Adipocytes promote pancreatic cancer migration and invasion through fatty acid metabolic reprogramming.

Locally advanced and metastatic pancreatic cancer (PC) frequently grows in adipose tissue and has a poor prognosis. Although adipose tissue is largely composed of adipocytes, the mechanisms by which adipocytes impact PC are poorly understood. Using an in vitro coculture model, it was shown that adipocytes promoted tumor progression, and an intricate metabolic network between PC cells and adipocytes was identified and elucidated. First, the proteome of Panc­1 PC cells cultured with or without mature adipocytes was identified. This revealed activated hypoxia signaling in cocultured Panc­1 cells, which was confirmed by the increased expression of factors downstream of hypoxia signaling, such as ANGPTL4 and glycolytic genes, as determined by reverse transcription­quantitative PCR and western blot analysis. In addition, it was demonstrated that coculture with cancer cells activated STAT3 and induced an insulin­resistant phenotype in adipocytes. Furthermore, enhanced fatty acid ß­oxidation and increased lipid droplets (LDs) were observed in the cocultured cancer cells. In contrast, downregulated lipid metabolism and a decrease in the size of LDs were found in cocultured adipocytes. Finally, it was shown that the increase in LDs contributed to the increased metastatic capacity of the cocultured PC cells. These data demonstrated that interrupting the mechanisms of lipid uptake from adipocytes in the microenvironment may offer a potential strategy for attenuating PC metastasis.

ARBUSCULAR MYCORRHIZA-INDUCED KINASES AMK8 and AMK24 associate with the receptor-like kinase KINASE3 to regulate arbuscular mycorrhizal symbiosis in Lotus japonicus.

Arbuscular mycorrhizal (AM) symbiosis is a widespread, ancient mutualistic association between plants and fungi, and facilitates nutrient uptake into plants. Cell surface receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) play pivotal roles in transmembrane signaling, while few RLCKs are known to function in AM symbiosis. Here, we show that 27 out of 40 AM-induced kinases (AMKs) are transcriptionally upregulated by key AM transcription factors in Lotus japonicus. Nine AMKs are only conserved in AM-host lineages, among which the SPARK-RLK-encoding gene KINASE3 (KIN3) and the RLCK paralogues AMK8 and AMK24 are required for AM symbiosis. KIN3 expression is directly regulated by the AP2 transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), which regulates the reciprocal exchange of nutrients in AM symbiosis, via the AW-box motif in the KIN3 promoter. Loss of function mutations in KIN3, AMK8, or AMK24 result in reduced mycorrhizal colonization in L. japonicus. AMK8 and AMK24 physically interact with KIN3. KIN3 and AMK24 are active kinases and AMK24 directly phosphorylates KIN3 in vitro. Moreover, CRISPR-Cas9-mediated mutagenesis of OsRLCK171, the sole homolog of AMK8 and AMK24 in rice (Oryza sativa), leads to diminished mycorrhization with stunted arbuscules. Overall, our results reveal a crucial role of the CBX1-driven RLK/RLCK complex in the evolutionarily conserved signaling pathway enabling arbuscule formation.

Scaled Sea Surface Design and RCS Measurement Based on Rough Film Medium.

The electromagnetic (EM) scattering characteristics of the rough sea surface is very important for target surveying and detection in a sea environment. This work proposes a scaled sea surface designing method based on a rough thin-film medium. For the prototype sea surface, the permittivity is calculated with the seawater temperature, salinity, and EM wave frequency according to the Debye model. The scale film material is mixed with carbon black and epoxy, whose volume ratio is optimized with the genetic algorithm through the existing electromagnetic parameter library. This method can overcome the previous difficulties of adjusting the same permittivity of the prototype sea water. According to the EM scaled theory, the scaled geometric sample is numerically generated with the D-V spectrum for the given wind speed, and is fabricated using 3D printing to keep the similar seawater shape. Then, the sample is sprayed with a layer of film material for EM scattering measurement. The simulated and measured radar cross-section (RCS) results show good consistency for the prototype seawater and scaled materials, which indicates the proposed scaled method is a more efficient method to get the seawater scattering characteristics.

Application of omental interposition to reduce pancreatic fistula and related complications in pancreaticoduodenectomy: A propensity score-matched study.

BACKGROUND: The life-threatening complications following pancreatoduodenectomy (PD), intra-abdominal hemorrhage, and postoperative infection, are associated with leaks from the anastomosis of pancreaticoduodenectomy. Although several methods have attempted to reduce the postoperative pancreatic fistula (POPF) rate after PD, few have been considered effective. The safety and short-term clinical benefits of omental interposition remain controversial. AIM: To investigate the safety and feasibility of omental interposition to reduce the POPF rate and related complications in pancreaticoduodenectomy. METHODS: In total, 196 consecutive patients underwent PD performed by the same surgical team. The patients were divided into two groups: An omental interposition group (127, 64.8%) and a non-omental interposition group (69, 35.2%). Propensity score-matched (PSM) analyses were performed to compare the severe complication rates and mortality between the two groups. RESULTS: Following PSM, the clinically relevant POPF (CR-POPF, 10.1% vs 24.6%; P = 0.025) and delayed postpancreatectomy hemorrhage (1.4% vs 11.6%; P = 0.016) rates were significantly lower in the omental interposition group. The omental interposition technique was associated with a shorter time to resume food intake (7 d vs 8 d; P = 0.048) and shorter hospitalization period (16 d vs 21 d; P = 0.031). Multivariate analyses showed that a high body mass index, nonapplication of omental interposition, and a main pancreatic duct diameter < 3 mm were independent risk factors for CR-POPF. CONCLUSION: The application of omental interposition is an effective and safe approach to reduce the CR-POPF rate and related complications after PD.

Biochemical Tools for Tracking Proteolysis.

All living organisms depend on tightly regulated cellular networks to control biological functions. Proteolysis is an important irreversible post-translational modification that regulates most, if not all, cellular processes. Proteases are a large family of enzymes that perform hydrolysis of protein substrates, leading to protein activation or degradation. The 473 known and 90 putative human proteases are divided into 5 main mechanistic groups: metalloproteases, serine proteases, cysteine proteases, threonine proteases, and aspartic acid proteases. Proteases are fundamental to all biological systems, and when dysregulated they profoundly influence disease progression. Inhibiting proteases has led to effective therapies for viral infections, cardiovascular disorders, and blood coagulation just to name a few. Between 5 and 10% of all pharmaceutical targets are proteases, despite limited knowledge about their biological roles. More than 50% of all human proteases have no known substrates. We present here a comprehensive list of all current known human proteases. We also present current and novel biochemical tools to characterize protease functions in vitro, in vivo, and ex vivo. These tools make it achievable to define both beneficial and detrimental activities of proteases in health and disease.

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy.

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.

Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti.

Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia.

The anti-breast cancer property of physcion via oxidative stress-mediated mitochondrial apoptosis and immune response.

CONTEXT: Physcion (Phy) exerts several pharmacological effects including anti-inflammatory, antioxidant, and antitumor properties. OBJECTIVE: This study investigates the cytotoxicity and its underlying mechanisms of Phy on breast cancer. MATERIALS AND METHODS: Human breast cancer cell MCF-7 was treated with 5-400 µM Phy for 24 h, MCF-7-xenografted BALB/c nude mice and immunosuppressive mice model induced by cyclophosphamide were intraperitoneally injected with 0.1 mL/mouse normal saline (control group) and 30 mg/kg Phy every other day for 14 or 28 days, and pathological examination, ELISA and western blot were employed to investigate the Phy anti-breast cancer property in vitro and in vivo. RESULTS: In MCF-7 cells, Phy 24 h treatment significantly reduced the cell viability at dose of 50-400 µM and 24 h, with an IC50 of 203.1 µM, and 200 µM Phy induced 56.9, 46.9, 36.9, and 46.9% increment on LDH and caspase-3, -8 and -9. In MCF-7-xenograft tumour nude mice and immunosuppressive mice, 30 mg/kg Phy treatment inhibited tumour growth from the 8th day, and reduced Bcl-2 and Bcl-xL >50%, HO-1 and SOD-1 > 70% in tumour tissues of immunosuppressive mice. In addition, Phy reduced nuclear factor erythroid 2-related factor 2 > 30% and its downstream proteins, and enhanced the phosphorylation of nuclear factor-kappa B > 110% and inhibitor of NF-кB α > 80% in the tumour tissues of BALB/c mice. DISCUSSION AND CONCLUSIONS: This research demonstrated that Phy has an anti-breast cancer property via the modulation of oxidative stress-mediated mitochondrial apoptosis and immune response, which provides a scientific basis for further research on its clinical applications.

Modeling of Nucleation and Growth in the Synthesis of PbS Colloidal Quantum Dots Under Variable Temperatures.

Lead sulfur colloidal quantum dots (PbS CQDs) are a kind of IV-VI semiconductor nanocrystals which have attracted enormous interest in recent years because of their unique physicochemical properties. Controlling size, size distribution, and yield of PbS CQDs plays key priorities in order to improve their properties when they are applied in the photovoltaics and energy storage applications. Despite many systematical studies in PbS CQD syntheses with various perspectives, details of the formation mechanism impacted on the size, concentration, and size distribution of PbS CQDs in complicated reaction conditions remain poorly understood. In this work, an improved kinetic rate equation (IKRE) model is employed to describe PbS CQD formation under variable solution temperatures. After establishing the necessary discretized equations and reviewing the link between model parameters and experimental information, a parametric study is performed to explore the model's feature. In addition, a set of experimental data has been compared with the result of IKRE model fits, which would be used to obtain corresponding thermodynamic and kinetic parameters that can further affect the CQD growth over longer timescales. This method builds up the relationship between the nucleation and Ostwald ripening stage that would provide the possibility for future large-scale manufacturing of CQDs.

A Review on the Effects of ZnO Nanowire Morphology on the Performance of Interpenetrating Bulk Heterojunction Quantum Dot Solar Cells.

Interpenetrating bulk heterojunction (IBHJ) quantum dot solar cells (QDSCs) offer a direct pathway for electrical contacts to overcome the trade-off between light absorption and carrier extraction. However, their complex three-dimensional structure creates higher requirements for the optimization of their design due to their more difficult interface defect states control, more complex light capture mechanism, and more advanced QD deposition technology. ZnO nanowire (NW) has been widely used as the electron transport layer (ETL) for this structure. Hence, the optimization of the ZnO NW morphology (such as density, length, and surface defects) is the key to improving the photoelectric performance of these SCs. In this study, the morphology control principles of ZnO NW for different synthetic methods are discussed. Furthermore, the effects of the density and length of the NW on the collection of photocarriers and their light capture effects are investigated. It is indicated that the NW spacing determines the transverse collection of electrons, while the length of the NW and the thickness of the SC often affect the longitudinal collection of holes. Finally, the optimization strategies for the geometrical morphology of and defect passivation in ZnO NWs are proposed to improve the efficiency of IBHJ QDSCs.

An MAP kinase interacts with LHK1 and regulates nodule organogenesis in Lotus japonicus.

Symbiosis receptor-like kinase (SymRK) is a key protein mediating the legume-Rhizobium symbiosis. Our previous work has identified an MAP kinase kinase, SIP2, as a SymRK-interacting protein to positively regulate nodule organogenesis in Lotus japonicus, suggesting that an MAPK cascade might be involved in Rhizobium-legume symbiosis. In this study, LjMPK6 was identified as a phosphorylation target of SIP2. Stable transgenic L. japonicus with RNAi silencing of LjMPK6 decreased the numbers of nodule primordia (NP) and nodule, while plants overexpressing LjMPK6 increased the numbers of nodule, infection threads (ITs), and NP, indicating that LjMPK6 plays a positive role in nodulation. LjMPK6 could interact with a cytokinin receptor, LHK1 both in vivo and in vitro. LjMPK6 was shown to compete with LHP1 to bind to the receiver domain (RD) of LHK1and to downregulate the expression of two LjACS (1-aminocyclopropane-1-carboxylic acid synthase) genes and ethylene levels during nodulation. This study demonstrated an important role of LjMPK6 in regulation of nodule organogenesis and ethylene production in L. japonicus.

[High-throughput screening and quantitative analysis method of 112 pharmaceutical and personal care products in water based on ultra-high performance liquid chromatography with high-resolution mass spectrometry].

An effective method based on ultra-high performance liquid chromatography-triple quadrupole/electrostatic field orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) was developed for high-throughput screening and quantitative analysis of 112 pharmaceutical and personal care products (PPCPs) in water. The water samples were separately extracted under alkaline and acidic conditions and cleaned on an SPE column (Cleanert PEP-2). The sample was separated and detected by UHPLC-Q-Orbitrap MS, and the data were collected in full MS scan/date dependent MS2 mode. All the 112 PPCPs in water were qualitatively screened and quantified on the basis of the peak area of the precursor ion. The PPCPs showed a good linear response in the mass concentration range of three orders of magnitude (r2 ≥ 0.9901), and the limits of quantification were between 0.002 µg/L and 0.8 µg/L. The recoveries of 60.1%-129.5% were achieved at different spiked levels of 0.2, 0.4, and 0.8 µg/L, and the RSDs were 1.1%-17.8% (n=6). Moreover, the method was used to screen the PPCPs in water samples acquired from seven regions of Dalian city (China), and 16 PPCPs were identified and quantified. Therefore, this simple method with high sensitivity and wide screening range can be applied to water quality management and environmental risk monitoring.

DOMe: A deduplication optimization method for the NewSQL database backups.

Reducing duplicated data of database backups is an important application scenario for data deduplication technology. NewSQL is an emerging database system and is now being used more and more widely. NewSQL systems need to improve data reliability by periodically backing up in-memory data, resulting in a lot of duplicated data. The traditional deduplication method is not optimized for the NewSQL server system and cannot take full advantage of hardware resources to optimize deduplication performance. A recent research pointed out that the future NewSQL server will have thousands of CPU cores, large DRAM and huge NVRAM. Therefore, how to utilize these hardware resources to optimize the performance of data deduplication is an important issue. To solve this problem, we propose a deduplication optimization method (DOMe) for NewSQL system backup. To take advantage of the large number of CPU cores in the NewSQL server to optimize deduplication performance, DOMe parallelizes the deduplication method based on the fork-join framework. The fingerprint index, which is the key data structure in the deduplication process, is implemented as pure in-memory hash table, which makes full use of the large DRAM in NewSQL system, eliminating the performance bottleneck problem of fingerprint index existing in traditional deduplication method. The H-store is used as a typical NewSQL database system to implement DOMe method. DOMe is experimentally analyzed by two representative backup data. The experimental results show that: 1) DOMe can reduce the duplicated NewSQL backup data. 2) DOMe significantly improves deduplication performance by parallelizing CDC algorithms. In the case of the theoretical speedup ratio of the server is 20.8, the speedup ratio of DOMe can achieve up to 18; 3) DOMe improved the deduplication throughput by 1.5 times through the pure in-memory index optimization method.

Use of CRISPR/Cas9 for Symbiotic Nitrogen Fixation Research in Legumes.

Nitrogen-fixing rhizobia have established a symbiotic relationship with the legume family through more than 60 million years of evolution. Hundreds of legume host genes are involved in the SNF (symbiotic nitrogen fixation) process, such as recognition of the bacterial partners, nodulation signaling and nodule development, maintenance of highly efficient nitrogen fixation within nodules, regulation of nodule numbers, and nodule senescence. However, investigations of SNF-related gene functions and dissecting molecular mechanisms of the complicated signaling crosstalk on a genomic scale were significantly restricted by insufficient mutant resources of several representative model legumes. Targeted genome-editing technologies, including ZFNs, TALENs, and CRISPR-Cas systems, have been developed in recent years and rapidly revolutionized biological research in many fields. These technologies were also applied to legume plants, and significant progress has been made in the last several years. Here, we summarize the applications of these genome-editing technologies, especially CRISPR-Cas9, toward the study of SNF in legumes, which should greatly advance our understanding of the basic mechanisms underpinning the legume-rhizobia interactions and guide the engineering of the SNF pathway into nonlegume crops to reduce the dependence on the use of nitrogen fertilizers for sustainable development of modern agriculture.

Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9.

The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.

Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus.

SymRK-interacting protein 1 (SIP1) has previously been shown to interact with the symbiosis receptor kinase, SymRK, in Lotus japonicus. A longer variant of the SIP1 transcript, SIP1L, was isolated and characterized. SIP1L contains an additional 17 amino acids that make its C-terminus a complete heat shock protein 20 (Hsp20)-like domain. In contrast to SIP1S, the longer splicing variant SIP1L could not interact with SymRK. Both SIP1L and SIP1S transcripts could be detected in developing nodules and other plant tissues, although the former was always more abundant than the latter. SIP1L and SIP1S formed heteromeric protein complexes, which were co-localized in the plasma membrane, cytoplasm and nuclei. Expression of SIP1-RNAi in transgenic hairy roots resulted in impairment in the nodule and arbuscular mycorrhizal development, suggesting an important role of SIP1 in the common symbiosis pathway. Overexpression of either SIP1L or SIP1S increased the number of nodules formed on transgenic hairy roots, indicating a positive role of SIP1 in nodulation. The SIP1S-like transcript was not detected in other higher plants tested, and the SIP1L-like proteins of these plants were capable of interacting with the SymRK orthologs. It is proposed that the loss of the ability of SIP1L to interact with SymRK in Lotus is compensated by the expression of a shorter splicing variant, SIP1S, which binds SymRK and may play a role in relaying the symbiosis signals to downstream cellular events.

Copper-catalyzed aldol-type addition of ketones to aromatic nitriles: a simple approach to enaminone synthesis.

An efficient method for the synthesis of enaminones is described. The aldol-type addition of ketones to aromatic nitriles proceeded smoothly in the presence of a simple copper catalyst system (CuI-2,2'-bipyridine-NaO(t)Bu) in N,N-dimethylformamide. Enaminones in satisfactory to excellent yields were produced using this technique.

Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell.

The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH ( 1 )) to selectively detect the fluorescence from donor, and a FRET channel (CH ( 2 )) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell.

Quantitative analysis of caspase-3 activation by fitting fluorescence emission spectra in living cells.

Confocal fluorescence imaging and fluorescence resonance energy transfer (FRET) technology have been widely used to study protein-protein interactions in living cells. However, it is very difficult to quantitatively analyze FRET efficiency due to the excitation spectral crosstalk and emission spectral crosstalk between donor and acceptor. In this study, we developed a novel method to quantitatively obtain the FRET efficiency by fitting the emission spectra (FES) of donor-acceptor pair, and this method is free from both excitation and emission spectral crosstalk. We used the FES method to quantitatively monitor the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by FES are consistent with that by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) in living cells stably expressing SCAT3. In this study, the FES was also used to analyze the caspase-3 activation in living cells during anti-cancer drug such as taxol, Artesunate (ART) or Dihydroartemisinin (DHA) treatment. Our results showed that ART or DHA induced apoptosis by a caspase-3-dependent manner, while caspase-3 was not involved in taxol-induced cell death.

[Fluorescence emission analysis of caspase-3 activation induced by Xiao-Ai-Ping (XAP) inside living human lung adenocarcinoma cells].

The CCK-8 was used to measure the inhibition effect of Xiao-Ai-Ping (XAP), a traditional medicine, on the human lung adenocarcinoma (ASTC-a-1) cells viability. The ASTC-a-1 cells expressing stably with SCAT3, a fluorescence resonance energy transfer (FRET) plasmid based on the green fluorescent protein mutants (GFPs), was verified using confocal fluorescence scanning microscopy imaging, fluorescence emission spectra and FRET acceptor photobleaching techniques. The caspase-3 activation can be monitored by the fluorescence emission spectra of SCAT3 inside living cells. The cells expressing stably with SCAT3 were cultured with XAP for 96 hours, and the fluorescence emission spectra of the SCAT3 inside living cells were measured at the time of 0, 24, 72, and 96 hours, respectively. Experimental results showed that: (1)XAP inhibited obviously the proliferation of ASTC-a-1 cells and induced the cell death. The inhibition of XAP on the cells was dose-dependent; (2)the SCAT3 inside living cells was cleaved completely 72 hours after the XAP treatment, implying that a great deal of pro-caspase-3 was activated by XAP; (3)24 hours after XAP treatment, the emission spectra of SCAT3 inside living cells cultured in DMEM without XAP for 48 and 72 hours did not change greatly, implying that XAP did not activate obviously caspase-3 within 24 hours.