Genetic switch selectively kills hepatocellular carcinoma cell based on microRNA and tissue-specific promoter.

The clinical treatment of hepatocellular carcinoma (HCC) is still a heavy burden worldwide. Intracellular microRNAs (miRNAs) commonly express abnormally in cancers, thus they are potential therapeutic targets for cancer treatment. miR-21 is upregulated in HCC whereas miR-122 is enriched in normal hepatocyte but downregulated in HCC. In our study, we first generated a reporter genetic switch compromising of miR-21 and miR-122 sponges as sensor, green fluorescent protein (GFP) as reporter gene and L7Ae:K-turn as regulatory element. The reporter expression was turned up in miR-21 enriched environment while turned down in miR-122 enriched environment, indicating that the reporter switch is able to respond distinctly to different miRNA environment. Furthermore, an AAT promoter, which is hepatocyte-specific, is applied to increase the specificity to hepatocyte. A killing switch with AAT promoter and an apoptosis-inducing element, Bax, in addition to miR-21 and miR-122 significantly inhibited cell viability in Huh-7 by 70 % and in HepG2 by 60 %. By contrast, cell viability was not affected in five non-HCC cells. Thus, we provide a novel feasible strategy to improve the safety of miRNA-based therapeutic agent to cancer.

Novel Semiconductive Ternary Hybrid Heterostructures for Artificial Optoelectronic Synapses.

Synaptic devices that mimic biological synapses are considered as promising candidates for brain-inspired devices, offering the functionalities in neuromorphic computing. However, modulation of emerging optoelectronic synaptic devices has rarely been reported. Herein, a semiconductive ternary hybrid heterostructure is prepared with a D-D'-A configuration by introducing polyoxometalate (POM) as an additional electroactive donor (D') into a metalloviologen-based D-A framework. The obtained material features an unprecedented porous 8-connected bcu-net that accommodates nanoscale [α-SiW12 O40 ]4- counterions, displaying uncommon optoelectronic responses. Besides, the fabricated synaptic device based on this material can achieve dual-modulation of synaptic plasticity due to the synergetic effect of electron reservoir POM and photoinduced electron transfer. And it can successfully simulate learning and memory processes similar to those in biological systems. The result provides a facile and effective strategy to customize multi-modality artificial synapses in the field of crystal engineering, which opens a new direction for developing high-performance neuromorphic devices.

TEM8 functions as a receptor for uPA and mediates uPA-stimulated EGFR phosphorylation.

BACKGROUND: TEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown. RESULTS: Here we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections. CONCLUSIONS: Taken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.

Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc.

Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity.

AIM: To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis. METHODS: Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K(+) efflux and Na(+) influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively. RESULTS: Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 µg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K(+) efflux and Na(+) influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect. CONCLUSION: Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections.

The Chimeric Chaoyang-Zika Vaccine Candidate Is Safe and Protective in Mice.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital syndromes including microcephaly and fetal demise in pregnant women. No commercial vaccines against ZIKV are currently available. We previously generated a chimeric ZIKV (ChinZIKV) based on the Chaoyang virus (CYV) by replacing the prME protein of CYV with that of a contemporary ZIKV strain GZ01. Herein, we evaluated this vaccine candidate in a mouse model and showed that ChinZIKV was totally safe in both adult and suckling immunodeficient mice. No viral RNA was detected in the serum of mice inoculated with ChinZIKV. All of the mice inoculated with ChinZIKV survived, while mice inoculated with ZIKV succumbed to infection in 8 days. A single dose of ChinZIKV partially protected mice against lethal ZIKV challenge. In contrast, all the control PBS-immunized mice succumbed to infection after ZIKV challenge. Our results warrant further development of ChinZIKV as a vaccine candidate in clinical trials.

Rapid Generation of Recombinant Flaviviruses Using Circular Polymerase Extension Reaction.

The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

Complete genome sequence of the bacterium Methylovorus sp. strain MP688, a high-level producer of pyrroloquinolone quinone.

Methylotrophic bacteria are widespread microbes which can use one carbon compound as their only carbon and energy sources. Here we report the finished, annotated genome sequence of the methylotrophic bacterium Methylovorus sp. strain MP688, which was isolated from soil for high-level production of pyrroloquinolone quinone (PQQ) in our lab.

[The quality control of 4C-clone screening assay].

Circular chromosome conformation capture (4C) can be used to analyze the high-resolution interaction map of cis-regulatory elements in genome-wide studies. In this study, we optimized the condition of PCR with the mimical 4C sample in order to establish a specific and efficient assay, which was validated practically. This 4C-clone screening assay can be used as the quality control in the application of 4C assay.

[Conserved non-coding elements in human genome].

Study of comparative genomics has revealed that about 5% of the human genome are under purifying selection, 3.5% of which are conserved non-coding elements (CNEs). While the coding regions comprise of only a small part. In human, the CNEs are functionally important, which may be associated with the process of the establishment and maintain of chromatin architecture, transcription regulation, and pre-mRNA processing. They are also related to ontogeny of mammals and human diseases. This review outlined the identification, functional significance, evolutionary origin, and effects on human genetic defects of the CNEs.

[In vivo delivery of siRNA].

RNA interference (RNAi) is a mechanism of posttranscriptional gene silencing mediated by small interfering RNA (siRNA). The ability of synthetic siRNA to silence genes in vivo has made it well suited as therapeutic drug, but the instability and polarity of siRNA and the complexity of in vivo circumstances retarded rapid development of RNAi-based therapies. In this review, a summary of the advances on in vivo siRNA delivery is presented and discussed.

Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins.

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.

[Proteomic analysis of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells].

OBJECTIVE: To investigate the mechanism of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells. METHODS: In this study, the proteins extracted from paclitaxel-induced apoptotic MCF-7 cells were analyzed by 2-dimentional gel electrophoresis (2-DE), and compared with those from untreated MCF-7 cells. The differential proteins were identified by mass spectrometry. RESULTS: At 24 hour after paclitaxel (100 nmol/L) treatment, MCF-7 cells were collected and extracted the whole proteins. Seventeen up-regulated or down-regulated proteins were found by analysis of the differential proteomic 2-DE map. Six of them were identified by mass spectrometry. They were enolase 1, chloride intracellular channel 1, keratin 8, ribosomal protein S12, galectin-1 and histidine triad nucleotide binding protein, respectively. CONCLUSION: We effectively found the changed proteins in the process of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells by proteomic techniques. These up-regulated or down-regulated proteins are important molecules for our further research about the mechanism of paclitaxel-induced apoptosis.

Receptor-binding domain of SARS-Cov spike protein: soluble expression in E. coli, purification and functional characterization.

AIM: Spike protein of coronavirus is responsible for virus binding, fusion and entry, and is a major inducer of neutralizing antibodies. This paper was to find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS: Three fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST/RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST/RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

[Development of toxin targeting to VEGF-KDR].

OBJECTIVE: To develop toxin targeting vascular endothelial growth factor receptor II (VEGF-II/KDR) fused with a KDR-binling peptide screened from peptide library. METHODS: By affinity to KDR molecular which expressed specifically by new born vascular endothelial cell, peptides to KDR were screened from C7 peptide library by phage display. Among them, a peptide binding to KDR with high affinity termed as P5 was selected and fused to the N-terminal of Shiga toxin subunit A (StxA). The protein (P5-StxA) was expressed in E. coli. RESULTS: ELISA and Western blot were applied to characterize the binding interaction between the fusion protein, P5-StxA and KDR. Cytotoxicity assay showed that P5-StxA maintained similar toxicity to cell as StxA. In the model of angiogenesis, P5-StxA inhibited selectively VEGF-induced growth of preexisting vessels of the chick chorioallantoic membrane. CONCLUSION: These studies demonstrate the small peptide, P5, maybe be used as carrier of toxin targeting to KDR.

Inhibition of tumor growth and metastasis by ATF-Fc, an engineered antibody targeting urokinase receptor.

Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumors. In this study, ATF-Fc, an antibody-like molecule comprising the amino-terminal fragment of human uPA (ATF) linked to the Fc fragment of human IgG1 via a flexible linker was developed. Its antitumor activities were evaluated in vitro and in vivo. The results showed that ATF-Fc had obvious cytotoxic effect on several types of tumor cells, which is dependent on cellular expression of uPAR and its Fc fragment. Treatment with ATF-Fc caused a significant suppression on tumor growth and metastasis of xenograft human tumors (MCF-7 breast cancer and BGC-823 gastric cancer) in athymic nude mice. Furthermore, we demonstrated that ATF-Fc had an anti-angiogenesis activity both in vitro and in vivo. In conclusion, we provided a novel therapeutic antibody-like molecule in the management of a variety of solid tumors by disrupting the uPA/uPAR interaction.

[Expression, purification and characterization of rat procarboxypeptidase B in Pichia pastoris].

Carboxypeptidase B is a metalloenzyme, which is widely used for commercial and research purposes. Commercially available CPB purified from porcine or bovine pancreas is very expensive, and is not totally free from other proteases. In order to express the rat proCPB in Pichia pastoris, total RNA extracted from SD rat pancreas cells was reversely transcripted to synthesize cDNA, and the proCPB ORF was synthesized by PCR. After digestion with Xho I and EcoR I , the fragment was inserted into pPIC9, and the recombinant plasmid was named as pPIC9-proCPB. By digestion with Sac I , the lined pPIC9-proCPB was transformed into Pichia pastoris strains GS115 with PEG1000 and integrated into their genomes. In the inducement of methanol, recombinant proCPB was successfully expressed in Pichia pastoris, and could be secreted into the supernatant in the culture. After optimizing the fermentation conditions, a higher production could be obtained when GS115-proCPB was induced in BMGY (pH6.0) at 28CC, with addition of 0.5% casein. The yield of recombinant protein reached 500mg/L, achieving over 94% of total protein in the culture supernatant. The purity of recombinant CPB can reach 96% after two step phenyl sepharose F F purification, and 38% of total protein can obtained after optimizing the pufication method. Comparing to the specific activity 180u/mg of CPB purchased from Sigma, the specific activity of recombinant CPB is 110u/mg. Mass spectrometry analyses showed the mass of the recombinant CPB was 35.1 kD, which is very close to the theory value 35.2 kD. Amino acid sequencing of N-terminal of recombinant CPB further indicated proCPB was expressed successfully and modificated correctly after translation.

[Recombinant batroxobin expressed highly in Pichia pastoris].

Methylotrophic yeast, Pichia pastoris was used to express recombinant batroxobin, and a technology route of producing recombinant protein was finally established. We synthesized batroxobin gene artificially by means of recursive PCR. pPIC9-batroxobin was constructed and transformed into Pichia pastoris GS115 (his4). Recombinant batroxobin was expressed in yeast engineering strain and it was purified from the culture supernatant. 10 mg of recombinant batroxobin was purified from 1 liter fermentation media, it exhibited specific activity of 238 NIH units/mg and had molecular weight of 30.55 kD. The purified recombinant protein converted fibrinogen into fibrin clot in vitro, and shortened bleeding time in vivo. This study laid a foundation of development of hemostatic of recombinant snake venom thrombin-like enzyme.

Antitumor activities of TEM8-Fc: an engineered antibody-like molecule targeting tumor endothelial marker 8.

Tumor endothelial marker 8 (TEM8) was discovered as a cell membrane protein that is predominantly expressed in tumor endothelium and identified as a receptor for anthrax toxin. We developed an antibody-like molecule that consists of the protective antigen (PA)-binding domain of human TEM8 linked to the Fc portion of human immunoglobulin G1 (TEM8-Fc). This engineered protein bound to PA in a divalent cation-dependent manner and efficiently protected J774A.1 macrophage-like cells against anthrax toxin challenge in a dose-dependent manner. TEM8-Fc suppressed the growth and metastasis of xenograft human tumors in athymic nude mice (control versus 10 mg/kg TEM8-Fc, mean tumor weight: LS-180, 1.72 versus 0.16 g, difference = 1.56 g, 95% confidence interval [CI] = 0.96 to 2.16 g; P<.001; MCF-7, 1.12 versus 0.08 g, difference = 1.04 g, 95% CI = 0.77 to 1.31 g; P<.001; HepG2, 1.28 versus 0.35 g, difference = 0.93 g, 95% CI = 0.60 to 1.25 g; P<.001). Furthermore, TEM8 interacted with the M2 isoenzyme of pyruvate kinase (M2-PK), which has an important role in tumor growth and metastasis. TEM8-Fc is a novel therapeutic antibody-like agent in the management of solid tumors that may act by trapping M2-PK.

[Expression of ATR-Fc fusion protein in CHO cells].

ATR-Fc is a fusion protein consisting of extracellular domain of human anthrax toxin receptor (ATR) and a fragment (hinge, CH2, and CH3 domains) of the Fc of human IgG1. The aim of ATR-Fc expression is to get an antibody-like molecule binding to protective antigen (PA), a component of anthrax toxins, this fusion protein may compete with cell surface receptor for PA binding, and block the transport of lethal factor (LF) and edema factor (EF) into cells, thereby act as an antitoxin to prevent and treat anthrax infection. A DNA fragment encoding N-terminal amino acids 1-227 of ATR and human IgG1 Fc was inserted into the Hind III and Not I sites of pcDNA3.1 to generate the eukaryotic vector pcDNA3.1/ATR-Fc for expression of ATR-Fc fusion protein. Using lipofectine-mediated gene transfer technique, pcDNA3.1/ATR-Fc was transfected into CHO-K1 cells. After selected with G418, a recombinant CHO cell line, ATR-Fc-1D5, whose expression level was about 10 - 15 microg/(10(6) cells x d), was established. The recombinant protein expressed by the ATR-Fc-1D5 cells was purified with protein A chromatography. The experimental results demonstrated a direct and specific interaction between ATR-Fc and PA assessed by ELISA.