Self-Assembled L-DNA Linkers for Rapid Construction of Multi-Specific Antibody-Drug Conjugates Library.

One of the key challenges of improving clinical outcomes of antibody drug conjugates (ADCs) is overcoming cancer resistance to the antibody and/or drug components of ADCs, and hence the need for ADC platforms with high combinatory flexibility. Here, we introduce the use of self-assembled left-handed DNA (L-DNA) oligonucleotides to link combinatory single-domain antibodies and toxin payloads for tunable and adaptive delivery of ADCs. We demonstrate that the method allows convenient construction of a library of ADCs with multi-specific targeting, multi-specific payloads, and exact drug-antibody ratio. The newly constructed ADCs with L-DNA scaffold showed favorable properties of in vitro cell cytotoxicity and in vivo suppression and eradication of solid tumors. Collectively, our data suggest that the L-DNA based modular ADC (MADC) platform is a viable option for generating therapeutic ADCs and for potentially expanding ADC therapeutic window via multi-specificity.

Amelioration of Experimental Autoimmune Encephalomyelitis in Alzheimer's Disease Mouse Models: A Potential Role for Aß.

Emerging data have highlighted the coexistence of multiple sclerosis (MS) and Alzheimer's disease (AD), both of which are common central nervous system degenerative diseases with a heavy burden on patients, their families, and society. However, it is unclear how MS progresses under an AD pathological background. We aimed to address the question of how MS progresses under an AD pathological background. We induced the experimental autoimmune encephalomyelitis (EAE) model of MS in two types of AD mouse models, Tg6799 and APP/PS1 mice. We found that, compared with wild-type mice, the clinical symptoms of EAE were significantly ameliorated in APP/PS1 mice but not in Tg6799 mice. Moreover, a much lower level of serum Aß was observed in Tg6799 mice. EAE clinical symptoms in Tg6799 and C57BL/6J mice were ameliorated by intraperitoneal injection of Aß42. Peripheral administration of Aß42 peptides was able to inhibit Th17 development in vivo, which is likely to occur through the inhibition of IL-6 production in dendritic cells. Our findings revealed that AD and EAE could coexist in the same mouse, and Aß residing in peripheral circulation likely plays an anti-inflammatory role in preventing EAE progression. These findings reveal the potential benefit of Aß, one of the supervillains of AD, at least in certain contexts.

Neonatal cortical astrocytes possess intrinsic potential in neuronal conversion in defined media.

Astrocytes are multifunctional brain cells responsible for maintaining the health and function of the central nervous system. Accumulating evidence suggests that astrocytes might be complementary source across different brain regions to supply new neurons during adult neurogenesis. In this study, we found that neonatal mouse cortical astrocytes can be directly converted into neurons when exposed to neurogenic differentiation culture conditions, with insulin being the most critical component. Detailed comparison studies between mouse cortical astrocytes and neuronal progenitor cells (NPCs) demonstrated the converted neuronal cells originate indeed from the astrocytes rather than NPCs. The neurons derived from mouse cortical astrocytes display typical neuronal morphologies, express neuronal markers and possess typical neuronal electrophysiological properties. More importantly, these neurons can survive and mature in the mouse brain in vivo. Finally, by comparing astrocytes from different brain regions, we found that only cortical astrocytes but not astrocytes from other brain regions such as hippocampus and cerebellum can be converted into neurons under the current condition. Altogether, our findings suggest that neonatal astrocytes from certain brain regions possess intrinsic potential to differentiate/transdifferentiate into neurons which may have clinical relevance in the future.

Hepatitis B Virus DNA Polymerase Displays an Anti-Apoptotic Effect by Interacting with Elongation Factor-1 Alpha-2 in Hepatoma Cells.

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

MiR-194 promotes hepatocellular carcinoma through negative regulation of CADM1.

Aberrant expression of microRNAs may contribute to the initiation and progression of various types of human cancer and they may also constitute biomarkers for cancer diagnosis and treatment. However, the specific function of miR-194 in hepatocellular carcinoma (HCC), and the potential mechanism of its involvement in HCC were unclear. In the present study, we found that miR-194 inhibited CADM1 protein level expression by inhibiting mRNA translation of CADM1; the expression of CADM1 was low in liver cancer cells and tumor tissues, and the high expression of miR-194 was closely related to HCC. MiR-194 promoted proliferation, invasion, migration, and cell cycle progression of HCC cells, and such promotion effect was inhibited by CADM1. In addition, miR-194 may play a tumor-promoting action in a HCC xenograft tumor model. These results suggested that miR-194 may promote the occurrence and development of HCC by inhibiting CADM1. Therefore, miR-194 may be a promising novel therapy for diagnosis of hepatocellular carcinoma.

Research on promoting liver fibrosis injury by the targeted regulation of miR-202 for HGF to activate HSC.

BACKGROUND: Liver fibrosis is the primary cause of liver cirrhosis and hepatocellular carcinoma and leads to considerable morbidity and mortality. Recent studies have shown that microRNAs are associated with fibrotic processes in liver disorders, but the exact role of miR-202 is still unclear, and its related mechanisms are not fully understood. AIMS: The aim of this research is to analyze the regarded regulation of miR-202 on HGF and its role in the pathological progress of liver fibrosis. METHODS: In the present study, qRT-PCR was used to detect the expression level of miR-202 in serum of patients with liver fibrosis and to compare its expression in patients with different pathological stages. HGF was predicted to be the target gene of miR-202 by TargetScan and was verified by Dual-luciferase reporter gene assay. qRT-PCR and western blot were used to detect the regulatory effect of mir-202 on the mRNA and protein of HGF; effect of miR-202 on the expression of fibrosis factors α-smooth muscle actin (α-SMA), FSP1, and collagen was detected; effect of miR-202 on liver fibrosis in mice was detected by establishing CCL4-induced mouse model. RESULTS: We found that the expression level of miR-202 in serum of patients with liver fibrosis was significantly higher than that of healthy people, and increased with the increase of fibrosis; miR-202 inhibited the expression level of mRNA and protein of HGF by combining with the 3'-UTR of HGF; the expression level of miR-202 significantly increased after hepatic stellate cells (HSC) were stimulated by AngII; the overexpression of miR-202 could up-regulate the expression of fibrotic factors α-SMA, FSP1, and collagen I. In addition, miR-202 up-regulated the expression of collagen I and collagen III in liver tissue of mice with liver fibrosis and promoted the progress of liver fibrosis. CONCLUSIONS: miR-202 could negatively regulate the expression of target gene HGF, activated HSC, and increased the expression levels of various fibrosis factors, and the pathological process of liver fibrosis injury was promoted.

Visualization of Ligand-Bound Ectodomain Assembly in the Full-Length Human IGF-1 Receptor by Cryo-EM Single-Particle Analysis.

Tyrosine kinase receptor of insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) bind to hormones, such as insulin, IGF-1, and IGF-2, and transduces the signals across the cell membrane. However, the complete structure of the receptor and the signal transduction mechanism remains unclear. Here, we report the cryo-EM structure of the ligand-bound ectodomain in the full-length human IGF-1R. We reconstructed the IGF-1R/insulin complex at 4.7 Å and the IGF-1R/IGF-1 complex at 7.7 Å. Our structures reveal that only one insulin or one IGF-1 molecule binds to and activates the full-length human IGF-1R receptor.

Self-cleaning isotype g-C3N4 heterojunction for efficient photocatalytic reduction of hexavalent uranium under visible light.

Photocatalysis is a promising method to eliminate hexavalent uranium (U(Ⅵ)) and recycle it from wastewater. However, most of researched photocatalysts are metal-contained, inactive in visible light, and inconvenient to recycle, which unfortunately impedes the further utilization of photocatalytic technology in U(Ⅵ) pollution treatment. Herein, g-C3N4 isotype heterojunction with interpenetrated tri-s-triazine structure (ipCN) was prepared by inserting urea into the interlayer of tri-s-triazine planes of thiourea-derived g-C3N4 and in-site thermal treating. The synthesized nanocomposites were used to convert soluble U(Ⅵ) ions into U(Ⅳ) sediment under visible light. Experimental and characterization results reveal that ipCN possess larger BET surface area, more negative-charged surface, higher U(Ⅵ) adsorption capability, and more efficient mass diffusion and charges transfer properties. With these excellent characteristics, nearly 98% U(Ⅵ) could be removed within 20 min over ipCN5:1 and 92% photoreduction efficiency could also be kept after 7 cycle uses, which were equal to or even superior than most reported metal-based photocatalysts. It is also proven that the configuration of U(Ⅵ) and photogenerated ·O2- play a significant role in the photocatalytic U(Ⅵ) reduction process, with (UO2)x(OH)y2x-y are more prone to be adsorbed and the photoinduced process of ·O2- will steal electrons from photocatalysts. Furthermore, with the self-generated ·O2- and H2O2, a green and facile regeneration process of photocatalysts was proposed This work provides a promising scheme to extract U(Ⅵ) from the perspectives of photocatalysts exploitation, photocatalytic reduction, and photocatalysts regeneration, which is meaningful for the sustainable U(Ⅵ) resource recovery and U(Ⅵ) pollution purification.

Crystal structure of PppA from Pseudomonas aeruginosa, a key regulatory component of type VI secretion systems.

The Type VI secretion system (T6SS) is a membrane protein complex related to inter-bacterial competitions and host-pathogen interactions in Pseudomonas aeruginosa. The T6SS is regulated by a great variety of regulatory mechanisms at multiple levels, including post-translational modification with threonine phosphorylation mediated by Ser/Thr protein kinase PpkA and phosphatase PppA. The T6SS is activated by PpkA via Thr phosphorylation of Fha, and PppA can antagonize PpkA. PppA is a PP2C-family protein phosphatase and plays a key role in the disassembly and reassembly of T6SS organelles. Herein, we report the first crystal structure of PppA from Pseudomonas aeruginosa, which was determined at a resolution of 2.10 Å. The overall structure consists of a bacteria PPM structural core and a flexible flap subdomain. PppA harbors a catalytic pocket containing two manganese ions which correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C. The flexibility and the diversity of the sequence of flap subdomain across the homologues might provide clues for substrates specific recognition of phosphatases.

[Cell proteins that potentially interact with HBV polymerase were identified by co-immunoprecipitation-based LC-MS/MS identification and IPA].

Hepatitis B virus (HBV) is a major cause of chronic liver disease, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. HBV polymerase (Pol) is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma. Protein-protein interactions appears to be crucial for its role. The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique. The HBV Pol gene was amplified by polymerase chain reaction (PCR) and cloned into pCDNA3.1(+). The recombinant plasmid pCDNA3. 1(+)-Pol-flag was transfected into HeLa cells. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol. Eleven of these have previously been reported as proteins that interact with HBV Pol. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol. Among these proteins, four proteins may play a role in three major molecular cellular networks, and are therefore worthy of further investigation.

Enhanced immune response of a bicistronic DNA vaccine expressing fusion antigen Hsp65-Esat-6 of Mycobacterium Tuberculosis with GM-CSF as a molecular adjuvant

This study aimed to construct a bicistronic DNA vaccine expressing fusion antigen Hsp65-Esat-6 of Mycobacterium tuberculosis with cytokine GM-CSF as a molecular adjuvant (pIRES-Hsp65-ESAT-6-GM-CSF, pIRHEG), and the immune response in mice. C57BL/6 mice were immunized with the recombinant plasmid to detect the titer of antibodies, lymphocyte proliferation, the ratio of CD4+, CD8+T cell and IFN ~ γﻌIL-2 secretion. The titer of antibody, lymphocyte proliferation, the ratio of CD4+T and CD8+T cells and IFN ~ γ, IL-2 secretion of pIRHEG group was significant higher than other recombinant plasmid groups, which significant differed by statistical mean. The bicistronic DNA vaccine could induce an effective immune response in mice and could be used as vital ingredient of a new tuberculosis vaccine candidate.

Construction and expression of a recombinant eukaryotic expression plasmid containing the preS1-preS2-S genes of hepatitis B virus and the granulocyte-macrophage colony stimulating factor gene: A study of its immunomodulatory effects.

A total of 10-20% of the population remains unresponsive or weakly responsive to hepatitis B vaccine, which is composed of hepatitis B surface antigen HBsAg (S protein). Therefore, it is necessary to develop a hepatitis B vaccine with a better penetrating and responsive rate. In the present study, a plasmid pVAX1-L-GM was constructed and its immunomodulatory effect of as hepatitis B virus (HBV) DNA vaccine was analyzed through the immunization of BALB/c mice. Immune responses were measured after immunization by anti-HBsAg, proliferation of splenocytes, the number of CD4+ and CD8+ molecules, CTL cytotoxicity, cytokines of IFN-γ and IL-2 secretion assays. Following the immunization, mice in the pVAX1-L-GM group produced antibody 2 weeks earlier compared to the control plasmid pVAX1 and pVAX1HBsAg groups and antibody levels showed significant differences. Enhanced HBsAg-specific splenocyte proliferation as well as specific cytotoxic activities of splenic CTLs were also detected. Furthermore, pVAX1-L-GM plasmid increased the number of CD4+ and CD8+ molecules on the surface of the spleen T cell and the level of IFN-γ, IL-2 secretion. pVAX1-L-GM induced a specific immune response in mice and enhanced the immune effect. Thus, a foundation was laid for developing immunogenicity of a better prevention and treatment of HBV via a hepatitis B vaccine.

Construction of a fusion expression plasmid containing the G250 gene and human granulocyte-macrophage colony stimulating factor and its significance in renal cell carcinoma.

This study aimed to construct a eukaryotic expression plasmid containing the G250/MN/CA IX (G250) and human granulocyte-macrophage colony stimulating factor (hGM-CSF) genes, and to detect the expression of these proteins in vitro by recombinant plasmids in eukaryotic cells. pORF-hGM-CSF and pcDNA3.0-G250 were used as the template to amplify G250 and hGM-CSF by routine polymerase chain reaction (PCR). The two PCR products were cloned into the eukaryotic vector pVAX1, in order to construct a recombinant plasmid pVAX1-G250-hGM, and the plasmid was transfected into human embryonic kidney 293 cells. The protein expression was then determined by immunocytochemistry, atomic force microscopy, ELISA and Western blotting. DNA sequencing showed that the cloned G250 and hGM-CSF sequences were consistent with the reported Gene Bank ones. Moreover, a high expression was noted following recombinant plasmid transfection of the G250 and hGM-CSF proteins. Thus, the eukaryotic expression vector pVAX1-G250-hGM containing G250 and hGM-CSF was constructed, allowing for the investigation of the anti-G250 antigen vaccine and immune response mechanisms of biological immunotherapy in renal cell carcinoma.