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Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic resonance (EPR) spectroscopy and dynamic nuclear polarization (DNP). In this work, we have demonstrated that two semiquinone methide radicals (OXQMâ and CTQMâ ) can be easily produced from the trityl-based quinone methides (OXQM and CTQM) via reduction by various reducing agents including biothiols and ascorbate under anaerobic conditions. Both radicals have relatively low pKa's and exhibit EPR single line signals at physiological pH. Moreover, the bioreduction of OXQM in three cell lysates enables quantitative generation of OXQMâ which was most likely mediated by flavoenzymes. Importantly, the resulting OXQMâ exhibited extremely high stability in the E.coli lysate under anaerobic conditions with 76- and 14.3-fold slower decay kinetics as compared to the trityl OX063 and a gem-diethyl pyrrolidine nitroxide, respectively. Intracellular delivery of OXQM into HeLa cells was also achieved by covalent conjugation with a cell-permeable peptide as evidenced by the stable intracellular EPR signal from the OXQMâ moiety. Owing to extremely high resistance of OXQMâ towards bioreduction, OXQM and its derivatives show great application potential in in-cell EPR and in-cell DNP studies for various cells which can endure short-term anoxic treatments.
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Indolquinonas , Oxidación-Reducción , Humanos , Células HeLa , Espectroscopía de Resonancia por Spin del Electrón , Indolquinonas/química , Anaerobiosis , Benzoquinonas/química , Benzoquinonas/metabolismo , Compuestos de Tritilo/química , Escherichia coli/metabolismo , Marcadores de SpinRESUMEN
Despite limited research on refractory and/or endocrine therapy failure in elderly metastatic breast cancer (MBC) patients, a prior study showed that low-dose oral cyclophosphamide (CY) can improve the overall survival rate of MBC patients, possibly through the immunoregulation of regulatory T cells (Tregs). We preliminarily investigated the combination of endocrine therapy (ET) with oral low-dose CY as salvage therapy in elderly patients via peripheral blood regulatory T-cell analyses. In addition, we evaluated the associations of tumor tertiary lymphoid structures (TLSs) with therapeutic outcomes. HR+/HER2- advanced breast cancer patients who received low-dose CY combined with ET or ET only from April 2015 to August 2021 were enrolled in this retrospective study. The primary outcome was the clinical control rate (CCR), and the secondary outcome was progression-free survival (PFS). Circulating T lymphocyte subpopulations represented by Tregs were monitored during treatment by flow cytometry methods. TLSs wereconfirmed by hematoxylin-eosin staining of pretreatment specimens, and CD3, CD4, and Foxp3 were detected using Opal multicolor immunofluorescence. A total of 85 patients who received CY + ET and 50 patients who received ET only were enrolled, the percentage of patients who received CCR was 73% (62/85) vs. 70% (45/50), and the objective response rate (ORR) was 28% (24/85) vs. 24% (12/50). No deaths occurred during the study period. The mean PFS time was 13 vs. 11 months (P = 0.03). In the CY + ET group, decreases in CD4+/CD25+/Foxp3+ T cells (P < 0.001) were favorable for both clinical control and prolonged PFS (P < 0.001). Compared with patients without TLSs, those with TLSs were more likely to have better clinical control and PFS (mean time = 6 months), and a greater number of Treg cells during TLS pretreatment correlated with longer PFS (P = 0.043). Oral low-dose CY combined with standard ET exerts immunological effects by decreasing Treg levels to achieve improved clinical responses. Moreover, patients with TLSs might benefit more from such therapy than those without TLSs, and a high Treg cell count in TLSs before treatment predicts better therapeutic efficacy.
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Neoplasias de la Mama , Ciclofosfamida , Linfocitos T Reguladores , Humanos , Ciclofosfamida/administración & dosificación , Ciclofosfamida/uso terapéutico , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/mortalidad , Anciano , Estudios Retrospectivos , Administración Oral , Persona de Mediana Edad , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Metástasis de la Neoplasia , Resultado del TratamientoRESUMEN
In the original publication [...].
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To boost the enzyme-like activity, biological compatibility, and antiaggregation effect of noble-metal-based nanozymes, folic-acid-strengthened Ag-Ir quantum dots (FA@Ag-Ir QDs) were developed. Not only did FA@Ag-Ir QDs exhibit excellent synergistic-enhancement peroxidase-like activity, high stability, and low toxicity, but they could also promote the lateral root propagation of Arabidopsis thaliana. Especially, ultratrace cysteine or Hg2+ could exclusively strengthen or deteriorate the inherent fluorescence property with an obvious "turn-on" or "turn-off" effect, and dopamine could alter the peroxidase-like activity with a clear hypochromic effect from blue to colorless. Under optimized conditions, FA@Ag-Ir QDs were successfully applied for the turn-on fluorescence imaging of cysteine or the stress response in cells and plant roots, the turn-off fluorescence monitoring of toxic Hg2+, or the visual detection of dopamine in aqueous, beverage, serum, or medical samples with low detection limits and satisfactory recoveries. The selective recognition mechanisms for FA@Ag-Ir QDs toward cysteine, Hg2+, and dopamine were illustrated. This work will offer insights into constructing some efficient nanozyme sensors for multichannel environmental analyses, especially for the prediagnosis of cysteine-related diseases or stress responses in organisms.
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Mercurio , Puntos Cuánticos , Puntos Cuánticos/toxicidad , Cisteína , Dopamina , Ácido Fólico , Imagen Óptica , Peroxidasas , Raíces de PlantasRESUMEN
Hydrogen sulfide (H2S) plays a critical role in cancer biology. Herein, we developed a series of glycosidase-triggered hydrogen sulfide (H2S) donors by connecting sugar moieties (including glucose, galactose and mannose) to COS donors via a self-immolative spacer. In the presence of corresponding glycosidases, H2S was gradually released from these donors in PBS buffer with releasing efficiencies from 36 to 67 %. H2S release was also detected by H2S probe WSP-1 after treatment HepG2 cells with Man1. Cytotoxicities of these glycosylated H2S donors were evaluated against HepG2 by MTT assay. Among them, Man1 and Man2 exhibited an obvious reduction of cell viability in HepG2 cells, with cell viability as 37.6 % for 80 µM of Man. Consistently, significant apoptosis was observed in HepG2 cells after treatment with Man1 and Man2. Finally, We evaluated the potential of Man1 for combination therapy with doxorubicin. A synergistic effect was observed between Man1 and Doxorubicin in HepG2 and Hela cells. All these results indicated glycosidase-activated H2S donorshave promising potential for cancer therapy.
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Sulfuro de Hidrógeno , Humanos , Células HeLa , Sulfuro de Hidrógeno/farmacología , Óxidos de Azufre , Doxorrubicina/farmacología , Glicósido HidrolasasRESUMEN
Alfalfa (Medicago sativa L.), a crucial and widely grown forage legume, faces yield and quality challenges due to salinity stress. The defender against apoptotic death (DAD) gene, recognized initially as an apoptosis suppressor in mammals, plays a pivotal role in catalyzing N-glycosylation, acting as a positive regulator for protein folding and endoplasmic reticulum (ER) export. Here, we found that the MsDAD2 gene was specially induced in the salt-tolerant alfalfa cultivar (DL) under salinity stress, but not in the salt-sensitive cultivar (SD). Overexpression of MsDAD2 enhanced the salinity resistance of transgenic alfalfa by promoting NAD(P)H-quinone oxidoreductase (NQO1) and cytochrome b6f complex subunit (Cyt b6/f) expression, thereby mitigating reactive oxygen species (ROS) production. ChIP-qPCR analysis suggested that the differential expression of MsDAD2 in DL and SD under salinity stress may be linked to dynamic histone modifications in its promoter. Therefore, our findings elucidate a novel regulatory mechanism of MsDAD2 in alfalfa's response to salinity stress, underscoring its significance as a target for alfalfa breeding to enhance salt tolerance.
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Regulación de la Expresión Génica de las Plantas , Medicago sativa , Medicago sativa/genética , Medicago sativa/metabolismo , Estrés Salino/genética , Tolerancia a la Sal/genética , SalinidadRESUMEN
Systematic assessment of adverse side effects of Adoptive T cell therapy, especially cytokine-induced killer cell and dendric cell treatment Dendritic cells-Cytokine-induced killer (DC-CIK) therapy, especially when combined with chemotherapy, has not been reported. Totally 1100 consecutive patients (2504 trail cycles) enrolled in DC-CIK treatment trials at Beijing Shijitian Hospital between August 2012 and August 2022 were retrospectively reviewed. The 370 patients (34%)/815 cycles enrolled in our trial combined with chemotherapy. In total, 548 (cases)/870 (cycles) patients experienced AEs. The AE class was mainly composed of Neurological 34 cycles (4%), Musculoskeletal 28 cycles (3%), Immunopathies 5 cycles (1%), Hematological 521 cycles (60%), 224 general disorders and administration site conditions cycles (26%), Gastrointestinal 209 cycles (24%), Skin 15 cycles (2%), and 119 Metabolism and Nutrition disorders cycles (14%). The AE class of gastrointestinal (vomiting, P=0.025), nutritional (anorexia, P=0.016), and hematological disorders (anemia P<0.0001, leukopenia P<0.0001) appeared in the DC-CIK treatment and were mainly correlated with chemotherapy. Multiple logistic regression analysis suggested that regardless of whether DC-CIK was combined with chemotherapy, multi-line treatment was more prone to nausea, anorexia, fatigue, anemia, and leukopenia than first-line treatment. However, correlation analysis verified that increasing the number of cycles of DC-CIK treatment alone could reduce the incidence rate of fatigue (P=0.001), anorexia (P<0.0001), and anxiety (P=0.01). Most of the adverse side effects that occurred during autologous DC-CIK treatment were associated with combined or previously applied chemotherapeutic treatment, which also indicated that autologous DC-CIK anti-tumor therapy was safe.
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Electron paramagnetic resonance (EPR) spectroscopy and imaging coupled with the use of suitable probes is a promising tool for assessment of the tumor microenvironment (TME). Measurement of multiple TME parameters by EPR is very desirable but challenging. Herein, we designed and synthesized a class of negative-charged trityl quinodimethane MTPs as unimolecular triple-function extracellular probes for redox, pH, and oxygen (O2) levels. Using the deuterated analogue, dMTP5, which has an optimal pKa as well as high sensitivity to bioreduction and O2, we reasonably evaluated pH effects on efflux of reducing agents from HepG2 cells and cellular O2 consumption.
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Oxígeno , Sustancias Reductoras , Espectroscopía de Resonancia por Spin del Electrón/métodos , Oxígeno/química , Oxidación-Reducción , Concentración de Iones de HidrógenoRESUMEN
Nonsteroidal anti-inflammatory drugs (NSAIDs) increase risks of severe small intestinal injuries. Development of effective therapeutic strategies to overcome this issue remains challenging. Nitric oxide (NO) as a gaseous mediator plays a protective role in small intestinal injuries. However, small intestine-specific delivery systems for NO have not been reported yet. In this study, we reported a small intestine-targeted polymeric NO donor (CS-NO) which was synthesized by covalent grafting of α-glucosidase-activated NO donor onto chitosan. In vitro and in vivo experiments demonstrated that CS-NO could be activated by intestinal α-glucosidase to release NO in the small intestine. Pre-treatment of mice with CS-NO significantly alleviated small intestinal damage induced by indomethacin, as demonstrated by down-regulation of the levels of pro-inflammatory cytokines and chemokines CXCL1/KC. Moreover, CS-NO also attenuated indomethacin-induced gut barrier dysfunction as evidenced by up-regulation of the levels of tight junction proteins and restoration of the levels of goblet cells and MUC2 production. Meanwhile, CS-NO effectively restored the defense function of Paneth cells against pathogens in small intestine. Our present study paves the way to develop NO-based therapeutic strategy for NSAIDs-induced small intestinal injuries.
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Óxido Nítrico , alfa-Glucosidasas , Ratones , Animales , Óxido Nítrico/metabolismo , alfa-Glucosidasas/metabolismo , alfa-Glucosidasas/farmacología , Antiinflamatorios no Esteroideos/efectos adversos , Indometacina/efectos adversos , Indometacina/metabolismo , Intestino Delgado/lesiones , Intestino Delgado/metabolismoRESUMEN
Cellular dynamic nuclear polarization (DNP) has been an effective means of overcoming the intrinsic sensitivity limitations of solid-state nuclear magnetic resonance (ssNMR) spectroscopy, thus enabling atomic-level biomolecular characterization in native environments. Achieving DNP signal enhancement relies on doping biological preparations with biradical polarizing agents (PAs). Unfortunately, PA performance within cells is often limited by their sensitivity to the reductive nature of the cellular lumen. Herein, we report the synthesis and characterization of a highly bioresistant and hydrophilic PA (StaPol-1) comprising the trityl radical OX063 ligated to a gem-diethyl pyrroline nitroxide via a rigid piperazine linker. EPR experiments in the presence of reducing agents such as ascorbate and in HeLa cell lysates demonstrate the reduction resistance of StaPol-1. High DNP enhancements seen in small molecules, proteins and cell lysates at 18.8 T confirm that StaPol-1 is an excellent PA for DNP ssNMR investigations of biomolecular systems at high magnetic fields in reductive environments.
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The adoptive cell therapy (ACT) and delivery of ex vivo activated cellular products, such as dendritic cells (DCs), NK cells, and T cells, have shown promise for the treatment of gastric cancer (GC). However, it is unknown which cells can improve patient survival. This study was focused on the antitumour activity of a subset of these cellular products and their relationships with clinical outcomes. Nineteen patients were enrolled at the Capital Medical University Cancer Center, Beijing Shijitan Hospital, from June 1, 2013, to May 30, 2016. CD8+PD1+ T-cell sorting was carried out using flow cytometry, and the T-cell receptor (TCR) repertoire during ex vivo expansion for 15 days was analyzed by next-generation sequencing. After 15 days of culture, the number of CD8+ T cells had increased significantly, and the number of CD4+ T cells had increased correspondingly. After ex vivo expansion, CD8+ T cells exhibited significantly enhanced expression of PD-1, LAG-3, and TIM-3 but not 4-1BB. Survival analysis showed that patients with a pro/pre value of CD8+PD-1+ T cells >2.4 had significantly favorable overall survival (OS) (median OS time, 248 days versus 96 days, P=0.02) and progression-free survival (PFS) (median PFS time, 183 days vs. 77 days, P=0.002). The sorted CD8+PD-1+ T cells displayed enhanced antitumor activity and increased IFN-γ secretion after coculture with autologous tumor cell lines. TCR repertoire diversity was decreased after ex vivo expansion, which decreased the Shannon index and increased the clonality value. The prognosis of patients was significantly improved and was associated with the extent of CD8+PD-1+ T-cell expansion. In summary, this study showed that after ex vivo expansion for 15 days, CD8+PD-1+ T cells could be identified as tumor-reactive cells in patients treated for GC. Changing TCR species can predict the extent of CD3+CD8+PD1+ T-cell growth and the effect of ACT treatment.
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Purpose: We aimed to investigate the restoration of CD8+PD-1+ T cells through adoptive T-cell therapy (ACT) in relation to the prognosis and the therapeutic response to anti-PD-1 in patients with advanced pancreatic cancer (APC). Methods: A total of 177 adult patients who underwent tumor resection as initial treatment for pancreatic ductal adenocarcinoma (PDAC) from February 2013 to July 2019 at Zhongnan Hospital of Wuhan University were enrolled in this study. Another cohort of 32 patients with APC was prospectively enrolled from Capital Medical University Cancer Center between June 1, 2013, and May 30, 2019. Results: Of the 177 patients who received tumor resection, 67 tumor samples showed overexpression of PD-L1 and 110 patients with low expression of PD-L1. We found that overexpressed PD-L1 was a significant prognostic factor related to overall survival (OS). Furthermore, we tested the percentage of peripheral CD8+PD-1+ T cells in all patients and found that it was significantly correlated with the PD-L1 expression and the prognosis of patients with PDAC. The peripheral blood T lymphocyte subtypes were tracked for 30 months, and CD8+PD-1+ cells were shown to decrease. After that, we performed ACT for patients with APC in another cancer center. We found that the ratios of posttreatment of ACT/pre-ACT CD8+PD-1+ T cells were significantly related to the prognosis of patients with APC. Moreover, patients with combined treatment of ACT with anti-PD-1 had significantly favorable OS. Conclusions: This study showed that the CD8+PD-1+ T-cell level was related to the expression of PD-L1. Restoring CD8+PD-1+ T cells in patients with APC by treatment of ACT significantly benefits the prognosis and facilitates the response to anti-PD-1.
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The human epidermal growth factor receptor-2 (ERBB2; formerly HER2)isoform ERBB2ΔEx16 (ERBB2d16) was oncogenic by mediating epithelial-mesenchymal transition (EMT), immune evasion, and resistance cell death to the anti-HER2 (trastuzumab) therapy. However, its physiological implications in gastric cancer were unclear. In this study, we examined a total of 110 patients with either locally advanced or metastatic HER2+ gastric cancer for the expression of ERBB2d16 and EMT markers, and the infiltration of CD3+ T cells in tumor tissues, and evaluated their relevance with the responses to the standard chemotherapy plus trastuzumab according to the RECIST criteria. We found that the ERBB2d16 isoform was present at a relatively high level in about half of the tumor samples examined (53/110) and an elevated ERBB2d16/ERBB2 ratio was positively associated with the expression of high E-cadherin and low vimentin indicating EMT, and with poor CD3+ T cell infiltration and strong intratumoral expression of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) as well as reduced diversity of T cell receptor clones. Moreover, the progression-free survival and overall survival of patients treated with trastuzumab were substantially shorter in those with a high ERBB2d16/ERBB2 ratio. In agreement, analysis by Cox proportional hazards models confirmed that high ERBB2d16 expression was a risk factor associated with an adverse prognosis. Thus, our data fit well with an oncogenic role of ERBB2d16 in gastric cancer by promoting EMT and immunosuppression. We also found that ERBB2d16 expression resists gastric cell death in patients treated with trustuzumab, and the ERBB2d16/ERBB2 ratio may serve as a novel prognostic maker for patients with gastric cancer that receive trastuzumab therapy.
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In e-commerce, sellers can disclose product information (such as quality, size information, function, and so on) to make consumers understand the products. However, in the process of information disclosure, consumers often fall into information distortion or information loss. Because of its immutability and traceability, blockchain can help e-commerce sellers improve information disclosure and ensure the efficiency of information transmission. We study a duopoly competitive e-commerce market in which two e-commerce sellers compete in information disclosure. According to whether to apply blockchain, we divide the sellers' decision-making into four research scenarios (NN, BN, NB, BB). Based on the above four scenarios, we get the market demand of different products depending on the consumer utility, and further establish the game model in the competitive environment. This paper explores the impact of blockchain on information disclosure and consumer surplus, and achieves the Nash equilibrium of blockchain application for both sides. In the expansion model, we study e-commerce sellers' risk aversion and capital constraints, and further explore their impact on blockchain in practice. Finally, combining with blockchain's characteristics, we also analyze the impact of the application of blockchain at other aspects on the supply chain. We find that when consumers' trust in information is low or the cost of blockchain applications is low, all e-commerce sellers in competition will adopt blockchain. In addition, when consumers have low trust in information, it will be difficult to achieve complete equilibrium in the application of blockchain as their risk aversion increases. For capital constrained sellers, when the cost of blockchain application is low, it will be difficult to achieve full equilibrium for blockchain applicants as the bank financing rate increases.
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Epigenetic modifications within promoter sequences can act as regulators of gene expression. Shoot regeneration is influenced by both DNA methylation and histone methylation, but the mechanistic basis of this regulation is obscure. Here, we identified 218 genes related to the regeneration capacity of callus that were differentially transcribed between regenerable calli (RC) and non-regenerable calli (NRC) in Arabidopsis thaliana. An analysis of the promoters of five of the differentially expressed genes (FWA, ACC1, TFL1, MAX3, and GRP3) pointed to an inverse relationship between cytosine methylation and transcription. The FWA promoter was demethylated and highly expressed in NRC, whereas it was methylated and expressed at low levels in RC. Explants of the hypomethylation mutants fwa-1 and fwa-2 showed strong levels of FWA expression and regenerated less readily than the wild type, suggesting that FWA inhibits direct in vitro shoot regeneration. WUSCHEL-RELATED HOMEOBOX 9 (WOX9), which is required for shoot apical meristem formation, was directly repressed by FWA. Overexpressing WOX9 partly rescued the shoot regeneration defect of fwa-2 plants. These findings suggest that cytosine methylation of the FWA promoter forms part of the regulatory system governing callus regenerability and direct in vitro shoot regeneration.
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Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Citosina/metabolismo , Metilación de ADN/genética , Proteínas de Homeodominio/genética , Brotes de la Planta/fisiología , Regiones Promotoras Genéticas , Regeneración/fisiología , Factores de Transcripción/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Mutación/genética , Brotes de la Planta/citología , Brotes de la Planta/ultraestructura , Unión Proteica , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Dynamic nuclear polarization (DNP) is a powerful method to enhance the sensitivity of solid-state magnetic nuclear resonance (ssNMR) spectroscopy. However, its biomolecular applications at high magnetic fields (preferably>14â T) have so far been limited by the intrinsically low efficiency of polarizing agents and sample preparation aspects. Herein, we report a new class of trityl-nitroxide biradicals, dubbed SNAPols that combine high DNP efficiency with greatly enhanced hydrophilicity. SNAPol-1, the best compound in the series, shows DNP enhancement factors at 18.8 T of more than 100 in small molecules and globular proteins and also exhibits strong DNP enhancements in membrane proteins and cellular preparations. By integrating optimal sensitivity and high resolution, we expect widespread applications of this new polarizing agent in high-field DNP/ssNMR spectroscopy, especially for complex biomolecules.
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Campos Magnéticos , Óxidos de Nitrógeno , Espectroscopía de Resonancia Magnética , Proteínas de la MembranaRESUMEN
Tetrathiatriarylmethyl (trityl) radicals have been recently shown to react with biological oxidoreductants including glutathione (GSH), ascorbic acid (Asc), and superoxide anion radical (O2â¢-). However, how the substituents affect the reactivity of trityl radicals is still unknown. In this work, five asymmetric trityl radicals were synthesized and their reactivities with GSH, Asc, and O2â¢- investigated. Under aerobic conditions, GSH induces fast decays for the thioether- (TSA) and N-methyleneglycine-substituted (TGA) derivatives and slow decay for the 4-carboxyphenyl-containing one (TPA). Under anaerobic conditions, the direct reduction of these radicals by GSH also occurs with rate constants (kGSH) from 1.8 × 10-4 M-1 s-1 for TPA to 1.0 × 10-2 M-1 s-1 for TGA. Moreover, these radicals can also react with O2â¢- with rate constants (kSO) from 1.2 × 103 M-1 s-1 for ET-01 to 1.6 × 104 M-1 s-1 for TGA. Surprisingly, these radicals are completely inert to Asc in both aerobic and anaerobic conditions. Additionally, the substituents exert an important effect on redox potentials of these trityl radicals. This work demonstrates that the redox properties of the trityl radicals strongly depend on their substituents, and TPA with high stability toward GSH shows great potential for intracellular applications.
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Compuestos de Tritilo , Agua , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Oxidación-ReducciónRESUMEN
Recurrence and progression of non-muscle-invasive bladder cancer (NMIBC), frequent despite the availability of multiple treatment modalities, may be partly explained by the presence of immunosuppressive cell populations. We hypothesized that progression of disease could be prevented by the administration of an activated T cell immunotherapy (ACT) at time points when immunosuppressive populations increased in peripheral blood. In an N-of-1 study, a patient with multiple primary bladder high grade urothelial carcinomas, previously treated with standard local resection and chemotherapy but with evidence of progression, received ACT consisting of dendritic cells mixed with cytokine induced killer cells (DC/CIK), intravenously 18 times over a 6 year period at indicated time of observed increases in peripheral blood immunosuppressive CD8+/CD28- cells. Peripheral blood was analyzed for T cell phenotype by flow cytometry, T cell receptor (TCR) repertoire, and circulating tumor DNA (ctDNA) by next generation sequencing (NGS) at the time of each infusion. Cystoscopy and pelvic CT scans were performed at routine intervals to assess clinical status of disease. There has been no recurrence or metastasis of urothelial carcinoma. Peripheral blood cytotoxic T cells and unique TCR clones increased and suppressive T cell populations decreased after DC/CIK infusions evidenced by the two more proof-of concept cases. ctDNA analysis detected mutations in six genes (ARID1B, MYCN, CDH23, SETD2, NOTCH4 and FAT1) which appeared at different times, but all of them disappeared after the DC-CIK infusions. These data suggest that DC/CIK infusions may be associated with beneficial changes in T cell phenotype, TCR repertoire, decreases in circulating tumor DNA and sustained recurrence-free survival.
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Fluorescent imaging of cellular superoxide anion radical (O2â¢-) is of great significance to investigate reactive oxygen species-related pathophysiological processes and drug metabolism. However, the application of this technique is far away from maximum partially due to the lack of suitable probes. In this work, we propose a new strategy for design of near-infrared (NIR) O2â¢- fluorescent probes in which p-cresol is used as a self-immolative linker to conjugate the NIR fluorophore DDAO (9H-1,3-Dichloro-7-hydroxy-9,9-dimethylacridine-2-one) with the O2â¢--sensing group (i.e., trifluoromethanesulfonate). The introduction of self-immolative linker effectively increases the self-stability of these probes under physiological conditions. Importantly, the electron-withdrawing halogen substituents on the linker greatly enhance the sensitivity of the probes to O2â¢-. As such, the representative probe DLS4 exhibits high self-stability over a broad range of pHs (5.0-8.5), high selectivity as well as excellent sensitivity to O2â¢- with a detection limit (LOD) of 7.3 nM and 720-fold fluorescence enhancement upon reaction with O2â¢-. Moreover, DLS4 enables imaging of O2â¢- generation in PMA-stimulated RAW 264.7 cells and HeLa cells, and the fluorescence intensities are proportional to the PMA concentrations. In addition, the doxorubicin-induced cytotoxicity of H9c2 cells was also evaluated using DLS4. The present study provides a novel strategy for molecular design of small-molecule O2â¢- fluorescent probes and the resulting probes show great potential as reliable tools to study the development and progression of O2â¢--related diseases and drug metabolism in various systems.
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Colorantes Fluorescentes , Superóxidos , Fluorescencia , Células HeLa , Humanos , Especies Reactivas de OxígenoRESUMEN
An efficient method for the synthesis of difluoroalkylated 2-azaspiro[4.5]decanes via copper-catalyzed difluoroalkylation of N-benzylacrylamides with ethyl bromodifluoroacetate has been established. The reaction experienced a tandem radical addition and dearomatizing cyclization process. In addition, the resultant products can be smoothly converted into a difluoroalkylated quinolinone and saturated spirocyclohexanone scaffold.