Enhanced mapping of small-molecule binding sites in cells.

Photoaffinity probes are routinely utilized to identify proteins that interact with small molecules. However, despite this common usage, resolving the specific sites of these interactions remains a challenge. Here we developed a chemoproteomic workflow to determine precise protein binding sites of photoaffinity probes in cells. Deconvolution of features unique to probe-modified peptides, such as their tendency to produce chimeric spectra, facilitated the development of predictive models to confidently determine labeled sites. This yielded an expansive map of small-molecule binding sites on endogenous proteins and enabled the integration with multiplexed quantitation, increasing the throughput and dimensionality of experiments. Finally, using structural information, we characterized diverse binding sites across the proteome, providing direct evidence of their tractability to small molecules. Together, our findings reveal new knowledge for the analysis of photoaffinity probes and provide a robust method for high-resolution mapping of reversible small-molecule interactions en masse in native systems.

Anti-CD19 CAR T-cell consolidation therapy combined with CD19+ feeding T cells and TKI for Ph+ acute lymphoblastic leukemia.

We conducted a single-arm, open-label, single-center phase 1 study to assess the safety and efficacy of multicycle-sequential anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in combination with autologous CD19+ feeding T cells (FTCs) and tyrosine kinase inhibitor (TKI) as consolidation therapy in patients under the age of 65 years with de novo Ph-positive CD19+ B-cell acute lymphoblastic leukemia. Participants were given induction chemotherapy as well as systemic chemotherapy with TKI. Afterward, they received a single cycle of CD19 CAR T-cell infusion and another 3 cycles of CD19 CAR T-cell and CD19+ FTC infusions, followed by TKI as consolidation therapy. CD19+ FTCs were given at 3 different doses. The phase 1 results of the first 15 patients, including 2 withdrawals, are presented. The most common adverse events were cytopenia (13/13) and hypogammaglobinemia (12/13). There was no incidence of cytokine release syndrome above grade 2 or immune effector cell-associated neurotoxicity syndrome or grade 4 nonhematological toxicities. All 13 patients achieved complete remission, including 12 patients with a complete molecular response (CMR) at the data cutoff. The relapse-free survival was 84%, and the overall survival was 83% with a median follow-up of 27 months. The total number of CD19-expressing cells decreased with an increasing CMR rate. CD19 CAR T cells survived for up to 40 months, whereas CD19+ FTCs vanished in 8 patients 3 months after the last infusion. These findings could form the basis for the development of an allo-HSCT-free consolidation paradigm. This trial was registered at www.clinicaltrials.gov as #NCT03984968.

A heterobifunctional molecule system for targeted protein acetylation in cells.

Protein acetylation is a vital biological process that regulates myriad cellular events. Despite its profound effects on protein function, there are limited research tools to dynamically and selectively regulate protein acetylation. To address this, we developed an acetylation tagging system, called AceTAG, to target proteins for chemically induced acetylation directly in live cells. AceTAG uses heterobifunctional molecules composed of a ligand for the lysine acetyltransferase p300/CBP and a FKBP12F36V ligand. Target proteins are genetically tagged with FKBP12F36V and brought in proximity with p300/CBP by AceTAG molecules to subsequently undergo protein-specific acetylation. Targeted acetylation of proteins in cells using AceTAG is selective, rapid, and can be modulated in a dose-dependent fashion, enabling controlled investigations of acetylated protein targets directly in cells. In this protocol, we focus on (1) generation of AceTAG constructs and cell lines, (2) in vitro characterization of AceTAG mediated ternary complex formation and cellular target engagement studies; and (3) in situ characterization of AceTAG induced acetylation of targeted proteins by immunoblotting and quantitative proteomics. The robust procedures described herein should enable the use of AceTAG to explore the roles of acetylation for a variety of protein targets.

Protocol for 3D screening of lung cancer spheroids using natural products.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for ∼84% of all lung cancer cases. NSCLC remains one of the leading causes of cancer-associated death, with a 5-year survival rate less than 25%. This type of cancer begins with healthy cells that change and start growing out of control, leading to the formation of lesions or tumors. Understanding the dynamics of how the tumor microenvironment promotes cancer initiation and progression that leads to cancer metastasis is crucial to help identify new molecular therapies. 3D primary cell tumor models have received renewed recognition due to their ability to better mimic the complexity of in vivo tumors and as a potential bridge between traditional 2D culture and in vivo studies. Vast improvements in 3D cell culture technologies make them much more cost effective and efficient largely because of the use of a cell-repellent surfaces and a novel angle plate adaptor technology. To exploit this technology, we accessed the Natural Products Library (NPL) at UF Scripps, which consists of crude extracts, partially purified fractions, and pure natural products (NPs). NPs generally are not very well represented in most drug discovery libraries and thus provide new insights to discover leads that could potentially emerge as novel molecular therapies. Herein we describe how we combined these technologies for 3D screening in 1536 well format using a panel of ten NSCLC cells lines (5 wild type and 5 mutant) against ∼1280 selected members of the NPL. After further evaluation, the selected active hits were prioritized to be screened against all 10 NSCLC cell lines as concentration response curves to determine the efficacy and selectivity of the compounds between wild type and mutant 3D cell models. Here, we demonstrate the methods needed for automated 3D screening using microbial NPs, exemplified by crude extracts, partially purified fractions, and pure NPs, that may lead to future use targeting human cancer.

Transcriptome analysis revealed the role of mTOR and MAPK signaling pathways in the white strain of Hypsizygus marmoreus extracts-induced cell death of human hepatoma Hep3B cells.

The aim of this study was to investigate the anticancer mechanisms of white genius mushroom (WGM). WGM is a popular edible mushroom in Taiwan and has been demonstrated to mediate potent antiproliferation effects against human Hep3B liver cancer cells in our previous study. According to next generation sequencing technology and KEGG pathway enrichment analysis, mTOR and MAPK signaling pathways were markedly changed during treatment with WGM extracts in Hep3B cells. Therefore, this study examined the effects of WGM extracts on the expression of mTOR and MAPK signaling pathway-related proteins, such as PI3K, Akt, mTOR, Ras, Raf, MEK, ERK, p38 and JNK in Hep3B cells. According to the results of immunoblotting, we demonstrated that the protein expression of the members of PI3K/Akt/mTOR and MAPK signaling pathways were involved in WGM extracts-induced cell death. Furthermore, the inhibitors of PI3K/Akt/mTOR and MAPK signaling pathways such as rapamycin, MK2206, LY3214996 and SB202190, blocked the induction of cell death and vacuoles formation induced by WGM extracts. This study also demonstrated that WGM extracts is able to inhibit Hep3B cell migration and colony formation in a dose-dependent manner. In addition to being a very popular food, WGM should be a pharmacologically safe natural agent for cancer treatment. Therefore, WGM might be designed to develop into a dietary chemopreventive agent for the cancer treatment.

Investigation of the risk factors to predict cytokine release syndrome in relapsed or refractory B-cell acute lymphoblastic leukemia patients receiving IL-6 knocking down anti-CD19 chimeric antigen receptor T-cell therapy.

CD19 chimeric antigen receptor-T (CAR-T) cell therapy has achieved remarkable results in patients with relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). However, the cytokine release syndrome (CRS) was presented in most patients as common toxicity and severe CRS (sCRS) characterized by the sharp increase in interleukin-6 (IL-6) could be life-threatening. We conducted a phase II clinical trial of ssCAR-T-19 cells, anti-CD19 CAR-T cells with shRNA targeting IL-6, in 61 patients with r/r B-ALL. This trial was registered at www.clinicaltrials.gov as #NCT03275493. Fifty-two patients achieved CR while nine patients were considered NR. The median duration of response (DOR) and overall survival (OS) were not reached (>50 months). CRS developed in 81.97% of patients, including 54.10% with grades 1 to 2 (grade 1, 31.15%; grade 2, 22.95%) and 27.87% with grades 3 to 4 (grade 3, 26.23%; grade 4, 1.64%). sCRS occurs earlier than mild CRS (mCRS). A multivariable analysis of baseline characteristics identified high bone marrow disease burden and poor genetic risk before infusion as independent risk factors for sCRS. After infusion, patients with sCRS exhibited larger expansion of ssCAR-T-19 cells, higher peak levels of IL-6, IL-10, and IFN-γ, and suffered more severe hematological and non-hematological toxicities compared with those with mCRS.

[Scraping needling technique combined with western medication for neurogenic tinnitus of kidney essence deficiency：a randomized controlled trial].

OBJECTIVE: To compare the clinical efficacy of scraping needling technique combined with western medication and simple western medication for neurogenic tinnitus of kidney essence deficiency. METHODS: A total of 68 patients with neurogenic tinnitus of kidney essence deficiency were randomly divided into an observation group and a control group, 34 cases in each group. In the control group, oral methylcobalamin tablets were given, 0.5 mg each time, 3 times a day; oral flunarizine hydrochloride capsules were given before bed, 5 mg each time, once a day, 4 weeks in total. On the basis of the treatment as the control group, scraping needling technique was applied at Tinghui (GB 2), Yifeng (TE 17), Yangchi (TE 4) on the affected side and Shenshu (BL 23), Lieque (LU 7), 5 min each acupoint, once a day, 5 times a week for 4 weeks. Before treatment, 2, 4 weeks into treatment and 4 weeks after treatment (follow-up), the tinnitus severity score, tinnitus visual analogue scale (VAS) score and pure tone average (PTA) were observed, and the clinical efficacy was evaluated in the two groups. RESULTS: The tinnitus severity scores, VAS scores and PTA of each time point after treatment in the two groups were lower than those before treatment (P<0.05), and those in the observation group were lower than the control group (P<0.05). The total effective rates of each time point after treatment in the observation group were 50.0% (17/34), 79.4% (27/34), 79.4% (27/34), which were higher than 26.5% (9/34), 64.7% (22/34), 61.8% (21/34) in the control group (P<0.05). CONCLUSION: Scraping needling technique combined with western medication could improve tinnitus severity, tinnitus volume and hearing in patients with neurogenic tinnitus of kidney essence deficiency, and its curative effect is better than simple western medication.

Identification of Key Pathways Involved in White Strain of Hypsizygus marmoreus Extracts-Induced Cell Death of Human Hepatoma Hep3B Cells by Next Generation Sequencing.

White strain of Hypsizygus marmoreus is named as white genius mushroom (WGM) and is a popular food in Taiwan. We have confirmed the cytotoxicity of WGM extracts on human Hep3B liver cancer cells. A total of 8711 significantly differential genes were identified through large-scale transcriptome sequencing. According to the KEGG pathway enrichment analysis, autophagy, mitophagy and apoptosis pathways were identified as significant in WGM extracts-treated cells. WGM extracts induced a dose-dependent generation of reactive oxygen species (ROS) and membrane-enclosed vacuoles in Hep3B cells. The inhibition of ROS by the ROS scavengers blocked the induction of cell death and vacuoles formation. We suggested that the cell death and membrane-enclosed vacuoles induced by WGM extracts are dependent on ROS production in Hep3B cells. (2E,6E)-3,7,11,15,19,23,27,31,35-Nonamethylhexatriaconta-2,6,34-triene-1,11,15,19,23,27,31-heptol and (18:2) lysophosphatidylcholine were identified in WGM extracts. In addition to being a very popular edible mushrooms, WGM may be developed into a dietary supplement or dietary chemopreventive agent for the cancer treatment.

A Phase II Trial of the Double Epigenetic Priming Regimen Including Chidamide and Decitabine for Relapsed/Refractory Acute Myeloid Leukemia.

OBJECTIVE: To explore the role of chidamide, decitabine plus priming regimen in the salvage treatment of relapsed/refractory acute myeloid leukemia. METHODS: A clinical trial was conducted in relapsed/refractory acute myeloid leukemia patients using chidamide, decitabine, cytarabine, idarubicin, and granulocyte-colony stimulating factor, termed CDIAG, a double epigenetic priming regimen. RESULTS: Thirty-five patients were recruited. Three patients received 2 treatment cycles. In 32 evaluable patients and 35 treatment courses, the completed remission rate (CRR) was 42.9%. The median OS time was 11.7 months. The median OS times of responders were 18.4 months, while those of nonresponders were 7.4 months (P = 0.015). The presence of RUNX1 mutations was associated with a high CRR but a short 2-year OS (P = 0.023) and PFS (P = 0.018) due to relapse after treatment. The presence of IDH mutations had no effect on the remission rate (80.0% vs. 73.3%), but showed a better OS (2-year OS rate: 100.0% vs. 28.9%). Grade 3/4 nonhematological adverse events included pneumonia, hematosepsis, febrile neutropenia, skin and soft tissue infection and others. CONCLUSION: The double epigenetic priming regimen (CDIAG regimen) showed considerably good antileukemia activity in these patients. Adverse events were acceptable according to previous experience. The study was registered as a clinical trial. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/, identifier:NCT03985007.

Targeted Protein Acetylation in Cells Using Heterobifunctional Molecules.

Protein acetylation is a central event in orchestrating diverse cellular processes. However, current strategies to investigate protein acetylation in cells are often nonspecific or lack temporal and magnitude control. Here, we developed an acetylation tagging system, AceTAG, to induce acetylation of targeted proteins. The AceTAG system utilizes bifunctional molecules to direct the lysine acetyltransferase p300/CBP to proteins fused with the small protein tag FKBP12F36V, resulting in their induced acetylation. Using AceTAG, we induced targeted acetylation of a diverse array of proteins in cells, specifically histone H3.3, the NF-κB subunit p65/RelA, and the tumor suppressor p53. We demonstrate that targeted acetylation with the AceTAG system is rapid, selective, reversible and can be controlled in a dose-dependent fashion. AceTAG represents a useful strategy to modulate protein acetylation and should enable the exploration of targeted acetylation in basic biological and therapeutic contexts.

Successful application of anti-CD19 CAR-T therapy with IL-6 knocking down to patients with central nervous system B-cell acute lymphocytic leukemia.

Few studies have described chimeric antigen receptor-modified T cell (CAR-T) therapy for central nervous system (CNS) B-cell acute lymphocytic leukemia (B-ALL) patients due to life-threatening CAR-T-related encephalopathy (CRES) safety issues. In this study, CAR-Ts targeting CD19 with short hairpin RNA (shRNA)-IL-6 gene silencing technology (ssCART-19s) were prepared. We conducted a phase 1 clinical trial (ClinicalTrials.gov number, NCT03064269). Three patients with relapsed CNS B-ALL were enrolled, conditioned with the fludarabine and cyclophosphamide for lymphocyte depletion and infused with ssCART-19s for three consecutive days. Clinical symptoms and laboratory examinations were monitored. After ssCART-19 treatment, three patients' symptoms resolved almost entirely. Brain leukemic infiltration reduced significantly based on magnetic resonance imaging (MRI), and there were no leukemic blasts in cerebrospinal fluid (CSF), which was confirmed by cytological and molecular examinations. Additionally, increases in the levels of cytokines and immune cells were observed in the CSF of all patients. Only grade 1 cytokine release syndrome (CRS) manifesting as fever was noted in patients. In conclusion, CAR-Ts with shRNA-IL-6 gene knockdown migrated into the CNS, eradicated leukemic cells and elevated cytokines in CSF with mild, acceptable side effects.

Successful treatment of acute B lymphoblastic leukemia relapse in the skin and testicle by anti-CD19 CAR-T with IL-6 knocking down: a case report.

BACKGROUND: Extramedullary relapse is an important cause of treatment failure among patients with acute lymphoblastic leukemia (ALL). This type of relapse is commonly observed in the central nervous system, while it is rare in the testicles and skin. Chimeric antigen receptor-modified T cell (CAR-T) therapy targeting CD19 has shown to be a beneficial treatment approach for relapsed/refractory B cell acute lymphoblasticleukemia (r/r B-ALL). Yet, few studies have reported data regarding the treatment of extramedullary B-ALL relapse, especially both in skin and testicle, with CAR-T therapy. CASE PRESENTATION: Here we reported a single case of a patient with relapsed B-ALL in skin and testicle who was successfully treated by the shRNA-IL6-modified anti-CD19 CAR-T(ssCAR-T-19) therapy. A 29-year-old man with relapsed B-ALL in skin and testicle was enrolled in clinal trial involving the shRNA-IL6-modified anti-CD19 CAR-T(ssCAR-T-19) therapy (ClinicalTrials.gov number, NCT03919240). The patient had toxicity consistent with the grade 1 cytokine release syndrome. CONCLUSIONS: ssCART-19 therapy may be used to effectively eliminate infiltrating leukemia cells in the skin and testicle with mild toxicity, which could be a much safer approach to bridge allo-HSCT, thus further improving the patient's outcome. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT03919240, Registered 18 April 2019, retrospectively registered.

Fenofibrate induces human hepatoma Hep3B cells apoptosis and necroptosis through inhibition of thioesterase domain of fatty acid synthase.

This study demonstrated that fenofibrate, a lipid-lowering drug, induced a significant time-dependent cytotoxicity of hepatoma Hep3B cells. Hep3B cells are significantly more sensitive to cell killing by fenofibrate than hepatoma HepG2, lung cancer CH27 and oral cancer HSC-3 cells. From the result of docking simulation, fenofibrate can bind excellently to the thioesterase domain of fatty acid synthase (FASN) binding site as orlistat, a FASN inhibitor, acts. The fenofibrate-induced cell cytotoxicity was protected by addition of palmitate, indicating that the cytotoxic effect of fenofibrate is due to starvation of Hep3B cells by inhibiting the formation of end product in the FASN reaction. Inhibition of lipid metabolism-related proteins expression, such as proteins containing thioesterase domain and fatty acid transport proteins, was involved in the fenofibrate-induced Hep3B cell death. Fenofibrate caused S and G2/M cell cycle arrest by inducing cyclin A/Cdk2 and reducing cyclin D1 and E protein levels in Hep3B cells. The anti-tumor roles of fenofibrate on Hep3B cells by inducing apoptosis and necroptosis were dependent on the expression of Bcl-2/caspase family members and RIP1/RIP3 proteins, respectively. These results suggest that fenofibrate has an anti-cancer effect in Hep3B cells and inhibition of lipid metabolism may be involved in fenofibrate-induced Hep3B cells apoptosis and necroptosis.

Orlistat Displays Antitumor Activity and Enhances the Efficacy of Paclitaxel in Human Hepatoma Hep3B Cells.

Orlistat has been proved to be an effective fatty acid synthase inhibitor that is able to inhibit the proliferation and induce apoptosis in many cancer cell types. However, the anticancer effects of orlistat on hepatocellular carcinoma are undefined. We found that orlistat inhibited cell growth and induced G0/G1 cell cycle arrest with increased cyclin D, cyclin E, and p21 expression in human hepatoma Hep3B cells. Furthermore, protein expression of cyclin A, cyclin B, Cdk1, Cdk2, and Cdk4 was reduced by orlistat. This study investigated the role of lipid metabolism on orlistat-induced human hepatoma Hep3B cell death. The decrease in the expression of key enzymes in fatty acid metabolism, including FASN, ACOT8, PPT1, FABP1, CPT1 and CPT2, was observed after orlistat treatment. We also demonstrated that peroxisomal activity was involved in the orlistat-induced Hep3B cell death. In this study, we established an in vitro model to investigate the effect of orlistat on lipid accumulation. We found that orlistat significantly inhibited the cellular lipid content when administered in fatty acid overload conditions in Hep3B cells. Combination treatment of orlistat and paclitaxel was able to induce a synergistic effect on growth inhibition and cell apoptosis in Hep3B cells. Our data suggested that orlistat displays antitumor activity and enhances the efficacy of paclitaxel in Hep3B cells.

Combination of rituximab and mammalian target of rapamycin inhibitor everolimus (RAD001) in diffuse large B-cell lymphoma.

Abstract We evaluated the efficacy of the anti-CD20 monoclonal antibody rituximab in combination with the mammalian target of rapamycin (mTOR) inhibitor everolimus for treating diffuse large B-cell lymphoma (DLBCL). The combination of rituximab and everolimus was more effective for inhibiting cell growth compared with single-agent therapy. An increase in G0/G1 cell cycle arrest and an increased population of cells in apoptosis were observed in the combination treatment group. The addition of rituximab reduced the overexpression of p-AKT caused by the negative feedback loop of everolimus and had an enhanced effect on inhibition of mTOR signaling, thus providing a rationale for this synergistic effect. Furthermore, combination treatment was also more effective than treatment with either agent alone for inhibiting the growth of DLBCL xenografts. Our study provides preclinical evidence and a theoretical basis for combination therapy with rituximab and everolimus in DLBCL.

Epidemiology and clinical characteristics of hand foot, and mouth disease in a Shenzhen sentinel hospital from 2009 to 2011.

BACKGROUND: We investigated the epidemiological and clinical data of all hand, foot, and mouth disease (HFMD) cases in a sentinel hospital of Shenzhen, China from 2009 to 2011. METHODS: HFMD cases diagnosed in our institution were assessed from 2009 to 2011. Both epidemiological and clinical features were analyzed retrospectively. All the fatal cases were reported. RESULTS: A total of 12132 patients were diagnosed with HFMD, of which 2944 (24.3%) were hospitalized. Of the 2944 hospitalized patients, the highest proportion of diagnosed cases were admitted in May and July (989/2944, 33.6%). In 2009 all severe HFMD cases were diagnosed with enterovirus 71 (EV71). In 2010 and 2011, some of the severe HFMD were diagnosed with Coxsackievirus A16 (CA16). Incidence was highest in 0-4-year old children, with males being predominant. There were sporadic cases with HFMD the whole year except in February. All cases were cured in 2009. Six deaths were reported during 2010 and 2011. CONCLUSIONS: EV71 can cause severe complications and deaths in our region. HFMD is an important public health problem in Shenzhen in spite of stringent measures taken in preschool centers. A high degree of vigilance should be maintained over the disease situation.

Activation of the PI3K/AKT/mTOR pathway in diffuse large B cell lymphoma: clinical significance and inhibitory effect of rituximab.

Diffuse large B cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma and accounts for approximately 30% of newly diagnosed lymphoid neoplasms in Western countries, and 40-50% in China. A better understanding of the biology of DLBCL is needed for the development of potential therapeutic agents that target specific intracellular pathways. In this study, expression of the important components of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway and their clinical significance were investigated in 73 DLBCL cases. The effect of rituximab alone or combined with the PI3K/AKT/mTOR pathway inhibitor rapamycin was further evaluated in the DLBCL cell lines. A total of 73 patients were identified, including 45 men and 28 women aged 18 to 78 years (median age 50 years). Of these patients, p-AKT was positive in 40 cases (54.8%), p-p70S6K in 34 cases (46.6%), and p-4E-BP1 in 33 cases (45.2%). Activation of the PI3K/AKT/mTOR pathway was related to poor disease outcome in DLBCL patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) but not in those treated with rituximab-CHOP. Rituximab combined with rapamycin synergically downregulated the PI3K/AKT/mTOR signaling pathway. Western blot analysis revealed a baseline activation status of the PI3K/AKT/mTOR pathway in DLBCL cell lines, with high levels of p-AKT, p-mTOR, in addition to downstream molecules p-p70S6K and p-4E-BP1. The results indicate that the PI3K/AKT/mTOR pathway is a potentially important signaling route and an unfavorable prognostic factor for DLBCL. Patients with PI3K/AKT/mTOR activation experience a more rapidly deteriorating clinical course with poor treatment response and decreased survival time. Addition of rituximab could downregulate PI3K/AKT/mTOR activation, reversing its negative effect on chemotherapy-treated patients. In addition, our results indicate that the combination of rituximab and inhibition of the activated PI3K/AKT/mTOR pathway could be a promising target for DLBCL therapeutic intervention in the future.

[Effects of phosphatidylinositol-3 kinase/protein kinase b/bone morphogenetic protein-15 pathway on the follicular development in the mammalian ovary].

In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.

[Expression of structural protein VP1 of enterovirus 71 in E.coli and preparation of anti-VP1 monoclonal antibodies].

AIM: To express recombinant structural protein VP1 of enterovirus 71 (EV71) in E.coli and prepare anti-VP1 monoclonal antibodies (mAbs). METHODS: With P1 gene as a template, EV71 VP1 gene was amplified by PCR and cloned into the expression vector pET-32a(+). After transformation into E.coli TG1, the positive clones were screened and sequenced. The recombinant plasmid was then transformed into BLgold (DE3) and the recombinant protein in inclusion bodies was induced by IPTG and detected by SDS-PAGE. After the inclusion bodies were solubilized with 6 mol/L guanidine hydrochloride, we purified VP1 protein using Ni-NTA affinity chromatography, and then immunized the mice with it to prepare the mAbs against VP1 protein. RESULTS: Recombinant VP1 protein was expressed in E.coli. We obtained totally 24 VP1 monoclonal hybridoma cell strains, of which one was EV71 positive determined by Western blotting and five were positive for IFA. CONCLUSION: These mAbs are valuable reagents for the development of new vaccines and detection kits for EV71.