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. METHODS: Immunohistochemistry analysis was carried out to examine fibroblast activation protein (FAP) and Gasdermin E (GSDME) expression in clinical specimens of lung adenocarcinoma and squamous cell carcinoma tissue. Then the engineered hFAP-CAR-NK-92 cells efficiency was determined in vitro with lactate dehydrogenase (LDH) cytotoxicity assay and the cell morphology of A549, H226, and cancer-related fibroblast (CAF) was observed by electron microscopy. After the co-culture of target cells and effect cells, flow cytometry was employed for examining the CD107a expression in the effect cells, and western blotting was conducted for the cleavage levels of Caspase 3 and GSDME proteins in the target cells. The safety and efficacy of hFAP-CAR-NK-92 cells adoptive transfer immunotherapy in a tumor-bearing mouse were evaluated. RESULTS: Clinical studies have shown FAP positivity in patients with NSCLC. Compared with A549 or H226 cells alone, FAP expression was notably raised in A549+CAF cells or H226+CAF cells in nude mice, respectively (p < 0.05). The killing efficiency of K562 cells was not significantly different between hFAP-CAR-NK-92 and NK-92 cells (p > 0.05). The hFAP-CAR-NK-92 cells presented a higher killing efficiency against the hFAP-target (A549-hFAP, H226-hFAP and CAF-hFAP) cells than the NK-92 cells (p < 0.05). The degranulation of CD107a and cleavage levels of GSDME and Caspase 3 protein in the hFAP-CAR-NK-92 group were higher than those in the NK-92 group (p < 0.05). The 300 nM Granzyme B also induced pyroptosis in hFAP- or GSDME-positive cells (p < 0.05). In vivo experiments revealed that hFAP-CAR-NK-92 cells inhibited tumor progression of hFAP-positive NSCLC (p < 0.05). CONCLUSIONS: In this study, we successfully constructed hFAP-CAR-NK-92 cells and confirmed that hFAP-CAR-NK-92 cells could target hFAP-positive NSCLC to inhibit the progression of NSCLC by activating the Caspase-3/GSDME pyroptosis pathway.

Evaluation of Xpert MTB/RIF for the Diagnosis of Extrapulmonary Tuberculosis in China.

We evaluate the performance of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis (EPTB) in China. The performance of Xpert was evaluated compared to the composite reference standard (CRS), drug susceptibility testing (DST), and imaging examination. The overall sensitivity and specificity of Xpert were 64.1% (195/304) and 100% (24/24), respectively, using CRS as the gold standard. The sensitivity was significantly higher than that of culture for pus (P<0.05). The proportion of EPTB-positive cases diagnosed by imaging was two times more than that diagnosed using Xpert; however, 6 out of 19 cases may have been overdiagnosed by imaging. Compared to phenotypic DST, the sensitivity and specificity of Xpert were 80% (12/15) and 100% (75/75), respectively. Considering its high sensitivity and specificity, Xpert MTB/RIF may be used as a rapid initial test for EPTB diagnosis, and may also support a quicker decision on the treatment regimen. The combination of imaging and Xpert testing could provide high efficiency and accurate diagnosis of suspected EPTB.

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.S. Drug Collection Library and identified 11 old drugs with antiangiogenic activity, including Closantel, an FDA-approved broad-spectrum salicylanilide antiparasitic drug for a variety of types of animals. Closantel was confirmed to have antiangiogenic activity in zebrafish with a half-inhibitory concentration (IC50) at 1.69 µM on the intersegmental vessels and 1.45 µM on the subintestinal vessels. Closantel also markedly suppressed cancer growth in zebrafish xenotransplanted with human lymphoma, cervical cancer, pancreatic cancer, and liver cancer cells, generally in a dose-dependent manner. These data reveal that Closantel has antiangiogenesis and anticancer effects and could be a potential drug candidate for animal and human cancer treatments. Further study is needed to clarify the mechanisms involved in the antiangiogenesis and anticancer effects of Closantel.

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. The cells were treated with sanguinarine (1 µmol/L) or its analogue berberine (1 µmol/L) for 4 d. Cell proliferation was evaluated by counting cell numbers and with a BrdU incorporation assay. The levels of cleaved PARP-89 (an apoptosis marker) and cyclin-D1 (a proliferative index) were measured using Western blotting. RESULTS: In 10% serum-containing media, overexpressing either Rac1 or Rac1b did not significantly change the cell proliferation. In the serum-starved media, however, the survival rate of Rac1b cells was significantly increased, whereas that of Rac1 cells was moderately increased. The level of cleaved PARP-89 was significantly increased in serum-starved Rac1 cells, but markedly reduced in serum-starved Rac1b cells. The level of cyclin-D1 was significantly increased in both serum-starved Rac1 and Rac1b cells. Treatment with sanguinarine, but not berberine, inhibited the proliferation of Rac1b cells, which was accompanied by significantly increased the level of PARP-89, and decreased both the level of cyclin-D1 and the percentage of BrdU positive cells. CONCLUSION: Rac1b enhances the cell proliferation under a growth-limiting condition via both anti-apoptotic and pro-proliferative mechanisms. Sanguinarine, as the specific inhibitor of Rac1b, is a potential therapeutic agent for malignant tumors with up-regulated Rac1b.

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. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.

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. Interestingly, the knockout mice were significantly resistant to anesthesia reagents. Upon injection in the feet with FITC-dextran, the mutant mice displayed a 2-fold delay of the absorption peak in the peripheral circulation. Examining the ear lymphatic vessels of the mutant mice revealed a reduction in the amount of fluid in the lumens of the lymphangions, suggesting an impairment of lymph formation. The Mylk-deficient lymphatic muscle exhibited a significant reduction of peristalsis and of myosin light chain phosphorylation in response to depolarization. We thus concluded that MLCK and myosin light chain phosphorylation are required for lymphatic vessel contraction. Lymphatic contractility is not an exclusive requirement for lymphatic absorption, and external force appears to be necessary for absorption.

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. A significant number of drug candidates have failed in preclinical tests due to their adverse effect on development and reproductivity. Compared to conventional mammal testing, zebrafish testing for assessing developmental and reproductive toxicity offers several compelling experimental advantages, including transparency of embryo and larva, higher throughput, shorter test period, lower cost, smaller amount of compound required, easier manipulation and direct compound delivery. Toxicity and safety assessments using zebrafish have also been accepted by the FDA and EMEA for investigative new drug (IND) approval.

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. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection.

[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. The aim of the present study was to investigate the influences of circadian rhythms on estrous cycle in female mice using clock gene Rev-erb-α knock out (Rev-erb-α(-/-)) mice. To test the fertility of Rev-erb-α(-/-) mice, litter sizes were counted after mating with C57BL/6J male mice. HE staining was used to observe the change of follicle development. The number of embryos of Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice was compared 1.5 d after mating with C57BL/6J male mice. Then Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice were housed to adult, and daily vaginal lavage with 0.9% saline was used to monitor estrous cycle for at least 30 days. Quantity of various cells was counted on specified smears views after staining. We observed estrous cycles of Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice using line plots and periodic spectrograms. The results showed that the Rev-erb-α(-/-) female mice were infertility, and the number of embryos of Rev-erb-α(-/-) females was less than that of Rev-erb-α(+/+) females. However, the follicle development of Rev-erb-α(-/-) female mice was normal. The estrous cycle of Rev-erb-α(-/-) female mice was 3.22 days longer than that of Rev-erb-α(+/+) female mice. The results suggest that loss of Rev-erb-α prolongs estrous cycle, which is probably one of the reasons for female mice infertility, and circadian rhythm is important for mammalian estrous cycle.

[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. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.

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). However, in vivo effects of RSV on the decline of brain function accompanying the aging process, especially those on cognitive loss, have not been not investigated. Here we report that, after intraventricular injection of RSV for one week in 8-9 month-old mice, the long-term memory formation and the LTP induction from hippocampus CA1 were improved. The RSV enhancement effects were blocked in SIRT1 mutant mice. Additional experiments suggest that RSV effects are likely to be mediated through reduced expressions of miR-134 and miR-124, which may in turn up-regulate CREB levels to subsequently promote BDNF synthesis. These findings demonstrate a role for RSV in cognition and a microRNA-CREB-BDNF mechanism by which RSV regulates these processes, demonstrating its value as a potential therapeutic target against CNS disorders in aging.

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. RESULTS: All the tested mammalian hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish, whereas the non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% (8/8) as compared with mammalian results, suggesting that hepatotoxic drugs in mammals also caused similar hepatotoxicity in zebrafish. DISCUSSION: Larval zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity. This convenient, reproducible animal model saves time and money for drug discovery and can serve as an intermediate step between cell-based evaluation and conventional animal testing of hepatotoxicity.

[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. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel. RESULTS: Suitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA. CONCLUSIONS: We have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

[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. The bladder irrigation was performed twice a week for three weeks. The CTL cytotoxicity and profile of CD8(+) tumor-infiltrating lymphocytes were analyzed. RESULTS: The IL-4-SA fusion protein was durably anchored to the biotinylated mucosal surface of bladder wall for up to 5 days.On day 80 after the implantation of MB49 cells, all of PBS-treated mice died, and 8 out of 10 mice in the GFP-SA-treated group died from tumor burden.In contrast, 5 out of 10 mice in the IL-4-SA-treated group were tumor-free. The MB49 tumor-specific cytotoxicity from mice in the IL-4-SA group was (11.3 ± 1.2)%, (22.7 ± 1.5)% and (31.0 ± 3.0)% at the effector to target ratios of 1:1, 25:1 and 50:1, respectively. But the corresponding cytotoxicity was (4.3 ± 0.6)%, (9.0 ± 1.0)% and (14.3 ± 1.5)% in the GFP-SA+IL-4 group, and (3.3 ± 0.6)%, (7.3 ± 0.6)%, (12.7 ± 2.1)% in the PBS group. The tumor-specific cytotoxicity in the SA-CD40L group was significantly higher than that in the control groups (P < 0.05). The infiltrating CD8(+) T cells in tumors in the IL-4-SA-treated group were increased compare with those in other groups. CONCLUSION: Intravesical anchoring of IL-4-SA elicites strong and long-lasting immunoprotection against superficial bladder cancer, and the novel immunotherapy may be an attractive therapeutic alternative in future.

[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. Then establish ELISA to detect the specific anti-VP2 IgG antibodies against HBoV1 and 2 in healthy children aged 3-6 years in Nanjing, China. RESULTS: The recombinant protein 6 x His-VP2 was produced in a larger quantity at 25 degrees C induced by IPTG (1 mmol/L) over night and purified by Ni-NTA column. Seropositive rates of HBoV1 and 2 were 62.2% and 55.5% and their mixed seropositivity was 37%. CONCLUSION: The optimizing expression of the capsid protein VP2 from human Bocavirus constructed successfully and get a high yield under certain conditions. The established ELISA could be used to further analyze seroepidemiology of HBoV in China.

[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. Anti-HCV ELISAs were developed when the fusion protein was used in the biotin-pre-coated microplate or ordinary microplate, and then the sensitivity and specificity of the ELISA were evaluated with confirmed human sera panels. RESULTS: The fusion protein was expressed in high yields and purified successfully, the ELISA detection of anti-HCV with human sera panel indicated that its sensitivity and specificity is higher when SA-tagged HCV antigen (C44P-SA) coated in biotin-pre-coated microplate, compared to C44P or C44P-SA coated in ordinary microplate. CONCLUSION: The sensitivity and specificity of anti-HCV ELISA can be improved when a novel HCV diagnostic antigen fused to SA combined with the biotin- pre-coated microplate. This study laid a foundation for improving the performance of HCV diagnostics.

[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. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.

[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. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA. RESULTS: CFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability. CONCLUSION: The reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.

[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. RESULTS: NCTD significantly inhibited the proliferation of the cells and caused degradation of Cdc6 protein to result in the inhibition of the DNA replication of the cells shown by BrdU incorporation assay. CONCLUSION: NCTD can induce the degradation of Cdc6 in cancer cells to produce an anti-cancer effect.

[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. Flow cytometry was used to detect hIL21-SA and SA- hIL21 fusion protein on the biotinylated MB49 tumor cells. MTT assay was used to evaluate the effect of the fusion protein on the proliferation of human peripheral blood lymphocytes (PBLs) stimulated by Anti-CD3. RESULTS: The recombinant fusion proteins were highly expressed in BL21(DE3) at about 30% of the total bacterial proteins. The two fusion proteins exhibited bifunctional activities, i.e. both biotin-binding property and hIL21 activity and SA-mediated high-affinity binding to biotinylated cell surfaces (with anchoring modified rate of about 95.18% and 96.91%). CONCLUSION: We have successfully obtained bifunctional fusion protein hIL21-SA and SA- hIL21,which will provide a basis for further study of tumor biotherapy using the proteins.