Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway that relies on the molecular chaperone heat shock cognate 70 kDa protein (HSC70) and the lysosomal receptor LAMP-2A. By recognizing substrate proteins containing KFERQ-like pentapeptide motif, CMA plays a central role in multiple infectious contexts. In host defense and cellular homeostasis, CMA contributes to organelle quality control by selectively degrading damaged or misfolded proteins, including stress- or organelle-associated substrates, thereby limiting pathogen replication while mitigating infection-induced stress and preserving cellular function. Although its detailed mechanisms remain incompletely defined, CMA is thought to involve coordinated steps in which molecular chaperones recognize specific target sequences, recruit autophagy-related components, and deliver substrates for lysosomal translocation and degradation. Recent studies have revealed substantial progress in understanding CMA during viral, bacterial, and fungal infections, identifying key regulatory nodes and signaling pathways. These advances underscore the therapeutic potential of CMA-targeted strategies, such as stabilizing LAMP-2A or enhancing HSC70-mediated substrate recognition. However, the spatiotemporal specificity of CMA's pro- or antiviral effects remains a major challenge for clinical translation. This review summarizes current progress in this emerging field and highlights unresolved questions, particularly whether tissue- or cell-type-specific regulation of CMA occurs during infection and how precise modulation of CMA activity might achieve optimal anti-infective outcomes.

Figure S3. Quantitative ChIP assay was used to detect the enrichment of CDKN1A, CXCR4 and negative control region by NCOR in SUDHL4 cells exposed to 5 μM WK692 for 24 h. (*, P < 0.05 versus control)

Figure S2. WK692 specifically inhibits BCL6 but not other BTB-ZF Proteins. Reporter assays were performed to test the activity of WK692 with BCL6, Kaiso and PLZF. (***, P < 0.001 versus control).

Traditional methodologies for assessing chemical toxicity are expensive and time-consuming. Computational modeling approaches have emerged as low-cost alternatives, especially those used to develop quantitative structure-activity relationship (QSAR) models. However, conventional QSAR models have limited training data, leading to low predictivity for new compounds. We developed a data-driven modeling approach for constructing carcinogenicity-related models and used these models to identify potential new human carcinogens. To this goal, we used a probe carcinogen dataset from the US Environmental Protection Agency's Integrated Risk Information System (IRIS) to identify relevant PubChem bioassays. Responses of 25 PubChem assays were significantly relevant to carcinogenicity. Eight assays inferred carcinogenicity predictivity and were selected for QSAR model training. Using 5 machine learning algorithms and 3 types of chemical fingerprints, 15 QSAR models were developed for each PubChem assay dataset. These models showed acceptable predictivity during 5-fold cross-validation (average CCR = 0.71). Using our QSAR models, we can correctly predict and rank 342 IRIS compounds' carcinogenic potentials (PPV = 0.72). The models predicted potential new carcinogens, which were validated by a literature search. This study portends an automated technique that can be applied to prioritize potential toxicants using validated QSAR models based on extensive training sets from public data resources.

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a co-stimulatory receptor and an important target for cancer immunotherapy. We herein present a potent FcγR-independent GITR agonist IBI37G5 that can effectively activate effector T cells and synergize with anti-programmed death 1 (PD1) antibody to eradicate established tumors. IBI37G5 depends on both antibody bivalency and GITR homo-dimerization for efficient receptor cross-linking. Functional analyses reveal bell-shaped dose responses due to the unique 2:2 antibody-receptor stoichiometry required for GITR activation. Antibody self-competition is observed after concentration exceeded that of 100% receptor occupancy (RO), which leads to antibody monovalent binding and loss of activity. Retrospective pharmacokinetics/pharmacodynamics analysis demonstrates that the maximal efficacy is achieved at medium doses with drug exposure near saturating GITR occupancy during the dosing cycle. Finally, we propose an alternative dose-finding strategy that does not rely on the traditional maximal tolerated dose (MTD)-based paradigm but instead on utilizing the RO-function relations as biomarker to guide the clinical translation of GITR and similar co-stimulatory agonists.

[This corrects the article on p. 2387 in vol. 10, PMID: 32905508.].

本研究目的在于使用哺乳动物真核表达系统获得具有高免疫原性重组人Jagged2胞外区C2-EGF2区段(aa27～309),为后续抗Jagged2抗体相关研究奠定基础.首先,合成JAG2胞外区基因并插入pcDNA3.1(+)质粒构建哺乳细胞真核表达载体.使用阳离子脂质体转染试剂瞬时转染HEK293F细胞,收集培养上清,使用镍离子亲和层析柱梯度洗脱纯化,纯化后蛋白样品经过12％胶浓度SDS-PAGE电泳,免疫Balb/c小鼠并利用间接ELISA法测试小鼠抗血清效价.结果显示,150 mmol/L咪唑浓度下洗脱出的Jagged2蛋白在相对分子质量35 000左右出现单一电泳条带,ImageJ分析纯度达90％以上.3次免疫后小鼠产生的抗血清效价最高可达106以上.此结果表明真核表达得到的Jagged2胞外区蛋白具有较高的纯度和免疫原性,可以作为免疫原蛋白支持后续的抗体筛选.

Multiple myeloma (MM) is a hematological malignancy that results from the malignant proliferation of plasma cells in the bone marrow. B cell maturation antigen (BCMA) is highly selectively expressed in malignant plasma cells and is a novel therapeutic target for MM. Here, we developed a bispecific T cell engager, IBI379, that targets BCMA and CD3, and investigated its antitumor efficacy against MM. IBI379 showed strong binding affinity with both BCMA and CD3, which triggered T cell activation, proliferation, and cytokine release. An in vitro study demonstrated that IBI379 induced the lysis of MM cells expressing differing levels of BCMA on the cell surface. Administration of IBI379 in H929 or Daudi-BCMA cell xenograft mouse models significantly inhibited tumor growth without inducing body weight loss. The mechanism of action study revealed the accumulation of CD4+CD8+ T cells and granzyme B-positive T cells in tumors that were treated with IBI379. Moreover, administration of low dose of IBI379 in cynomolgus monkeys was well-tolerated and induced the depletion of BCMA+ B cells and a mild transient increase of cytokines. Collectively, these results demonstrate that IBI379 is a highly potent therapeutic strategy for depleting BCMA-positive B cells and is a promising approach for the treatment of MM.

Recently, anticancer peptides (ACPs) have emerged as unique and promising therapeutic agents for cancer treatment compared with antibody and small molecule drugs. In addition to experimental methods of ACPs discovery, it is also necessary to develop accurate machine learning models for ACP prediction. In this study, features were extracted from the three-dimensional (3D) structure of peptides to develop the model, compared to most of the previous computational models, which are based on sequence information. In order to develop ACPs with more potency, more selectivity and less toxicity, the model for predicting ACPs, hemolytic peptides and toxic peptides were established by peptides 3D structure separately. Multiple datasets were collected according to whether the peptide sequence was chemically modified. After feature extraction and screening, diverse algorithms were used to build the model. Twelve models with excellent performance (Acc > 90%) in the ACPs mixed datasets were used to form a hybrid model to predict the candidate ACPs, and then the optimal model of hemolytic peptides (Acc = 73.68%) and toxic peptides (Acc = 85.5%) was used for safety prediction. Novel ACPs were found by using those models, and five peptides were randomly selected to determine their anticancer activity and toxic side effects in vitro experiments.

The humanized Delta-like 4 (DLL4) monoclonal antibody H3L2 with a quite high affinity for hrDLL4 inhibits the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype, inducing dysfunctional angiogenesis and tumour cell apoptosis, which effectively arrests breast cancer cell growth in vivo. To develop a more effective therapy, an engineered cysteine residue at alanine 121 (Kabat numbering) on each H3L2 heavy chain or at valine 207 (Kabat numbering) on each H3L2 light chain was established by site-directed mutagenesis. Three engineered antibodies, THL4, TH2 and TL2, were identified, and the specific-site antibody-drug conjugates (ADCs) THL4-mpeoDM1 (named HLmD4), TH2-mpeoDM1 (named HmD2), TL2-mpeoDM1 (named LmD2) and THL4-vcMMAE (named HLvM4), were produced, which exhibit much more potent antitumour activity than the naked antibody. The engineered ADCs can be directed against DLL4 and effectively internalized, followed by the release of small molecule cytotoxic agents, e.g., DM1 or MMAE, into the cytosol, which inhibit the synthesis of microtubules and induce G2/M phase growth arrest and cell death through the induction of apoptosis. ADC-conjugated DM1 was highly potent against DLL4 expressing cells in vitro. We systematically compared the in vitro potency and the in vivo preclinical efficacy and safety profiles of the heterogeneous conventional ADC, H3L2 mpeoDM1 (named JmD4) with that of the homogeneous engineered conjugate HLmD4. The engineered anti DLL4 ADCs, particularly HLmD4, showed more potent antitumour activity than Docetaxel and superior safety compared with JmD4 in two xenograft tumour models. Our findings indicate that engineered ADCs have promising potential as effective preclinical therapies for cancers.

Increasing preclinical and clinical studies revealed that many tumor models had resistance to anti-VEGF-A and anti-VEGF-R2 therapies. Studies have shown that simultaneously blocked DLL4-Notch and VEGF signaling pathways can synergistically inhibit density and function of tumor blood vessels and reduce tumor growth rate. We successfully developed a bispecific monoclonal antibody (named HB-32) that targeting both human DLL4 and human VEGF. HB-32 showed high binding affinity to VEGF and DLL4. Furthermore, HB-32 inhibited proliferation, migration and tube formation of HUVEC. Finally, in vivo xenograft studies demonstrated that HB-32 inhibited proliferation of breast cancer cells (MDA-MB-231) and induced tumor cell apoptosis more efficiently than an anti-VEGF antibody or anti-DLL4 antibody alone. These findings indicate that our bispecific antibody provide a potential treatment for breast cancer.

Hypersecretion of hepatic very LDL (VLDL)-associated triglyceride (TG) is the hallmark of hypertriglyceridemia. The estrogen-related receptor gamma (ERR gamma), an orphan nuclear receptor, plays crucial roles in the regulation of metabolic homeostasis, including TG formation in the liver. It remains unclear whether ERR gamma regulates hepatic VLDL-TG secretion. We demonstrated that knockdown of ERR gamma impairs hepatic VLDL-TG secretion in mice, whereas overexpression of ERR gamma favors the secretion, indicating a novel role of ERR gamma in hepatic TG metabolism. We found that ERR gamma transcriptionally regulates the expression of PLA2G12B by binding to the promoter region of the Pla2g12b gene. In Pla2g12b-null mice, ERR gamma fails to regulate hepatic VLDL-TG secretion. There is an apparent accumulation of large lipid droplets in the liver of Pla2g12b-null mice. These data suggest that ERR gamma is a novel regulator of hepatic VLDL-TG secretion, which is mediated through the action on PLA2G12B.-Chen, L., Wu, M., Zhang, S., Tan, W., Guan, M., Feng, L., Chen, C., Tao, J., Chen, L., Qu, L. Estrogen-related receptor gamma regulates hepatic triglyceride metabolism through phospholipase A2 G12B.

The anti-human Delta-like 4 (DLL4) monoclonal antibody MMGZ01 has a high affinity to hrDLL4 and arrests the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype, promotes immature vessels, and effectively reduces breast cancer cell growth in vivo. To develop a much more effective therapy, we conjugated MMGZ01 with two small-molecule cytotoxic agents, i.e., monomethyl auristatin E (MMAE) and doxorubicin (DOX), with different linkers to generate antibody drug conjugates (ADCs), i.e., MMGZ01-vc-MMAE (named MvM03) and MMGZ01-GMBS-DOX (named MGD03), that are more potent therapeutic agents than naked antibody therapeutic agents. The produced anti-DLL4 ADCs can be effectively directed against DLL4 and internalized. Then, the release of MMAE or DOX into the cytosol can induce G2/M or G0/G1 phase growth arrest and cell death through the induction of apoptosis. In vitro, MvM03 was highly potent and selective against DLL4 cell lines. The anti-DLL4 ADCs, particularly MvM03, showed more potent anti-tumour activity than Docetaxel, which is an inhibitor of the depolymerisation of microtubules, in two xenograft breast cancer tumour models. Our findings indicate that anti-DLL4 ADCs have promising potential as an effective therapy for breast cancer. (C) 2017 Elsevier B.V. All rights reserved.

The gene fragment encoding acyl-CoA ligase DptE and its mutant DptE-296 were amplified using polymerase chain reaction(PCR) and were inserted into the prokaryotic expression vector pet22b through the restriction enzymes bamH Ⅰ and Xho L The recombinant plasmid was identified by screening positive clones using ampicillin,bacteria PCR and DNA sequencing.The correct plasmid was transformed into E.coliBL21 (DE3),induced by IPTG,extracting periplasmic proteins by osmotic shock method,purified by nickel affinity chromatography,identified by SDS-PAGE and Western blot.The purified protein was connected to biotin to assay the affinity with decanoic acid by fortebio.The recombinant vector DptE-pet22b and DptE-296-pet22b was successfully constructed.SDS-PAGE and Western blot showed that the expressed protein was consistent with the expected.Fortebio experiments showed that both DptE and DptE-296 can bind decanoic acid,but the combination is not strong.

为探索高校生物制药卓越工程师人才培养模式,本文以中国药科大学生物制药卓越工程师专业为例,探讨了如何从优化课程体系和教学内容、改革教学方法和考核方法、完善教学管理制度、培育实践和创新能力师资等四个方面进行改革,建立起以工程实践和科研创新能力为核心的人才培养模式,为国家培养高层次应用型生物制药专业人才.

A pyrene derivative PYPA, namely, 1-(pyren-l-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine (PYPA), was synthesized to develop a chemosensor. The studies show that PYPA exhibits high sensitivity and selectivity toward Zn2+ over many other metal cations in the presence of pyrophosphatic acid (PPi). It has a low detection limit of 0.030 nmol/L. Detection of Zn2+ was not interfered by various cations or anions. In the presence of PPi, over a 8-fold fluorescence enhancement at 383 nm was observed within 20 s. Cell studies further demonstrated that PYPA can be a potential probe to detect Zn2+ in human liver cells (L-02).

FLTC was synthesized and used as a fluorescent chemosensor to detect Hg2+. It showed high selectivity toward Hg2+ over many heavy metal ions in an ethanol–H2O (3:2, v/v, HEPES buffer, 0.5mM, pH7.15) solution with a detection limit of 0.21μM. After complexation with Hg2+, FLTC showed extremely high selectivity toward Ag+ with a detection limit of 0.009μM. Therefore, detection of Hg2+ and Ag+ could be realized using FLTC and the FLTC–Hg2+complex, respectively. Cytotoxicity assays and fluorescence microscopy analysis showed that FLTC could be used as a fluorescent probe to detect Hg2+ and Ag+ in L-02 human liver cells.

Blockage of Delta-like 4 (DLL4)-directed Notch signaling induces excessive tip cell formation and endothelial proliferation resulting in dysfunctional angiogenesis in tumors. MMGZ01, as a murine antihuman DLL4 monoclonal antibody, specifically binds to human DLL4 and blocks Notch pathway. Here, the structure of MMGZ01 variable fragment (Fv) was established and framework region (FR) residues which supported complementarily determining region (CDR) loop conformation were identified. Important residues interactions were also identified through docking MMGZ01 Fv with antigen epitope in DLL4. To humanize the murine antibody, we modified MMGZ01 Fv through CDR grafting and the reconstructed antibody (H3L2) maintained similar structure and binding affinity to parental MMGZ01 after back mutation of 12 canonical murine residues in the FRs. Meanwhile, H3L2 promoted human umbilical vein endothelial cell (HUVEC) proliferation through inhibiting DLL4-directed Notch pathway. Moreover, in MDA-MB-231-bearing nude mice, H3L2 induced dysfunctional angiogenesis and tumor cell apoptosis and showed superior anti-tumor activity. In conclusion, H3L2 is an ideal humanized antibody that inhibits tumor growth through targeting DLL4-Notch pathway and has attracting potentials for clinical applications.

Increasing evidence suggests that DLL4 (Delta-like 4)-Notch signaling plays a critical role in cell fate determination and differentiation in tissues. Blocking DLL4-Notch signaling results in inhibition of tumor growth, which is associated with increased nonfunctional vessels and poor perfusion in the tumor. We successfully generated a human DLL4 monoclonal antibody MMGZ01 that binds specifically to DLL4 to disrupt the interaction between DLL4 and Notch1. MMGZ01 showed high affinity to DLL4 to inhibit the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype. Furthermore, MMGZ01 stimulated HUVEC vessel sprouting and tubule formation in vitro. In addition, MMGZ01 had a pronounced effect in promoting immature vessels and reduced breast cancer cell growth in vivo. Finally, MMGZ01 treatment inhibited the proliferation of breast cancer cells, induced tumor cell apoptosis, suppressed mammosphere formation, decreased CD44(+)/CD24(-) cell population, and reduced epithelial mesenchymal transition (EMT). These findings suggest that antagonism of the DLL4-Notch signaling pathway might provide a potential therapeutic approach for breast cancer treatment.