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Characterizing the profiles of proteome and metabolome at the single-cell level is of great significance in single-cell multiomic studies. Herein, we proposed a novel strategy called one-shot single-cell proteome and metabolome analysis (scPMA) to acquire the proteome and metabolome information in a single-cell individual in one injection of LC-MS/MS analysis. Based on the scPMA strategy, a total workflow was developed to achieve the single-cell capture, nanoliter-scale sample pretreatment, one-shot LC injection and separation of the enzyme-digested peptides and metabolites, and dual-zone MS/MS detection for proteome and metabolome profiling. Benefiting from the scPMA strategy, we realized dual-omic analysis of single tumor cells, including A549, HeLa, and HepG2 cells with 816, 578, and 293 protein groups and 72, 91, and 148 metabolites quantified on average. A single-cell perspective experiment for investigating the doxorubicin-induced antitumor effects in both the proteome and metabolome aspects was also performed.
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Proteoma , Espectrometría de Masas en Tándem , Humanos , Proteoma/metabolismo , Cromatografía Liquida , Metaboloma , Células HeLaRESUMEN
In this work, metal-organic frameworks (MOFs) are utilized as effective ECL coreactant accelerator to enhance the ECL responses of N-(aminobutyl)-N-(ethylisoluminol) (ABEI). Zn-based MOFs (MOF-Zn-1) were prepared by chelating Zn ions with melamine and thiophenedicarboxylic acid (TPDA), which observably accelerated the electrocatalytic oxidation of tripropylamine (TPA). Then, ABEI-MOF-Zn-1 as a high-performance ECL emitter was synthesized via an amide reaction between ABEI and mercaptopropionic acid (MPA) modified MOF-Zn-1. Strikingly, the ABEI-MOF-Zn-1 showed the 18-fold increase in the ECL signals relative to pure ABEI by using TPA as a coreactant. Moreover, ferrocene (Fc) as a quencher was first linked with capture DNA (cDNA), and then used to modify the ABEI-MOF-Zn-1, thereby constructing a label-free ECL biosensor. After the linkage between chloramphenicol (CAP) and aptamer DNA (aptDNA), the ECL response was definitely recovered by releasing L-DNA from double-stranded DNA (dsDNA, hybridization of aptDNA and L-DNA). The resultant sensor showed a wide linear range of 1.00 nM-0.10 mM (R2 = 0.99) and a low limit of detection (LOD) down to 0.11 nM for detecting CAP. This work developed a novel pattern to design an efficient ECL enhanced emitter, coupled by expanding its potential applications in clinical diagnosis.
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Técnicas Biosensibles , Nanopartículas del Metal , Estructuras Metalorgánicas , Cloranfenicol , Técnicas Electroquímicas , Límite de Detección , Mediciones LuminiscentesRESUMEN
The robust and strong electrochemiluminescence (ECL) emission of organic emitters in an aqueous solution is crucial for expanding their applications in early diagnosis. Herein, a Zn porphyrin-based metal-organic framework ((Zn)porphMOF) was facilely obtained by chelating Zn(ii)meso-tetra (4-sulfonatophenyl) porphine (Zn-TSPP) with Zn ions, showing substantially enhanced ECL radiation with K2S2O8 as the coreactant via the "reduction-oxidation" route in aqueous media. In contrast with Zn-TSPP, (Zn)porphMOF displayed 22-fold increase in the ECL intensity because of the agglomeration effect. By virtue of the dramatic confinement towards the energy and electron transfer of ascorbic acid (AA) during the ECL process, an ultrasensitive biosensor was developed with a wide linear range (3.77 to 26.4 µM) and ultra-low detection limit of 0.29 µM at 3 times of the signal-to-noise ratio (3S/N). This work offers a feasible avenue to harvest the steady and boosted ECL responses of organic molecules in aqueous media, also greatly expanding the MOF applications in bioanalysis.
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The autonomic nervous system plays a crucial role in regulating bone metabolism, with sympathetic activation stimulating bone resorption and inhibiting bone formation. We found that fractures lead to increased sympathetic tone, enhanced osteoclast resorption, decreased osteoblast formation, and thus hastened systemic bone loss in ovariectomized (OVX) mice. However, the combined administration of parathyroid hormone (PTH) and the ß-receptor blocker propranolol dramatically promoted systemic bone formation and osteoporotic fracture healing in OVX mice. The effect of this treatment is superior to that of treatment with PTH or propranolol alone. In vitro, the sympathetic neurotransmitter norepinephrine (NE) suppressed PTH-induced osteoblast differentiation and mineralization, which was rescued by propranolol. Moreover, NE decreased the PTH-induced expression of Runx2 but enhanced the expression of Rankl and the effect of PTH-stimulated osteoblasts on osteoclastic differentiation, whereas these effects were reversed by propranolol. Furthermore, PTH increased the expression of the circadian clock gene Bmal1, which was inhibited by NE-ßAR signaling. Bmal1 knockdown blocked the rescue effect of propranolol on the NE-induced decrease in PTH-stimulated osteoblast differentiation. Taken together, these results suggest that propranolol enhances the anabolic effect of PTH in preventing systemic bone loss following osteoporotic fracture by blocking the negative effects of sympathetic signaling on PTH anabolism.
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Anabolizantes , Resorción Ósea , Fracturas Osteoporóticas , Ratones , Animales , Hormona Paratiroidea/farmacología , Anabolizantes/farmacología , Fracturas Osteoporóticas/tratamiento farmacológico , Propranolol/farmacología , Factores de Transcripción ARNTL , Resorción Ósea/tratamiento farmacológico , Antagonistas Adrenérgicos beta/farmacologíaRESUMEN
The shotgun proteomic analysis is currently the most promising single-cell protein sequencing technology, however its identification level of ~1000 proteins per cell is still insufficient for practical applications. Here, we develop a pick-up single-cell proteomic analysis (PiSPA) workflow to achieve a deep identification capable of quantifying up to 3000 protein groups in a mammalian cell using the label-free quantitative method. The PiSPA workflow is specially established for single-cell samples mainly based on a nanoliter-scale microfluidic liquid handling robot, capable of achieving single-cell capture, pretreatment and injection under the pick-up operation strategy. Using this customized workflow with remarkable improvement in protein identification, 2449-3500, 2278-3257 and 1621-2904 protein groups are quantified in single A549 cells (n = 37), HeLa cells (n = 44) and U2OS cells (n = 27) under the DIA (MBR) mode, respectively. Benefiting from the flexible cell picking-up ability, we study HeLa cell migration at the single cell proteome level, demonstrating the potential in practical biological research from single-cell insight.
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Proteoma , Proteómica , Animales , Humanos , Células HeLa , Proteómica/métodos , Proteoma/metabolismo , Análisis de la Célula Individual , Flujo de Trabajo , Mamíferos/metabolismoRESUMEN
Although single-cell multi-omics technologies are undergoing rapid development, simultaneous transcriptome and proteome analysis of a single-cell individual still faces great challenges. Here, we developed a single-cell simultaneous transcriptome and proteome (scSTAP) analysis platform based on microfluidics, high-throughput sequencing, and mass spectrometry technology to achieve deep and joint quantitative analysis of transcriptome and proteome at the single-cell level, providing an important resource for understanding the relationship between transcription and translation in cells. This platform was applied to analyze single mouse oocytes at different meiotic maturation stages, reaching an average quantification depth of 19,948 genes and 2,663 protein groups in single mouse oocytes. In particular, we analyzed the correlation of individual RNA and protein pairs, as well as the meiosis regulatory network with unprecedented depth, and identified 30 transcript-protein pairs as specific oocyte maturational signatures, which could be productive for exploring transcriptional and translational regulatory features during oocyte meiosis.
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Proteoma , Transcriptoma , Animales , Ratones , Transcriptoma/genética , Proteoma/metabolismo , Oocitos/metabolismo , Oogénesis/genética , Perfilación de la Expresión Génica , MeiosisRESUMEN
PURPOSE: Adults with T-cell lymphoblastic lymphoma (T-LBL) generally benefit from treatment with acute lymphoblastic leukemia (ALL)-like regimens, but approximately 40% will relapse after such treatment. We evaluated the value of CpG methylation in predicting relapse for adults with T-LBL treated with ALL-like regimens. EXPERIMENTAL DESIGN: A total of 549 adults with T-LBL from 27 medical centers were included in the analysis. Using the Illumina Methylation 850K Beadchip, 44 relapse-related CpGs were identified from 49 T-LBL samples by two algorithms: least absolute shrinkage and selector operation (LASSO) and support vector machine-recursive feature elimination (SVM-RFE). We built a four-CpG classifier using LASSO Cox regression based on association between the methylation level of CpGs and relapse-free survival in the training cohort (n = 160). The four-CpG classifier was validated in the internal testing cohort (n = 68) and independent validation cohort (n = 321). RESULTS: The four-CpG-based classifier discriminated patients with T-LBL at high risk of relapse in the training cohort from those at low risk (P < 0.001). This classifier also showed good predictive value in the internal testing cohort (P < 0.001) and the independent validation cohort (P < 0.001). A nomogram incorporating five independent prognostic factors including the CpG-based classifier, lactate dehydrogenase levels, Eastern Cooperative Oncology Group performance status, central nervous system involvement, and NOTCH1/FBXW7 status showed a significantly higher predictive accuracy than each single variable. Stratification into different subgroups by the nomogram helped identify the subset of patients who most benefited from more intensive chemotherapy and/or sequential hematopoietic stem cell transplantation. CONCLUSIONS: Our four-CpG-based classifier could predict disease relapse in patients with T-LBL, and could be used to guide treatment decision.
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Islas de CpG/genética , Metilación de ADN , Recurrencia Local de Neoplasia/epidemiología , Nomogramas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Toma de Decisiones Clínicas/métodos , Supervivencia sin Enfermedad , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Femenino , Estudios de Seguimiento , Trasplante de Células Madre Hematopoyéticas , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/prevención & control , Selección de Paciente , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidad , Valor Predictivo de las Pruebas , Receptor Notch1/genética , Estudios Retrospectivos , Medición de Riesgo/métodosRESUMEN
We aimed to establish a discriminative gene-expression-based classifier to predict survival outcomes of T-cell lymphoblastic lymphoma (T-LBL) patients. After exploring global gene-expression profiles of progressive (n = 22) vs. progression-free (n = 28) T-LBL patients, 43 differentially expressed mRNAs were identified. Then an eleven-gene-based classifier was established using LASSO Cox regression based on NanoString quantification. In the training cohort (n = 169), high-risk patients stratified using the classifier had significantly lower progression-free survival (PFS: hazards ratio 4.123, 95% CI 2.565-6.628; p < 0.001), disease-free survival (DFS: HR 3.148, 95% CI 1.857-5.339; p < 0.001), and overall survival (OS: HR 3.790, 95% CI 2.237-6.423; p < 0.001) compared with low-risk patients. The prognostic accuracy of the classifier was validated in the internal testing (n = 84) and independent validation cohorts (n = 360). A prognostic nomogram consisting of five independent variables including the classifier, lactate dehydrogenase levels, ECOG-PS, central nervous system involvement, and NOTCH1/FBXW7 status showed significantly greater prognostic accuracy than each single variable alone. The addition of a five-miRNA-based signature further enhanced the accuracy of this nomogram. Furthermore, patients with a nomogram score ≥154.2 significantly benefited from the BFM protocol. In conclusion, our nomogram comprising the 11-gene-based classifier may make contributions to individual prognosis prediction and treatment decision-making.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Transcriptoma , Adulto , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nomogramas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Estudios RetrospectivosRESUMEN
New prognostic factors are needed to establish indications for haematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) for T-cell lymphoblastic lymphoma (T-LBL) patients. We used microarray to compare T-LBL tissue samples (n = 75) and fetal thymus tissues (n = 20), and identified 35 differentially expressed miRNAs. Using 107 subjects as the training group, we developed a five-miRNA-based classifier to predict patient survival with LASSO Cox regression: lower risk was associated with better prognosis (disease-free survival (DFS): hazard ratio (HR) 4.548, 95% CI 2.433-8.499, p < 0.001; overall survival (OS): HR 5.030, 95% CI 2.407-10.513, p < 0.001). This classifier displayed good performance in the internal testing set (n = 106) and the independent external set (n = 304). High risk was associated with more favorable response to HSCT (DFS: HR 1.675, 95% CI 1.127-2.488, p = 0.011; OS: HR 1.602, 95% CI 1.055-2.433, p = 0.027). When combined with ECOG-PS and/or NOTCH1/FBXW7 status, this classifier had even better prognostic performance in patients receiving HSCT (DFS: HR 2.088, 95% CI 1.290-3.379, p = 0.003; OS: HR 1.996, 95% CI 1.203-3.311, p = 0.007). The five-miRNA classifier may be a useful prognostic biomarker for T-LBL adults, and could identify subjects who could benefit from HSCT.
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MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Linfocitos T/metabolismo , Linfocitos T/patología , Supervivencia sin Enfermedad , Femenino , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Inducción de Remisión/métodosRESUMEN
OBJECTIVE: To investigate the efficacy of domestic decitabine (D) combined with pre-excitation chemotherapy consisted of Ara-c, THP and G-CSF(CTG) in treatment of middle-aged and elderly patients with MDS-transformed AML and prognosis-related factors. METHODS: Seventy-six patients with MDS-transformed AML treated in our hospital from June 2013 to June 2015 were selected according to treatment regimens, 76 patients were divided into 2 groups: CTG group(36 cases) and D+CTG group(40 cases). The patients in CTG group received treatment with Ara-C, THP and G-CSF; the patients received the treatment with decitabine plus CTG. The patients in 2 groups all received 4 course treatment, then received maintaining treatment. The therapeutic efficacy and incidence of adverse reactions in 2 group were compared, at the same time, the risk factors affecting the prognos of patients treated with D+CTG were analyzed. RESULTS: There were no siginificant differences in age, sex, initial blood cell count, bone marrow blast ratio, disease types, chromosome karyotypes and FLT3-ITD gene mutation between 2 groups. The efficacy analysis showed that the efficacy of D+CTG was superior to CTG, ORR in D+CTG group was significantly higher than that in CTG group (72ã52 vs 50%) (P<0.05), moreover, no significant differences in bone marrow inhibition digree infeetion, gastroinfestinal response and liver damage were found between 2 groups (P>0.05). The follow-np for 2 years showed that the median survival time in D+CTG group was significantly longer than that in CTG group (19.9 vs 11.0 months) (P<0.05). The multivariate analysis showed that the 1 course efficacy (RR=3.926, P=0.015) and FLT3-ITD gene mutation (RR=4.347, P=0.004) were independent risk factors affecting the efficacy of D+CTG treatment. CONCLUSION: The short-and long-term efficacy of domestic decitasine combined with preexcitation chenotherapy in treatment of middec-aged and eldery patients with MDS transformed AML is superior to single pre-excitation chenothrapy, moreover the incidence of adverse reactions did not increase. The 1 course efficacy and FLT-3 ITD gene mutation are the independent risk factors affecting the prognosis of patients. .
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos , Anciano , Decitabina/administración & dosificación , Humanos , Persona de Mediana Edad , PronósticoRESUMEN
The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.
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Citosina Desaminasa/genética , Genes Transgénicos Suicidas/genética , Vectores Genéticos/genética , Proteínas Tirosina Quinasas/genética , Flucitosina/farmacología , Ganciclovir/farmacología , Terapia Genética , Humanos , Células K562 , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes de Fusión/genética , Recombinación Genética , Retroviridae/genéticaRESUMEN
BACKGROUND & OBJECTIVE: The lentiviral vectors can integrate interest genes into genome of the target cells that allow for stable transgenic expression even in non-dividing cells without evoking an immune response of the host. All the features have promised them to be used in vivo gene therapy. This study was designed to explore the killing effect of double suicide genes mediated by lentivirus on lymphoma cells (Raji). METHODS: The three plasmids expressed lentivirus, packaging plasmid pCMV 8.2, envelope plasmid pCMV.VSVG and target plasmid (pHR'CS. GFP as control group, pHR'CS.CDglytk as experiment group) were packaged into 293T cells using lipofectine method. Supernatant was harvested and concentrated. The Raji cells were infected with the concentrated virus. The gene integration and expression were confirmed by fluorescence microscopy and RT-PCR. After prodrug GCV or/and 5-FC administration, MTT method was used to detect the growth inhibition rate (GIR) of Raji cells for evaluating the killing effect of CD and HSV-tk double suicide genes on Raji cells. RESULTS: The three plasmids were effectively transferred into 293T cells. Green fluorescence on the cell was observed through fluorescence microscopy and a lot of virus particles were observed through transmission electronic microscopy. Double suicide genes mediated by lentivirus were effectively and stably expressed in Raji cells. The GIR of Raji cells using GCV or 5-FC was 51% or 50%, respectively, and it was apparently higher than that of untransfected cells(P< 0.01). When using GCV and 5-FC together, the GIR was 73%, which was apparently higher than that of group using GCV or 5-FC alone (P< 0.01). CONCLUSION: Double suicide genes mediated by lentiviral vector could transfect lymphoma cells effectively and stably. The double suicide gene system enhanced killing effect remarkably on lymphoma cells than CD/5FC or HSV-tk/GCV system alone.
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Citosina Desaminasa/genética , Terapia Genética , Lentivirus/genética , Linfoma/terapia , Simplexvirus/enzimología , Timidina Quinasa/genética , Relación Dosis-Respuesta a Droga , Flucitosina/farmacología , Ganciclovir/farmacología , HumanosRESUMEN
OBJECTIVE: To explore the feasibility and efficiency of cytosine deaminase (CD)/thymidine kinase (TK) gene-modified donor T cells used in allogeneic bone marrow transplantation (allo-BMT) as an approach to mitigate GVHD without compromising engraftment. METHODS: The pseudotyped lentivirus vectors containing CD and TK double suicide genes were transfected with lipofectine to donor T cells. Lethally irradiated 615 leukemia mice were transplanted with BALB/c bone marrow plus CD(+)TK(+)T cells. GVHD prophylaxis was by administration of ganciclovir (GCV) and 5-Fluoride cytosine (5-FC). RESULTS: The pseudotyped lentivirus-mediated gene transfer system could efficiently transfer CD and TK double suicide genes into donor T cells. Administration of GCV and 5-FC to the mice could markedly potentiate the CFU-S and CFU-GM yields and raise the number of peripheral white blood cells. 1 x 10(7) CD(+)TK(+) allogeneic T cells caused GVHD of a similar magnitude and time course to that of fresh, naive T cells after allo-BMT. Administration of GCV and 5-FC in mice received CD(+)TK(+)T cells reduced the severity of GVHD and resulted in significantly longer survival as compared with non-administration mice, and the effect was stronger than that of administration of GCV or 5-FC alone. CONCLUSION: Administration of CD + TK gene-modified donor T cells to recipient in allo-BMT might be an approach to mitigate GVHD without compromising alloengraftment.
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Trasplante de Médula Ósea , Citosina Desaminasa/genética , Terapia Genética , Enfermedad Injerto contra Huésped/terapia , Lentivirus/genética , Timidina Quinasa/genética , Animales , Peso Corporal , Femenino , Enfermedad Injerto contra Huésped/patología , Ratones , Ratones Endogámicos BALB C , Trasplante HomólogoRESUMEN
To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus-mediated gene transfer system could transfer CD and TK double suicide genes into K562 cells with high efficiency and it had strong killing effects when giving 5-FC and/or GCV. The cytotoxic effects of double suicide genes were superior to that of single suicide gene. The lentivirus-mediated double suicide gene transfer system is a high-efficiency gene transfer vector.