Min Wang

Progressive loss and dysfunction of islet β-cells has not yet been solved in the treatment of diabetes. Regenerating protein (Reg) has been identified as a trophic factor which is demonstrated to be associated with pancreatic tissue regeneration. We previously produced recombinant Reg3α protein (rReg3α) and proved that it protects against acute pancreatitis in mice. Whether rReg3α protects islet β-cells in diabetes has been elusive. In the present study, rReg3α stimulated MIN6 cell proliferation and resisted STZ-caused cell death. The protective effect of rReg3α was also found in mouse primary islets. In BALB/c mice, rReg3α administration largely alleviated STZ-induced diabetes by the preservation of β-cell mass. The protective mechanism could be attributed to Akt/Bcl-2/-xL activation and GRP78 upregulation. Scattered insulin-expressing cells and clusters with small size, low insulin density, and exocrine distribution were observed and considered to be neogenic. In isolated acinar cells with wheat germ agglutinin (WGA) labeling, rReg3α treatment generated insulin-producing cells through Stat3/Ngn3 signaling, but these cells were not fully functional in response to glucose stimulation. Our results demonstrated that rReg3α resists STZ-induced β-cell death and promotes β-cell regeneration. rReg3α could serve as a potential drug for β-cell maintenance in anti-diabetic treatment.

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

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.

Purpose Bi-specific antibody (BsAb) is an emerging novel format of antibody. We aimed to develop the natural killer (NK) cell receptor NK group 2, member D (NKG2D)-mediated, immune surveillance system. In this system, the NKG2D ligand MHC class I-related chain A (MICA) was fused with BsAb, which targeted a cluster of differentiation 24 (CD24), a tumor-initiating cell marker that is over-expressed on hepatocellular carcinoma (HCC). Methods The Homo MICA extracellular domains (hMICA) were fused to the end of the heavy chain of cG7 with the flexible pentapeptide (Gly-Gly-Gly-Gly-Ser; G4S), which formed the cG7-MICA that was further identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting (WB). The targeting specificity was characterized using the Surface Plasmon Resonance (SPR) technology and a flow cytometry assay. Furthermore, the design of BsAb cG7-MICA that targeted CD24 and NKG2D was proven to enhance antibody-dependent, cell-mediated cytotoxicity (ADCC) in vitro by the CytoTox 96 Nonradioactive Cytotoxicity assay. Degranulation and a cytokine production assay of NK cells demonstrated that NK cells were activated effectively by cG7-MICA. Further, in HCC-bearing nude mice, the anti-tumor effects of cG7-MICA combined with sorafenib were verified again. Results We purified cG7-MICA successfully, and it has a high affinity. In vivo, cG7-MICA recruited NK cells to the tumor site and improved the anti-tumor efficacy of sorafenib. cG7-MICA also activated NK cells to release interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and it increased the CD107a expression on the surface of the NK cells in vitro. Conclusion NK cells play a major role in the natural, innate immune system, and they have the function of identifying and killing target cells. cG7-MICA remodels the function of MICA molecules to activate NK cells, which provides a possible strategy for HCC-targeting immunotherapy.

Aims: The potential effect of regenerating (Reg) proteins in the treatment of diabetes has been indicated in the past decade, but the clinical use of Reg proteins requires more advances in translational medicine. In the present study, we produced recombinant regenerating protein 2 (rReg2), to prove its protective effect against streptozocin (STZ)‐induced diabetes in BALB/c mice. Materials and Methods: rReg2 was administrated in STZ‐induced diabetic mice. Blood glucose, body weight, serum insulin and islet β‐cell loss were determined. However, Reg2 has also been reported to serve as an autoantigen that induces autoimmune attacks on islets and aggravates diabetic development in non‐obese diabetic mice. To address this contradiction, complete Freund's adjuvant was injected to generate a model that was hypersensitive to Reg2. In this model, islet CD8 T‐cell infiltration, serum Reg2 antibody and interleukin (IL)‐4 and IL‐10, and splenic CD4+/interferon (IFN)‐γ+ T cells were determined. Results: Direct rReg2 pretreatment preserved islet β‐cell mass against STZ and improved glycaemia, body weight and serum insulin content. The protection against cell death was further confirmed in cultured mouse islets and MIN6 cells. On the other hand, significant elevations of serum Reg2 antibody and splenic CD4+/IFN‐γ+ T cells, and decreases in serum IL‐4 and IL‐10 were detected in rReg2‐vaccinated mice, which may contribute to the accelerated diabetes. Interestingly, these mice, upon further rReg2 treatment, exhibited alleviated diabetic conditions with less islet CD8+ T‐cell infiltration. Conclusion: rReg2 treatment ameliorated STZ‐induced diabetes in normal BALB/c mice. By contrast, rReg2 vaccination exacerbated, but further rReg2 treatment alleviated, the severity of STZ‐induced diabetes. Thus, the protective effect of rReg2 is predominant over the autoantigenic β‐cell destruction, supporting the potential of rReg2 in the clinical treatment of diabetes.

Quorum sensing (QS) is a cell-to-cell communication that is used by bacteria to regulate collective behaviors. Quorum sensing controls virulence factor production in many bacterial species and it is regarded as an attractive target to combat bacterial pathogenicity, especially against antibiotic-resistant bacteria. Chlorogenic acid (CA), abundant in fruits, vegetables, and Chinese herbs, processes multiple activities. In this research, we explored its quorum sensing quenching activity. In Pseudomonas aeruginosa, CA significantly inhibited the formation of biofilm, the ability of swarming, and virulence factors including protease and elastase activities and rhamnolipid and pyocyanin production. CA showed similar inhibitory effects in Chromobacterium violaceum on its biofilm formation, swarming motility, chitinolytic activity and violacein production. We examined the expression of QS-related genes in P.aeruginosa and found these genes were all downregulated by CA treatment. Computational modeling revealed that CA can form hydrogen bonds with all three QS receptors. Caenorhabditis elegans and mouse infection models were employed to explore the anti-virulence ability of CA and its effect on pathogenesis process in vivo. CA extended the survival period and reduced the quantity of P. aeruginosa in nematode gut, showing a moderate protective effect on C. elegans. In mice wound model, CA-treated groups showed an accelerating healing rate and the bacteria number in wound area was also decreased by CA treatment. It is suggested by our research that CA has potential to be used as an anti-virulence factor in P. aeruginosa infection.

The expression level of folate receptor alpha (FRα) is located highly rate in ovarian cancer though it is remained absent in normal tissues. This highly tumor restricted expression profile makes FRα a promising target for tumor therapy and diagnosis. In this research we report a FRα binding peptide C7(Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser) discovered by phage display and this peptide showed specific binding to FRα expressing cells by cell ELISA and flow cytometry. Tumor targeting ability of C7 was proved in vivo by both phage homing experiment and fluorescence imaging. C7 can be internalized by SKOV3 cells and its affinity to FRα was determined by MST. The molecular recognition was revealed by structure modeling, suggesting its binding mode with FRα.