Photochromic Polyoxoniobates with Photoinduced "D-f-A" Electron Transfer Mechanism.

Compounds with redox activities have appealing applications in catalytic, electronic and magnetic properties, but the redox inert of polyoxoniobates (PONbs) significantly limits their applications for a long time. In this work, we are able to integrate organophosphate and lanthanide cluster into PONb to create the first family of inorganic-organic hybrid organophosphate-Ln-PONb composite clusters. These novel species not only present the first family of redox active PONbs that can be reduced to form long-lived "heteropoly blues" under ambient conditions, but also a new photochromic system. More importantly, the analyses of the electronic configurations and photochromic properties for a series of designed proof-of-concept PONbs models allow us to discover a D-f-A electron transfer mechanism, that is, photoinduced electron is transferred from a photosensitive organophosphate electron donor (D) to the NbV electron acceptor (A) through the unoccupied 4 f-orbitals of Ln (f). This work paves the way for developing diverse PONb-based redox materials and expanding the possibility of the applications of PONbs in the redox chemistry.

CircDUSP1 regulates tumor growth, metastasis, and paclitaxel sensitivity in triple-negative breast cancer by targeting miR-761/DACT2 signaling axis.

Triple-negative breast cancer TNBC) is a malignant tumor with high incidence and high mortality that threaten the health of women worldwide. Circular RNAs (circRNAs) are a new class of noncoding RNAs that participate in the biological processes of various tumors, but the regulatory roles of circRNAs in TNBC have not been fully elucidated. In this study, the expression and characterization of circDUSP1 was detected via quantitative real-time PCR, nuclear-cytoplasmic fractionation assay, and fluorescence in situ hybridization. Then, in vitro and in vivo functional experiments were performed to evaluate the effects of circDUSP1 in TNBC. The interaction among circDUSP1, miR-761, DACT2 were confirmed by dual luciferase reporter assay, RNA pull-down, and RNA immunoprecipitation experiments. We identified the circRNA named circDUSP1 that was inversely correlated with tumorigenesis and progression in TNBC. Overexpression of circDUSP1 significantly attenuated cell proliferation, migration, invasion, and epithelial-mesenchymal transition, while increased the sensitivity of TNBC cells to paclitaxel. In-depth mechanism analysis indicated that circDUSP1 acts as an endogenous sponge of miR-761 to reduce its suppression on target gene DACT2 expression in TNBC. Upregulation of miR-761 or downregulation of DACT2 partially reversed the biological process of TNBC and the prognosis of paclitaxel affected by circDUSP1. Taken together, our findings revealed a role for the regulation of the miR-761/DACT2 axis by circDUSP1 in the biological process of TNBC. These results provided new insights into the biological mechanism and targeted therapy of TNBC.

LncRNA SNHG5 promotes the glycolysis and proliferation of breast cancer cell through regulating BACH1 via targeting miR-299.

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors in women. Accumulating studies have been reported that long non-coding RNA (lncRNA) SNHG5 is highly expressed in BC. However, the specific molecular mechanism of SNHG5 in BC is unclear. METHODS: Gene and protein expressions in BC cell were detected by qRT-PCR and western blotting. The proliferation and cell cycle were measured using colony formation assay and flow cytometry analysis, separately. The glucose consumption and lactate production were determined by using the glucose assay kit and lactate assay kit. A dual-luciferase reporter assay was performed to measure the interaction between miR-299 and SNHG5 or BACH1. RESULTS: SNHG5 and BACH1 expressions were increased in BC cell while miR-299 level was decreased. SNHG5 increased BACH1 expression by directly targeting miR-299. SNHG5 silencing or miR-299 overexpression suppressed the proliferation of BC cell, arrested the cell cycle in the G1 cell phase, and decreased the glucose consumption and lactate production of BC cell. However, inhibition of miR-299 or overexpression of BACH1 could reverse the inhibitory effects of sh-SNHG5 on cell proliferation and glycolysis in BC. CONCLUSION: SNHG5 promoted the BC cell growth and glycolysis through up-regulating BACH1 expression via targeting miR-299. These findings may improve the diagnostic and therapeutic approaches to BC.

Establishment of a Suspension MDBK Cell Line in Serum-Free Medium for Production of Bovine Alphaherpesvirus-1.

The Madin-Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 107 cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log10TCID50/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine.

Exosomal microRNA-503-3p derived from macrophages represses glycolysis and promotes mitochondrial oxidative phosphorylation in breast cancer cells by elevating DACT2.

MicroRNAs (miRNAs) are emerging drivers in tumor progression, while the role of miR-503-3p in breast cancer (BC) remains largely unknown. We aimed to explore the impact of macrophage-derived exosomal miR-503-3p in the development of BC by regulating disheveled-associated binding antagonist of beta-catenin 2 (DACT2). miR-503-3p and DACT2 expression in BC tissues and cells was assessed, and the expression of Wnt/ß-catenin signaling pathway-related proteins in BC cells was also evaluated. Macrophages were induced and exosomes were extracted. The screened BC cell lines were, respectively, treated with exosomes, miR-503-3p inhibitor/mimic or upregulated/inhibited DACT2, and then the phenotypes, glucose intake, oxygen consumption rate, and adenosine-triphosphate (ATP) level of BC cells were determined. Cell growth in vivo was also observed. MiR-503-3p was elevated, DACT2 was reduced, and Wnt/ß-catenin signaling pathway was activated in BC cells. Macrophage-derived exosomes, upregulated miR-503-3p or inhibited DACT2 promoted malignant behaviors of BC cells, glucose intake, and activity of the Wnt/ß-catenin signaling pathway, while repressed oxygen consumption rate and ATP level in BC cells. Reversely, reduced miR-503-3p or upregulated DACT2 exerted opposite effects. This study revealed that reduction of macrophage-derived exosomal miR-503-3p repressed glycolysis and promoted mitochondrial oxidative phosphorylation in BC by elevating DACT2 and inactivating Wnt/ß-catenin signaling pathway. Our research may provide novel targets for BC treatment.

The Immune Cell Landscape in Renal Allografts.

Immune cell infiltration plays an important role in the pathophysiology of kidney grafts, but the composition of immune cells is ill-defined. Here, we aimed at evaluating the levels and composition of infiltrating immune cells in kidney grafts. We used CIBERSORT, an established algorithm, to estimate the proportions of 22 immune cell types based on gene expression profiles. We found that non-rejecting kidney grafts were characteristic with high rates of M2 macrophages and resting mast cells. The proportion of M1 macrophages and activated NK cells were increased in antibody-mediated rejection (ABMR). In T cell-mediated rejection (TCMR), a significant increase in CD8 T cell and γδT cell infiltration was observed. CD8 positive T cells were dramatically increased in mixed-ABMR/TCMR. Then, the function of ABMR and TCMR prognostic molecular biomarkers were identified. Finally, we described the gene expression of molecular markers for ABMR diagnosis was elevated and related to the ratio of monocytes and M1 macrophages in ABMR biopsies, while the expression of TCMR diagnosis markers was increased too and positively correlated with γδT cells and activated CD4 memory T cells in TCMR biopsies. Our data suggest that CIBERSORT's deconvolution analysis of gene expression data provides valuable information on the composition of immune cells in renal allografts.

Inhibition of melanoma by survivin-specific lymphocytes combined with CCL17 and granulocyte-macrophage colony-stimulating factor in a mouse syngeneic model.

As a new generation of treatment, tumor immunotherapy targeting tumor-associated antigens (TAA) has attracted widespread attention. The survivin antigen belongs to TAA. It is a key inhibitor of apoptosis and a key regulator of cell cycle progression; furthermore, it may be a candidate target for tumor therapy. In addition, studies have confirmed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and CCL17 significantly affect local anti-tumor immunity in the tumor microenvironment. The mouse survivin gene was screened by BIMAS and SYFPEITHI to obtain the highest scored mouse survivin epitope peptide, which was synthesized into a peptide vaccine to immunize normal mice. Subsequently, spleen lymphocytes were isolated to induce survivin-specific cytotoxic T lymphocytes (CTL). Next, genetic engineering was used to construct the B16F10 cell line that stably expressed CCL17 and GM-CSF genes. A mouse melanoma model was used to observe the effects of the combination of the three on tumor volume and tumor weight. In-vitro survivin-specific CTL combined with CCL17 gene had a stronger inhibitory effect on B16F10 cells, while combined GM-CSF gene did not enhance the inhibitory effect of CTL on B16F10 cells. In-vivo experiments demonstrated that survivin-specific CTL combined with GM-CSF and CCL17 genes can inhibit the growth of mouse melanoma. HE staining and immunohistochemistry showed that the tumor had more necrotic cells and more infiltrating lymphocytes. The results showed that survivin-specific CTL combined with CCL17 and GM-CSF genes could inhibit tumor growth better.

Elevated estrogen receptor ß expression in triple negative breast cancer cells is associated with sensitivity to doxorubicin by inhibiting the PI3K/AKT/mTOR signaling pathway.

Based on its pathological characteristics, breast cancer is a highly heterogeneous disease. Triple negative breast cancer (TNBC) is an aggressive subtype, and due to a lack of effective therapeutic targets, patients with TNBC do not significantly benefit from endocrine or anti-HER2 therapy. Conventional chemotherapy has been regarded as the only systemic therapy option for TNBC, but its therapeutic efficacy remains limited. Estrogen receptor ß (ERß) has been identified as a tumor suppressor in TNBC. Therefore, the aim of the present study was to identify the role of ERß in regulating the response to chemotherapy, and to investigate its underlying mechanism in TNBC. MDA-MB-231 and BT549 cells were treated with doxorubicin (DOX), liquiritigenin [Liq, (Chengdu Biopurify Phytochemicals, Ltd.); a specific ERß agonist], or a combination of DOX and Liq in vitro. The effects of various treatments on cell viability and proliferation were measured using the Cell Counting Kit-8 and colony-formation assays, respectively. MDA-MB-231 and ERß knockdown (ERß-KD) MDA-MB-231 cells were selected for the establishment of ERα-/ERß+ and ERα-/ERß- cell models, respectively. The two cell models were treated with DOX, Liq or a combination of DOX and Liq. The effects of the treatment on the PI3K/AKT/mTOR signaling pathway were evaluated by assessing the protein expression levels of AKT and mTOR using western blot analysis. Low Liq concentrations increased the sensitivity of MDA-MB-231 and BT549 cells to DOX. Moreover, the synergistic effect of Liq and DOX treatment was associated with the inhibition of the PI3K/AKT/mTOR signaling pathway in MDA-MB-231 cells, and the effect was ERß-dependent. The results suggested that elevated ERß expression was associated with sensitivity to doxorubicin by inhibiting the PI3K/AKT/mTOR signaling pathway; therefore, the combined use of conventional chemotherapeutic drugs with ERß agonists may serve as an effective therapy for TNBC.

Discovery and characterization of a novel splice variant of the p53 tumor suppressor gene in a human T cell leukemia cellline.

Alternative splicing produces multiple mRNA variants of TP53 which have diverse biologic functions. In this study, we identified a novel splice variant of TP53 lacking a 200 nt portion of exon 4 (p53ΔE4p) from a human leukemia T cell line. No protein product of p53ΔE4p was identifiable by western blot; however, forced expression of the variant in HEK-293T cells expressing wild-type p53 could inhibit cell proliferation and promote cell death. Interestingly, this novel variant also significantly enhances the expression of reporter genes. Moreover, transcriptome analysis showed that genes related to DNA binding and regulation of transcription by RNA polymerase II function were significantly upregulated following p53ΔE4p transfection, suggesting a role for this variant in the regulation of gene expression.

Clinical study on surgical treatment of granulomatous lobular mastitis.

BACKGROUND: The etiology and pathogenesis of granulomatous lobular mastitis (GLM) remain unknown, with no unified evaluation criteria or standard treatments. This study aimed to assess the etiology and features of GLM, as well as the effects of surgery (lesion excision + stage I breast reconstruction; LE + BR) for GLM. METHODS: This study evaluated 178 female GLM patients retrospectively in 2006-2015. The surgery and non-surgery groups included 164 and 14 patients, respectively. All patients received conservative therapy (traditional Chinese medicine combined with regional wet compress and pus drainage). In addition, the surgery group (n=164) underwent LE + BR. Clinical data, including disease course, causes, lesion size, marital status, and treatment approaches, were assessed. RESULTS: Follow-up was 13-117 months. Seventy-five of the 178 patients had no overt causes (42.1%); meanwhile, 63 (35.4%) and 16 (9.0%) had congenital nipple retraction and a history of psychotropic drugs for >1 year, respectively. The surgery group showed lesions significantly shrunk (≤1 quadrant) with acute inflammation fully controlled; 8 showed recurrence, indicating a cure rate of 95.1% (156/164). In the non-surgery group, 4 cases showed relapse after 6-14 months (cure rate =71.4%; 10/14). Therefore, surgical treatment was significantly more efficient than non-surgical treatment (P=0.001). Kaplan-Meier survival curves for the two treatment types showed a significant difference in recurrence (log rank =11.84, P<0.001). CONCLUSIONS: In GLM patients, LE + BR is safe and effective with respect to cosmetic results, recovery time, and recurrence. Successful surgery should be performed for patients whose lesions ≤1 quadrant, aim to achieve optimal GLM treatment.

microRNA biomarkers in colorectal cancer liver metastasis.

Liver metastasis is a primary factor of prognosis and long-term survival for patients diagnosed with colorectal cancer (CRC). Colorectal cancer liver metastasis (CRCLM), is a complex biological process involving multiple factors and steps, and its mechanisms are yet to be discovered. In recent years, small noncoding RNAs, especially microRNAs (miRNAs) have been proven to play an important role in tumorigenesis, progression and metastasis in a variety of cancers, including CRC. Increasing evidence suggests that miRNAs, including those from exosomes secreted by tumor cells in circulation, could be used as promising biomarkers in early cancer detection, treatment, and prognosis. In this review, we focus on the functional roles and clinical applications of miRNAs, especially those from circulating exosomes secreted by tumor cells related to CRCLM.

Identification of an HLA-A2-restricted CD147 epitope that can induce specific CTL cytotoxicity against drug resistant MCF-7/Adr cells.

Cluster of differentiation (CD)147 is highly expressed in drug-resistant tumor cell lines and is involved in the formation of tumor drug resistance. Therefore, immunotherapy utilizing CD147 epitope peptides is a promising approach for the elimination of drug-resistant tumor cells. However, like most tumor-associated antigens (TAAs), CD147 belongs to the autoantigen category, and T cells that recognize high affinity, immunodominant epitopes from autoantigens are deleted though thymic negative selection. Furthermore, wild-type autoantigen peptides cannot effectively activate and expand T lymphocytes with lower affinity T cell receptors in vivo. However, mutations of TAA peptides have been demonstrated to increase the affinity of major histocompatibility complex molecules and their binding to T cell receptor molecules, leading to activation of T lymphocytes in vitro. In the present study, a high-affinity point mutation peptide, CD147126-134L2, was predicted by the human leukocyte antigen (HLA) binding prediction algorithm and its affinity was testified using a T2 binding assay. In addition, when peptide-specific cytotoxic T lymphocytes (CTLs) were stimulated with dendritic cells loaded with the CD147126-134L2 peptide under HLA-A*02:01 restriction, interferon-γ release and cytotoxicity assays showed that peptide-specific CTLs effectively cross-recognized and lysed T2 target cells loaded either with the wild-type (CD147126-134) or mutated peptide (CD147126-134L2). Moreover, the CD147126-134L2 peptide-specific CTLs exerted strong cytotoxic activity against drug-resistant MCF-7/Adr cells, which express a high level of CD147 and are HLA-A*02:01-positive, but not against normal MCF-7 cells. Thus, this suggests that the wild-type peptide (CD147126-134) is naturally presented on HLA-A*02:01 of CD147-expressing MCF-7/Adr cells and is cross-recognized by CTLs. In conclusion, an HLA-A*02:01-restricted CD147-point mutant epitope peptide was identified that induces CTLs to efficiently lyse drug-resistant MCF-7 cells that highly express CD147. Therefore, this immunotherapeutic approach should be explored as a potential treatment for drug-resistant tumors.

γδTCR immunoglobulin constant region domain exchange in human αßTCRs improves TCR pairing without altering TCR gene-modified T cell function.

The adoptive genetic transfer of T cell receptors (TCRs) has been shown to be overall feasible and offer clinical potential as a treatment for different types of cancer. However, this promising clinical approach is limited by the serious potential consequence that exogenous TCR mispairing with endogenous TCR chains may lead to the risk of self-reactivity. In the present study, domain­exchange and three­dimensional modeling strategies were used to create a set of chimeric TCR variants, which were used to exchange the partial or complete constant region of αßTCR with corresponding γδTCR domains. The expression, assembly and function of the chimeric TCR variants were examined in Jurkat T cells and peripheral mononuclear blood cells (PBMCs). Genetically­encoded chimeras were fused with a pair of fluorescent proteins (ECFP/EYFP) to monitor expression and the pairing between chimeric TCRα chains and TCRß chains. The fluorescence energy transfer based on confocal laser scanning microscopy showed that the introduction of γδTCR constant sequences into the αßTCR did not result in a global reduction of mispairing with endogenous TCR. However, the TCR harboring the immunoglobulin­like domain of the γδTCR constant region (i.e., TCR∆IgC), showed a higher expression and preferential pairing, compared with wild­type (wt)TCR. The function analysis showed that TCR∆IgC exhibited the same levels of interferon-γ production and cytotoxic activity, compared with wtTCR. Furthermore, these modified TCR-transduced T cells retained the classic human leukocyte antigen restriction of the original TCR. The other two chimeric TCRs, had either exchange of the cp+tm+ic domain or exchange of the whole C domain (Fig. 1). Ultimately, exchange of these domains demonstrated defective function in the transduced T cells. Taken together, these findings may provide further understanding of the γδTCR constant domain with implications for the improvement of TCR gene transfer therapy.

[Homology modeling and eukaryotic expression of a modified αß TCR harboring the immunoglobulin-like domain of γδ TCR].

Objective To design, construct and express a chimeric αß TCR harboring the immunoglobulin-like (Ig) domain of γδ TCR in Jurkat T cells. Methods The fusion sites of TCR δIg were determined by bioinformatics analysis. Then the protein structures of TCR α δIg and TCR ß Î´Ig were predicted by homology modeling. Furthermore, the structures of TCR α δIg and TCR ß Î´Ig were compared with the wild type (wt) TCR α and TCR ß respectively by combinatorial extension (CE). After that, the TCR α δIg and TCR ß Î´Ig were fused to fluorescent protein ECFP and EYFP respectively via the overlap PCR, and then the fusion genes (TCR α δIg-ECFP and TCR ß Î´Ig-EYFP) were cloned into pIRES2-EGFP vector and respectively located at the upstream and downstream of an internal ribosome entry site (IRES). The recombinant prokaryotic expression vector pIRES-TCR ßδIg-EYFP/TCR αδIg-ECFP was transferred into Jurkat T cells. Finally, the expression of TCR δIg in Jurkat T cells was monitored by confocal laser scanning microscopy (CLSM). Results The variable region structure of the TCR δIg did not change and the antigen recognition active regions remained stable compared to the wtTCR. The recombinant expression plasmid was successfully constructed as confirmed by PCR identification and sequencing analysis. CLSM showed that TCR δIg was expressed and located at the plasma membrane of Jurkat T cells. Conclusion The design of TCR δIg was reasonable and the TCR δIg could be expressed on Jurkat T cell surface.

Influence of cell physiological state on gene delivery to T lymphocytes by chimeric adenovirus Ad5F35.

Adoptive transfer of genetically-modified T cells is a promising approach for treatment of both human malignancies and viral infections. Due to its ability to efficiently infect lymphocytes, the chimeric adenovirus Ad5F35 is potentially useful as an immunotherapeutic for the genetic modification of T cells. In previous studies, it was found that the infection efficiency of Ad5F35 was significantly increased without enhanced expression of the viral receptor after T cell stimulation; however, little is known about the underlying mechanism. Nonetheless, cell physiology has long been thought to affect viral infection. Therefore, we aimed to uncover the physiologic changes responsible for the increased infection efficiency of Ad5F35 following T cell stimulation. Given the complexity of intracellular transport we analyzed viral binding, entry, and escape using a Jurkat T cell model and found that both cell membrane fluidity and endosomal escape of Ad5F35 were altered under different physiological states. This, in turn, resulted in differences in the amount of virus entering cells and reaching the cytoplasm. These results provide additional insight into the molecular mechanisms underlying Ad5F35 infection of T cells and consequently, will help further the clinical application of genetically-modified T cells for immunotherapy.

P-glycoprotein Mediates Postoperative Peritoneal Adhesion Formation by Enhancing Phosphorylation of the Chloride Channel-3.

P-glycoprotein (P-gp) is encoded by the multidrug resistance (MDR1) gene and is well studied as a multi-drug resistance transporter. Peritoneal adhesion formation following abdominal surgery remains an important clinical problem. Here, we found that P-gp was highly expressed in human adhesion fibroblasts and promoted peritoneal adhesion formation in a rodent model. Knockdown of P-gp expression by intraperitoneal injection of MDR1-targeted siRNA significantly reduced both the peritoneal adhesion development rate and adhesion grades. Additionally, we found that operative injury up-regulated P-gp expression in peritoneal fibroblasts through the TGF-ß1/Smad signaling pathway and histone H3 acetylation. The overexpression of P-gp accelerated migration and proliferation of fibroblasts via volume-activated Cl(-) current and cell volume regulation by enhancing phosphorylation of the chloride channel-3. Therefore, P-gp plays a critical role in postoperative peritoneal adhesion formation and may be a valuable therapeutic target for preventing the formation of peritoneal adhesions.

[A novel assay for analysis of adenovirusmediated endosome lysis of T lymphocytes].

Objective: To analyze adenovirus-mediated endosome lysis of T cells, we developed a novel approach based on pHrodo dextran (pH-sensitive fluorescent dye). Methods: After incubating Jurkat cells (T cell leukemia) with serotype 5 adenovirus (Ad5) and pHrodo dextran, we determined the optimal incubation time and concentration of pHrodo dextran. To assess viral lysis of the endosome, we monitored the ratio changes of mean fluorescence intensity in different time points by laser scanning confocal microscopy. Results: After incubating Jurkat cells with Ad5 and 80 µg/mL pHrodo dextran for 10 minutes, we observed the fluorescence intensity was significantly reduced at 30 minutes compared with that of endosomes at 0 minute. However, we found the mean fluorescence intensity was only slightly reduced by inhibiting V-ATPase with the bafilomycin A1 treatment. Conclusion: The method based on pH-sensitive dye can be used to analyze the adenovirus-mediated endosome lysis of T cells.

Identification of peptide-specific TCR genes by in vitro peptide stimulation and CDR3 length polymorphism analysis.

Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells.

Pharmacokinetic study of 14-(3-methylbenzyl)matrine and 14-(4-methylbenzyl)matrine in rat plasma using liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS) was developed and validated to determine the 14-(3-methylbenzyl)matrine (3MBM) and 14-(4-methylbenzyl)matrine (4MBM) levels in rat plasma in the present study. The analytes were separated using a C18 column (1.9 µm, 2.1 mm × 100 mm) equipped with a Security Guard C18 column (5 µm, 2.1 mm × 10 mm), followed by detection via triple-quadrupole mass spectrometry using an electrospray ionization (ESI) source. Sample pretreatment involved one-step protein precipitation with isopropanol:ethyl acetate (v/v, 25:75), and pseudoephedrine hydrochloride was used as an internal standard. The method was linear in the concentration range of 5-2000 ng/ml for both compounds. The intra-day and inter-day relative standard deviations (RSDs) were less than 15%, and all relative errors (REs) were within 15%. The proposed method enables the unambiguous identification and quantification of these two compounds in vivo. This study is the first to determine the 3MBM and 4MBM levels in rat plasma after oral administration of these compounds. These results provide a meaningful basis for evaluating the clinical applications of these medicines.

[Extraction of miRNA from Glycyrrhiza uralensis Decoction and Its Effect on Immune Cells].

OBJECTIVE: To extract microRNA(miRNA) from Glycyrrhiza uralensis(liquorice) decoction and to explore its effect on mmune cells. METHODS: With dried processed liquorice, the water decoction was prepared according to the conventional method and subsequently concentrated by rotary evaporation. The concentrated decoction was further freeze-dried by freeze dryer, and miRNAs were extracted with Plant MicroRNA Extraction Kit. The extracted miRNA was digested by DNase I and then analyzed through the agarose gell electrophoresis. The PBMC was isolated from healthy volunteers and treated respectively by liquorice water extract, glycyrrhizic acid, glycyrrhetic acid and liquorice miRNAs. After 24 hours, the cells numbers were counted, and the changes of cell morphology were observed. The expression of CD3, CD56 and HLA-DR were analyzed by flow cytometry to identify the change of cell subsets in PBMC. RESULTS: miRNAs could be extracted from the decoction of dried liquorice which further confirmed the stability of miRNAs. The in vitro culture experiment showed that,compared with the controls, PBMC treated with liquorice miRNAs appeared apparent cell aggregation and increased cell number and HLA-DR+ cells proportion. CONCLUSION: The miRNAs are successfully extracted from the freeze-dried decoction of dried liquorice. It is indicated that liquorice miRNAs have significant stimulative effects on the growth of PBMC and HLA-DR+ cells subset.