RESUMEN
In order to explore a new mode for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL), 31 cases of AITL and 28 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) were used as the study subjects. Identifying T follicular helper (TFH) cells with CD4, CD10, Bcl-6, and PD-1, identifying proliferative B cells with CD20 and EZH2, identifying proliferative follicular dendritic cells (FDCs) with CD21 and CD23, and analyzing the value of TFH/B/FDC proliferation and immunolocalization in the diagnosis of AITL. (1) Outside the inherent lymphoid follicles, simultaneous proliferation of TFH/B/FDC (a new diagnostic mode) were observed in AITL [83.87%; 26/31], with their immunolocalizations in the same site [83.87%; 26/31], while this phenomenon was not observed in 28 cases of PTCL-NOS (P<0.05). (2) The sensitivity and specificity of using this new mode to diagnose AITL were both high (83.87%, 100%), which was superior to CD2 (100%, 0%), CD3 (100%, 0%), CD4 (100%, 32.14%), CD5 (100%, 25%), CD10 (61.9%, 100%), Bcl-6 (42.86%, 100%), PD-1 (83.87%, 96.43%), and its Youden Index (0.84) was the highest. The areas under the curve (AUC) of CD10, Bcl-6, PD-1, and new mode to diagnosis AITL were 0.81, 0.71, 0.90, and 0.92, respectively, while the new mode had the highest AUC. The simultaneous proliferation of TFH/B/FDC cells outside the inherent lymphoid follicles can be used to assist in the diagnosis of AITL, and the simultaneous spatiotemporal proliferation of TFH/B/FDC cells is a specific immunomorphology of AITL.
Asunto(s)
Proteínas Proto-Oncogénicas c-bcl-6 , Humanos , Femenino , Masculino , Persona de Mediana Edad , Anciano , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Neprilisina/metabolismo , Linfadenopatía Inmunoblástica/diagnóstico , Linfadenopatía Inmunoblástica/patología , Células Dendríticas Foliculares/patología , Células Dendríticas Foliculares/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Adulto , Linfoma de Células T/diagnóstico , Linfoma de Células T/patología , Linfoma de Células T/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proliferación Celular , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células T Auxiliares Foliculares/inmunología , Células T Auxiliares Foliculares/metabolismo , Receptores de Complemento 3d/metabolismo , Receptores de Complemento 3d/análisis , Antígenos CD20/metabolismo , Antígenos CD20/análisis , Linfoma de Células T Periférico/diagnóstico , Linfoma de Células T Periférico/patología , Antígenos CD4/metabolismo , Sensibilidad y Especificidad , Anciano de 80 o más Años , Inmunohistoquímica/métodos , Curva ROCRESUMEN
On the basis of our previous comparative studies on the DNA binding of a pair of ruthenium(II) complex enantiomers, Δ-[Ru(bpy)2PBIP]2+ and Λ-[Ru(bpy)2PBIP]2+ {bpy = 2,2'-bipyridine, PBIP = 2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline}, in this study, their antitumor activities and mechanisms were further investigated comparatively. The cytotoxicity assay demonstrated that both the enantiomers exerted selective antiproliferative effects on cancer cell lines A2780 and PC3. Fluorescence localization experiments suggested that both the enantiomers effectively permeated the nucleus of HeLa cells and co-localized with DNA, resulting in their DNA damage and apoptosis. Flow cytometry experiments showed that the apoptosis was enhanced by increasing the concentration of each enantiomer. Western blotting analyses indicated that both extrinsic and intrinsic apoptosis pathways were activated by the two enantiomers. miRNA microarray analyses displayed that both the enantiomers up- and downregulated multiple miRNAs, some of which were predicted to be associated with carcinogenesis. The above experimental results also showed that the Δ-enantiomer exerted a more potent antitumor activity, a higher efficiency of entering cancer cells and a stronger apoptosis-inducing effect compared with the Λ-enantiomer. Combined with the previously published research results, experimental results from this study implied that the antitumor activity of a metal complex might have originated from the conformation change of DNA in tumor cells caused by the intercalation of the complex, that the antitumor mechanism of a metal complex could be related to its DNA-binding mode, and that the antitumor efficiency of a metal complex could result from its DNA-binding strength.
RESUMEN
A pair of ruthenium(II) complex enantiomers, Δ- and Λ-[Ru(bpy)2MBIP]2+ (bpy = 2,2'-bipyridine, MBIP = 2-(3-bromophenyl)imidazo[5,6-f]phenanthroline), were designed, synthesized, and characterized. Comparative studies between the enantiomers on their binding behaviors to calf thymus DNA (CT-DNA) were conducted using UV-visible, fluorescence, and circular dichroism spectroscopies, viscosity measurements, isothermal titration calorimetry, a photocleavage experiment, and molecular simulation. The experimental results indicated that both the enantiomers spontaneously bound to CT-DNA through intercalation stabilized by the van der Waals force or the hydrogen bond and driven by enthalpy and that Δ-[Ru(bpy)2MBIP]2+ intercalated into DNA more deeply than Λ-[Ru(bpy)2MBIP]2+ did and exhibited a better DNA photocleavage ability. Molecular simulation further indicated that Δ-[Ru(bpy)2MBIP]2+ more preferentially intercalated between the base pairs of CT-DNA to the major groove, and Λ-[Ru(bpy)2MBIP]2+ more favorably intercalated to the minor groove. These research findings should be very helpful to the understanding of the stereoselectivity mechanism of DNA-bindings of metal complexes, and be useful for the design of novel metal-complex-based antitumor drugs with higher efficacy and lower toxicity.
Asunto(s)
Compuestos Organometálicos , Rutenio , ADN/química , Estructura Molecular , Compuestos Organometálicos/química , Fenantrolinas/química , Rutenio/química , EstereoisomerismoRESUMEN
Presynaptic syntaxin binding protein 1 (STXBP1) is essential for neurotransmitter release. Heterozygous mutations in this protein cause STXBP1 encephalopathy (STXBP1-E), which is characterized by intellectual disabilities and epilepsies. Since nonhuman primates closely resemble humans, monkey models may advance studies on the pathogenesis and therapeutic treatments of STXBP1-E. We generated cynomolgus monkeys carrying STXBP1 (R292H) mutation through base editing of in vitro fertilized embryos to mimic a clinical condition. The newborn STXBP1-edited monkeys exhibited focal epilepsy, and the animal that survived beyond the first week postpartum presented typical EEG phenotypes. Biochemical analysis of brain biopsy samples showed reduced levels of STXBP1 (MUNC18-1) and SNARE complex proteins. Single-cell sequencing identified one specific cell cluster that may contribute to encephalopathy. Thus, our case report shows that base-edited STXBP1 mutant monkeys are a good animal model for STXBP1-E, and that a base-editing approach is useful for generating primate models of human genetic disorders.
Asunto(s)
Encefalopatías , Epilepsia , Animales , Encéfalo/metabolismo , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Femenino , Macaca fascicularis/metabolismo , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , MutaciónRESUMEN
Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and â¼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.