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Ginger is an important cooking spice and herb worldwide, and scientific research has gradually confirmed the effect of ginger on preventing hair loss. Cedrol (CE) is a small sesquiterpene molecule in ginger and its external administration (EA) has shown hope in promoting hair growth, and alternative administration mode has become a potential treatment scheme to improve the efficacy of CE. The purpose of this study is to evaluate the effects of oral administration (OA) and EA of CE on hair regeneration of C57BL/6 alopecia areata (AA) mice induced by cyclophosphamide (CP) and to clarify the potential hair growth mechanism of CE in AA model in vitro and in vivo. The results showed that CE-OA has a shorter hair-turning black time and faster hair growth rate, and can lessen hair follicle damage induced by CP and promote hair follicle cell proliferation. Its effect is superior to CE-EA. At the same time, CE can increase the cytokines IFN-γ, IL-2, and IL-7 in the serum of mice, and decrease the expression of adhesion factors ICAM-1 and ELAM-1, thus alleviating the immunosuppression induced by CP. Mechanism research shows that CE regulates the JAK3/STAT3 signaling pathway, activates the Wnt3α/ß-catenin germinal center, and ameliorates oxidative stress induced by CP, thus promoting the proliferation of hair follicle cells and reversing AA. These results provide a theoretical basis for understanding the anti-AA mechanism of CE-OA, indicating that CE can be used as raw material for developing oral hair growth drugs.
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Based on the significant biological activities and the remarkable physical and chemical properties of 1H-1,2,3-triazole pharmacophore, we herein adopted the strategy of click chemistry to combine the triazole fragment and the unique scaffold of 25-OCH3-PPD (AD-1) to design a series of potent compounds inducing apoptosis and DNA damage. The anti-proliferative effect was verified by MTT assay and colony formation assay. DNA double-stand breaks (DSBs) were obtained by observing the nuclear focus formation and the protein expression of γ-H2AX. Cell cycle arrest was evaluated by the cycle-related proteins such as CDK2, CDK4, CDK6, Cyclin D1 and P21. Apoptosis was assessed by flow cytometry, mitochondrial membrane potential (MMP) detection and the expression of apoptosis-related proteins. Reactive oxygen species (ROS) generation was measured with 2', 7'-dichlorofluorescein diacetate (DCFH-DA) staining. According to SAR analysis, the most potent compound 6a exhibited great inhibitory effect against A549 cells, which IC50 value of 2.84 ± 0.68 µM. Furthermore, 6a remarkably induced DNA damage, cell cycle arrest and apoptosis in A549 cells. 6a treatment increased the levels of ROS. Network pharmacology and molecular docking predicted the potential signaling pathways and ligand-receptor interactions, and the results of western blotting showed that 6a inhibited the PI3K/Akt/Bcl-2 signaling pathway by decreasing PI3K and Bcl-2 and total level of Akt expression, while Bax and Cyt c were increasing in 6a-treated A549 cells. As mentioned above, 6a has a potent inhibitory effect in A549 cells through induction of DNA damage, apoptosis via ROS generation and modulation of PI3K/Akt/Bcl-2 signaling pathway.
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Antineoplásicos , Apoptosis , Proliferación Celular , Daño del ADN , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Especies Reactivas de Oxígeno , Triazoles , Humanos , Triazoles/farmacología , Triazoles/química , Triazoles/síntesis química , Especies Reactivas de Oxígeno/metabolismo , Daño del ADN/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Relación Estructura-Actividad , Apoptosis/efectos de los fármacos , Estructura Molecular , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Simulación del Acoplamiento Molecular , Células A549RESUMEN
In this study, we designed and synthesized 19 nitrogen-containing heterocyclic derivatives of panaxadiol (PD). We first reported the antiproliferative activity of these compounds against four different tumor cells. The results of the MTT assay showed that the PD pyrazole derivative (compound 12b) had the best antitumor activity and could significantly inhibit the proliferation of four tested tumor cells. For A549 cells, the IC50 value was as low as 13.44±1.23â µM. Western blot analysis showed that the PD pyrazole derivative was a bifunctional regulator. On the one hand, it can down-regulate the expression of HIF-1α by acting on PI3â K/AKT signaling pathway in A549 cells. On the other hand, it can induce the decrease of CDKs protein family and E2F1 protein expression levels, thus playing a crucial role in cell cycle arrest. According to the results of molecular docking, we found that multiple hydrogen bonds were formed between the PD pyrazole derivative and two related proteins, and the docking score of the derivative was also significantly higher than that of the crude drug. In summary, the study of the PD pyrazole derivative laid a foundation for the development of ginsenoside as an antitumor agent.
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Antineoplásicos , Ginsenósidos , Relación Estructura-Actividad , Ginsenósidos/química , Línea Celular Tumoral , Simulación del Acoplamiento Molecular , Proliferación Celular , Antineoplásicos/química , Pirazoles/farmacología , Pirazoles/química , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , ApoptosisRESUMEN
Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.
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Sistemas CRISPR-Cas , Edición Génica , ADN/genéticaRESUMEN
We present a novel genome-wide off-target prediction method named Extru-seq and compare it with cell-based (GUIDE-seq), in vitro (Digenome-seq), and in silico methods using promiscuous guide RNAs with large numbers of valid off-target sites. Extru-seq demonstrates a high validation rate and retention of information about the intracellular environment, both beneficial characteristics of cell-based methods. Extru-seq also shows a low miss rate and could easily be performed in clinically relevant cell types with little optimization, which are major positive features of the in vitro methods. In summary, Extru-seq shows beneficial features of cell-based and in vitro methods.
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Sistemas CRISPR-Cas , Genoma , Edición Génica , ARN Guía de Sistemas CRISPR-CasRESUMEN
Prime editors (PEs) are powerful tools that widen the possibilities for sequence modifications during genome editing. Although methods based on the analysis of Cas9 nuclease or nickase activity have been used to predict genome-wide off-target activities of PEs, no tool that directly uses PEs for this purpose has been reported yet. In this study, we present a cell-based assay, named TAgmentation of Prime Editor sequencing (TAPE-seq), that provides genome-wide off-target candidates for PEs. TAPE-seq analyses are successfully performed using many different versions of PEs. The TAPE-seq predictions are compared with results from two other off-site prediction methods, Cas9 nuclease-based GUIDE-seq and Cas9 nickase-based Digenome-seq (nDigenome-seq). TAPE-seq shows a lower miss rate, and a higher area under the receiver operating characteristic curve compared to the other methods. TAPE-seq also identified valid off-target sites that were missed by the other methods.
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Edición Génica , Genoma , Edición Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Desoxirribonucleasa I/genética , Sistemas CRISPR-CasRESUMEN
Based on the well-known cytotoxicity of indole compounds, we used the 'Fisher indole synthesis' method to introduce appropriately substituted indole rings into panaxadiol (PD), generating eighteen novel Panaxadiol indole derivatives. Six human cancer cell lines (A549, HepG-2, HCT-116, SGC-7901, MDA-MB-231, PC-3 cells) and one normal ovarian cell lines (IOSE144) were designed to evaluate the anti-proliferative activity of the PD derivatives. The results showed that the majority of PD derivatives showed enhanced anti-proliferative activity, when compared with PD, with P-Methylindolo-PD exhibiting the highest cytotoxicity. In A549 cells, IC50 value was 5.01±0.87â µM, which is roughly 12â times higher than the activity of PD and 5â times that of 5-FU. Moreover, cell morphology analysis and Annexin V-FITC/PI assays exhibited that P-Methylindolo-PD could induce A549 cell apoptosis (55.7 % of apoptotic cells at 20â µM). Moreover, molecular docking experiments were performed to explore the molecular mechanism underlining the binding of P-Methylindolo-PD to the active site of EGFR. The results support that P-Methylindolo-PD might be a promising lead compound for further studies.
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Antineoplásicos , Antineoplásicos/química , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Ginsenósidos , Humanos , Indoles/farmacología , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Ginsenoside Re is a protopanaxatriol-type saponin extracted from the berry, leaf, stem, flower bud, and root of Panax ginseng. In recent years, ginsenoside Re (Re) has been attracting attention as a dietary phytochemical. In this review, studies on Re were compiled by searching a combination of keywords, namely "pharmacology," "pharmacokinetics," and "toxicology," in the Google Scholar, NCBI, PubMed, and Web of Science databases. The aim of this review was to provide an exhaustive overview of the pharmacological activities, pharmacokinetics, and toxicity of Re, focusing on clinical evidence that has shown effectiveness in specific diseases, such as diabetes mellitus, nervous system diseases, inflammation, cardiovascular disease, and cancer. Re is also known to eliminate virus, enhance the immune response, improve osteoporosis, improve skin barrier function, enhance intracellular anti-oxidant actions, regulate cholesterol metabolism, alleviate allergic responses, increase sperm motility, reduce erectile dysfunction, promote cyclic growth of hair follicles, and reduce gastrointestinal motility dysfunction. Furthermore, this review provides data on pharmacokinetic parameters and toxicological factors to examine the safety profile of Re. Such data will provide a theoretical basis and reference for Re-related studies and future applications.
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The development of a compliant neural probe is necessary to achieve chronic implantation with minimal signal loss. Although fiber-based neural probes fabricated by the thermal drawing process have been proposed as a solution, their long-term effect on the brain has not been thoroughly investigated. Here, we examined the mechanical interaction of thermally drawn fiber implants with neural tissue through computational and histological analyses. Specifically, finite element analysis and immunohistochemistry were conducted to evaluate the biocompatibility of various fiber implants made with different base materials (steel, silica, polycarbonate, and hydrogel). Moreover, the effects of the coefficient of friction and geometric factors including aspect ratio and the shape of the cross-section on the strain were investigated with the finite element model. As a result, we observed that the fiber implants fabricated with extremely softer material such as hydrogel exhibited significantly lower strain distribution and elicited a reduced immune response. In addition, the implants with higher coefficient of friction (COF) and/or circular cross-sections showed a lower strain distribution and smaller critical volume. This work suggests the materials and design factors that need to be carefully considered to develop future fiber-based neural probes to minimize mechanical invasiveness.
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Schizophrenia is a severe mental disorder with an unclear pathophysiology. Increased expression of the immune gene C4 has been linked to a greater risk of developing schizophrenia; however, it is not known whether C4 plays a causative role in this brain disorder. Using confocal imaging and whole-cell electrophysiology, we demonstrate that overexpression of C4 in mouse prefrontal cortex neurons leads to perturbations in dendritic spine development and hypoconnectivity, which mirror neuropathologies found in schizophrenia patients. We find evidence that microglia-mediated synaptic engulfment is enhanced with increased expression of C4. We also show that C4-dependent circuit dysfunction in the frontal cortex leads to decreased social interactions in juvenile and adult mice. These results demonstrate that increased expression of the schizophrenia-associated gene C4 causes aberrant circuit wiring in the developing prefrontal cortex and leads to deficits in juvenile and adult social behavior, suggesting that altered C4 expression contributes directly to schizophrenia pathogenesis.
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Complemento C4/genética , Neuronas/fisiología , Corteza Prefrontal/citología , Esquizofrenia/genética , Conducta Social , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Animales Recién Nacidos , Comunicación Celular/genética , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vías Nerviosas/metabolismo , Corteza Prefrontal/patología , Esquizofrenia/patología , Regulación hacia Arriba/genéticaRESUMEN
Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5' end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.
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Proteína 9 Asociada a CRISPR/fisiología , División del ADN , ADN/química , ADN/metabolismo , Mutación con Ganancia de Función , ARN Guía de Kinetoplastida/farmacología , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , ADN/efectos de los fármacos , ADN Helicasas/fisiología , Edición Génica/métodos , Conformación de Ácido Nucleico/efectos de los fármacos , Ingeniería de Proteínas , ARN Guía de Kinetoplastida/análisis , ARN Guía de Kinetoplastida/metabolismo , Imagen Individual de Molécula , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/genéticaRESUMEN
The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.
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Sistemas CRISPR-Cas/genética , Biblioteca de Genes , HumanosRESUMEN
The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.
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Sistemas CRISPR-Cas , Evolución Molecular Dirigida , ADN/genética , Escherichia coli/genética , Edición Génica , Células HEK293 , Humanos , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/genética , Proteínas Recombinantes/química , Ribonucleoproteínas/química , Especificidad por SustratoRESUMEN
In this study, temporal and spatial variations in five defined molecular size fractions of dissolved organic matter (DOM) were examined for a well preserved wetland (Upo Wetland) and its surrounding areas, and the influencing factors were explored with many biotic and abioic parameters. For each DOM sample, the five size fractions were determined by size-exclusion chromatography coupled with organic carbon detector (SEC-OCD). For 2-year long monthly monitoring, bio-polymers (BP), humic substances (HS), building blocks (BB), low molecular-weight (LMW) neutrals, and LMW acids displayed the median values of 264, 1884, 1070, 1090, and 11 µg-CL(-1), respectively, accounting for 6.2%, 41.7%, 24.5%, 26.4%, and 0.4% of dissolved organic carbon (DOC). The dominant presence of HS indicated that terrestrial input played important roles in DOM composition of the freshwater ecosystem, which contrasted with coastal wetlands in other reports. Both seasonal and periodic patterns in the variations were found only for HS and BB among the size fractions. It was also notable that the sources of HS were seasonally shifted from aquagenic origin in winter to pedogenic origin in summer. The correlations among the size fractions revealed that BB and LMW neutrals might be degradation products from HS and humic-like substances (HS+BB), respectively, while LMW acids, from LMW neutrals. Principle component analysis revealed that the humic-like substances and the aromaticity of DOM were associated with temperature, chlorophyll a, phosphorous, and rainfall, whereas the other fractions and the molecular weight of HS were primarily affected by solar irradiation. Significant correlations between DOM composition and some biotic factors further suggested that DOM may even affect the biological communities, which provides an insight into the potential coupling effects of biotic and abiotic factors on DOM molecular composition in freshwater wetlands.
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Monitoreo del Ambiente , Agua Dulce/química , Sustancias Húmicas , HumedalesRESUMEN
Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.
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Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/genética , Ingeniería Genética , HumanosRESUMEN
We report here a new small molecule fluorogen and RNA aptamer pair for RNA labeling. The small-molecule fluorogen is designed on the basis of fluorescently quenched sulforhodamine dye. The SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure and fluorescence screening in E. coli have been applied to discover the aptamer that can specifically activate the fluorogen with micromolar binding affinity. The systematic mutation and truncation study on the aptamer structure determined the minimum binding domain of the aptamer. A series of rationally modified fluorogen analogues have been made to probe the interacting groups of fluorogen with the aptamer. These results led to the design of a much improved fluorogen ASR 7 that displayed a 33-fold increase in the binding affinity for the selected aptamer in comparison to the original ASR 1 and an 88-fold increase in the fluorescence emission after the aptamer binding. This study demonstrates the value of combining in vitro SELEX and E. coli fluorescence screening with rational modifications in discovering and optimizing new fluorogen-RNA aptamer labeling pairs.
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Colorantes Fluorescentes/química , Hibridación Fluorescente in Situ/métodos , ARN/análisis , Técnica SELEX de Producción de Aptámeros , Xantenos/química , Escherichia coli/química , Escherichia coli/genética , Fluorescencia , Mutación , ARN/química , ARN/genética , Xantenos/síntesis químicaRESUMEN
Geldanamycin and its analogs are important anticancer agents that inhibit the newly targeted, heat-shock protein (Hsp) 90, which is a chaperone protein in eukaryotic cells. To resolve which geldanamycin biosynthetic genes are responsible for particular post-polyketide synthase (PKS) processing steps and in which order the reactions occur, we individually inactivated candidate genes in Streptomyces hygroscopicus subsp. duamyceticus JCM4427, and isolated and elucidated the structures of intermediates from each mutant. The results indicated that gel7 governs at least one of the benzoquinone ring oxidation steps. In addition, gel16 was found to be involved in double-bond formation between C-4 and C-5 of 4,5-dihydrogeldanamycin, which confirmed our previous findings that this double bond reduced during the post-PKS modification of the polyketide assembly. In addition, pro-geldanamycin, which does not possess a double bond at C-4/5, was purified from the gel7 and 8 double-gene-inactivated mutant.
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Proteínas Bacterianas/metabolismo , Benzoquinonas/metabolismo , Lactamas Macrocíclicas/metabolismo , Familia de Multigenes , Sintasas Poliquetidas/metabolismo , Streptomyces/genética , Secuencia de Aminoácidos , Clonación Molecular , Genes Bacterianos , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Streptomyces/metabolismoRESUMEN
Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A. We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M A, verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.
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Intrones/genética , Metales/química , Animales , Catálisis , Cristalografía por Rayos X , Iones/química , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , ARN Catalítico/química , ARN Catalítico/metabolismo , Especificidad por Sustrato , Tetrahymena/enzimologíaRESUMEN
A new quinazolinedione alkaloid, wuchuyuamide IV (1) was isolated from the fruits of Evodia officinalis.1 showed moderate cytotoxicity against Hela and HT1080 cell lines.
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Alcaloides/química , Evodia/química , Frutas/química , Quinazolinonas/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Humanos , Estructura MolecularRESUMEN
The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains.