RESUMO
Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development.
Assuntos
Macrófagos , Humanos , Diferenciação Celular , Linhagem da Célula , Macrófagos/citologia , Microglia , Especificidade de ÓrgãosRESUMO
Turmeric (Curcuma longa), a typical source with recognized anti-inflammatory activity, is one such medicine-food homology source, yet its anti-inflammatory mechanisms and specific component combinations remain unclear. In this study, a net fishing method combining bio-affinity ultrafiltration and ultra-high performance liquid chromatography-mass spectrometry (AUF-LC/MS) was employed and 13 potential COX-2 inhibitors were screened out from C. longa. 5 of them (C1, 17, 20, 22, 25) were accurately isolated and identified. Initially, their IC50 values were measured (IC50 of C1, 17, 20, 22 and 25 is 55.08, 48.26, 29.13, 111.28 and 150.48 µM, respectively), and their downregulation of COX-2 under safe concentrations (400, 40, 120, 50 and 400 µM for C1, 17, 20, 22 and 25, respectively) was confirmed on RAW 264.7 cells. Further, in transgenic zebrafish (Danio rerio), significant anti-inflammatory activity at safe concentrations (15, 3, 1.5, 1.5 and 3 µg/mL for C1, 17, 20, 22 and 25, respectively) were observed in a dose-dependent manner. More importantly, molecular docking analysis further revealed the mode of interaction between them and the key active site residues of COX-2. This study screened out and verified unreported COX-2 ligands, potentially accelerating the discovery of new bioactive compounds in other functional foods.
Assuntos
Curcuma , Inibidores de Ciclo-Oxigenase 2 , Ciclo-Oxigenase 2 , Ultrafiltração , Peixe-Zebra , Animais , Curcuma/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/química , Camundongos , Ciclo-Oxigenase 2/metabolismo , Cromatografia Líquida de Alta Pressão , Células RAW 264.7 , Relação Dose-Resposta a Droga , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Espectrometria de Massas , HumanosRESUMO
DNA methylation alters gene expression in numerous biological processes, including embryonic development. It is little known about the effect of cryopreservation on sperm DNA methylation. The present study has investigated whether cryopreservation causes abnormal DNA methylation in cynomolgus macaque sperm for five critical genes that includes the maternally imprinted gene (SNRPN), genes associated with male infertility (HSPA1L, MTHFR) and genes involved in embryonic development (TET3, LZTR1). Our results showed that sperm motility, the percentage of acrosomal integrity, DNA integrity and mitochondrial membrane potential were decreased after cryopreservation either being frozen with penetrating cryoprotectant, glycerol (Gly) or ethylene glycol (EG), compared to fresh sperm (p = 0.000), but the methylation patterns of the five target genes from cynomolgus macaque sperm samples were not affected after cryopreservation as evaluated by the Bisulfite Sequencing PCR (BSP) method. The data indicates that the current protocol for sperm cryopreservation of cynomolgus macaque is safe in terms of DNA methylation levels in these genes related to critical sperm functions.
Assuntos
Criopreservação , Preservação do Sêmen , Animais , Criopreservação/métodos , Metilação de DNA , Desenvolvimento Embrionário , Etilenoglicol , Feminino , Fertilização , Glicerol , Macaca fascicularis/genética , Masculino , Gravidez , Sêmen , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides/genética , Espermatozoides , Proteínas Centrais de snRNPRESUMO
The volatile oil of Curcumae Rhizoma has many active components,which are the key to the quality of Curcumae Rhizoma. Exploring the difference between volatile oil of different kinds of Curcumae Rhizoma facilitates the quality control and rational application of resources. In this study,GC-MS was applied to realize online qualitative and semi-quantitative analysis of the chemical composition spectrum of volatile oil from Curcuma wenyujin( CW),C. phaeocaulis( CP),and C. kwangsiensis( CK). Forty components were identified and their fingerprints were compared and evaluated. Hierarchical cluster analysis( HCA),principal component analysis( PCA),and orthogonal partial least squares discrimination analysis( OPLS-DA) were adopted to analyze the overall and outlier data. The results showed that the whole data could be divided into three kinds according to each analysis mode,and the volatile components of Curcumae Rhizoma vary greatly among species. PCA explored the difference between outliers and the mean value of the group and found that some volatile oils from CW may be greatly affected by the origin. By OPLS-DA,the samples from Zhejiang were able to gather,but those from Guizhou remained isolated,indicating the influence of growing environment on Curcumae Rhizoma metabolites. Based on VIP results combined with the heat map,characteristic volatile oil components of Curcumae Rhizoma from different varieties were screened out: curdione and linalool for CW; 2-undecanone for CP; humulene,γ-selinene,and zederone for CK. The GCMS method established in this study describes Curcumae Rhizoma samples comprehensively and accurately,and the characteristic components screened based on chemometrics can be used to distinguish Curcumae Rhizoma from different varieties and give them unique pharmacodynamic significance,which is fast,convenient,stable,and reliable and supports the rational application of Curcu-mae Rhizoma resources. It is found that the region of origin has great influence on CW,which is worthy of further study.
Assuntos
Óleos Voláteis , Curcuma , Cromatografia Gasosa-Espectrometria de Massas , Análise de Componente Principal , RizomaRESUMO
This study aimed to establish a rapid and accurate method for identification of raw and vinegar-processed rhizomes of Curcuma kwangsiensis, in order to predict the content of curcumin compounds for scientific evaluation. A complete set of bionics recognition mode was adopted. The digital odor signal of raw and vinegar-processed rhizomes of Curcuma kwangsiensis were obtained by e-nose, and analyzed by back propagation(BP) neural network algorithm, with the accuracy, the sensitivity and specificity in discriminant model, correlation coefficient as well as the mean square error in regression model as the evaluation indexes. The experimental results showed that the three indexes of the e-nose signal discrimination model established by the neural network algorithm were 100% in training set, correction set and prediction set, which were obviously better than the traditional decision tree, naive bayes, support vector machine, K nearest neighbor and boost classification, and could accurately differentiate the raw and vinegar products. Correlation coefficient and mean square error of the regression model in prediction set were 0.974 8 and 0.117 5 respectively, and could well predict curcumin compounds content in Curcuma kwangsiensis, and demonstrate the superiority of the simulation biometrics model in the analysis of traditional Chinese medicine. By BP neural network algorithm, e-nose odor fingerprint could quickly, conveniently and accurately realize the discrimination and regression, which suggested that more bionics information acquisition and identification patterns could be combined in the field of traditional Chinese medicine, so as to provide ideas and methods for the rapid evaluation and stan-dardization of the quality of traditional Chinese medicine.
Assuntos
Curcumina , Nariz Eletrônico , Ácido Acético , Teorema de Bayes , Curcuma , Redes Neurais de Computação , RizomaRESUMO
Colorectal cancer (CRC) is responsible for one of the major cancer incidence and mortality worldwide. It is well known that MicroRNAs (miRNAs) play vital roles in maintaining the cell development and other physiological processes, as well as, the aberrant expression of numerous miRNAs involved in CRC progression. MiRNAs are a class of small, endogenous, non-coding, single-stranded RNAs that bind to the 3'-untranslated region (3'-UTR) complementary sequences of their target mRNA, resulting in mRNA degradation or inhibition of its translation as a post-transcriptional regulators. Moreover, miRNAs also can target the long non-coding RNA (lncRNA) to regulate the expression of its target genes involved in proliferation and metastasis of CRC. The functions of these dysregulated miRNAs appear to be context specific, with evidence of having a dual role in both oncogenes and tumor suppression depending on the cellular environment in which they are expressed. Therefore, the unique expression profiles of miRNAs relate to the diagnosis, prognosis, and therapeutic outcome in CRC. In this review, we focused on several oncogenic and tumor-suppressive miRNAs specific to CRC, and assess their functions to uncover the molecular mechanisms of tumor initiation and progression in CRC. These data promised that miRNAs can be used as early detection biomarkers and potential therapeutic target in CRC patients.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Animais , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Progressão da Doença , Humanos , Prognóstico , RNA Longo não Codificante/genética , RNA Mensageiro/genéticaRESUMO
The immune responses during the initiation and invasion stages of human lung adenocarcinoma (LUAD) development are largely unknown. Here, we generated a single-cell RNA sequencing map to decipher the immune dynamics during human LUAD development. We found that T follicular helper (Tfh)-like cells, germinal center B cells, and dysfunctional CD8+ T cells increase during tumor initiation/invasion and form a tertiary lymphoid structure (TLS) inside the tumor. This TLS starts with an aggregation of CD4+ T cells and the generation of CXCL13-expressing Tfh-like cells, followed by an accumulation of B cells, and then forms a CD4+ T and B cell aggregate. TLS and its associated cells are correlated with better patient survival. Inhibiting TLS formation by Tfh or B cell depletion promotes tumor growth in mouse models. The anti-tumoral effect of the Tfh-dependent TLS is mediated through interleukin-21 (IL-21)-IL-21 receptor signaling. Our study establishes an anti-tumoral role of the Tfh-dependent TLS in the development of LUAD.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Estruturas Linfoides Terciárias , Animais , Camundongos , Humanos , Linfócitos T Auxiliares-Indutores , Estruturas Linfoides Terciárias/patologia , Linfócitos T CD8-Positivos/patologiaRESUMO
A sensitive and selective Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for the identification and quantification of two potential genotoxic impurities (PGIs) - viz. methyl N-((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)-N-nitroso-L-valinate (PGI-1) and N-nitroso Valsartan (PGI-2) - in the angiotensin II receptor blocker valsartan. Among these impurities, PGI-1 is a distinctive compound which has never been reported. For this, chromatographic separation was performed using a Waters XBridge BEH C18 column (150 mm × 4.6 mm, 2.5 µm), with ammonium acetate aqueous solution (0.01 mol/L) as mobile phase A and acetonitrile as mobile phase B, in a gradient elution mode at a 0.5 mL/min flow rate. Mass spectrometric conditions were optimized using electrospray ionization (ESI) in positive mode. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying 2 PGIs at 0.016 ppm in samples at 50 mg/mL concentration. This validated approach presented good linearity over the concentration range of 0.016-0.06 ppm for 2 PGIs. The correlation coefficient of each impurity was observed greater than 0.999. The accuracy of this method was in the range of 83-113% for the aforementioned PGIs. In addition, expert knowledge rules (Derek-based) and statistical (Q) SAR evaluation system (Sarah-based) were used to evaluate and classify the genotoxicity of both valsartan-related PGIs as well as to define their standard limits. The predicted results were positive and classified into the third category, and the total nitrosamine limit was set to 0.03 ppm. As such, this approach represents a good quality control system for the simultaneous and precise quantitation of PGIs in valsartan.
Assuntos
Dano ao DNA , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , ValsartanaRESUMO
Recent studies concerning products that originate from natural plants have sought to clarify active ingredients, which both explains the mechanisms of the function and aids in quality control during production. As a traditional functional plant, Curcumae Rhizoma (CR) has been proven to be effective in promoting blood circulation and removing blood stasis. However, the components that play a role in its huge compound library are still unclear. The present study aimed to develop a high-throughput screening method to identify thrombin inhibitors in CR and validate them by in vitro and in vivo experiments. The effect of CR on thrombin in HUVECs cells was determined by ELISA, then an affinity-ultrafiltration-UPLC-Q-Exactive Orbitrap/MS approach was applied. Agatroban and adenosine were used as positive and negative drugs respectively to verify the reliability of the established method. The in vitro activity of the compounds was determined by specific substrate S-2238. The in vivo effect of the active ingredients was determined using zebrafish. Molecular docking was used to understand the internal interactions between compounds and enzymes. ELISA results showed that CR had an inhibitory effect on thrombin. The screening method established in this paper is reliable, by which a total of 15 active compounds were successfully identified. This study is the first to report that C7, 8, and 11 have in vitro thrombin-inhibitory activity and significantly inhibit thrombosis in zebrafish models at a safe dose. Molecular docking studies were employed to analyze the possible active binding sites, with the results suggesting that compound 16 is likely a better thrombin inhibitor compared with the other compounds. Based on the affinity-ultrafiltration-UPLC-Q-Exactive Orbitrap/MS approach, a precisely targeted therapy method using bio-active compounds from CR might be successfully established, which also provides a valuable reference for targeted therapy, mechanism exploration, and the quality control of traditional herbal medicine.
RESUMO
It's far from enough to describe the blood circulation promoting effect of Curcumae Rhizoma (CR), which is widely grown as a functional vegetable or spice in south and southeast Asian countries, and processed Curcumae Rhizoma (PCR), only by disclosing the content of a couple of relative compounds. In this study, the thrombin inhibitory effect as well as 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-picrylhydrazyl (DPPH) free radical scavenging ability of CR/PCR extracts was investigated, and TANGO Fourier transform near-infrared (FT-NIR, Bruker, Germany)-a benchtop instrument allowing the full range NIR wavelength scanning-and handheld-NIR spectrometer (Micro-NIR, NIR-S-G1, InnoSpectra corporation, China) that can be connected to smart phones were used to realize the rapid detection of pharmacodynamic indicators. model was evaluated based on the determination coefficient (R2), root mean square error (RMSE), standard error of test set (SEP) and ratio of performance to deviation (RPD). The results of pharmacodynamics experiment confirmed for the first time that CR has significant inhibitory effect on thrombin, and the modeling results revealed that Micro-NIR had a good prediction on the antioxidant capacity (ABTS and DPPH free radical clearance) with RPD greater than 3, but showed a general predictive performance on thrombin inhibition ability (RPDâ¯=â¯2.434). In contrast, FT-NIR provided a good prediction for all the three indicators, with R2 greater than 0.9 and RPD greater than 4.5. Further insights into the capability of the two devices were obtained by analyzing the wavebands selection work. In the full wavelength range, wavebands related to thrombin inhibition were mainly distributed in the combination area which is out of the reach of handheld Micro-NIR, thus resulting in a decrease in the prediction ability. Therefore, compared to the benchtop-NIR, the detection range of the handheld-NIR is the main factor limiting its capability Based on an overall assessment, handheld NIR spectrometer, by greatly expanding the application scenario of NIR technology, is considered as a useful device with a satisfying predictive ability through model construction.
Assuntos
Antioxidantes , Rizoma , Antioxidantes/farmacologia , China , AlemanhaRESUMO
Curcumae Rhizoma (CR) and vinegar processed Curcumae Rhizoma (PCR) are common medicinal materials widely used in clinical practice in China. There are sliced CR (SCR) and two kinds of PCR products which are processed by different methods: WPCR-prepared with the whole CR root boiled in vinegar and then sliced, and SPCR-prepared with the whole CR root steamed and sliced before boiled with vinegar. In this study, the feasibility of Fourier transform near infrared spectrum (FT-NIR) used to determine the main discrepant components of SCR, WPCR and SPCR were investigated. High performance liquid chromatography (HPLC) was used to identified five discrepant compounds in the three kinds of CR products-curzerene, curcumenol, curdione, furanodienone and demethoxycurin. Pretreatment of NIR qualitative data by different methods revealed that the second derivative in combination with 9 points of Savitzky-Golay smooth (2D9S) could accurately distinguish SCR, SPCR and WPCR from each other, and the discrimination ability was improved significantly by wavebands selection. Then a model with great accuracy was established by combining with wavebands selection and partial least squares regression (PLSR). Compared with the competitive adaptive reweighted sampling (CARS) selection method, 2D9S- variable combination population analysis (VCPA)-Genetic algorithm (GA)-PLSR model was evidently more accurate in prediction of the content of curzerene, curcumenol, curdione and furanodienone, with an R2p of 0.9558, 0.9129, 0.9098 and 0.9350, as well as a ratio of performance to deviation (RPD) of 4.8454, 3.4640, 3.3020 and 4.0082, respectively. Whereas, the content of demethoxycurin failed to be well predicted. The correlation analysis revealed that the results of wavebands selection were consistent with the trend of changes in the content of these target compounds and the findings of NIR absorption analysis, and the characteristic chemical bonds of these compounds corresponded to the areas with significant correlation in the heat map. It can be concluded that the NIR system, combined with appropriate variable selection and linear regression method, can precisely distinguish SCR, SPCR and WPCR from each other, and can accurately and rapidly determine the four discrepant compounds in the three CR products, suggesting a potential of being routinely used for a more diversified analysis in medicinal herbs study.
Assuntos
Algoritmos , Rizoma , China , Análise dos Mínimos Quadrados , TecnologiaRESUMO
The aim of this study was to establish a robust and rapid method for identification of the changes of Curcumae Rhizoma (CR) in its physical properties and the components after processing. Color and odor used as the indexes for physical property evaluation in this study were quantified by non-targeted bionic detectors colorimeter and e-nose according to Chinese pharmacopoeia, and all chemical changes of the samples were observed by NIR. The technique of mid-level data fusion was adopted to simulate the recognition mode of human, and the prediction accuracy was 100%. A modified NIR band extraction method was used for feature selection after removing the redundant data which otherwise may compromise the accuracy of analysis, with a result showing bisdemethoxycurcumin and curcumol as the key factors most correlated with the changes during processing. The fused matrix was analyzed to evaluate the weight of different sensors and precisely describe the external changes of the samples, followed by a pairwise correlation analysis to investigate the role of colorimeter, e-nose and NIR in identifying the changes caused by processing as well as their correlation with each other. The results of data fusion revealed that aromatic derivatives produced during processing were closely associated with the changes in external characteristics, i.e., color and smell of the samples, while the changes in proteins did not cause significant differences. Correlation analysis demonstrated that bionic sensors could be classified into two groups: a*, WW and WS sensors, which were related to NIR band at about 6500-6700 cm-1 formed by NH vibration of amide and protein degradation, were sensitive to the processed CR; while L*, b* and WC sensors were found to be correlated to NIR band around 8000 cm-1 which was caused by first overtone of C-H combination of aromatic derivatives, thus could be employed as the detectors for raw CR.
Assuntos
Medicamentos de Ervas Chinesas , Nariz Eletrônico , Curcuma , Humanos , Odorantes , RizomaRESUMO
The prognosis of patients with colorectal cancer (CRC) remains poor owing to diagnosis typically occurring at advanced stages of the disease. The understanding of the molecular regulatory signatures of CRC may lead to the identification of biomarkers for the early detection, prevention and clinical intervention of CRC. Epidemiological studies have indicated that cyclooxygenase1 (COX1) serves an active function in colon carcinogenesis. However, the molecular mechanism underlying COX1 regulation in CRC remains unknown. In the present study, COX1 was identified to be markedly upregulated in colorectal tissues of patients with CRC, and in the CRC cell lines HCT116 and HT29. To determine the function of COX1 in cancer development, short hairpin RNA knockdown of COX1 was employed in HCT116 and HT29 CRC cells in the present study. The results demonstrated that silencing of COX1 depolarized the mitochondrial membrane potential, inhibited adenosine triphosphate production, induced the generation of intracellular reactive oxygen species and triggered caspasedependent mitochondrial apoptosis. Furthermore, depletion of COX1 suppressed antiapoptotic Bcell lymphoma 2 and enhanced proapoptotic Bcl2associated X protein expression by inhibiting the p65 subunit phosphorylation of nuclear factor κB (NFκB). Taken together, the results of the present study indicated that COX1 inhibition significantly triggered cell death by destroying the mitochondrial function that is associated with deactivation of the NFκB signaling pathway. These results suggest COX1 as a potential anticancer target in CRC.
Assuntos
Apoptose/genética , Neoplasias Colorretais/genética , Ciclo-Oxigenase 1/genética , Regulação para Baixo/genética , Mitocôndrias/genética , NF-kappa B/genética , Transdução de Sinais/genética , Adulto , Idoso , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , Potencial da Membrana Mitocondrial/genética , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Chemokines represent a superfamily of small secretion proteins that functionally mediate immune cell transmigration in normal or inflammatory conditions. Although anatomic and polygenetic evidence suggests that tree shrews are primate-like species, understanding of the structure and function of tree shrew chemokines has only just commenced. In this study, we cloned tree shrew chemokine CXCL8 and its cognate receptors. Predicted three-dimensional (3D) structures showed that binding domains in CXCL8 and CXCR1/2 were highly conserved between tree shrews and humans. We found that the human CXCL8 (hCXCL8) protein induced migration of tree shrew peripheral blood mononuclear cells (PBMCs) expressed by CXCR1/2 (tsCXCR1/2). Blocking interaction between hCXCL8 and tsCXCR1/2 with allosteric antagonists (reparixin and SB265610) significantly decreased tree shrew PBMC transmigration. Over-expressing tree shrew CXCR1 in human HEK 293 T cells further enhanced cellular in vitro transmigration. Similar to primate species, our findings suggest that CXCL8 and CXCR1/2 constitute a structurally- and functionally-conserved chemotaxis responsible for tree shrew immune activities.
Assuntos
Sequência Conservada/genética , Interleucina-8/genética , Tupaiidae/genética , Animais , Linhagem Celular , Células HEK293 , Humanos , MasculinoRESUMO
Bcl-2 inhibits cell proliferation by delaying G0/G1 to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G0/G1 phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G0/G1 arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1-3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.