The combination of breast cancer PDO and mini-PDX platform for drug screening and individualized treatment.

The majority of advanced breast cancers exhibit strong aggressiveness, heterogeneity, and drug resistance, and currently, the lack of effective treatment strategies is one of the main challenges that cancer research must face. Therefore, developing a feasible preclinical model to explore tailored treatments for refractory breast cancer is urgently needed. We established organoid biobanks from 17 patients with breast cancer and characterized them by immunohistochemistry (IHC) and next generation sequencing (NGS). In addition, we in the first combination of patient-derived organoids (PDOs) with mini-patient-derived xenografts (Mini-PDXs) for the rapid and precise screening of drug sensitivity. We confirmed that breast cancer organoids are a high-fidelity three-dimension (3D) model in vitro that recapitulates the original tumour's histological and genetic features. In addition, for a heavily pretreated patient with advanced drug-resistant breast cancer, we combined PDO and Mini-PDX models to identify potentially effective combinations of therapeutic agents for this patient who were alpelisib + fulvestrant. In the drug sensitivity experiment of organoids, we observed changes in the PI3K/AKT/mTOR signalling axis and oestrogen receptor (ER) protein expression levels, which further verified the reliability of the screening results. Our study demonstrates that the PDO combined with mini-PDX model offers a rapid and precise drug screening platform that holds promise for personalized medicine, improving patient outcomes and addressing the urgent need for effective therapies in advanced breast cancer.

Assessing regional employment effects of the national emission trading scheme in China: Does Okun's law work?

Employment creation and climate change mitigation are core tasks for achieving sustainable development goals. Whether or not carbon mitigation policy facilitate employment deserves deep exploration. Through the construction of multi-regional dynamic computable general equilibrium model (CGE) with more scientific energy & environment block, this paper first evaluates regional employment effects of the national emission trading scheme (ETS) in China. Furthermore, we explore the Okun's law of the national ETS based on the mediating effect model. The results show that whether in carbon-intensive industries (CIIs) or non-carbon-intensive industries (NCIIs), employment effects of the national ETS are differentiated across regions. Specifically, the national ETS generally promotes CIIs' employment in Southern, Eastern, Middle Yangtze River and Southwest regions, and has negative effects on CIIs' employment in other regions. Meanwhile, the national ETS brings employment creation to NCIIs of Southern region, while there are opposite results in NCIIs of Northeast region and mixed results in NCIIs of other regions. Moreover, the Okun's law of the national ETS holds in CIIs of each region, but it not fits the data for NCIIs. Therefore, it is important for the Chinese government to consider the differentiated employment effects in different regions carefully rather than adopt one-size-fit-all solution when constructing the national carbon market.

Early evaluation of circulating tumor DNA as marker of therapeutic efficacy and prognosis in breast cancer patients during primary systemic therapy.

BACKGROUND: We assessed the potential role of serial circulating tumor DNA (ctDNA) as a biomarker to monitor treatment response to primary systemic therapy (PST) in breast cancer and evaluated the predictive value of ctDNA to further identify patients with residual disease. METHODS: We prospectively enrolled 208 plasma samples collected at three time points (before PST, after 2 cycles of treatment, before surgery) of 72 patients with stage Ⅱ-III breast cancer. Somatic mutations in plasma samples were identified using a customized 128-gene capture panel with next-generation sequencing. The correlation between early change in ctDNA levels and treatment response or long-term clinical outcomes was assessed. RESULTS: 37 of 72 (51.4%) patients harbored detectable ctDNA alterations at baseline. Patients with complete response showed a larger decrease in ctDNA levels during PST. The median relative change of variant allele fraction (VAF) was -97.4%, -46.7%, and +21.1% for patients who subsequently had a complete response (n = 11), partial response (n = 11), and no response (n = 15) (p = 0.0012), respectively. In addition, the relative change of VAF between the pretreatment and first on-treatment blood draw exhibited the optimal predictive value to tumor response after PST (area under the curve, AUC = 0.7448, p = 0.02). More importantly, early change of ctDNA levels during treatment have significant prognostic value for patients with BC, there was a significant correlation between early decrease of VAF and longer recurrence-free survival compared to those with an VAF increase (HR = 12.54; 95% CI, 2.084 to 75.42, p = 0.0063). CONCLUSION: Early changes of ctDNA are strongly correlated with therapeutic efficacy to PST and clinical outcomes in BC patients. The integration of preoperative ctDNA evaluation could help improving the perioperative management for BC patients receiving PST.

Biological properties and potential application of extracts and compounds from different medicinal parts (bark, leaf, staminate flower, and seed) of Eucommia ulmoides: A review.

Eucommia ulmoides (E. ulmoides) is a relict plant belonging to the Eucommiaceae family. Each part of E. ulmoides including the bark, leaf, staminate flower, and seed, contains various active ingredients, that are edible and have medicinal value. This review summarizes the literatures on the functional properties of flavonoids, phenols, terpenoids, and polysaccharides in different parts of E. ulmoides. The prospects for application of the different parts of E. ulmoides as functional foods are also discussed. This review provides a reference for further research and development of the medicinal application of E. ulmoides.

PROTACs Targeting BRM (SMARCA2) Afford Selective In Vivo Degradation over BRG1 (SMARCA4) and Are Active in BRG1 Mutant Xenograft Tumor Models.

The identification of VHL-binding proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) in SW1573 cell-based experiments is described. These molecules exhibit between 10- and 100-fold degradation selectivity for BRM over the closely related paralog protein BRG1 (SMARCA4). They also selectively impair the proliferation of the H1944 "BRG1-mutant" NSCLC cell line, which lacks functional BRG1 protein and is thus highly dependent on BRM for growth, relative to the wild-type Calu6 line. In vivo experiments performed with a subset of compounds identified PROTACs that potently and selectively degraded BRM in the Calu6 and/or the HCC2302 BRG1 mutant NSCLC xenograft models and also afforded antitumor efficacy in the latter system. Subsequent PK/PD analysis established a need to achieve strong BRM degradation (>95%) in order to trigger meaningful antitumor activity in vivo. Intratumor quantitation of mRNA associated with two genes whose transcription was controlled by BRM (PLAU and KRT80) also supported this conclusion.

Novel disulfidptosis-derived gene blueprint stratifying patients with breast cancer.

INTRODUCTION: Breast cancer remains the predominant cancer among females, accounting for about 24.2% of all cancer cases. Alarmingly, it is the primary cause of cancer-related mortality in women under 45. METHODS: This research analyzed RNA sequencing data from 1082 TCGA-BRCA and 107 GSE58812 breast cancer patients. Single-cell RNA data from five patients in the GSE118389 data set were also studied. Using Random forest and COX regression, we developed a prognostic model. Pathway analysis employed GSVA and GO, while immune profiles were assessed via ssGSEA and MCPcounter. Mutation patterns utilized maftools, and drug sensitivity scores were derived from the GDSC database with oncoPredict. RESULTS: Analysis of the GSE118389 data set identified three distinct cell types: immune, epithelial, and stromal. P53 and VEGF were notably enriched. Five key genes (TMEM251, ADAMTSL2, CDC123, PSMD1, TLE1) were pinpointed for their prognostic significance. We introduced a disulfidptosis-associated score as a novel risk factor for breast cancer prognosis. Survival outcomes varied significantly between training and validation sets. Comprehensive immune profiling revealed no difference in activated CD8-positive T cells between risk groups, but a positive correlation of NK cells, neutrophils, cytotoxic lymphocytes, and monocytic cells with the riskscore was noted. Importantly, a negative association between the drug Nelarabine and riskscore was identified. CONCLUSION: This research underscores the significance of a disulfidptosis-associated gene signature in breast cancer prognosis.

Multi-object detection for crowded road scene based on ML-AFP of YOLOv5.

Aiming at the problem of multi-object detection such as target occlusion and tiny targets in road scenes, this paper proposes an improved YOLOv5 multi-object detection model based on ML-AFP (multi-level aggregation feature perception) mechanism. Since tiny targets such as non-motor vehicle and pedestrians are not easily detected, this paper adds a micro target detection layer and a double head mechanism to improve the detection ability of tiny targets. Varifocal loss is used to achieve a more accurate ranking in the process of non-maximum suppression to solve the problem of target occlusion, and this paper also proposes a ML-AFP mechanism. The adaptive fusion of spatial feature information at different scales improves the expression ability of network model features, and improves the detection accuracy of the model as a whole. Our experimental results on multiple challenging datasets such as KITTI, BDD100K, and show that the accuracy, recall rate and mAP value of the proposed model are greatly improved, which solves the problem of multi-object detection in crowded road scenes.

Update on the relationship between the SLC4A7 variant rs4973768 and breast cancer risk: a systematic review and meta-analysis.

OBJECTIVE: This meta-analysis aimed to update knowledge about the association between the SLC4A7 variant rs4973768 and breast cancer incidence. METHODS: Studies were identified from relevant digital databases. Fixed- or random-effects models were used to calculate odds ratios and 95% confidence intervals. Statistical Q and I2 tests and sensitivity analyses were used to detect interstudy heterogeneity and test the statistical stability of overall estimates, respectively. Egger's tests were applied to detect publication bias among included studies. In silico analysis was used to ascertain increased expression of SLC4A7 mRNA in rs4973768 with the mutant allele. Trial sequential analysis was used to calculate the study's sample size. RESULTS: The overall odds ratios reflected a positive correlation between the SLC4A7 rs4973768 polymorphism and susceptibility to breast cancer in five genetic comparisons of alleles T and C, and tests revealed significant heterogeneity in the allele comparison. After stratification by ethnicity, heterogeneity in Asian and White populations substantially decreased (Ph = 0.984, I2 = 0%) and remained stable (Ph = 0.083, I2 = 46.3%), respectively. The mutant allele was associated with increased expression of SLC4A7 mRNA in rs4973768. The cumulative z curve indicated that our conclusions were robust. CONCLUSIONS: Our updated consequence shows that the SLC4A7 rs4973768 polymorphism is associated with increased breast cancer risk.

Bioinformatics analysis reveals lipid metabolism may play an important role in the SiO2-stimulated rat model.

Silicosis is a progressive and irreversible common occupational disease caused by long-term inhalation of a large amount of free silica dust. Its pathogenesis is complex, and the existing prevention and treatment methods can not effectively improve silicosis injury. To uncover potential differential genes in silicosis, SiO2-stimulated rats and their control original transcriptomic data sets GSE49144, GSE32147 and GSE30178 were downloaded for further bioinformatics analysis. We used R packages to extract and standardize transcriptome profiles, then screened differential genes, and enriched GO and KEGG pathways through clusterProfiler packages. In addition, we investigated the role of lipid metabolism in the progression of silicosis by qRT-PCR validation and transfection with si-CD36. A total of 426 differential genes were identified in this study. Based on GO and KEGG enrichment analysis, it was found that lipid and atherosclerosis were significantly enriched. qRT-PCR was used to detect the relative expression level of differential genes in this signaling pathway of silicosis rat models. mRNA levels of Abcg1, Il1b, Sod2, Cyba, Cd14, Cxcl2, Ccl3, Cxcl1, Ccl2 and CD36 increased, mRNA levels of Ccl5, Cybb and Il18 decreased. In addition, at the cellular level, SiO2-stimulated lead to lipid metabolism disorder in NR8383, and silencing CD36 inhibited SiO2-induced lipid metabolism disorder. These results indicate that lipid metabolism plays an important role in the progression of silicosis, and the genes and pathways reported in this study may provide new ideas for the pathogenesis of silicosis.

Chronic Eosinophilic Pneumonia: Unraveling Recurrent Chest Infections in a Female Patient With a Family History of Atopy.

Eosinophilic pneumonia is a rare condition characterized by the infiltration of eosinophils in the lungs. We present a case of eosinophilic pneumonia in a 51-year-old British Caucasian female with a history of lichen sclerosus, deranged liver function tests, and a family history of atopy. The patient presented with fever, shortness of breath, lethargy, dry cough, and weight loss over a three-month period. Initial treatment with antibiotics did not yield improvement, and further investigations revealed marked eosinophilia on blood count. Bronchoscopy and biopsies confirmed the diagnosis of eosinophilic pneumonia, and the patient responded well to a tapering dose of prednisolone. This study highlights the importance of considering eosinophilic pneumonia in patients with unexplained respiratory symptoms and eosinophilia and emphasizes the role of bronchoscopy in establishing a definitive diagnosis.

USF1-CHCHD4 axis promotes lung adenocarcinoma progression partially via activating the MYC pathway.

BACKGROUND: This study aimed to identify genes related to lung adenocarcinoma (LUAD) and investigate the effects and molecular mechanisms of coiled-coil-helix-coiled-coil-helix domain containing 4 (CHCHD4) in the progression of LUAD. METHODS: The GEPIA database was used to evaluate the differential expression of CHCHD4 and the survival data of LUAD patients compared to controls. TCGA-LUAD database, JASPAR website, and GSEA were used to analyse the relationship between CHCHD4 and the upstream stimulating factor 1 (USF1) or MYC pathways. The proliferation, apoptosis, migration, and invasion of LUAD cells were evaluated using cell counting kit-8, 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, wound healing, and transwell assays. qRT-PCR, western blotting, and immunohistochemistry were used to detect the mRNA and protein expression, respectively. Furthermore, xenograft tumours from nude mice were used to verify the effect of CHCHD4 on LUAD in vivo. RESULTS: CHCHD4 overexpression was found in LUAD tumor tissues and cells, and high CHCHD4 was associated with a poor prognosis. Interestingly, CHCHD4 knockdown suppressed the malignant phenotype of the LUAD cells. Moreover, we found that USF1 upregulated CHCHD4 and promoted LUAD progression. CHCHD4 knockdown also inhibited the progression of LUAD. In addition, CHCHD4 knockdown suppressed xenograft tumour growth. CONCLUSION: USF1-CHCHD4 axis can promote LUAD progress, which may be through activating MYC pathway.

Preparation of Cemented Carbide and Study of Copper-Accelerated Salt Spray Corrosion and Erosion Behavior.

(1) Mud pulser carbide rotors, as a core component of ground communication in crude oil exploration, are often subjected to mud erosion and acid corrosion, resulting in pitting pits on the surface, which affects the accuracy. The purpose of this study was to investigate the acid corrosion and erosion behavior of cemented carbide materials and provide a reference for the wider application of cemented carbide materials in the petrochemical industry. (2) Experimental samples of tungsten-cobalt carbide were sintered at a low pressure by powder metallurgy. The petrochemical application environment was simulated by accelerated salt spray corrosion and solid slurry erosion with the aid of acidic copper, and the experimental phenomena were analyzed by SEM (scanning electron microscope), EDS (Energy Dispersive Spectroscopy), and XRD (X-ray diffraction). (3) The experimental results show that the coercivity of the pitted cobalt-cemented tungsten carbide prepared in this study was 17.89 KA/m, and the magnetic saturation strength was 14.42 G·cm3/g. The corrosion rate was the fastest during the acidic copper acceleration experiments from 4 h to 16 h, and the corrosion products of WCo3 and Co3O4 were generated on the corrosion surface. The maximum erosion rate of 0.00104 in the erosion experiment corresponds to a corrosion sample with a corrosion time of 36 h. (4) Therefore, the coercive magnetic force and magnetic saturation strength could be derived from the prepared carbide hard phase grains and carbon content in the appropriate range. The corrosion product in the corrosion process slowed the corrosion rate, and a large amount of cobalt and a small amount of tungsten was lost by oxidation during the corrosion process. The corrosion time had the greatest effect on the erosion performance of the carbide, and the long corrosion time led to surface sparseness, which reduced the erosion resistance.

Turning the Other Cheek: Influence of the cis-Tetrafluorocyclohexyl Motif on Physicochemical and Metabolic Properties.

The targeted introduction of substituents in order to tailor a molecule's pharmacologic, physicochemical, and metabolic properties has long been of interest to medicinal chemists. The all-cis tetrafluorocyclohexyl motif-dubbed Janus face, due to its electrostatically polarized cyclohexyl ring-represents one such example where chemists might incorporate a metabolically stable, polar, lipocompatible motif. To better understand its potential utility, we have synthesized three series of matched molecular pairs (MMPs) where each MMP differs only in the cyclohexane unit, i.e., with a tetrafluorocyclohexyl or a standard cyclohexyl motif. With the introduction of the facially polarized all-cis tetrafluorocyclohexyl ring, the resulting compounds have significantly modified physicochemical properties (e.g., kinetic solubility, lipophilicity and permeability) and metabolic stabilities. These results further speak to the promise of this substituent as a tactic to improve the drug-like properties of molecules.

Optimization of AlCrSiWN Coating Process Parameters and Performance Study by the Matrix Analysis Method.

An AlCrSiWN coating was prepared on a cemented carbide substrate by the arc ion plating technology. The optimization of the coating process was carried out by matrix analysis of orthogonal experiments to calculate the influence of the process parameters on the hardness, bonding and roughness indexes of the coating, determine the optimal coating process parameters, and focus on the influence of the bias voltage on the microscopic morphology, mechanical properties and friction properties of the coating. The results showed that the influence of the process parameters on the indexes of the orthogonal experiments was in the following order: bias voltage > arc current > N2 flow rate. The optimal solution was achieved with an arc current of 160 A, a bias voltage of −80 V, and a N2 flow rate of 600 sccm. Properly increasing the bias voltage improved the microscopic morphology, mechanical properties and wear resistance of the coating. When the bias voltage was −80 V, the coating surface presented fewer large particles with a less uniform size and no obvious crater defects; in addition, the cross-sectional structure changed from grape-like to columnar, and the coating had higher hardness, lower roughness and better bond strength. In the friction performance test, coating at a −80 V bias voltage showed better wear resistance, which was reflected in lower friction coefficient and wear, and the wear mechanism mainly consisted of adhesion and oxidation wear.

Can green industrial policy promote the total factor productivity of manufacturing enterprises?

The Green industrial policy is implemented to promote the coordinative development of the environment and industry, while there has been limited research involved in the policy effect evaluation. Taking China's Green Manufacturing Engineering Implementation Guide (GMEIG) released especially for manufacturing industry in 2016 as a quasi-natural experiment, this paper identifies the policy effect on the total factor productivity (TFP) of manufacturing enterprises, and the mechanisms and heterogeneities are further evaluated. The baseline results show that green industrial policy has significant promotion on the TFP of manufacturing enterprises. The mechanism is analyzed from two perspectives, including incentive effect and supervision effect. Moreover, mediating effects of different time points of government subsidy are investigated within the incentive effect. The results reveal that GMEIG promotes the TFP of manufacturing enterprises through incentive effect and the promotion on enterprises' TFP mainly benefits from government subsidies afterwards rather than government subsidies beforehand. When considering the heterogeneity at regional, industrial and enterprise levels, the policy effect is more significant in non-state-owned enterprises, as well as enterprises with smaller scale. What is more, market competitiveness is conducive to the promotion on enterprises' TFP, while current level of green preference fails to positively moderate the policy effect. This study provides theoretical and empirical support for the construction of green industrial policy and further facilitates China's green development.

Apolipoprotein C1 (APOC1), A Candidate Diagnostic Serum Biomarker for Breast Cancer Identified by Serum Proteomics Study.

Purpose - The present study aimed to identify differently expressed peptides involved in BC as potential biomarkers. Experimental Design - The serum proteomic profiling of 128 serum samples from 64 BC patients and 64 healthy controls (HC), using magnetic beads based immobilized metal ion affinity chromatography (MB-IMAC-Cu) separation followed by MALDI-TOF MS. ClinProTools software identified a number of distinct markers. Then, we performed liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to identify the candidate serum biomarker based on serum proteomics analysis. Finally, enzyme-linked immunosorbent assays (ELISAs) were used to verify the expression of the candidate serum biomarker in BC patients. Results - BC patients could be identified with sensitivity of 87.32% and specificity of 89.46%. Of 41 m/z peaks that differed between BC and HC, six peaks were significantly different between BC and HC (P < 0.01, fold change > 1.5), with peak 1 upregulated and peaks 2-6 downregulated in the BC group. The upregulated peak 1 (m/z: 6638.63) is identified as a region of apolipoprotein C1 (APOC1), and validation showed that APOC1 expression increased from healthy controls to those with FA as well as mastopathy, and finally BC patients. Conclusions and Clinical Relevance - The present study indicates that APOC1 could serve as a candidate serum diagnostic biomarker for BC.

Ultrasound targeted microbubble destruction-mediated SOCS3 attenuates biological characteristics and epithelial-mesenchymal transition (EMT) of breast cancer stem cells.

SOCS3 is low-expressed in breast cancer and may be a potential target. Ultrasound targeted microbubble destruction (UTMD) improved the efficiency of gene transfection. We explored the effects of UTMD-mediated transfection of SOCS3 on the biological characteristics and epithelial-mesenchymal transition (EMT) of breast cancer stem cells (BCSCs). The expression of SOCS3 in breast cancer (BC) and its association with prognosis were evaluated by GEPIA and The Cancer Genome Atlas (TCGA) websites. BCSCs were sorted by flow cytometry and immunomagnetic bead method, followed by sphere formation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and xenograft assays to test their effects in vitro and in vivo. The levels of SOCS3, EMT- and STAT3 pathway-related genes were determined by RT-qPCR and Western blot, respectively. The effects of liposome and UTMD on BCSCs and mice were compared by the gain-of-function experiments. Low expression of SOCS3 was associated with poor prognosis of BC patients, and found in BC and BCSCs. BCSCs were successfully sorted, with high viability and tumorigenicity. UTMD increased the transfection rate of SOCS3. Moreover, UTMD- and liposome-mediated SOCS3 reduced cell viability, proliferation, migration and invasion, blocked cell cycle, inhibited sphere formation in BCSCs, and retarded tumor growth in mice. Mechanistically, overexpressed SOCS3 inhibited the expressions of EMT-related genes and the activation of STAT3 pathway in BCSCs and mice. The regulatory effects of UTMD-mediated SOCS3 on the above-mentioned biological characteristics were better than liposome-mediated SOCS3. UTMD-mediated SOCS3 has a better therapeutic effect in BC, providing new experimental evidence for the treatment of BC.

Mesenchymal stem cell-derived exosome mir-342-3p inhibits metastasis and chemo-resistance of breast cancer through regulating ID4.

BACKGROUND: The mesenchymal stem cell-derived exosome (MSCs-exo) carrying microRNAs have been proved to regulate tumor biological activities. Clarifying molecular mechanism and identifying predictive microRNAs will be of great value in anti-tumor therapy improvement. OBJECTIVE: We aimed to investigate the regulatory role of microRNA-342-3p (miR-342-3p) in MSCs-exo on breast cancer. METHODS: Breast cancer tissues and cell lines were used to evaluate miR-342-3p expression in patients with or without lymph node/distal organ metastasis. The impact of MSCs-exo expression on tumor cell chemo-resistance and invasion/migration was measured. Dual-luciferase reporter gene assay was applied to identify binding site. Inhibitor of differentiation 4 (ID4) siRNA and miR-342-3p inhibitor transfection was conducted to further explore the miR-342-3p/ID4 axis on chemo-resistance and metastasis of breast cancer cells. RESULTS: Breast cancer cells revealed significantly lower level of miR-342-3p in patients with metastatic diseases. miR-342-3p suppressed invasive and chemo-resistant behavior of breast cancer tumor cells. Binding site between miR-342-3p and ID4 was proved. ID4 could reverse the influence of miR-342-3p on chemo-resistance. The tumor inhibition effect of IDA siRNA in vivo was also identified. CONCLUSIONS: This study demonstrated that miR-342-3p acted as potential tumor suppressor by inhibiting metastasis and chemo-resistance of breast cancer cells through targeting ID4. This study might provide potential therapy targets for the treatment of breast cancer.

Chemical approaches for the preparation of ubiquitinated proteins via natural linkages.

Ubiquitination is an important posttranslation modification (PTM) that regulates a variety of cellular processes, including protein degradation, DNA repair, and viral infections. In this process, the C-terminal carboxyl group of ubiquitin (Ub) or poly-Ub is attached to the ε-amine of lysine (Lys) side chain of an acceptor protein through an isopeptide bond. Studying a molecular mechanism of ubiquitination and deubiquitination is fundamental for unraveling its precise role in health and disease and hence crucial for drug development. Enzymatic approaches for protein ubiquitination possess limited ability to selectivity install Ub or Ub chain on the desired position of an acceptor protein and often lead to heterogeneous mixtures. In the past decades, chemical protein (semi)synthesis has been proved to be an efficient tool to facilitate site-specific protein ubiquitination, which significantly contributes to decode the Ub signal at molecular and structural levels. In this review, we summarize the synthetic strategies developed for protein ubiquitination, and the achievements to generate monoubiquitinated, di-ubiquitinated, and tetraubiquitinated proteins with native isopeptide and ester bonds.