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Surveys based on western populations have identified many risk factors for dysphagia in older people, but the potential risk factors consistent with the demographic characteristics of older, hospitalized Chinese patients require further study. This single-center prospective study aimed to determine the incidence of dysphagia in western China, and to develop and validate a model to predict the risk of dysphagia among older patients. A total of 343 inpatients (aged ≥ 65 years without dysphagia and cognitive impairment) were included. A score ≥ 2 on the Eating Assessment Tool-10 was defined as dysphagia. After a six-month follow-up, 70 (20.4%) patients were found to have dysphagia. The final model included age, wearing dentures, activities of daily living, cerebral vascular disease, coronary heart disease, and malignancy. The developed model has high predictive accuracy and can be easily implemented in daily practice.

Flow cytometric dot plots showing PD-1 expression on CD3+ T cells in PBMCs (left) or CD3+ T cells purified from PBMCs

The DOCK-AND-LOCK (DNL) method is a platform technology that combines recombinant engineering and site-specific conjugation to create multispecific, multivalent antibodies of defined composition with retained bioactivity. We have applied DNL to generate a novel class of trivalent bispecific antibodies (bsAb), each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of different antitumor Fabs. Here, we report the further characterization of two such constructs, (E1)-3s and (14)-3s, which activate T cells and target Trop-2– and CEACAM5-expressing cancer cells, respectively. (E1)-3s and (14)-3s, in the presence of human T cells, killed target cells grown as monolayers at subnanomolar concentrations, with a similar potency observed for drug-resistant cells. Antitumor efficacy was demonstrated for (E1)-3s coadministered with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice harboring xenografts of MDA-MB-231, a triple-negative breast cancer line constitutively expressing Trop-2 and PD-L1. Growth inhibition was observed following treatment with (E1)-3s or (14)-3s combined with human PBMC in 3D spheroids generated from target cell lines to mimic the in vivo behavior and microenvironment of these tumors. Moreover, addition of an antagonistic anti–PD-1 antibody increased cell death in 3D spheroids and extended survival of MDA-MB-231-bearing mice. These preclinical results emphasize the potential of combining T-cell–redirecting bsAbs with antagonists or agonists that mitigate T-cell inhibition within the tumor microenvironment to improve immunotherapy of solid cancers in patients. They also support the use of 3D spheroids as a predictive alternative to in vivo models for evaluating T-cell functions. Cancer Res; 77(19); 5384–94. ©2017 AACR.

Combined file of Table S1 thru S4. Table S1. Blockade of PD-1 binding to PD-L1 on MDA-MB-231 by anti-PD-1. Table S2. Surface expression of CEACAM5 and Trop-2 on selective cell lines. Table S3. Expression of Trop-2 and PD-L1 on MDA-MB-231 and its Trop-2-transfected sublines, including the IC50 of (E1)-3s determined with T cells purified from PBMCs of the same donor. Table S4. Effect of cPD-1 on IC50/EC50 of (E1)-3s to kill MDA-MB-231 or MDA-MB-231-S120 with redirected T cells.

Head and neck squamous cell carcinomas (HNSCC) are characterized by resistance to chemotherapy and overexpression of antiapoptotic Bcl-2 family members, including Bcl-XL and Bcl-2. Molecular targeting of Bcl-XL and/or Bcl-2 in HNSCC cells has been shown to promote apoptosis signaling and to sensitize cells to chemotherapy drugs, including cisplatin, which is commonly used in the treatment of HNSCC. We report that induction of HNSCC apoptosis by the proteasome inhibitor bortezomib is accompanied by up-regulation of the proapoptotic proteins Bik and Bim, natural cellular inhibitors of Bcl-XL and Bcl-2. Additionally, bortezomib treatment of HNSCC cells caused up-regulation of antiapoptotic Mcl-1L. Inhibition of Bik or Bim up-regulation using small interfering RNA markedly attenuated bortezomib-induced cell death. By contrast, small interfering RNA–mediated inhibition of Mcl-1L expression resulted in enhanced killing by bortezomib. Further investigation showed that the combination of bortezomib and cisplatin led to synergistic killing of HNSCC cells, with calculated combination indexes well below 1.0. Taken together, these results delineate a novel mechanism of HNSCC killing by bortezomib that involves up-regulation of Bik and Bik. Moreover, our findings suggest that the combination of bortezomib plus cisplatin, or bortezomib plus an inhibitor of Mcl-1L, may have therapeutic value in the treatment of HNSCC. [Mol Cancer Ther 2008;7(6):1647–55]

Supplementary Fig. S1 from Bortezomib induces apoptosis via Bim and Bik up-regulation and synergizes with cisplatin in the killing of head and neck squamous cell carcinoma cells

Combined file of Fig.S1 thru Fig.S5 - Fig. S1. Biochemical characterization of 5G9.G1.B11. Fig. S2. Binding of 5G9.G1.B11 to native PD-1. Fig. S3. Blocking activities of 5G9.G1.B11. Fig. S4. Biochemical characterization of cPD-1. Fig. S5. Binding of cPD-1 to SpESF-X10-2D1 cells transfected to overexpress human PD-1.

Systemic juvenile idiopathic arthritis (sJIA) is a severe autoinflammatory disorder with a still not clearly defined molecular mechanism. To better understand the disease, we used scattered datasets from public domains and performed a weighted gene coexpression network analysis (WGCNA) to identify key modules and hub genes underlying sJIA pathogenesis. Two gene expression datasets, GSE7753 and GSE13501, were used to construct the WGCNA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to the genes and hub genes in the sJIA modules. Cytoscape was used to screen and visualize the hub genes. We further compared the hub genes with the genome-wide association study (GWAS) genes and used a consensus WGCNA to verify that our conclusions were conservative and reproducible across multiple independent datasets. A total of 5,414 genes were obtained for WGCNA, from which highly correlated genes were divided into 17 modules. The red module demonstrated the highest correlation with the sJIA module ( r = 0.8 , p = 3 e − 29 ), whereas the green-yellow module was found to be closely related to the non-sJIA module ( r = 0.62 , p = 1 e − 14 ). Functional enrichment analysis demonstrated that the red module was mostly enriched in the activation of immune responses, infection, nucleosomes, and erythrocytes, and the green-yellow module was mostly enriched in immune responses and inflammation. Additionally, the hub genes in the red module were highly enriched in erythrocyte differentiation, including ALAS2, AHSP, TRIM10, TRIM58, and KLF1. The hub genes from the green-yellow module were mainly associated with immune responses, as exemplified by the genes KLRB1, KLRF1, CD160, and KIRs. We identified sJIA-related modules and several hub genes that might be associated with the development of sJIA. Particularly, the modules may help understand the mechanisms of sJIA, and the hub genes may become biomarkers and therapeutic targets of sJIA in the future.

The occurrence of metastasis is a serious risk for renal cell carcinoma (RCC) patients. In order to develop novel therapeutic approaches to control the progression of metastatic RCC, it is of urgent need to understand the molecular mechanisms underlying RCC metastasis and identify prognostic markers of metastatic risk. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been known to be closely associated with extracellular matrix (ECM) turnover, which plays a highly active role in tumor metastasis. Recent studies have shown that immunophilin FK-506-binding protein 51 (FKBP51) may be important for the regulation of ECM function, and exert effects on the invasion and migration of tumor cells. However, the mechanisms underlying these activities remain unclear. The present study detected the role of FKBP51 in clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC, and found that FKBP51 significantly promotes ccRCC invasion and migration by binding with the TIMP3, connecting TIMP3 with Beclin1 complex and increasing autophagic degradation of TIMP3. Given the important roles that TIMPs/MMPs play in ECM regulation and remodeling, our findings will provide new perspective for future investigation of the regulation of metastasis of kidney cancer and other types of cancer.

Engagement of programmed death 1 receptor (PD-1) and its ligand PD-L1/2 induces a signal transduction pathway that inhibits the activity of tumor-infiltrating cytotoxic T lymphocytes and promotes tumor growth and metastasis. Antibodies blocking PD-1 or PD-L1 can restore antitumor T cell responses and cause long-term remission in a subset of cancer patients with advanced or refractory tumors. In this study, we asked whether PD-L1 vaccination could confer tumor control in mouse tumor models. To address the central tolerance toward self-molecules, we fused the extracellular domain of PD-L1 (PD-L1E) to the C-terminal of the translocation domain of diphtheria toxin (DTT). DTT is able to elicit CD4+ T cell responses required for inducing robust immune responses against self-molecules. The fusion molecule is called DPDL1E. When formulated with incomplete Freund’s adjuvant (IFA), DPDL1E elicited robust immune responses biased toward the Th1 type and inhibited tumor growth in both preventive and therapeutic mouse tumor models. We further showed that the anti-DPDL1E sera blocked PD-L1 binding to PD-1 in vitro. The DPDL1E vaccination increased the levels of tumor-infiltrating T lymphocytes (TILs) and reduced the levels of myeloid-derived suppressor cells (MDSCs) as well as exhausted LAG3+PD-1+ CD8+ T cells. All of these data suggest that DPDL1E vaccination reverses the suppressive phenotype of the tumor microenvironment and that it is a promising strategy for cancer therapy. Keywords: PD-L1, myeloid-derived suppressor cell, tumor-infiltrating lymphocytes, cancer immunotherapy, tumor microenvironment

Anti-TNF inhibitors are efficacious in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA), Crohn's disease (CD), juvenile idiopathic arthritis (JIA), and ankylosing spondylitis (AS). However, more and more clinical case reports revealed that anti-TNF inhibitors could increase the risk of viral, fungal, and bacterial (especially intracellular) infection. In this study, based on Immune Epitope Database (IEDB) online B cell epitope prediction and the knowledge of TNF three dimensional (3D) structure we developed a novel vaccine (DTNF114-TNF114) that targeting TNF epitope 1-14, which produced antibodies only partially binding to trans-membrane TNF (tmTNF), therefore partially sparing tmTNF-TNFR1/2 interaction. Immunization with DTNF114-TNF114 significantly protected and prolonged the survival rate of mice challenged with lipopolysaccharide (LPS); and in the mCherry expressing Mycobacterium bovis Bacillus Calmette-Guerin (mCherry-BCG) infection model, DTNF114-TNF114 immunization significantly decreased soluble TNF (solTNF) level in serum, meanwhile did not suppress host immunity against infection. Thus, this novel and infection concern-free vaccine provides a potential alternative or supplement to currently clinically used anti-TNF inhibitors.

Blocking inhibitory signaling and engaging stimulatory signaling have emerged as important therapeutic modalities for cancer immunotherapy. This study aimed to investigate immunomodulatory features of three recombinant costimulatory ligand proteins in a mouse model, which are extracellular domains of OX40-ligand (OX40L), 4-1BB-ligand (4-1BBL), or two domains in tandem, fused with the transmembrane domain of diphtheria toxin (DTT), named DTT-COS1, DTT-COS2, and DTT-COS12, respectively. In vitro study showed that DTT-COS1 and DTT-COS12 had immunological activity increasing the ratio of CD8/CD4 T cells. Treatments with DTT-COS1 and DTT-COS12 dramatically generated immune protection against the B16F10 tumor challenge in both prophylactic and therapeutic efficacy. Furthermore, regarding tumor microenvironment (TME) immunomodulation, DTT-COS1 treatment increased the proportion of CD4+ effector T cells (Teff) and decreased the expression of a suppressive cytokine. Meanwhile, DTT-COS12 reduced regulatory T cells (Treg) and improved the level of stimulatory cytokines. In addition, endogenous antibodies against OX40L/4-1BBL were generated, which may help with antitumor responses. Unexpectedly, DTT-COS2 lacked antitumor effects in vitro and in vivo. Importantly, serum analysis of liver-function associated factors and pro-inflammatory cytokines demonstrated that treatments were safe formulations in mice without signs of systemic toxicity. Remarkably, DTT-COS1 and DTT-COS12 are functional immunomodulators for mouse B16F10 melanoma, creating practical preclinical value in cancer immunotherapy.

BACKGROUND: The commercial success of monoclonal antibodies (Mabs) has made biological therapeutics attractive to pharmaceutical companies. The priority of biopharmaceutical companies is to acquire and develop cell lines that enable them to manufacture biologics quickly, consistently, and economically. Clone selection is a critical process for cell line development. However, the traditional clone selection process requires the evaluation of large numbers of clones using cell growth rate, cell densities and titer, product quality, and so on. METHODS: To improve efficiency of the clone selection strategies, we developed a relative titer (RT) prediction model by the quantitative information extracted from microscope images during the cell line development process. The performance of this RT prediction model was further evaluated with 50 clones from 5 different cell lines. RESULTS: The RT prediction model was able to predict high producers from a given data set when the same host cells were used. Although inaccurate prediction occurred when different host cell was used, this RT prediction model may serve as an excellent proof of concept study that quantitative information from cell line development images provides valuable information to facilitate the cell line development process. CONCLUSIONS: Here, we present the first predictive model that can be used to estimate the relative productivity of Chinese hamster ovaries (CHO) clones during the cell line development. Additional experiments are currently in process to further improve the RT predictive model. Nevertheless, our current study will serve as a foundation for more prediction models for cell line development that can facilitate the selection of clones.

The DOCK-AND-LOCK (DNL) method is a platform technology that combines recombinant engineering and site-specific conjugation to create multispecific, multivalent antibodies of defined composition with retained bioactivity. We have applied DNL to generate a novel class of trivalent bispecific antibodies (bsAb), each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of different antitumor Fabs. Here, we report the further characterization of two such constructs, (E1)-3s and (14)-3s, which activate T cells and target Trop-2– and CEACAM5-expressing cancer cells, respectively. (E1)-3s and (14)-3s, in the presence of human T cells, killed target cells grown as monolayers at subnanomolar concentrations, with a similar potency observed for drug-resistant cells. Antitumor efficacy was demonstrated for (E1)-3s coadministered with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice harboring xenografts of MDA-MB-231, a triple-negative breast cancer line constitutively expressing Trop-2 and PD-L1. Growth inhibition was observed following treatment with (E1)-3s or (14)-3s combined with human PBMC in 3D spheroids generated from target cell lines to mimic the in vivo behavior and microenvironment of these tumors. Moreover, addition of an antagonistic anti–PD-1 antibody increased cell death in 3D spheroids and extended survival of MDA-MB-231-bearing mice. These preclinical results emphasize the potential of combining T-cell–redirecting bsAbs with antagonists or agonists that mitigate T-cell inhibition within the tumor microenvironment to improve immunotherapy of solid cancers in patients. They also support the use of 3D spheroids as a predictive alternative to in vivo models for evaluating T-cell functions. Cancer Res; 77(19); 5384–94. ©2017 AACR.

Bispecific antibodies (bsAbs) for redirecting T cells to cancers have shown promise in both pre-clinical and clinical studies. However, clinical success has been minimal for solid cancers to-date. Previously, we reported highly effective T-cell redirected therapy of pancreatic and gastric tumors in xenograft models using a trivalent bsAb, designated (E1)-3s, which comprises an anti-CD3 scFv covalently conjugated to a stabilized dimer of a Trop-2-targeting humanized Fab. Trop-2 is highly expressed in diverse epithelial cancers, including breast, lung, gastric, colorectal, pancreatic, bladder, ovarian, uterine, and prostate carcinomas, with limited presence on normal human tissues. Compared to first generation bsAbs (e.g., BiTE), which induce high-level cytokine production that can lead to serious side effects, efficient T-cell killing is mediated by (E1)-3s with minimal cytokine release. Herein, we report the potential utility of (E1)-3s for therapy of breast cancers, including TNBC. Additionally, we show that addition of a checkpoint inhibitor can enhance bsAb-redirected T-cell therapy. IMMU-cPD1 is a chimeric mAb that binds with high affinity to human PD-1 and efficiently blocks binding to its ligand, PD-L1. The MDA-MB-231 human TNBC cell line has relatively low levels of surface Trop-2 (36,000/cell) and expresses PD-L1 constitutively. In ex-vivo assays, where MDA-MB-231 cells were mixed with purified human T cells, (E1)-3s mediated potent T-cell redirected killing (IC50< 10 pM). Addition of IMMU-cPD1 enhanced (E1)-3s-mediated T-cell killing. The advantage of combining the checkpoint inhibitor with redirected T-cell therapy was supported with in-vivo xenograft studies. NOD-SCID mice were co-injected with purified human T cells (2.5 x 106) and MDA-MB-231 (5 x 106). Groups were administered: 1) five daily injections of (E1)-3s; 2) cPD-1 twice weekly for 4 weeks; or 3) a combination of (E1)-3s and cPD1 treatments. An untreated control group comprising T cells and tumor cells reached the endpoint (tumors >1 cm3) with a median survival time (MST) of 35 days. The group treated with (E1)-3s had significantly smaller tumors (P=0.0023, AUC) and improved MST (42 days, P=0.0019). The group treated with the combination of (E1)-3s and IMMU-cPD1 had significantly smaller tumors (P=0.0121, AUC) and longer MST (49 days, P=0.008), compared to those treated with (E1)-3s alone. Treatment with IMMU-cPD1 alone did not retard tumor growth or improve MST, compared to the untreated control group. In conclusion, (E1)-3s is an attractive candidate for T-cell redirected therapy of breast cancer due to its potent activity with potentially reduced side effects and the prevalence of Trop-2 expression associated with this disease. Tumor microarrays representing 117 breast cancer patients showed >85% positivity for Trop-2. Our immunohistochemical analysis of more than 50 individual TNBC patient specimens demonstrated 92% positivity, with 80% having moderate to strong Trop-2-staining. Combining checkpoint inhibitors with redirected T-cell therapy may represent a new paradigm for the management of solid cancers, including breast, and is worthy of further investigation. Citation Format: Rossi EA, Chang K, Cardillo TM, Rossi DL, Li R, Mostafa A, Sharkey RM, Goldenberg DM. Enhanced efficacy of redirected T-cell therapy of TNBC with a Trop-2/CD3 bispecific antibody in combination with a checkpoint inhibitor [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-14-01.

Essentials Coagulation Factor (F) XI is a safe target for the development of antithrombotics. We designed an antigen comprising the human FXI catalytic domain and diphtheria toxin T domain. Antigen immunization reduced plasma FXI activity by 54% and prevented thrombosis in mice. FXI vaccination can serve as an effective strategy for thrombosis prevention. SummaryBackground Coagulation factor XI serves as a signal amplifier in the intrinsic coagulation pathway. Blockade of FXI by mAbs or small-molecule inhibitors inhibits thrombosis without causing severe bleeding, which is an inherent risk of currently available antithrombotic agents. Objectives To design an FXI vaccine and assess its efficacy in inhibiting FXI activity and preventing thrombosis. Methods An FXI antigen was generated by fusing the catalytic domain of human FXI to the C-terminus of the transmembrane domain of diphtheria toxin. The anti-FXI antibody response, plasma FXI activity and antithrombotic efficacy in mice immunized with the FXI antigen were examined. Results The antigen elicited a significant antibody response against mouse FXI, and reduced the plasma FXI activity by 54.0% in mice. FXI vaccination markedly reduced the levels of coagulation and inflammation in a mouse model of inferior vena cava stenosis. Significant protective effects were also observed in mouse models of venous thrombosis and pulmonary embolism. Conclusions Our data demonstrate that FXI vaccination can serve as an effective strategy for thrombosis prevention.

Proteins can be inactivated by high temperature commonly encountered during manufacture, delivery, and storage. Therefore, an accurate determination of the onset temperature (Ton) at which proteins are irreversibly inactivated is of great importance to both protein science and the pharmaceutical industry. The commonly used techniques for monitoring the process of protein denaturation including intrinsic fluorescence spectroscopy, CD, and DSC are often not sensitive enough to reflect Ton at which proteins start to loss their bioactivities. In this study, we found that Ton of irreversible inactivation of lysozyme and trypsin can be more accurately determined by analysis of the heating and cooling fluorescence-temperature courses (FTCs) of proteins labeled with FITC fluorescence dye, compared with the intrinsic fluorescence reporter. Moreover, we have evaluated the storage buffers for trypsin using this method. We speculate that this method could find practical utility in biopharmaceutical high-throughput screening for evaluation of stabilizing buffers and enzyme formulation.

Induction of cytotoxic T lymphocytes (CTL) is critical to cancer vaccine based immunotherapy. Efforts to elicit CTLs against tumor MUC1 with peptide based vaccine have not been successful in clinical application. We have design a MUC1 vaccine by replacing B cell epitope of CTB with MUC1 VNTR peptide. Immunization with hybrid CTB-MUC1 plus aluminum hydroxide and CpG adujuvant (CTB-MUC1-Alum-CpG) induce MUC1-specific CTLs in mice. Moreover, this vaccination can prevent tumor growth and reduce tumor burden in MUC1+B16 mice model. Meanwhile, CTB-MUC1-Alum-CpG vaccination can promote Th1 cells and CD8+ T cells inflate to tumor tissue. Our approach might be applicable to other cancer vaccine design.

Sanjin Tablets are completely natural preparation with significant efficacy in treating urinary tract infection. To identify the bioactive compounds from Sanjin Tablets, we separated components capable of binding to the soluble proteins of uropathogenicEscherichia coli(UPEC) by affinity binding and characterized their identities using liquid chromatography-mass spectrometry (LC-MS) analysis. Our study discovered eight compounds withE. coliprotein-binding capabilities, and all these compounds were tracked back to the original natural ingredients of Sanjin Tablets. These compounds presented essentially no antibacteria activity, indicating that they affect UPEC by means other than directly killing the cells. Further molecular modeling analysis predicted molecular targets for these compounds and mapped the residues potentially involved in compound-target interactions. All the predicted targets turned out to be critical proteins regulating the metabolisms ofE. coli, suggesting that these compounds may affect metabolic pathways in UPEC and inhibit pathogenesis. These data will benefit future design of drugs with higher efficacy and specificity on targeting pathogenic bacteria.

The profiling of abnormally-expressed proteins in host cells using mass spectrometry (MS) analysis is a classical approach for screening disease-associated biomarkers in clinical diagnosis. However, few pathogen-specific antigens can currently be detected in serum using this proteomic approach, since these are very low-abundant proteins that are easily masked by host high-abundant proteins. Identification of pathogen-specific antigens in the sera of tuberculosis patients is crucial for the clinical diagnosis of this infectious disease, especially in immune-compromised patients. In the present study, two biomimetic affinity chromatography (BiAC) media, At-23 and A115-94, were selected from a library of BiAC media and used to selectively fractionate Albumin and Immunoglobulin from sera, respectively, prior to MS analyses. Each fraction was collected and screened against the proteomic database of M. tuberculosis complex. Three antigens, FbpA, FbpB and BfrB, were identified with two distinct peptides in BiAC-fractionated sera from tuberculosis patients, which were confirmed by Western blotting. Moreover, the identification of pathogen-specific antigens in sera by BiAC in conjunction with ESI-CID-MS/MS represents a promising strategy for the discovery of disease-associated biomarkers in other diseases.

The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis (MTB) have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines and diagnostics. The constituents of the MTB CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel-based pre-fractionation is usually performed before MS analysis. In this study, we describe a novel pre-fractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six-column cascade. The LC-MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first-time identifications. Notably, ∼1/3 (20/61) of these novel CFPs are membrane proteins, among which 9 proteins have 2–14 transmembrane domains (TMD). In addition, ∼1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that BiAC pre-fractionation markedly improves protein detection by LC-MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased. This article is protected by copyright. All rights reserved

Vascular endothelial growth factor (VEGF) plays an important role in the progression of various cancers. The VEGF-specific antibody bevacizumab combined with chemotherapy was shown to significantly improve progression-free survival in certain cancers. However, repeated administration is necessary for effective suppression of VEGF, thereby making the therapy expensive and cumbersome. Thus, it is urgent to develop alternative reagents such as VEGF vaccines. Here we report that DTT-VEGF, a VEGF-based antigen consisting of the receptor-binding domain of VEGF and diphtheria toxin T domain (DTT), not only stimulated neutralizing antibody response, but also induced type 1 immune response as well as anti-tumor cytotoxic T lymphocytes in mice when administered with aluminum hydroxide adjuvant. The antibodies triggered by DTT-VEGF immunization inhibited the binding of VEGF to VEGF receptor and downregulated the serum VEGF levels in tumor-bearing mice. VEGF-specific IgG2a and IgG2b antibodies as well as type 1 cytokines were stimulated by DTT-VEGF vaccination. The splenocytes from DTT-VEGF-immunized mice showed cytotoxic activity against B16-F10 cells expressing VEGF. Extensive necrosis with severe hemorrhage and enhanced CD8+ T cell infiltration were observed in tumors from DTT-VEGF-immunized mice. The percentages of CD31+ vascular areas in the tumor sections from DTT-VEGF-immunized mice were significantly lower than those of control mice. DTT-VEGF significantly inhibited tumor growth in preventive and therapeutic vaccination settings in mouse models. Our data suggest that DTT is an effective antigen carrier to break immune self-tolerance and our vaccine design has potential to be used for human cancer therapy.

Additional file 1: Figure S1. Assessment of IL-15Rα expression. (a) Blood samples in WT B6 or irradiated B6 mice (n = 4) at day 5 post T-cells transfer were stained with OVA-tetramer, anti-CD8 Ab and anti-IL-15Rα Ab, and analyzed by flow cytometry. OVA-tetramer and CD8 double positive T-cells were gated for further assessing the expression of IL-15Rα (solid lines). Gray shaded histograms represent isotype Ab controls. **p

Candida antarctica lipase B (CalB) is one of the most widely used biocatalysts in organic synthesis. The traditional method for purification of CalB is a multi-step, high cost and low recovery procedure. Biomimetic affinity purification had high efficiency purification. We selected 298 ligand columns from a 700-member library of synthetic ligands to screen Pichia pastoris protein extract. Of the 298, three columns (named as A9-14, A9-10, and A11-33) had one-step purification effect, and A9-14 of these affinity ligands, had both high purification and recovery. The one-step recovery of CalB reached 73% and the purification reached 91% upon purification. The active groups of A9-14 were cyclohexylamine and propenylamine. Furthermore, both A9-14 and A9-10 had the same R1 active group of cyclohexylamine which might act the main binding role for CalB. The synthetic ligand A9-14 had a binding capacity of 0.4 mg/mL and had no negative effects on its hydrolytic activity. Unlike a natural affinity ligand, this synthetic ligand is highly stable to resist 1 M NaOH, and thus has great potential for industrial scale production of CalB.

Affinity chromatography has played an increasingly important role both in the pharmaceutical industry and academic research. In the present study, we report our preliminary investigation on the relationship between the affinity ligand structure and its adsorption to multi-protein samples. The structure of the ligands, including the size of the ring (cyclic group) and the length of the chain (linear group), has a great impact on the adsorption of ligands to proteins. Meanwhile, the functional groups that the ligands carry are also closely related to the adsorption of ligands to proteins. This research provides good guidance for the design and synthesis of affinity materials in affinity chromatography. It is also useful to other protein-ligand interaction-related research.

Purpose To detect serum and vitreous transthyretin (TTR) in high myopia patients and to evaluate potential associations between TTR and clinical parameters and ocular pathologies, including different ocular pathologies. Design and methods Serum samples from 16 high myopia patients and 4 controls were analyzed by LTQ-MASS. Serum samples from 116 high myopia patients and 86 healthy controls were tested by Western blots and ELISA. Eight healthy and 40 pathologic vitreous samples were analyzed by ELISA. And corresponding serum samples were also analyzed by ELISA. Results Significant increased TTR serum levels were detected in high myopia patients compared to healthy controls. The high levels of serum TTR were associated with ocular pathologies, long axial length, and low visual acuity. TTR in high myopia patients with macular hole and macular detachment was upregulated in both vitreous and the corresponding serum samples. TTR levels in serum samples of high myopia patients with long axial lengths were higher than in the vitreous. Conclusions Serum TTR may be a biomarker for high myopia patients with ocular pathologies.

Rice straw hydrolysate was used as lignocellulose-based carbon source forChlorella pyrenoidosacultivation and the feasibility ofin situbiodiesel production was investigated. 13.7 g/L sugar was obtained by enzymatic hydrolyzation of rice straw.Chlorella pyrenoidosashowed a rapid growth in the rice straw hydrolysate medium, the maximum biomass concentration of 2.83 g/L was obtained in only 48 hours. The lipid content of the cells reached as high as 56.3%.In situtransesterification was performed for biodiesel production. The optimized condition was 1 g algal powder, 6 mLn-hexane, and 4 mL methanol with 0.5 M sulfuric acid at the temperature of 90°C in 2-hour reaction time, under which over 99% methyl ester content and about 95% biodiesel yield were obtained. The results suggested that the method has great potential in the production of biofuels with lignocellulose as an alternative carbon source for microalgae cultivation.

In the microbial fermentation bioreactor, the processes of mRNA transcription, protein translation, and enzyme-catalyzed biosynthesis remain as “black boxes” of industrial monitoring and process control. Monitoring the kinetics of these “black boxes” is very helpful for optimizing and controlling the microbial fermentation process. This study first applied a molecular beacon (MB) to monitor the changes in the mRNA level of the phzC gene during antibiotic phenazine-1-carboxylic acid fermentation. Seven typical MB hybridization buffers were compared, and the effect of formamide on MBs was also studied. The results showed that rapid monitoring of the mRNA level using MBs was feasible. The optimal hybridization buffer for phzC MB was 100 mM Tris, 1 mM MgCl2, pH 8.0. The optimal hybridization temperature was 35 °C, and formamide proved unsuitable for MB hybridization. The limit of detection of phzC MB was 1.67 nM and MB hybridization was complete by 7 min. Given that the time for RNA extraction is 12 min, it is possible that monitoring of phzC mRNA can be completed in less than 20 min. Since production of most amine acids, organic acids, wines, antibiotics, and proteins relies on microbial fermentation, our method may have some potential for application in these other microbial industries.

Biomass pretreatment is one of major bottlenecks to convert biomass to bioethanol at present. We found that cereal straw could be completely dissolved in a 10-fold volume excess of trifluoroacetic acid (≥99%) (TFA). Pretreatment with TFA completely disrupted the dense cellulose crystallinity of the biomass. Further research showed that the TFA dissolved, but did not degrade, the cellulose in the straw. TFA did, however, degrade 65.65% of xylan in the straw, as well as reduce 20.0–23.3% of the acid-insoluble lignin. Isopropanol could precipitate 92.7% of the material dissolved in the TFA solution. These results led us to design a procedure for pretreatment of cellulosic biomass involving a dissolving step with TFA and a precipitation step with isopropanol. Experiments show that the procedure is technically feasible. Moreover, TFA and isopropanol could be completely evaporated off the supernatant and precipitate, and recycled back into the process.

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.

In this report, the proteomics of adult mouse testis were analyzed by the combined usage of cascade affinity fractionation and LC-MS/MS. The differences between the selected affinity ligands in size, shape, structure, and biochemical characteristics, result in each ligand exhibiting a specific affinity to some protein groups. Therefore, a cascade composition of different ligands can be applied to the fractionation of complex tissue proteins. Ultimately, the fractions collected from cascade affinity fractionation were analyzed by LC-MS/MS, which resulted in high confidence identification of a total of 1378 non-redundant mouse testis protein groups, over 2.6 times as many proteins as were detected in the un-fractionated sample (526). All detected proteins were bioinformatically categorized according to their physicochemical characteristics (such as relative molecular mass, pI, grand average hydrophobicity value, and transmembrane helices), subcellular location, and function annotation. This approach highlighted the sensitivity of this method to a wide variety of protein classes. Utilizing a combination of cascade affinity fractionation and LC-MS/MS, we have established the largest proteomic database for adult mouse testis at the present time.

High effective acidic transesterification catalyzed by trifluoroacetic acid for biodiesel production was studied in the present research. The results showed that the oil could be converted to biodiesel directly by one-step trifluoroacetic acid catalyze process without extreme temperature and pressure conditions. The optimum process combination was 2.0 M catalyst concentration with 20:1 M ratio of methanol to oil at temperature of 120 °C. It reduced product specific gravity from an initial value of 0.965 to a value of 0.878 in about 5 h of reaction time, and the methyl ester content reached as high as 98.4%. The present procedure represents a simple and mild method for biodiesel production in short reaction time and with high conversion rate, which would offer potential for an industrial process.

A new method using molecularly imprinted polymers (MIPs) as specific adsorbent materials coupled with ELISA analysis is being reported for the first time for the detection of clenbuterol (CLB) residue in the pig muscles. After optimization of the posttreatments, the total amount of template bleeding in the CLB MIPs was decreased to only 3.0 ng CLB/60 mg MIPs, which is 10 times lower than that of the previous report. Moreover, compared to the methods of C18-ELISA and single ELISA, the combined molecularly imprinted solid-phase extraction (MISPE)–ELISA exhibited high precision and robust accuracy for CLB at all three spiked levels of 0.5, 5.0, and 10.0 ng g−1.

Chlorpromazine is extensively used as a sedative hypnotic in veterinary application, however, improper and over usage of chlorpromazine pose a serious threat to human health and animal production. Due to the difficulty in monitoring trace level of chlorpromazine residues in complicated biological matrices, specific adsorption materials for preconcentration and clean-up of chlorpromazine are indispensable for sample preparation before further determination. In this study, a new molecularly imprinted polymer (MIP) for recognition of chlorpromazine was obtained by bulk polymerization. Then the imprinting effect of MIP was confirmed via chromatographic evaluation by compared with the corresponding non-imprinted polymer (NIP). Furthermore, the adsorption selectivity and capacity analysis revealed that the MIP had high specification for chlorpromazine. Finally, with the optimized protocol of molecularly imprinted solid-phase extraction (MISPE), the target analyte in pig urine at different spiked levels were selectively separated and purified for routine HPLC-UV analysis, with the improved recoveries above 73.3% and the intraday RSD of less than 8.7%, proving that the obtained MIP as specific SPE sorbents for extraction of chlorpromazine is applicable.

Six molecularly imprinted polymers (MIPs) of erythromycin (ERY) were prepared by noncovalent bulk polymerization using methacrylic acid (MAA) as the functional monomer. On the basis of binding analysis, the MIPs with 1:2 optimum ratio of template to MAA were selected for subsequent scanning electron microscopy and Brunauer-Emmett-Teller analyses, which indicated that the MIPs had more convergent porous structures than the nonimprinted polymers. The equilibrium binding experiments showed that the binding sites of MIPs were heterogeneous, with two dissociation constants of 0.005 and 0.63 mg mL(-1), respectively. Furthermore, the performance of the MIPs as solid-phase extraction (SPE) sorbents was evaluated, and the selectivity analysis showed that the MIPs could recognize ERY with moderate cross-reactivity for other macrolides. The overall investigation of molecularly imprinted SPE for cleanup and enrichment of the ERY in pig muscle and tap water confirmed the feasibility of utilizing the MIPs obtained as specific SPE sorbents for ERY extraction in real samples. [figure: see text]