Development of FAP-Targeted Chimeric Antigen Receptor NK-92 Cells for Non-Small Cell Lung Cancer.

Objectives: Over the past two decades, great progress has been made in advancing the early detection and multimodal treatment of non-small cell lung cancer (NSCLC). However, overall cure rates and survival rates of NSCLC are still not satisfactory, and research into new therapies is needed. This study attempted to construct human Fibroblast Activation Protein-Chimeric Antigen Receptor Natural killer (NK)-92 cells (hFAP-CAR-NK-92 cells) and explore their potential therapeutic effects in NSCLC.

Closantel Suppresses Angiogenesis and Cancer Growth in Zebrafish Models.

Angiogenesis has emerged as an important therapeutic target in several major diseases, including cancer and age-related macular degeneration. The zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. In this study, we have taken advantage of the transgenic Tg (fli1a:EGFP) zebrafish line to screen the U.

Sanguinarine inhibits Rac1b-rendered cell survival enhancement by promoting apoptosis and blocking proliferation.

Aim: Small GTPase Rac1 is a member of the Ras superfamily, which plays important roles in regulation of cytoskeleton reorganization, cell growth, proliferation, migration, etc. The aim of this study was to determine how a constitutively active Rac1b regulated cell proliferation and to investigate the effects of the Rac1b inhibitor sanguinarine.

Methods: Three HEK293T cell lines stably overexpressing GFP, Rac1-GFP or Rac1b-GFP were constructed by lentiviral infection.

Myosin phosphatase target subunit 1 (MYPT1) regulates the contraction and relaxation of vascular smooth muscle and maintains blood pressure.

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists.

Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption.

Lymphatic absorption is a highly regulated process driven by both an extrinsic mechanism (external force) and an intrinsic mechanism (lymphatic vessel contractility). The lymphatic muscle is a specialized smooth muscle with unique mechanical properties. To understand the molecular mechanism and relative contribution of smooth muscle contraction in lymphatic absorption, we analyzed mice with a smooth muscle-specific deletion of Mylk, a critical gene for smooth muscle contraction.

[Expression and purification of four single-stranded DNA-binding proteins and their binding on HCV RNA].

Objective: Express and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

Methods: The expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE.

Zebrafish models for assessing developmental and reproductive toxicity.

The zebrafish is increasingly used as a vertebrate animal model for in vivo drug discovery and for assessing chemical toxicity and safety. Numerous studies have confirmed that zebrafish and mammals are similar in their physiology, development, metabolism and pathways, and that zebrafish responses to toxic substances are highly predictive of mammalian responses. Developmental and reproductive toxicity assessments are an important part of new drug safety profiling.

[Circadian clock gene Rev-erb-α deficiency prolongs female estrous cyclicity].

The circadian clock has been linked to female reproductive physiology and endocrine in mammals. Epidemiological studies of female shift workers have shown increased rates of abnormal reproduction and adverse pregnancy. But little is known how the circadian rhythms affect reproduction.

[Prokaryotic expression and characterization of two recombinant receptor-binding domain(RBD) proteins of human coronavirus NL63(HcoV-NL63)].

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting.

Resveratrol improves learning and memory in normally aged mice through microRNA-CREB pathway.

Resveratrol (RSV) is a natural compound found in grapes and red wine. It has been well known for its beneficial effects as a dietary supplement in prevention of cardiovascular diseases and cancer. Recently, in vitro studies have reported the neuroprotective role of RSV in neurodegenerative process in Alzheimer's disease (AD).

Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish.

Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment.

A zebrafish phenotypic assay for assessing drug-induced hepatotoxicity.

Introduction: Numerous studies have confirmed that zebrafish and mammalian toxicity profiles are strikingly similar and the transparency of larval zebrafish permits direct in vivo assessment of drug toxicity including hepatotoxicity in zebrafish.

Methods: Hepatotoxicity of 6 known mammalian hepatotoxic drugs (acetaminophen [APAP], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. Zebrafish liver degeneration was originally screened visually, quantified using an image-based morphometric analysis and confirmed by histopathology.

[Optimizing expression of the capsid protein VP2 from human Bocavirus and establish it's seroepidemiology assying methord].

Objective: To obtain sufficient recombinant VP2 protein of human Bocavirus and establish it's seroepidemiology assying metbord. METHORD: Tbe capsid protein VP2 DNA genes of HBoV1 and 2 were optimized in accordance with tbe usage of the favorite codons in K coil so as to enhance its protein expression in prokaryotic expressing system. The protein was purified by Ni-NTA column, and its antigenicity was determined by Western Blot.

[Intravesical anchoring of streptavidin-tagged interleukin-4 fusion protein for immunotherapy of mouse superficial bladder cancer].

Objective: To evaluate the antitumor efficacy of streptavidin-tagged interleukin-4 (IL-4-SA) bifunctional fusion protein in the immunotherapy of mouse model of superficial bladder cancer.

Methods: IL-4-SA fusion protein was prepared and its biological activity was determined. One day after MB49 cell implantation, 100 µl of 1 mg/ml NHS-PEO4-biotin was instilled into the bladder for 30 minutes, followed by intravesical instillation of 100 µl PBS, GFP-SA+IL-4 or IL-4-SA and incubation for 1 hour.

[Preparation and application of a novel HCV diagnostic antigen fused to streptavidin].

Objective: To prepare streptavidin-tagged hepatitis C virus (HCV) fusion protein and explore its application for the detection of antibody against HCV infection.

Methods: A recombinant plasmid pET-11d-C44P-SA was constructed, which coding a novel HCV diagnostic antigens (C44P) and streptavidin (SA) fusion protein, and the fusion protein was generated with BL21 (DE3) E Coli and identified by Western Blot analysis. Then the fusion protein was purified through the Ni-NTA affinity chromatography and over 90% purity has been achieved.

[Expression and purification of different segments from HCoV-NL63 nucleocapsid protein and their application in detection of antibodies].

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA.

[Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay].

Objective: To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).

Methods: The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis.

[Norcantharidin inhibits DNA replication initiation protein Cdc6 in cancer cells].

Objective: To explore the inhibitory effect of norcantharidin (NCTD) on the expression of DNA replication initiation protein Cdc6 in cancer cells.

Methods: MTT assay was performed to detect the inhibitory effect on different cancer cell lines, including HeLa, HepG2, Jurkat and Ramos cells. The effect of NCTD on Cdc6 protein level was detected by Western blotting, and BrdU incorporation assay was used to evaluate the DNA replication of the cells.

[Expression, purification and bioactivity evaluation of streptavidin-tagged human interleukin-21 fusion protein].

Objective: To obtain streptavidin-tagged human interleukin-21 (hIL21) fusion protein and evaluate its bioactivities.

Methods: hIL21-SA-pET21 and pET24a-SA- hIL21 plasmids were constructed and expressed in BL21(DE3) host bacteria. The hIL21-SA and SA- hIL21 fusion protein were purified through Ni-NTA affinity chromatography and refolded by dialysis.

A novel immunotherapy for superficial bladder cancer by the immobilization of streptavidin-tagged bioactive IL-2 on the biotinylated mucosal surface of the bladder wall.

Background And Objective: Intravesical administration of Bacillus Calmette-Guerin (BCG) after transurethral resection is by far the most effective local therapy for superficial bladder cancer, the fifth most common cancer in the world. However, approximately one-third of patients fail to respond and most patients eventually relapse. In addition, there are pronounced side effects of BCG therapy, such as BCG sepsis and a high frequency of BCG-induced cystitis.

[Immobilization of streptavidin-tagged bioactive hTNF-alpha on biotinylated mucosal surface of the bladder wall for treatment of superficial bladder cancer in mice].

Objective: To investigate a novel immunotherapy through immobilization of streptavidin-tagged hTNF-alpha on the biotinylated mucosal surface of the bladder wall for bladder cancer treatment in mice.

Methods: A total of 120 female C57BL/6j mice were randomized into 5 equal groups, namely blank control, PBS, soluble hTNF-alpha, SA-GFP, and SA-hTNF-alpha treatment groups. Twenty-four hours after establishment of a mouse model of orthotopic superficial bladder cancer, SA-hTNF-alpha fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall, which was repeated every 4 days for a total of 6 sessions.

[Preparation and bioactivity evaluation of SA-mIL15 fusing protein].

Aim: To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins.

Methods: pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate SA/mIL15 fusion proteins. SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography, and mIL15-SA fusion protein through anion exchange chromatography, followed by refolding.

[A simple and efficient method for establishing a mouse model of orthotopic MB49 bladder cancer].

Objective: To establish a simple and efficient method for establishing a mouse model of orthotopic superficial bladder cancer.

Methods: C57BL/6 mice were anesthetized with sodium pentobarbital and catheterized with modified IV catheter (24 G). The mice were intravesically pretreated with HCl and then with NaOH, and after washing the bladders with phosphate-buffered saline (PBS), 100 microl (1 x 10(7)) MB49 cells were infused and allowed to incubate in the bladder for 2 h followed intravesical mitomycin C (MMC) administration.

[Artemin and GFRalpha3 expressions and their relevance to perineural invasiveness and metastasis of pancreatic carcinoma].

Objective: To investigate the association of artemin and GFRalpha3 expressions with perineural invasion and metastasis of pancreatic carcinoma.

Methods: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression of artemin and GFRalpha3 in pancreatic carcinoma tissues, adjacent tissues and normal pancreas tissues, and the relevance of artemin and GFRalpha3 expressions to the perineural invasion and metastasis of pancreatic carcinoma were analyzed.

Results: The positivity rates of artemin and GFRalpha3 expressions were 72.