovative decellularization technique for whole porcine ovaries by integrating liquid nitrogen with chemical agents to reduce the contact time between the scaffolds and chemical reagents. MATERIALS AND METHODS: Porcine ovaries were randomly assigned to three groups: novel decellularized group, conventional decellularized group and fresh group. The ovaries in the novel decellularized group underwent three cycles of freezing by liquid nitrogen and thawing at temperatures around 37 degree C before decellularization. The efficiency of the decellularization procedure was assessed through histological staining and DNA content analysis. The maintenance of ovarian decellularized extracellular matrix(ODECM) constituents was determined by analyzing the content of matrix proteins. Additionally, we evaluated the biocompatibility of the decellularized extracellular matrix(dECM) by observing the growth of granulosa cells on the ODECM scaffold in vitro. RESULTS: Hematoxylin and eosin staining, DAPI staining and DNA quantification techniques collectively confirm the success of the novel decellularization methods in removing cellular and nuclear components from ovarian tissue. Moreover, quantitative assessments of ODECM contents revealed that the novel decellularization technique preserved more collagen and glycosaminoglycan compared to the conventional decellularized group (P<0.05). Additionally, the novel decellularized scaffold exhibited a significantly higher number of granulosa cells than the conventional scaffold during in vitro co-culture (P<0.05). CONCLUSION: The novel decellularized method demonstrated high efficacy in eliminating DNA and cellular structures while effectively preserving the extracellular matrix. As a result, the novel decellularized method holds significant promise as a viable technique for ovarian decellularization in forthcoming studies. Doi.org/10.54680/fr24310110212.


Asunto(s)
Criopreservación , Matriz Extracelular Descelularizada , Nitrógeno , Ovario , Andamios del Tejido , Animales , Femenino , Nitrógeno/química , Porcinos , Ovario/citología , Andamios del Tejido/química , Criopreservación/métodos , Matriz Extracelular Descelularizada/química , Ingeniería de Tejidos/métodos , Células de la Granulosa/citología , Preservación de la Fertilidad/métodos , Matriz Extracelular/química , ADN/análisis , ADN/química
19.
Methods Mol Biol ; 2800: 35-53, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38709476

RESUMEN

Clustering of type II tumor necrosis factor (TNF) receptors (TNFRs) is essential for their activation, yet currently available drugs fail to activate signaling. Some strategies aim to cluster TNFR by using multivalent streptavidin or scaffolds based on dextran or graphene. However, these strategies do not allow for control of the valency or spatial organization of the ligands, and consequently control of the TNFR activation is not optimal. DNA origami nanostructures allow nanometer-precise control of the spatial organization of molecules and complexes, with defined spacing, number and valency. Here, we demonstrate the design and characterization of a DNA origami nanostructure that can be decorated with engineered single-chain TNF-related apoptosis-inducing ligand (SC-TRAIL) complexes, which show increased cell killing compared to SC-TRAIL alone on Jurkat cells. The information in this chapter can be used as a basis to decorate DNA origami nanostructures with various proteins, complexes, or other biomolecules.


Asunto(s)
ADN , Nanoestructuras , Nanoestructuras/química , Humanos , Células Jurkat , ADN/química , ADN/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/química , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores del Factor de Necrosis Tumoral/química , Nanotecnología/métodos , Conformación de Ácido Nucleico
20.
Methods Mol Biol ; 2800: 103-113, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38709481

RESUMEN

The spatial resolution of conventional light microscopy is restricted by the diffraction limit to hundreds of nanometers. Super-resolution microscopy enables single digit nanometer resolution by circumventing the diffraction limit of conventional light microscopy. DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) belongs to the family of single-molecule localization super-resolution approaches. Unique features of DNA-PAINT are that it allows for sub-nanometer resolution, spectrally unlimited multiplexing, proximity detection, and quantitative counting of target molecules. Here, we describe prerequisites for efficient DNA-PAINT microscopy.


Asunto(s)
ADN , Imagen Individual de Molécula , ADN/química , Imagen Individual de Molécula/métodos , Microscopía Fluorescente/métodos , Proteínas/química , Nanotecnología/métodos
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