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Aim: The oleanolic acid derivatives containing electrophilic warheads were synthesized, and their antitumor activities were investigated. Materials & methods: The cytotoxicity of compounds against tumor cells were determined by the MTT method. The antitumor effects of compounds 27a, Y03 and Y04 were evaluated in vitro through a wound-healing assay, apoptosis and cell circle analysis, and cellular reactive oxide species determination. The levels of related proteins in MCF-7 cells treated with Y03 was determined through Western blot analysis. Results & conclusion: Compounds 27a, Y03 and Y04 displayed high cytotoxicity against breast cancer cells and inhibited cell migration, induced apoptosis, arrest cell circle at G0/G1 and promoted cellular reactive oxide species generation. The antitumor mechanism involved inhibition of Akt/mTOR and induction of ferroptosis.

Oleanolic acid (OA) and its derivatives show potent anticancer function. Pancreatic cancer (PC) is the fourth core motive of cancer-related deaths worldwide. Epidermal growth factor receptor (EGFR) has been implicated in PC and has been validated as a therapeutic target. Our study demonstrated that K73-03, an OA derivative, was identified as a potent inhibitor of EGFR by using reverse pharmacophore screening and molecular dynamics simulation assays. Moreover, Western blot analysis showed that K73-03 markedly suppressed the levels of phosphorylated-EGFR (p-EGFR) and phosphorylated-Akt (p-Akt). The inhibitory effect of K73-03 on PC cells was assessed in vitro and in vivo. Mechanistically, K73-03 effectively inhibited the cell proliferation of PC cells, and induced apoptosis and autophagy of ASPC-1 cells in a dose-dependent manner. Additionally, pretreatment with chloroquine, an autophagy inhibitor, significantly inhibited K73-03-induced autophagy and enhanced K73-03-induced apoptotic cell death. K73-03 also strongly repressed ASPC-1 cells xenograft growth in vivo. Thus, all these findings provided new clues about OA analog K73-03 as an effective anticancer agent targeted EGFR against ASPC-1 cells, it is worth further evaluation in the future.

The metal-polyphenol network has been widely employed in the field of nanodrugs delivery due to its pH-responsiveness.

Efficient visualization helps researchers obtain valuable mechanistic insights and present interesting results with regard to the functional enrichment analysis of omics data. However, the functions of existing published tools used to implement relevant visualization are neither sufficiently comprehensive nor easily accessible. Most of these tools require users to have professional programming skills. This study alleviates this issue by proposing EnrichVisBox, a web application developed for integrative and versatile data visualization, including bubble plots, UpSet plots, polar bar plots, rectangle plots, ridgeline plots, network plots, and variant chord plots. Specifically, scientists can use these insightful plots to conveniently present functional enrichment analysis results of omics data with a simple mouse click through a user-friendly interface.

Evolutionary profiling has been largely limited to the nucleotide level. Using consistent proteomic methods, we quantified proteomic and phosphoproteomic layers in fibroblasts from 11 common mammalian species, with transcriptomes as reference. Covariation analysis indicates that transcript and protein expression levels and variabilities across mammals remarkably follow functional role, with extracellular matrix–associated expression being the most variable, demonstrating strong transcriptome-proteome coevolution. The biological variability of gene expression is universal at both interindividual and interspecies scales but to a different extent. RNA metabolic processes particularly show higher interspecies versus interindividual variation. Our results further indicate that while the ubiquitin-proteasome system is strongly conserved in mammals, lysosome-mediated protein degradation exhibits remarkable variation between mammalian lineages. In addition, the phosphosite profiles reveal a phosphorylation coevolution network independent of protein abundance.

Chemotherapeutic multidrug resistance (MDR) is the major hindrance for clinical therapy of colorectal cancer (CRC). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with selective cytotoxicity might overcome MDR of CRC cells. Unfortunately, cross-resistance to TRAIL has been detected in many CRC cells, suggesting the need to combine TRAIL with sensitizers to combat refractory CRC. Our purpose is to explore the potential of combination therapy of TRAIL and tumor-cell targeted photodynamic therapy (PDT) in combating CRC with both chemotherapeutic MDR and TRAIL resistance. Methods: Tumor cell-targeted PDT was performed using a Ze-IR700 photosensitizer with high affinity for epidermal growth factor receptor (EGFR). The impact of PDT on the gene expression of CRC cells was revealed by RNA sequencing. The synergistic antitumor effect of long-acting TRAIL and PDT was evaluated in mice bearing tumor grafts of CRC cells with both chemotherapeutic MDR and TRAIL resistance. Results: Chemotherapeutic MDR and TRAIL resistance are common in CRC cells. Pretreatment of CRC cells with tumor cell-targeted PDT significantly (10-60 times) increased the sensitivity of these CRC cells to TRAIL by upregulating death receptors. Combination therapy, but not monotherapy, of long-acting TRAIL and PDT greatly induced apoptosis of CRC cells, thus efficiently eradicated large (~150 mm3) CRC tumor xenografts in mice. Conclusions: Tumor cell-targeted PDT extensively sensitizes CRC cells to TRAIL. Combination therapy of long-acting TRAIL and PDT is promising to combat CRC with both chemotherapeutic MDR and TRAIL resistance, which might be developed as a novel strategy for precision therapy of refractory CRC.

Rationale: Over 50% of patients with heart failure have preserved ejection fraction (HFpEF), rather than reduced ejection fraction. Complexity of its pathophysiology and the lack of animal models hamper the development of effective therapy for HFpEF. Objective: This study was designed to investigate the metabolic mechanisms of HFpEF and test therapeutic interventions using a novel animal model. Methods and Results: By combining the age, long-term high-fat diet, and desoxycorticosterone pivalate challenge in a mouse model, we were able to recapture the myriad features of HFpEF. In these mice, mitochondrial hyperacetylation exacerbated while increasing ketone body availability rescued the phenotypes. The HFpEF mice exhibited overproduction of IL (interleukin)-1β/IL-18 and tissue fibrosis due to increased assembly of NLPR3 inflammasome on hyperacetylated mitochondria. Increasing β-hydroxybutyrate level attenuated NLPR3 inflammasome formation and antagonized proinflammatory cytokine–triggered mitochondrial dysfunction and fibrosis. Moreover, β-hydroxybutyrate downregulated the acetyl-CoA pool and mitochondrial acetylation, partially via activation of CS (citrate synthase) and inhibition of fatty acid uptake. Conclusions: Therefore, we identify the interplay of mitochondrial hyperacetylation and inflammation as a key driver in HFpEF pathogenesis, which can be ameliorated by promoting β-hydroxybutyrate abundance.

Evolutionary profiling has been largely limited to the nucleotide level. Using consistent proteomic methods, we quantified proteomic and phosphoproteomic layers in fibroblasts from 11 common mammalian species, with transcriptomic variability as reference. The co-variation analysis indicates that transcript and protein expression robustness across mammals remarkably follows functional role, with extracellular matrix-associated expressions being most variable, demonstrating strong transcriptome-proteome co-evolution. Interestingly, the variability control of gene expression is universal at both inter-individual and inter-species scales, but of different extent. RNA metabolism processes particularly show the higher inter-species versus inter-individual variations. Our results further uncover that while ubiquitin-proteasome system is extremely conserved in mammals, the lysosome-mediated protein degradation exhibits a remarkable variation between mammalian lineages. Additionally, the phosphosite profiles reveals phosphorylation co-evolution network independent of protein abundance.

Three derivatives of chlorin e6 (1–3) were synthesized by introduction of maleimide, cysteine and glutathione at C-13 carboxyl of the chlorin scaffold. The evaluation of their PDT effects showed that compound 1, the derivative with a maleimide group, exhibited more potent photocytotoxicity against HepG2 cells (IC[Formula: see text] 3.2 [Formula: see text]M) than 2 (IC[Formula: see text] 6.7 [Formula: see text]M) and 3 (IC[Formula: see text] 10.2 [Formula: see text]M), although the cellular uptake of 1 was slightly lower than that of 2 and 3. The high PDT effect of 1 was found to be in agreement with the high level of intracellular singlet oxygen. Further investigation of the mechanism revealed that 1 can significantly lower the GSH level in HepG2 cells due to the addiction reaction of maleimide and GSH, thus resulting in the reduction of ROS scavenging and the enhancement of cellular oxidative stress. This approach to improve PDT effects of photosensitizers by means of interfering with the cellular redox system and enhancing cellular oxidative stress offers a new strategy for development of photosensitizers in cancer therapy.

The discovery of novel non-invasive biomarkers for discriminating between prostate carcinoma (PCa) patients and benign prostatic hyperplasia (BPH) patients is necessary to reduce the burden of biopsies, avoid overdiagnosis and improve quality of life. Previous studies suggest that abnormal glycosylation of immunoglobulin gamma molecules (IgGs) is strongly associated with immunological diseases and prostate diseases. Hence, characterizing N-linked intact glycopeptides of IgGs that correspond to the N-glycan structure with specific site information might enable a better understanding of the molecular pathogenesis and discovery of novel signatures in preoperative discrimination of BPH from PCa. In this study, we profiled N-linked intact glycopeptides of purified IgGs from 51 PCa patients and 45 BPH patients by our developed N-glycoproteomic method using hydrophilic interaction liquid chromatography enrichment coupled with high resolution LC-MS/MS. The quantitative analysis of the N-linked intact glycopeptides using pGlyco 2.0 and MaxQuant software provided quantitative information on plasma IgG subclass-specific and site-specific N-glycosylation. As a result, we found four aberrantly expressed N-linked intact glycopeptides across different IgG subclasses. In particular, the N-glycopeptide IgG2-GP09 (EEQFNSTFR (H5N5S1)) was dramatically elevated in plasma from PCa patients, compared with that in BPH patients (PCa/BPH ratio = 5.74, p = 0.001). Additionally, the variations in these N-linked intact glycopeptide abundances were not caused by the changes in the IgG concentrations. Furthermore, IgG2-GP09 displayed a more powerful prediction capability (auROC = 0.702) for distinguishing PCa from BPH than the clinical index t-PSA (auROC = 0.681) when used alone or in combination with other indicators (auROC = 0.853). In conclusion, these abnormally expressed N-linked intact glycopeptides have potential for non-invasive monitoring and pre-stratification of prostate diseases.

Zinc(ii)-catalyzed intramolecular hydroarylation-redox cross-dehydrogenative coupling of N-propargylanilines with two types of carbon pronucleophiles (nitromethane as a sp3 carbon pronucleophile and phenylacetylenes as sp carbon pronucleophiles) proceeded to give the 2-substituted tetrahydroquinolines in good yields with 100% atomic utilization without any additional external oxidants.

Blood–brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti–ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Breast cancer is of the leading causes of cancer-related deaths and the most frequently diagnosed cancer among females worldwide. Despite advancements in breast cancer therapy, the disease eventually progresses in most patients because of de novo or secondary resistance. Thus, discovering novel drugs with high effectiveness and low toxicity for systemic therapy is essential. In this study, we investigated whether a new oleanolic derivative N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methylene-3-oxo-olean-12-en-28-amide (ZQL-4c) exhibits potential anticancer effects against breast cancer. We determined that ZQL-4c strongly inhibited cell proliferation and invasion and induced G2/M phase arrest and apoptosis in breast cancer cells. We then found that ZQL-4c induced the production of reactive oxygen species (ROS). We then found that ZQL-4c significantly inhibited Notch-AKT signaling pathways that are related to oxidative stress. Taken together, this study is the first to show that ZQL-4c can significantly suppress the growth and invasion of breast cancer by blocking Notch-Akt signaling pathways, which are mainly regulated by ROS-mediated oxidative stress. Thus, ZQL-4c might be considered a novel and potential anticancer drug for breast cancer treatment.

In order to study calculation technology of global structure strength for the deep water typical Spar platform its global structural strength analysis is completed. The dynamic-part and a low frequency-part loads are considered in this analysis. First, according to the 100-year return storm design wave parameters are obtained through search. wave loads with design wave parameters are calculated, and are applied to the structural model built. The stresses of global structure are gotten by finite element structural analysis. Then, low frequency-part loads which include wind, current and mooring forces also are applied to the structural model. The stresses produced by low frequency-part loads are gotten by finite element structural analysis. Finally the stresses produced by dynamic-part and a low frequency-part loads are combined to form total stresses of structure of Spar, and evaluation of structural strength of Spar is made in term of the rule. Analysis method for the structural strength of the deepwater typical Spar platform can be used as reference for relative technical people.

Proteomics analysis is often troubled by high-abundance proteins in samples such as plasma. However, many surgical tissue samples inevitably have got contaminated with blood before cryopreservation. Selection of an appropriate method to minimize the effect of high-abundance proteins is important for proteomics analysis of blood contaminated tissues. Here, we investigated and compared the abilities of data-independent acquisition (DIA) and data-dependent acquisition (DDA) strategies for the proteomics analysis of blood contaminated clinical tissue samples. Twelve pairs of carcinoma and para-carcinoma tissue samples from lung cancer patients were used for proteomics assays separately by DIA and DDA, and the blood contamination level in samples was evaluated by contamination index (CI). Compared with the DDA strategy, DIA in whole exhibited much better analytical capabilities in proteomics analysis of these samples with more identified protein groups and a higher discovery of differential proteins. With CI value increasing, whether DIA or DDA showed decreasing analysis ability. However, for samples with high CI values, the DIA strategy still shows acceptable analytical capability and indicates better blood pollution resistance than the DDA strategy. Our results implied that for clinical tissue samples, particularly for those contaminated with blood, DIA strategy should be a preferred method in proteomics studies.

Description, The “molecular machine” C34 could suppress tumor systemically via unimolecule-mediated sono/immunotherapy., Exploring facile and effective therapeutic modalities for synergistically controlling primary tumor and metastasis remains a pressing clinical need. Sonodynamic therapy (SDT) offers the possibility of noninvasively eradicating local solid tumors, but lacks antimetastatic activity because of its limited ability in generating systemic antitumor effect. Here, we exploited a previously unidentified ultrasound-driven “molecular machine,” DYSP-C34 (C34 for short), with multiple attractive features, emerging from preferential tumor accumulation, potent ultrasound-triggered cytotoxicity, and intrinsic immune-boosting capacity. Driven by the ultrasound, C34 functioned not only as a tumor cell killing reagent but also as an immune booster that could potentiate robust adaptive antitumor immunity by directly stimulating dendritic cells, resulting in the eradication of the primary solid tumor along with the inhibition of metastasis. This molecular machine, C34, rendered great promise to achieve systemic treatment against cancer via unimolecule-mediated SDT.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is recognized for its promising therapeutic effects against cancer. However, mechanisms underlying the effect of TRAIL on protein expression, signal transduction, and apoptosis induction remain unclear. We surmised that a systematic analysis of the proteome and phosphoproteome associated with TRAIL signaling may help elucidate the mechanisms involved and facilitate the development of therapeutics. Therefore, we investigated the proteome and phosphoproteome of non-small cell lung cancer cell line A549 treated with TRAIL. Our results indicated that 126 proteins and 1684 phosphosites were markedly differentially expressed between the phosphate-buffered saline- and TRAIL-treated groups. The expression at protein and phosphosite levels were not completely consistent. Gene ontology functional analysis revealed that metal ion (zinc) binding was highly affected by TRAIL treatment. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that almost all pathways that involved differentially expressed phosphosites were associated with apoptosis. We also identified an important kinase, AKT1, and its series of substrates in TRAIL signaling. The results of this study may provide guidance for future research on tumor therapy using TRAIL.

In-box LOCA accident is considered as a Design Basis Accident (DBA) of a fusion blanket because of its frequency and serious consequence. For a liquid metal blanket such as Dual-Functional Lithium Lead (DFLL) blanket, in-box LOCA not only causes pressurization in breeder box, but also propagates transient high pressure to Lithium Lead (PbLi) loop system. Similar to the water hammer effect in the coolant loop of a fission power plant, a high pressure wave in PbLi loop might damage components and threaten the integrity of loop structure. In this paper, the 3D Computational Fluid Dynamics (CFD) code FLUENT was coupled with the systems code RELAP5/MOD4.0 to simulate pressure propagation behavior triggered by an in-box LOCA accident in the PbLi loop system of a DFLL blanket. Reflection and superposition of the pressure wave in complex channels of a DFLL blanket module were accurately simulated with FLUENT, and propagation of pressure waves in the PbLi loop system was simulated by RELAP5 code. The coupling code, which linked FLUENT and RELAP5, transferred the pressure at the inlet/outlet interface of the blanket module to the PbLi loop. Simulation results showed that at the initial stage, the transient peak pressure at the monitoring points in the blanket module reached 14.454 MPa, and the transient peak pressure at the inlet/outlet of the blanket module were 9.612 MPa and 14.454 MPa respectively. Then the high pressure waves propagated to the PbLi loop, and triggered the opening of a blasting valve which connected the low-pressure PbLi tank to the PbLi main loop. The low-pressure PbLi tank acted as a water hammer arrestor and eliminated high pressure in the main loop pipe, which protected the downstream components.

The selaginellin derivatives are a type of novel natural pigments with an unusual alkynyl phenol skeleton from the genus Selaginella. Some of these natural compounds were previously reported to show important bioactivities, including anticancer activity, cardiovascular protection and phosphodiesterase-4 inhibition. We designed and synthesized fifteen biphenyl-containing diaryl acetylene derivatives mimicking the skeleton of natural alkynyl phenols. In MTT assay in cancer cells, compounds 1c, 2d, 2g, 2h, 2i and 2j exhibited potent antiproliferative activity. The evaluation of Hypoxia Inducible Factor-1 (HIF-1) pathway inhibitory activity in dual luciferase assay demonstrated that most tested compounds exhibited moderate to good activities. Compounds 1a, 2f and 2h displayed high HIF-1 inhibitory activities and relatively low cytotoxicity, demonstrating great potential as HIF-1 inhibitors. These results afford a new strategy for the discovery of new HIF-1 inhibitors and anti-proliferative agents from natural or synthetic diaryl acetylene derivatives.

Blanket is the strategic component in hydrogen fusion energy system, converting nuclear energy of hydrogen nuclei to thermal energy which could be further transported for the production of electricity. In order to investigate blanket transient behaviors induced by the in-box LOCA (Loss of Coolant Accident), a code named LBBFoam for high pressure compressible multi-phase flow is developed based on OpenFOAM, which is capable of simulating accident-induced pressure wave propagation in the blanket module. Two classic one dimension shock tube cases were simulated to verify the code. With this code, three scenarios with 8 MPa helium injection into PbLi in a blanket-like container were analyzed to capture pressure oscillation and bubble transportation characteristics. It is found that the maximum pressure in the PbLi zone exceeds and even doubles the helium injection pressure due to the reflection and superposition of pressure waves. This suggests that the traditional structural safety design limits of helium cooled PbLi blanket, which is usually set as the operating pressure of helium coolant, would be too low considering pressure wave superposition. This code will provide an assessment tool for the structural safety of blanket.

Shexiang Baoxin Pill (SBP) and Suxiao Jiuxin Pill (SJP) are traditional Chinese medicines used to treat cardiovascular disease (CVD) in China. However, the mechanism of their therapeutic effect on CVD has not been clearly elucidated yet.The aim of this study is to investigate the cardioprotective effect of SBP and SJP in the treatment of acute myocardial infarction (AMI) model rats by applying serum proteomic approach.The rat model of AMI was generated by ligating the left anterior descending coronary artery. 42 rats were randomly divided into four groups: sham-operating (Sham, n = 10) group, model (Mod, n = 8) group, Shexiang Baoxin pills pretreatment (SBP, n = 12) group and Suxiao Jiuxin pills pretreatment (SJP, n = 12) group. Data Independent Acquisition (DIA) proteomic approach was utilized to investigate the serum proteome from the rat individuals. The differentially expressed proteins were subsequently obtained with bioinformatic analysis.DIA-MS identified 415 proteins within 42 samples, and 84 differentially expressed proteins may contribute to the therapeutic effects of SBP and SJP. GOBP and KEGG pathway analysis of 84 differentially expressed proteins revealed that the proteins were mainly involved in platelet activation and adhesion processes. All 84 differentially expressed proteins presented the same changing tendency in the SBP and SJP groups when compared with the Mod group. Among these 84 proteins, 25 proteins were found to be related to CVD. Among these 25 proteins, ACTB, ACTG1, FGA, FGB, FGG, PF4 and VWF were found to be involved in platelet aggregation and activation. FN1, HSPA5 and YWHAZ were associated with adhesion.The results of our study suggest that the cardioprotective effects of SBP and SJP are achieved through the modulation of focal adhesion, platelet activation pathways.

This paper describes the flow field and impact load characteristics of a ventilated cylinder upon vertical and angled water entry. A high-speed camera is used to record the evolution of the cavity, and a pressure sensor placed on the head of the cylinder records the impact load. The results show that head ventilation produces an initial cavity before the cylinder enters the water. This changes the evolution of the cavity, reduces the volume of the cavity, and modifies the cavity closure mechanism. As the ventilation rate increases, the impact load decreases rapidly at first and then stabilizes at a certain level. The angle of attack also changes the flow field and impact load characteristics. In the case of angled water entry, an obvious asymmetric cavity is formed, and then cavity separation occurs. An increase in the angle of attack or the ventilation rate strengthens the cavity separation. The impact load decreases with increasing angle of attack at low ventilation rates but is independent of the angle of attack at high ventilation rates.

7-Hydroxyneolamellarin A (7-OH-Neo A, 1), a natural marine product derived from sponge Dendrilla nigra, was first synthesized with 10% overall yield under the instruction of convergent synthetic strategy. We found that 7-OH-Neo A could attenuate the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein and inhibit vascular epidermal growth factor (VEGF) transcriptional activity, showing well inhibitory effect on HIF-1 signaling pathway. Meantime, 7-OH-Neo A had the well anti-tumor activities, such as inhibiting tumor angiogenesis, proliferation, migration and invasion. More importantly, 7-OH-Neo A exhibited profound anti-tumor effect in mice breast cancer model by suppressing the accumulation of HIF-1α in tumor tissue. Mechanism study demonstrated that 7-OH-Neo A might target the protein with the ability of stabilizing HIF-1α in hypoxia. Due to the excellent water solubility, superior anti-tumor activity and good biocompatibility, 7-OH-Neo A shows the promising potential for being exploited as an anti-tumor agent in near future.

The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA规律性短重复回文序列簇（CRISPR）和 CRISPR 辅助蛋白 9（Cas9）构成的 CRISPR/Cas9 基因编辑技术快速推进了基因修饰猪作为医学研究模式动物的广泛应用。而高效的靶基因单链向导 RNA（sgRNA）是利用 CRISPR/Cas9 技术进行基因编辑成功的关键，对于猪等繁殖周期较长的大动物，则需要在实施动物实验前，在体外筛选出高效的 sgRNA 以避免时间和资源成本浪费。另外，如何高效获得阳性基因编辑单克隆细胞是目前尚待解决的难题。本研究建立了靶向猪基因组的 sgRNA 快速筛选方法，利用荧光载体富集基因编辑细胞，同时探索利用图案微阵列培养技术快速获得单克隆细胞的方法，在此基础上高效获得延胡索酰乙酰乙酸酶（

Background Gemcitabine (GCB) is a first-line chemotherapeutic drug for pancreatic cancer (PCa). However, the resistance begins developing within weeks of chemotherapy. SPINK1 overexpression enhances resistance to chemotherapy. In a recent study, our laboratory established that the oleanolic acid (OA) derivative, K73-03, had a strong inhibitory effect on a SPINK1 overexpressed PCa cells. Purpose In our current study, we studied the enhancement of GCB inhibitory effect by K73-03, a new novel OA derivative, alone or in combination with GCB on the GCB-resistant PCa cells by mitochondrial damage through regulation of the miR-421/SPINK1. Methods We detected the binding between miR-421 and SPINK1-3′-UTR in GCB-resistant PCa cells using Luciferase reporter assays. Cells viability, apoptosis, migration, and mitochondrial damage were investigated. Results The results demonstrated that the combination of K73-03 and GCB suppressed the growth of AsPC-1 and MIA PaCa-2 cells synergistically, with or without GCB resistance. Mechanistic findings showed that a combination of K73-03 and GCB silences SPINK1 epigenetically by miR-421 up-regulating, which leads to mitochondrial damage and inducing apoptosis in GCB-resistant PCa cells. Conclusion We found an interesting finding that the 73-03 in combination with GCB can improve GCB efficacy and decrease PCa resistance, which induced apoptosis and mitochondrial damage through epigenetic inhibition of SPINK1 transcription by miR-421 up-regulation. This was the first study that used OA derivatives on GCB-resistant PCa cells, so this combined strategy warrants further investigation.