Cloning of Bordetella pertussis putative outer protein D (BopD) and Leucin/Isoleucine/Valin binding protein (LivJ)

NASA Astrophysics Data System (ADS)

Öztürk, Burcu Emine Tefon

2017-04-01

Whooping cough also known as pertussis is a contagious acute upper respiratory disease primarily caused by Bordetella pertussis. It is known that this disease may be fatal especially in infants and recently, the number of pertussis cases has been increased. Despite the fact that there are numbers of acellular vaccines on the market, the current acellular vaccine compositions are inadequate for providing sustainable immunity and avoiding subclinical disease cases. Hence, exploring novel proteins with high immune protective capacities is essential to enhance the clinical efficacy of current vaccines. In this study, genes of selected immunogenic proteins via -omics studies, namely Putative outer protein D (BopD) and Leucin/Isoleucine/Valin Binding Protein (LivJ) were first cloned into pGEM-T Easy vector and transformed to into E. coli DH5α cells and then cloned into the expression vector pET-28a(+) and transformed into E. coli BL21 (DE3) cells to express the proteins.

The diagnostic performance of recombinant Trypanosoma cruzi ribosomal P2beta protein is influenced by its expression system.

PubMed

Marcipar, Iván S; Olivares, María Laura; Robles, Lucía; Dekanty, Andrés; Marcipar, Alberto; Silber, Ariel M

2004-03-01

In the present work, we have determined the effect of expression vectors and their corresponding host bacteria on the antigenic performance of Trypanosoma cruzi P2beta (TcP2beta) full-length recombinant protein. The gene encoding the TcP2beta ribosomal protein was cloned in pMAL-c2 and pET-32a vectors that allow the expression of high levels of soluble fusion proteins. A panel of 32 positive and 32 negative sera was assayed with the purified proteins expressed using pMal-c2 (TcP2beta-MBP) and pET-32a (TcP2beta-TRX) vectors and with MBP and TRX purified from pMAL-c2 and pET-32a vectors, respectively. The antigenic behavior of each TcP2beta recombinant protein differed in the diagnostic performance in terms of DI(+) (93.7 for TcP2beta-MBP vs 100% for TcP2beta-TRX), in DI(-) (90.5 for TcP2beta-MBP vs 100% for TcP2beta-TRX) and in cross-reaction with negative sera. To determine if the higher reactivity of expressed pMAL-c2 protein was due to folding during protein expression or to a steric effect related to the protein adsorption at the titration plate, the reactivity of sera against soluble proteins was assessed by ELISA inhibition assays. As each soluble protein preserved its level of reactivity, we concluded that differences in reactivity were due to intrinsic characteristics of the proteins and not to differences in patterns of adsorption to the plates.

[Cloning and sequence analysis of recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit and Actinobacillus actinomycetemcomitans fimbria associative protein].

PubMed

Li, Yi; Sun, Hong-chen; Guo, Xue-jun; Feng, Shu-zhang

2005-02-01

To clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap). Two couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5alpha. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced. The ltb-fap about 531bp in size was obtained successfully, and identified by PCR, restrictive enzyme and sequence analysis. The vector of pET28a ltb-fap was obtained.

Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Guan-Jing; Li, Lan-Fen; Li, Dan

2007-09-01

A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, withmore » unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.« less

[Prokaryotic expression and histological localization of the Taenia solium CDC37 gene].

PubMed

Huang, Jiang; Li, Bo; Dai, Jia-Lin; Zhang, Ai-Hua

2013-02-01

To express Taenia solium gene encoding cell division cycle 37 protein (TsCDC37) and investigate its antigenicity and localization in adults of Taenia solium. The complete coding sequence of TsCDC37 was amplified by PCR based on the recombinant plasmid clone from the cDNA library of adult Taenia solium. The PCR product was cloned into a prokaryotic expression vector pET-28a (+). The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21/DE3 and followed by expression of the protein induced by IPTG. The mice were immunized subcutaneously with purified recombinant TsCDC37 formulated in Freund's adjuvant. The antigenicity of the recombinant protein was examined by Western blotting. The localization of TsCDC37 in adult worms was demonstrated by immunofluorescent technique. The recombinant expression vector was constructed successfully. The recombinant protein was about M(r) 52 000, it was then purified and specifically recognized by immuno sera of SD rats and sera from patients infected with Taenia solium, Taenia saginata or Taenia asiatica. The immunofluorescence assay revealed that TsCDC37 located at the tegument of T. solium adult and the eggs. TsCDC37 gene has been expressed with immunoreactivity. The recombinant protein is mainly expressed in tegument and egg, and is a common antigen of the three human taenia cestodes.

[Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

PubMed

Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

2014-05-01

To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

Positron emission tomography and gene therapy: basic concepts and experimental approaches for in vivo gene expression imaging.

PubMed

Peñuelas, Iván; Boán, JoséF; Martí-Climent, Josep M; Sangro, Bruno; Mazzolini, Guillermo; Prieto, Jesús; Richter, José A

2004-01-01

More than two decades of intense research have allowed gene therapy to move from the laboratory to the clinical setting, where its use for the treatment of human pathologies has been considerably increased in the last years. However, many crucial questions remain to be solved in this challenging field. In vivo imaging with positron emission tomography (PET) by combination of the appropriate PET reporter gene and PET reporter probe could provide invaluable qualitative and quantitative information to answer multiple unsolved questions about gene therapy. PET imaging could be used to define parameters not available by other techniques that are of substantial interest not only for the proper understanding of the gene therapy process, but also for its future development and clinical application in humans. This review focuses on the molecular biology basis of gene therapy and molecular imaging, describing the fundamentals of in vivo gene expression imaging by PET, and the application of PET to gene therapy, as a technology that can be used in many different ways. It could be applied to avoid invasive procedures for gene therapy monitoring; accurately diagnose the pathology for better planning of the most adequate therapeutic approach; as treatment evaluation to image the functional effects of gene therapy at the biochemical level; as a quantitative noninvasive way to monitor the location, magnitude and persistence of gene expression over time; and would also help to a better understanding of vector biology and pharmacology devoted to the development of safer and more efficient vectors.

Soluble expression and one-step purification of recombinant mouse interferon-λ3 in Escherichia coli.

PubMed

Wang, Y Q; Zhou, M; Zeng, L M; Gao, Q Y; Yuan, X L; Li, Y; Li, M C

2015-02-01

Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.

[Expression, purification and protective antigen analysis of cell wall protein MRP of Streptococcus suis type 2].

PubMed

Wang, Ping-ping; Pian, Ya-ya; Yuan, Yuan; Zheng, Yu-ling; Jiang, Yong-qiang; Xiong, Zheng-ying

2012-02-01

To amplify the mrp gene of Streptococcus suis type 2 05ZYH33, express it in E.coli BL21 in order to acquire high purity recombinant protein MRP, then evaluate the protective antigen of recombinant protein MRP. Using PCR technology to obtain the product of mrp gene of 05ZYH33, and then cloned it into the expression vector pET28a(+). The recombinant protein was purified by affinity chromatography, later immunized New Zealand rabbit to gain anti-serum, then test the anti-serum titer by ELISA. The opsonophagocytic killing test demonstrated the abilities of protective antigen of MRP. The truncated of MRP recombinant protein in E.coli BL21 expressed by inclusion bodies, and purified it in high purity. After immunoprotection, the survival condition of CD-1 was significantly elevated. The survival rate of wild-type strain 05ZYH33 in blood was apparently decreased after anti-serum opsonophagocyticed, but the mutant delta; MRP showed no differences. MRP represent an important protective antigen activity.

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

Purification, Characterization, and Cloning of a Cold-Adapted Protease from Antarctic Janthinobacterium lividum.

PubMed

Kim, Hyun-Do; Kim, Su-Mi; Choi, Jong-Il

2018-03-28

In this study, a 107 kDa protease from psychrophilic Janthinobacterium lividum PAMC 26541 was purified by anion-exchange chromatography. The specific activity of the purified protease was 264 U/mg, and the overall yield was 12.5%. The J. lividum PAMC 25641 protease showed optimal activity at pH 7.0-7.5 and 40°C. Protease activity was inhibited by PMSF, but not by DTT. On the basis of the N-terminal sequence of the purified protease, the gene encoding the cold-adapted protease from J. lividum PAMC 25641 was cloned into the pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3) as an intracellular soluble protein.

Pyranose 2-oxidase from Phanerochaete chrysosporium : expression in E. coli and biochemical characterization

Treesearch

Ines Pisanelli; Magdalena Kujawa; Oliver Spadiut; Roman Kittl; Petr Halada; Jindrich Volc; Michael D. Mozuch; Philip Kersten; Dietmar Haltrich; Clemens Peterbauer

2009-01-01

The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in...

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

PubMed

Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

2017-02-01

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

PubMed Central

Sato, M; Figueiredo, ML; Burton, JB; Johnson, M; Chen, M; Powell, R; Gambhir, SS; Carey, M; Wu, L

2009-01-01

Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer. PMID:18305574

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Xiong-Zhuo; National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871; Li, Lan-Fen

The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction weremore » obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.« less

Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa.

PubMed

Catani, Cleide Ferreira; Azzoni, Adriano Rodrigues; Paula, Débora Pires; Tada, Susely Ferraz Siqueira; Rosselli, Luciana Kauer; de Souza, Anete Pereira; Yano, Tomomasa

2004-10-01

In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37 degrees C, with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy.

Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

PubMed

Salabert, Anne-Sophie; Vaysse, Laurence; Beaurain, Marie; Alonso, Mathieu; Arribarat, Germain; Lotterie, Jean-Albert; Loubinoux, Isabelle; Tafani, Mathieu; Payoux, Pierre

2017-01-01

Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression. A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo. Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft. The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

PubMed

Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

2014-12-01

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

[Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].

PubMed

Duan, Xiao-yi; Wang, Jian-sheng; Guo, You-min; Han, Jun-li; Wang, Quan-ying; Yang, Guang-xiao

2007-01-01

To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein. cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3). Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, andmore » enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.« less

Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Zhao, Yonggang; Tang, Xiaoqian; Zhan, Wenbin

2011-09-01

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea, Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors. They are thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR). We ligated the three genes into prokaryotic expression vector pET-28a (+), and transformed the recombinant plasmids into Escherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins, TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis. The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.

Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

PubMed Central

Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam

2014-01-01

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs. PMID:25192284

Adeno-associated virus type 8 vector–mediated expression of siRNA targeting vascular endothelial growth factor efficiently inhibits neovascularization in a murine choroidal neovascularization model

PubMed Central

Igarashi, Tsutomu; Miyake, Noriko; Fujimoto, Chiaki; Yaguchi, Chiemi; Iijima, Osamu; Shimada, Takashi; Takahashi, Hiroshi

2014-01-01

Purpose To assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (CNV), we generated an adeno-associated virus type 8 vector (AAV2/8) encoding an siRNA targeting vascular endothelial growth factor (VEGF), and determined the AAV2/8 vector’s ability to inhibit angiogenesis. Methods We initially transfected 3T3 cells expressing VEGF with the AAV2/8 plasmid vector psiRNA-VEGF using the H1 promoter and found that VEGF expression was significantly diminished in the transfectants. We next injected 1 μl (3 × 1014 vg/ml) of AAV2/8 vector encoding siRNA targeting VEGF (AAV2/8/SmVEGF-2; n = 12) or control vector encoding green fluorescent protein (GFP) (AAV2/8/GFP; n = 14) into the subretinal space in C57BL/6 mice. One week later, CNV was induced by using a diode laser to make four separate choroidal burns around the optic nerve in each eye. After an additional 2 weeks, the eyes were removed for flat mount analysis of the CNV surface area. Results Subretinal delivery of AAV2/8/SmVEGF-2 significantly diminished CNV at the laser lesions, compared to AAV8/GFP (1597.3±2077.2 versus 5039.5±4055.9 µm2; p<0.05). Using an enzyme-linked immunosorbent assay, we found that VEGF levels were reduced by approximately half in the AAV2/8/SmVEGF-2 treated eyes. Conclusions These results suggest that siRNA-VEGF can be expressed across the retina and that long-term suppression of CNV is possible through the use of stable AAV2/8-mediated siRNA-VEGF expression. In vivo gene therapy may thus be a feasible approach to the clinical management of CNV in conditions such as age-related macular degeneration. PMID:24744609

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

PubMed

Abdallah, M A; Pollenz, R S; Droog, F N; Nunamaker, R A; Tabachnick, W J; Murphy, K E

2000-12-01

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Delivery of Na/I symporter gene into skeletal muscle using nanobubbles and ultrasound: visualization of gene expression by PET.

PubMed

Watanabe, Yukiko; Horie, Sachiko; Funaki, Yoshihito; Kikuchi, Youhei; Yamazaki, Hiromichi; Ishii, Keizo; Mori, Shiro; Vassaux, Georges; Kodama, Tetsuya

2010-06-01

The development of nonviral gene delivery systems is essential in gene therapy, and the use of a minimally invasive imaging methodology can provide important clinical endpoints. In the current study, we present a new methodology for gene therapy-a delivery system using nanobubbles and ultrasound as a nonviral gene delivery method. We assessed whether the gene transfer allowed by this methodology was detectable by PET and bioluminescence imaging. Two kinds of reported vectors (luciferase and human Na/I symporter [hNIS]) were transfected or cotransfected into the skeletal muscles of normal mice (BALB/c) using the ultrasound-nanobubbles method. The kinetics of luciferase gene expression were analyzed in vivo using bioluminescence imaging. At the peak of gene transfer, PET of hNIS expression was performed using our recently developed PET scanner, after (124)I injection. The imaging data were confirmed using reverse-transcriptase polymerase chain reaction amplification, biodistribution, and a blocking study. The imaging potential of the 2 methodologies was evaluated in 2 mouse models of human pathology (McH/lpr-RA1 mice showing vascular disease and C57BL/10-mdx Jic mice showing muscular dystrophy). Peak luciferase gene activity was observed in the skeletal muscle 4 d after transfection. On day 2 after hNIS and luciferase cotransfection, the expression of these genes was confirmed by reverse-transcriptase polymerase chain reaction on a muscle biopsy. PET of the hNIS gene, biodistribution, the blocking study, and autoradiography were performed on day 4 after transfection, and it was indicated that hNIS expression was restricted to the site of plasmid administration (skeletal muscle). Similar localized PET and (124)I accumulation were successfully obtained in the disease-model mice. The hNIS gene was delivered into the skeletal muscle of healthy and disease-model mice by the ultrasound-nanobubbles method, and gene expression was successfully visualized with PET. The combination of ultrasound-nanobubble gene transfer and PET may be applied to gene therapy clinical protocols.

Interleukin 10 mediated by herpes simplex virus vectors suppresses neuropathic pain induced by human immunodeficiency virus gp120 in rats.

PubMed

Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C; Candiotti, Keith A; Lubarsky, David A; Hao, Shuanglin

2014-09-01

Human immunodeficiency virus (HIV)-associated sensory neuropathy is a common neurological complication of HIV infection affecting up to 30% of HIV-positive individuals. However, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments for HIV-related neuropathic pain (NP). In this study, we tested the hypothesis that inhibition of proinflammatory factors with overexpression of interleukin (IL)-10 reduces HIV-related NP in a rat model. NP was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. The hindpaws of rats were inoculated with nonreplicating herpes simplex virus (HSV) vectors expressing anti-inflammatory cytokine IL-10 or control vector. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The mechanical threshold response was assessed over time using the area under curves. The expression of phosphorylated p38 mitogen-activated kinase, tumor necrosis factor-α, stromal cell-derived factor-1α, and C-X-C chemokine receptor type 4 in both the lumbar spinal cord and the L4/5 dorsal root ganglia (DRG), was examined at 14 and 28 days after vector inoculation using Western blots. We found that in the gp120-induced NP model, IL-10 overexpression mediated by the HSV vector resulted in a significant elevation of the mechanical threshold that was apparent on day 3 after vector inoculation compared with the control vector (P < 0.001). The antiallodynic effect of the single HSV vector inoculation expressing IL-10 lasted >28 days. The area under curve in the HSV vector expressing IL-10 was increased compared with that in the control vector (P < 0.0001). HSV vectors expressing IL-10 reversed the upregulation of phosphorylated p38 mitogen-activated kinase, tumor necrosis factor-α, stromal cell-derived factor-1α, and C-X-C chemokine receptor type 4 expression at 14 and/or 28 days in the DRG and/or the spinal dorsal horn. Our studies demonstrate that blocking the signaling of these proinflammatory molecules in the DRG and/or the spinal cord using the HSV vector expressing IL-10 is able to reduce HIV-related NP. These results provide new insights on the potential mechanisms of HIV-associated NP and a proof of concept for treating painful HIV sensory neuropathy with this type of gene therapy.

A novel salt-inducible gene SbSI-1 from Salicornia brachiata confers salt and desiccation tolerance in E. coli.

PubMed

Yadav, Narendra Singh; Rashmi, Deo; Singh, Dinkar; Agarwal, Pradeep K; Jha, Bhavanath

2012-02-01

Salicornia brachiata is one of the extreme salt tolerant plants and grows luxuriantly in coastal areas. Previously we have reported isolation and characterization of ESTs from S. brachiata with large number of unknown gene sequences. Reverse Northern analysis showed upregulation and downregulation of few unknown genes in response to salinity. Some of these unknown genes were made full length and their functional analysis is being tested. In this study, we have selected a novel unknown salt inducible gene SbSI-1 (Salicornia brachiata salt inducible-1) for the functional validation. The SbSI-1 (Gen-Bank accession number JF 965339) was made full length and characterized in detail for its functional validation under desiccation and salinity. The SbSI-1 gene is 917 bp long, and contained 437 bp 3' UTR, and 480 bp ORF region encoding 159 amino acids protein with estimated molecular mass of 18.39 kDa and pI 8.58. The real time PCR analysis revealed high transcript expression in salt, desiccation, cold and heat stresses. However, the maximum expression was obtained by desiccation. The ORF region of SbSI-1 was cloned in pET28a vector and transformed in BL21 (DE3) E. coli cells. The SbSI-1 recombinant E. coli cells showed tolerance to desiccation and salinity stress compared to only vector in the presence of stress.

Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen

2007-09-01

A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous proteinmore » was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.« less

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

PubMed

Fu, L; Hou, Y L; Ding, X; Du, Y J; Zhu, H Q; Zhang, N; Hou, W R

2016-08-30

The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

Development of polyclonal antibodies against nucleocapsid protein of watermelon silver mottle virus and their application to diagnostic.

PubMed

Wu, Z; Wang, W; Li, Y; Rao, X

2014-01-01

Watermelon silver mottle virus (WSMoV) is an emerging disease of cucurbit crops in South China. Production of high-quality antibodies is necessary for the development of serological methods for detection of this virus. The nucleocapsid protein (NP) gene of WSMoV was amplified from WSMoV-infected watermelon leaves by RT-PCR and cloned into vector pET-28a (+) for prokaryotic expression. After identification via enzyme digestion and sequencing, the recombinant clone was transformed into Escherichia coli Rosetta (DE3) for protein expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular weight of the WSMoV NP fusion protein was 34.1 kDa. The fusion protein was purified and used as antigen for the preparation of polyclonal antisera in rabbits. Results of indirect ELISA and western blot analysis showed that the antisera reacted specifically with WSMoV NP. In addition, sensitivity and specificity of the antisera were examined on a number of infected field samples by indirect ELISA. These findings will facilitate further immunological and serological studies of WSMoV. .

[Cloning, prokaryotic expression and antibacterial assay of Tenecin gene encoding an antibacterial peptide from Tenebrio molitor].

PubMed

Liu, Ying; Jiang, Yu-xin; Li, Chao-pin

2011-12-01

To clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae. The antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction. Tenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction. Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively. Tenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.

Comparison of expression of monomeric and multimeric adenoregulin genes in Escherichia coli and Pichia pastorias.

PubMed

Zhou, Yuxun; Cao, Wei; Wang, Jinzhi; Ma, Yushu; Wei, Dongzhi

2005-05-01

Adenoregulin is a 33 amino acid antibiotic peptide who belongs to dermaseptin family which is the first vertebrate family to show lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. Synthetic adenoregulin gene was cloned in 2, 4 and 6 tandem repeats and subcloned in pET32a and pET22b vectors. Recombinant plasmids were transformed into E. coli BL21(DE3), Fusion proteins of Trx-ADR1, Trx-ADR2 and Trx-ADR4 could be expressed after the hosts were induced by IPTG, but the expression level decreased dramatically with the number of tandem repeats increased. ADR1, ADR4 and ADR6 could not be expressed by E. coli without carrier proteins. But for Pichia pastoris GS115, ADR1 and ADR6 in the fermentation broth of the hosts could be detected by ELISA, and the bactericidal activities could also be observed.

[Recombinant OspC identification and antigenicity detection from Borrelia burgdorferi PD91 in China].

PubMed

Chen, Jian; Wan, Kang-Lin

2003-10-01

To recombine OspC gene from Borrelia burgdorferi PD91 of China and expressed it in E. coli for early diagnosis of Lyme disease. The OspC gene was amplified from the genome of Borrelia burgdorferi PD91 strain by polymerase chain reaction and recombined with plasmid PET-11D. The recombinant plasmid PET-11D-OspC was identified with PCR, restriction endonuclease analysis and sequencing. The antigenicity was verified with Western Blot. OspC gene was cloned correctly into vector PET-11D. The resultant sequence was definitely different from the published sequence. The recombinant OspC seemed to have had strong antigenicity. The findings laid basis for the studies on early diagnosis of Lyme disease.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

[Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp. DS471].

PubMed

Ding, Linxian; Zhang, Pinghua; Hong, Huachang; Lin, Hongjun; Yokota, Akira

2012-01-01

The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene, secreted by Micrococcus luteus IAM 14879, in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria. Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified, cloned into a pET15b expression vector, and transformed into Escherichia coli BL21 (DE3). Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria, Rhodococcus sp. DS471, were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879. The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli, was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully, and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control. Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

PubMed

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

PubMed

Yasmin, Nusrat; Saleem, Mahjabeen; Naz, Mamoona; Gul, Roquyya; Rehman, Hafiz Muzzammel

2017-01-01

A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli . Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus ; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

PubMed Central

Yasmin, Nusrat; Naz, Mamoona; Gul, Roquyya; Rehman, Hafiz Muzzammel

2017-01-01

A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications. PMID:28875151

Imaging of glia activation in people with primary lateral sclerosis.

PubMed

Paganoni, Sabrina; Alshikho, Mohamad J; Zürcher, Nicole R; Cernasov, Paul; Babu, Suma; Loggia, Marco L; Chan, James; Chonde, Daniel B; Garcia, David Izquierdo; Catana, Ciprian; Mainero, Caterina; Rosen, Bruce R; Cudkowicz, Merit E; Hooker, Jacob M; Atassi, Nazem

2018-01-01

Glia activation is thought to contribute to neuronal damage in several neurodegenerative diseases based on preclinical and human post - mortem studies, but its role in primary lateral sclerosis (PLS) is unknown. To localize and measure glia activation in people with PLS compared to healthy controls (HC). Ten participants with PLS and ten age-matched HCs underwent simultaneous magnetic resonance (MR) and proton emission tomography (PET). The radiotracer [ 11 C]-PBR28 was used to obtain PET-based measures of 18 kDa translocator protein (TSPO) expression, a marker of activated glial cells. MR techniques included a structural sequence to measure cortical thickness and diffusion tensor imaging (DTI) to assess white matter integrity. PET data showed increased [ 11 C]-PBR28 uptake in anatomically-relevant motor regions which co-localized with areas of regional gray matter atrophy and decreased subcortical fractional anisotropy. This study supports a link between glia activation and neuronal degeneration in PLS, and suggests that these disease mechanisms can be measured in vivo in PLS. Future studies are needed to determine the longitudinal changes of these imaging measures and to clarify if MR-PET with [ 11 C]-PBR28 can be used as a biomarker for drug development in the context of clinical trials for PLS.

[Molecular cloning, expression and characterization of lysine decarboxylase gene of endophytic fungus Shiraia sp. Slf14 from Huperzia serrata].

PubMed

Peng, Silu; Yang, Huilin; Zhu, Du; Zhang, Zhibin; Yan, Riming; Wang, Ya

2016-04-14

Huperzine A (HupA) was approved as a drug for the treatment of Alzheimer's disease. The HupA biosynthetic pathway was started from lysine decarboxylase (LDC), which catalyzes lysine to cadaverine. In this study, we cloned and expressed an LDC gene from a HupA-producing endophytic fungus, and tested LDC activities. An endophytic fungus Shiraia sp. Slf14 from Huperzia serrata was used. LDC gene was obtained by RT-PCR, and cloned into pET-22b(+) and pET-32a(+) vectors to construct recombinant plasmids pET- 22b-LDC and pET-32a-LDC. These two recombinant plasmids were transformed into E. coli BL21, cultured for 8 h at 24 °C, 200 r/min with 1×10–3 mol/L IPTG into medium to express the LDC proteins, respectively. LDC proteins were purified by Ni2+ affinity chromatography. Catalytic activities were measured by Thin Layer Chromatography. At last, the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software. LDC and Trx-LDC were expressed in E. coli BL21 successfully. SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 kDa and 42.7 kDa respectively, which are consistent with bioinformatics analysis. In addition, TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities. This work can provide fundamental data for enriching LDC molecular information and reveal the HupA biosynthetic pathway in endophytic fungi.

Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

PubMed

Yue, Chang-Wu; Zhang, Yi-Zheng

2009-03-01

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

[Climate change - a pioneer for the expansion of canine vector-borne diseases?].

PubMed

Krämer, F; Mencke, N

2011-01-01

Vector-transmitted diseases are one of the major contributors to the global burden of disease in humans and animals. Climate change is consistently held responsible for the spread of parasitic acarid and insect vectors such as ticks, fleas, sand flies and mosquitoes, and their transmitted pathogens (in the case of the dog the so-called canine vector-borne diseases [CVBD]). Currently, there is only insufficient data available to prove whether climate change is a major driving force for vector and disease expansion, but the evidence is growing. Other reasons, such as ecological, demographic and socio-economic factors, e.g. pet travel into and pet import from endemic areas, also play a role in this development. Apart from all the controversial discussion of the factors leading to vector and disease expansion, preventative measures should include dog owners' education as they are responsible for individual parasite protection as well as for the minimisation of adverse risk behaviour, e.g. regarding pet travel. Broad-spectrum vector control should be practised by using parasiticides that repel and kill blood feeders in order to minimize the risk of CVBD-pathogen transmission.

Production and purification of a novel antibiotic peptide, adenoregulin, from a recombinant Escherichia coli.

PubMed

Zhou, Yu-Xun; Cao, Wei; Luo, Qing-Ping; Ma, Yu-Shu; Wang, Jin-Zhi; Wei, Dong-Zhi

2005-05-01

Adenoregulin is a member of dermaseptin family which are vertebrate antibiotic peptides having lethal effects against a broad spectrum of bacteria, fungi and protozoa. The 99 bp adenoregulin gene was cloned in the expression vector pET32a and transformed into Escherichia coli BL21(DE3). In fed-batch cultivation of BL21(DE3)/pET32a-adr, an exponential feeding strategy was applied to gain 60 g dry cells l-1. The recombinant fusion protein Trx-ADR was expressed in a soluble form. The fusion protein was isolated by Ni2+-chelating chromatography, cleaved with CNBr and purified to homogeneity through reverse phase-HPLC and size exclusion-HPLC. The purified recombinant adenoregulin had antibacterial activity against Escherichia coli K12D31 with apparent Mr of 3.4 kDa, identical to the anticipated value.

Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli.

PubMed

Chee Wei, T; Nurul Wahida, A G; Shaharum, S

2014-12-01

Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein.

PubMed

Li, Yifeng

2012-02-01

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount. Copyright © 2011 Elsevier Inc. All rights reserved.

Subunit Vaccine Preparation of Bovine Rotavirus and Its Efficacy in Mice.

PubMed

Suocheng, Wei; Tuanjie, Che; Changjun, Song; Fengling, Tian; Zhongren, Ma

2015-09-01

Rotaviruses (RV) are important viral diarrheal agents in calves. Vaccination is an optimum measure to prevent bovine rotaviruses (BRV) infection. However, little research on BRV VP7 vaccine has been done and currently there is no BRV vaccine. To prepare a subunit vaccine of BRV and investigate its efficacy. Total RNA was extracted from MA104 cells infected with bovine rotavirus (BRV) strain GSB01. BRV VP7 gene was amplified using real time fluorescence quantitative PCR (qPCR). The pEASY-T3-VP7 plasmid was digested using HindⅢ and BamHI restriction endonucleases, then recombined into the prokaryotic expression vector pET32a. The pET32a-VP7 and pET32a-VP7-LTB (heat-labile enterotoxin B subunit) were transformed into BL21 (DE3) competent cells of Escherichia coli, respectively, and induced with IPTG, then analyzed using SDS-PAGE. Sixty mice were randomly divided into three groups (n=20). Group A mice was used as His-tag control and mice in group B and C were inoculated with pET32a-VP7 and pET32a-VP7-LTB, respectively. VP7 IgG antibody titers and protection efficiency of pET32a-VP7-LTB were further determined in neonatal mice challenged with GSB01 BRV strain. SDS-PAGE analysis showed that the pET32a-VP7 was highly expressed in the BL21 (DE3) cells. PET32a-VP7 and pET32a-VP7-LTB protein could promote VP7 IgG antibody titer（8.33×103 vs. 17.26×103）in mice. Immunization protection ratios of pET32a-VP7 and pET32a-VP7-LTB proteins in the neonatal mice were 86.4% and 91.7%, respectively. The fusion protein of pET32a-VP7-LTB had excellent immunogenicity and protected mice from BRV infection. Our findings can be used for further developing of a high-efficiency subunit vaccine of BRV.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

PubMed

Schmitt, Christopher E; Morales, Blanca M; Schmitz, Ellen M H; Hawkins, John S; Lizama, Carlos O; Zape, Joan P; Hsiao, Edward C; Zovein, Ann C

2017-06-05

Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

Expression and characterization of duck enteritis virus gI gene

PubMed Central

2011-01-01

Background At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. Results The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. Conclusions The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene. PMID:21595918

Boosting isoprene production via heterologous expression of the Kudzu isoprene synthase gene (kIspS) into Bacillus spp. cell factory.

PubMed

Gomaa, Lamis; Loscar, Michael E; Zein, Haggag S; Abdel-Ghaffar, Nahed; Abdelhadi, Abdelhadi A; Abdelaal, Ali S; Abdallah, Naglaa A

2017-08-08

Isoprene represents a key building block for the production of valuable materials such as latex, synthetic rubber or pharmaceutical precursors and serves as basis for advanced biofuel production. To enhance the production of the volatile natural hydrocarbon isoprene, released by plants, animals and bacteria, the Kudzu isoprene synthase (kIspS) gene has been heterologously expressed in Bacillus subtilis DSM 402 and Bacillus licheniformis DSM 13 using the pHT01 vector. As control, the heterologous expression of KIspS in E. coli BL21 (DE3) with the pET28b vector was used. Isoprene production was analyzed using Gas Chromatography Flame Ionization Detector. The highest isoprene production was observed by recombinant B. subtilis harboring the pHT01-kIspS plasmid which produced 1434.3 μg/L (1275 µg/L/OD) isoprene. This is threefold higher than the wild type which produced 388 μg/L (370 μg/L/OD) isoprene, when both incubated at 30 °C for 48 h and induced with 0.1 mM IPTG. Additionally, recombinant B. subtilis produced fivefold higher than the recombinant B. licheniformis, which produced 437.2 μg/L (249 μg/L/OD) isoprene when incubated at 37 °C for 48 h induced with 0.1 mM IPTG. This is the first report of optimized isoprene production in B. licheniformis. However, recombinant B. licheniformis showed less isoprene production. Therefore, recombinant B. subtilis is considered as a versatile host for heterologous production of isoprene.

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

PubMed Central

Gerits, Annelies; Vancraeyenest, Pascaline; Vreysen, Samme; Laramée, Marie-Eve; Michiels, Annelies; Gijsbers, Rik; Van den Haute, Chris; Moons, Lieve; Debyser, Zeger; Baekelandt, Veerle; Arckens, Lutgarde; Vanduffel, Wim

2015-01-01

Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. PMID:26839901

Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

PubMed

Graham, L A; Bendena, W G; Walker, V K

1996-02-01

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

Cloning, Expression and Characteristics of a Novel Alkalistable and Thermostable Xylanase Encoding Gene (Mxyl) Retrieved from Compost-Soil Metagenome

PubMed Central

Verma, Digvijay; Kawarabayasi, Yutaka; Miyazaki, Kentaro; Satyanarayana, Tulasi

2013-01-01

Background The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes. Methodology/Principal Findings Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1) was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids) displayed homology with glycosyl hydrolase (GH) family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C. Conclusion/Significance This is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues. PMID:23382818

A PCR survey of vector-borne pathogens in different dog populations from Turkey.

PubMed

Guo, Huanping; Sevinc, Ferda; Ceylan, Onur; Sevinc, Mutlu; Ince, Ege; Gao, Yang; Moumouni, Paul Franck Adjou; Liu, Mingming; Efstratiou, Artemis; Wang, Guanbo; Cao, Shinuo; Zhou, Mo; Jirapattharasate, Charoonluk; Ringo, Aaron Edmond; Zheng, Weiqing; Xuan, Xuenan

2017-09-26

In the present study, a total of 192 blood samples were collected from pet dogs, kennel dogs and shepherd dogs in Konya district, Turkey, and tested by specific PCR for the presence of vector-borne pathogens. Several pathogens were identified, most of which can cause substantial morbidity in dogs. PCR results revealed that 54 (28.1%) dogs were infected with one or more pathogens. Positive results were obtained for Babesia spp. in 4 dogs (2.1%), Hepatozoon spp. in 8 dogs (4.2%) and Mycoplasma spp. in 46 dogs (24%). Three dogs (1.6%) were infected with two or three pathogens. The sequence analysis of the positive DNA samples revealed the presence of Babesia canis vogeli, Hepatozoon canis, Hepatozoon sp. MF, Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum. Ehrlichia canis and Anaplasma platys were not detected. Regardless of ownership status, vector-borne diseases were common in these dog populations. There was significant difference of pathogen prevalence among the different dog populations. Mycoplasma spp. was more frequent in the kennel dogs (31.9%) than in the pet (21.4%) and shepherd dogs (13.8%). Additionally, the frequency of Babesia spp. and Hepatozoon spp. was higher in the shepherd dogs which account for three quarters and half of the total number of Babesia spp. and Hepatozoon spp., respectively. To our knowledge, this is the first report of Mycoplasma infection in dogs in Turkey. The results of the present study provide a foundation for understanding the epidemiology of canine vector-borne diseases (CVBDs), and for strategies to control these diseases in Turkey.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

PubMed Central

Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

2009-01-01

Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

2013-09-01

Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Tianqing; Ming, Hongyan; Deng, Lili

Recombinant scorpion anti-excitation peptide (rANEP) has previously been expressed using the pET32a system and purified via affinity chromatography. However, rANEP is expressed in BL21(DE3) cells as an inclusion body, and the affinity tag can not be removed. To overcome this problem, we used a variety of protein, DsbA, MBP, TrxA, intein, and affinity tags in fusion and co-expression to achieve soluble and functional rANEP without any affinity tag. In the pCIT-ANEP expression vector, the highest soluble expression level was approximately 90% of the total cellular proteins in E. coli, and the rANEP was cleaved by the intein protein and subsequently purifiedmore » to obtain rANEP, which had the same activity as the natural ANEP. The purity of rANEP obtained using this method was over 95%, with a quantity of 5.1 mg from of purified rANEP from 1 L of culture. This method could expand the application of the soluble expression of disulfide-rich peptides in E. coli.« less

Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

PubMed

De Silva, Samantha R; Charbel Issa, Peter; Singh, Mandeep S; Lipinski, Daniel M; Barnea-Cramer, Alona O; Walker, Nathan J; Barnard, Alun R; Hankins, Mark W; MacLaren, Robert E

2016-11-01

Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4 -/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4 -/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

PET Imaging in Huntington's Disease.

PubMed

Roussakis, Andreas-Antonios; Piccini, Paola

2015-01-01

To date, little is known about how neurodegeneration and neuroinflammation propagate in Huntington's disease (HD). Unfortunately, no treatment is available to cure or reverse the progressive decline of function caused by the disease, thus considering HD a fatal disease. Mutation gene carriers typically remain asymptomatic for many years although alterations in the basal ganglia and cortex occur early on in mutant HD gene-carriers. Positron Emission Tomography (PET) is a functional imaging technique of nuclear medicine which enables in vivo visualization of numerous biological molecules expressed in several human tissues. Brain PET is most powerful to study in vivo neuronal and glial cells function as well as cerebral blood flow in a plethora of neurodegenerative disorders including Parkinson's disease, Alzheimer's and HD. In absence of HD-specific biomarkers for monitoring disease progression, previous PET studies in HD were merely focused on the study of dopaminergic terminals, cerebral blood flow and glucose metabolism in manifest and premanifest HD-gene carriers. More recently, research interest has been exploring novel PET targets in HD including the state of phosphodiesterse expression and the role of activated microglia. Hence, a better understanding of the HD pathogenesis mechanisms may lead to the development of targeted therapies. PET imaging follow-up studies with novel selective PET radiotracers such as 11C-IMA-107 and 11C-PBR28 may provide insight on disease progression and identify prognostic biomarkers, elucidate the underlying HD pathology and assess novel pharmaceutical agents and over time.

Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

PubMed

Petrzik, K; Mráz, I; Kubelková, D

2001-02-01

The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

PubMed

Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

2015-04-01

Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

[Prokaryotic expression of vp3 gene of Muscovy duck parvovirus, and its antiserum preparation for detection of virus multiplication].

PubMed

Huang, Yu; Zhu, Yumin; Dong, Shijuan; Yu, Ruisong; Zhang, Yuanshu; Li, Zhen

2015-01-01

New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

mRNA secondary structure engineering of Thermobifida fusca endoglucanase (Cel6A) for enhanced expression in Escherichia coli.

PubMed

Ali, Imran; Asghar, Rehana; Ahmed, Sajjad; Sajjad, Muhammad; Tariq, Muhammad; Waheed Akhtar, M

2015-03-01

The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5'-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5'-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = -2.7 kcal mol(-1)was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = -7.8 kcal mol(-1) and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg(-1) on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg(-1)), alkali treated bagass (1.7 Umg(-1)), filter paper (1.2 Umg(-1)) and BMCC (0.3 Umg(-1)). Metal ions affect endoglucanase activity in different ways. Only Fe(2+) exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2(+) (91.8 %).

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

PubMed

He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

2015-10-01

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning and expression of N22 region of Torque Teno virus (TTV) genome and use of peptide in developing immunoassay for TTV antibodies

PubMed Central

2014-01-01

Background Torque Teno Virus (TTV) is a DNA virus with high rate of prevalence globally. Since its discovery in 1997, several studies have questioned the role of this virus in causing disease. However, it still remains an enigma. Although methods are available for detection of TTV infection, there is still a need for simple, rapid and reliable method for screening of this virus in human population. Present investigation describes the cloning and expression of N22 region of TTV-genome and the use of expressed peptide in development of immunoassay to detect anti-TTV antibodies in serum. Since TTV genotype-1 is more common in India, the serum positive for genotype-1 was used as source of N22 for expression purpose. Methods Full length N22 region of ORF1 from TTV genotype-1 was amplified and cloned in pGEM®-T Easy vector. After cloning, the amplicon was transformed and expressed as a fusion protein containing hexa-histidine tag in pET-28a(+) vector using BL21 E. coli cells as host. Expression was conducted both in LB medium as well as ZYP-5052 auto-induction medium. The expressed peptide was purified using metal-chelate affinity chromatography and used as antigen in developing a blot immunoassay. Results Analysis of translated product by SDS-PAGE and western blotting demonstrated the presence of 25 kDa polypeptide produced after expression. Solubility studies showed the polypeptide to be associated with insoluble fraction. The use of this peptide as antigen in blot assay produced prominent spot on membrane treated with sera from TTV-infected patients. Analysis of sera from 75 patients with liver and renal diseases demonstrated a successful implication of N22 polypeptide based immunoassay in screening sera for anti-TTV antibodies. Comparison of the immunoassay developed using expressed N22 peptide with established PCR method for TTV-DNA detection showed good coherence between TTV-DNA and presence of anti-TTV antibodies in the sera analysed. Conclusions This concludes that TTV N22 region may be expressed and safely used as antigen for blot assay to detect anti-TTV antibodies in sera. PMID:24884576

Automated Whole-Body Bone Lesion Detection for Multiple Myeloma on 68Ga-Pentixafor PET/CT Imaging Using Deep Learning Methods.

PubMed

Xu, Lina; Tetteh, Giles; Lipkova, Jana; Zhao, Yu; Li, Hongwei; Christ, Patrick; Piraud, Marie; Buck, Andreas; Shi, Kuangyu; Menze, Bjoern H

2018-01-01

The identification of bone lesions is crucial in the diagnostic assessment of multiple myeloma (MM). 68 Ga-Pentixafor PET/CT can capture the abnormal molecular expression of CXCR-4 in addition to anatomical changes. However, whole-body detection of dozens of lesions on hybrid imaging is tedious and error prone. It is even more difficult to identify lesions with a large heterogeneity. This study employed deep learning methods to automatically combine characteristics of PET and CT for whole-body MM bone lesion detection in a 3D manner. Two convolutional neural networks (CNNs), V-Net and W-Net, were adopted to segment and detect the lesions. The feasibility of deep learning for lesion detection on 68 Ga-Pentixafor PET/CT was first verified on digital phantoms generated using realistic PET simulation methods. Then the proposed methods were evaluated on real 68 Ga-Pentixafor PET/CT scans of MM patients. The preliminary results showed that deep learning method can leverage multimodal information for spatial feature representation, and W-Net obtained the best result for segmentation and lesion detection. It also outperformed traditional machine learning methods such as random forest classifier (RF), k -Nearest Neighbors ( k -NN), and support vector machine (SVM). The proof-of-concept study encourages further development of deep learning approach for MM lesion detection in population study.

Automated Whole-Body Bone Lesion Detection for Multiple Myeloma on 68Ga-Pentixafor PET/CT Imaging Using Deep Learning Methods

PubMed Central

Tetteh, Giles; Lipkova, Jana; Zhao, Yu; Li, Hongwei; Christ, Patrick; Buck, Andreas; Menze, Bjoern H.

2018-01-01

The identification of bone lesions is crucial in the diagnostic assessment of multiple myeloma (MM). 68Ga-Pentixafor PET/CT can capture the abnormal molecular expression of CXCR-4 in addition to anatomical changes. However, whole-body detection of dozens of lesions on hybrid imaging is tedious and error prone. It is even more difficult to identify lesions with a large heterogeneity. This study employed deep learning methods to automatically combine characteristics of PET and CT for whole-body MM bone lesion detection in a 3D manner. Two convolutional neural networks (CNNs), V-Net and W-Net, were adopted to segment and detect the lesions. The feasibility of deep learning for lesion detection on 68Ga-Pentixafor PET/CT was first verified on digital phantoms generated using realistic PET simulation methods. Then the proposed methods were evaluated on real 68Ga-Pentixafor PET/CT scans of MM patients. The preliminary results showed that deep learning method can leverage multimodal information for spatial feature representation, and W-Net obtained the best result for segmentation and lesion detection. It also outperformed traditional machine learning methods such as random forest classifier (RF), k-Nearest Neighbors (k-NN), and support vector machine (SVM). The proof-of-concept study encourages further development of deep learning approach for MM lesion detection in population study. PMID:29531504

A Pretargeted Approach for the Multimodal PET/NIRF Imaging of Colorectal Cancer.

PubMed

Adumeau, Pierre; Carnazza, Kathryn E; Brand, Christian; Carlin, Sean D; Reiner, Thomas; Agnew, Brian J; Lewis, Jason S; Zeglis, Brian M

2016-01-01

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of strategies for the multimodal PET/NIRF imaging of cancer a very enticing prospect. Indeed, in the context of colorectal cancer, a single multimodal PET/NIRF imaging agent could be used to stage the disease, identify candidates for surgical intervention, and facilitate the image-guided resection of the disease. While antibodies have proven to be highly effective vectors for the delivery of radioisotopes and fluorophores to malignant tissues, the use of radioimmunoconjugates labeled with long-lived nuclides such as 89 Zr poses two important clinical complications: high radiation doses to the patient and the need for significant lag time between imaging and surgery. In vivo pretargeting strategies that decouple the targeting vector from the radioactivity at the time of injection have the potential to circumvent these issues by facilitating the use of positron-emitting radioisotopes with far shorter half-lives. Here, we report the synthesis, characterization, and in vivo validation of a pretargeted strategy for the multimodal PET and NIRF imaging of colorectal carcinoma. This approach is based on the rapid and bioorthogonal ligation between a trans -cyclooctene- and fluorophore-bearing immunoconjugate of the huA33 antibody (huA33-Dye800-TCO) and a 64 Cu-labeled tetrazine radioligand ( 64 Cu-Tz-SarAr). In vivo imaging experiments in mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts clearly demonstrate that this approach enables the non-invasive visualization of tumors and the image-guided resection of malignant tissue, all at only a fraction of the radiation dose created by a directly labeled radioimmunoconjugate. Additional in vivo experiments in peritoneal and patient-derived xenograft models of colorectal carcinoma reinforce the efficacy of this methodology and underscore its potential as an innovative and useful clinical tool.

Concurrent Respiratory Motion Correction of Abdominal PET and DCE-MRI using a Compressed Sensing Approach.

PubMed

Fuin, Niccolo; Catalano, Onofrio Antonio; Scipioni, Michele; Canjels, Lisanne P W; Izquierdo, David; Pedemonte, Stefano; Catana, Ciprian

2018-01-25

Purpose: We present an approach for concurrent reconstruction of respiratory motion compensated abdominal DCE-MRI and PET data in an integrated PET/MR scanner. The MR and PET reconstructions share the same motion vector fields (MVFs) derived from radial MR data; the approach is robust to changes in respiratory pattern and do not increase the total acquisition time. Methods: PET and DCE-MRI data of 12 oncological patients were simultaneously acquired for 6 minutes on an integrated PET/MR system after administration of 18 F-FDG and gadoterate meglumine. Golden-angle radial MR data were continuously acquired simultaneously with PET data and sorted into multiple motion phases based on a respiratory signal derived directly from the radial MR data. The resulting multidimensional dataset was reconstructed using a compressed sensing approach that exploits sparsity among respiratory phases. MVFs obtained using the full 6-minute (MC_6-min) and only the last 1 minute (MC_1-min) of data were incorporated into the PET reconstruction to obtain motion-corrected PET images and in an MR iterative reconstruction algorithm to produce a series of motion-corrected DCE-MRI images (moco_GRASP). The motion-correction methods (MC_6-min and MC_1-min) were evaluated by qualitative analysis of the MR images and quantitative analysis of maximum and mean standardized uptake values (SUV max , SUVmean), contrast, signal-to-noise ratio (SNR) and lesion volume in the PET images. Results: Motion corrected MC_6-min PET images demonstrated 30%, 23%, 34% and 18% increases in average SUV max , SUVmean, contrast and SNR, and an average 40% reduction in lesion volume with respect to the non-motion-corrected PET images. The changes in these figures of merit were smaller but still substantial for the MC_1-min protocol: 19%, 10%, 15% and 9% increases in average SUV max , SUVmean, contrast and SNR; and a 28% reduction in lesion volume. Moco_GRASP images were deemed of acceptable or better diagnostic image quality with respect to conventional breath hold cartesian VIBE acquisitions. Conclusion: We presented a method that allows the simultaneous acquisition of respiratory motion-corrected diagnostic quality DCE-MRI and quantitatively accurate PET data in an integrated PET/MR scanner with negligible prolongation in acquisition time compared to routine PET/DCE-MRI protocols. Copyright © 2018 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

PubMed

Zhang, Yuyao; Ma, Xiuli; Huang, Bing; Li, Yufeng; Yu, Kexiang; Li, Jianliang; Liu, Cunxia; Han, Hongyu; Cui, Yanshun

2015-04-04

To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

Emerging arthropod-borne diseases of companion animals in Europe.

PubMed

Beugnet, Frederic; Marié, Jean-Lou

2009-08-26

Vector-borne diseases are caused by parasites, bacteria or viruses transmitted by the bite of hematophagous arthropods (mainly ticks and mosquitoes). The past few years have seen the emergence of new diseases, or re-emergence of existing ones, usually with changes in their epidemiology (i.e. geographical distribution, prevalence, and pathogenicity). The frequency of some vector-borne diseases of pets is increasing in Europe, i.e. canine babesiosis, granulocytic anaplasmosis, canine monocytic ehrlichiosis, thrombocytic anaplasmosis, and leishmaniosis. Except for the last, these diseases are transmitted by ticks. Both the distribution and abundance of the three main tick species, Rhipicephalus sanguineus, Dermacentor reticulatus and Ixodes ricinus are changing. The conditions for such changes involve primarily human factors, such as travel with pets, changes in human habitats, social and leisure activities, but climate changes also have a direct impact on arthropod vectors (abundance, geographical distribution, and vectorial capacity). Besides the most known diseases, attention should be kept on tick-borne encephalitis, which seems to be increasing in western Europe, as well as flea-borne diseases like the flea-transmitted rickettsiosis. Here, after consideration of the main reasons for changes in tick vector ecology, an overview of each "emerging" vector-borne diseases of pets is presented.

Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies.

PubMed

Zhao, Mingzhi; Wu, Feilin; Xu, Ping

2015-12-01

Trypsin is one of the most important enzymatic tools in proteomics and biopharmaceutical studies. Here, we describe the complete recombinant expression and purification from a trypsinogen expression vector construct. The Sus scrofa cationic trypsin gene with a propeptide sequence was optimized according to Escherichia coli codon-usage bias and chemically synthesized. The gene was inserted into pET-11c plasmid to yield an expression vector. Using high-density E. coli fed-batch fermentation, trypsinogen was expressed in inclusion bodies at 1.47 g/L. The inclusion body was refolded with a high yield of 36%. The purified trypsinogen was then activated to produce trypsin. To address stability problems, the trypsin thus produced was acetylated. The final product was generated upon gel filtration. The final yield of acetylated trypsin was 182 mg/L from a 5-L fermenter. Our acetylated trypsin product demonstrated higher BAEE activity (30,100 BAEE unit/mg) than a commercial product (9500 BAEE unit/mg, Promega). It also demonstrated resistance to autolysis. This is the first report of production of acetylated recombinant trypsin that is stable and suitable for scale-up. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors.

PubMed

Schröder, R; Maassen, A; Lippoldt, A; Börner, T; von Baehr, R; Dobrowolski, P

1991-08-01

Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids.

Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11.

PubMed

Alnoch, Robson Carlos; Stefanello, Adriano Alves; Paula Martini, Viviane; Richter, Jeferson Luiz; Mateo, Cesar; Souza, Emanuel Maltempi de; Mitchell, David Alexander; Muller-Santos, Marcelo; Krieger, Nadia

2018-05-30

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5-10) and temperatures (25-45 °C), with the highest specific activity (1426 U mg -1 ) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n‑hexane with an activity of 4 U g -1 , maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis. Copyright © 2018 Elsevier B.V. All rights reserved.

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos.

PubMed

Gui, Tao; Zhang, Meiling; Chen, Jianwen; Zhang, Yuanliang; Zhou, Naru; Zhang, Yu; Tao, Jia; Sui, Liucai; Li, Yunsheng; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2012-08-01

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

[Cloning, expression and characterization of a novel esterase from marine sediment microbial metagenomic library].

PubMed

Xu, Shiqing; Hu, Yongfei; Yuan, Aihua; Zhu, Baoli

2010-07-01

To clone, express and characterize a novel esterase from marine sediment microbial metagenomic library. Using esterase segregation agar containing tributyrin, we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library. This fosmid was partially digested with Sau3A I to construct the sublibrary, from which the esterase positive subclone pFLS10 was obtained. The full length of the esterase gene was amplified and cloned into the expressing vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 cells. We analyse the enzyme activity and study the characterization of the esterase after its expression and purification. An ORF (Open Reading Frame) of 924 bp was identified from the subclone pFLS10. Sequence analysis indicated that it showed 71% amino acid identity to esterase (ADA70030) from a marine sediment metagenomic library. The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate (C4). The optimum temperature of the esterase was 20 degrees C, the optimum pH was 7.5. The esterase in this study had good thermostability at 20 degrees C and good pH stability at pH8 -10. Significant increase in lipolytic activity was observed with addition of K+ and Mg2+, while decrease with Mn2+ etc. We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library. The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions, which laid a basis for industrial application.

Neuroinflammatory component of gray matter pathology in multiple sclerosis.

PubMed

Herranz, Elena; Giannì, Costanza; Louapre, Céline; Treaba, Constantina A; Govindarajan, Sindhuja T; Ouellette, Russell; Loggia, Marco L; Sloane, Jacob A; Madigan, Nancy; Izquierdo-Garcia, David; Ward, Noreen; Mangeat, Gabriel; Granberg, Tobias; Klawiter, Eric C; Catana, Ciprian; Hooker, Jacob M; Taylor, Norman; Ionete, Carolina; Kinkel, Revere P; Mainero, Caterina

2016-11-01

In multiple sclerosis (MS), using simultaneous magnetic resonance-positron emission tomography (MR-PET) imaging with 11 C-PBR28, we quantified expression of the 18kDa translocator protein (TSPO), a marker of activated microglia/macrophages, in cortex, cortical lesions, deep gray matter (GM), white matter (WM) lesions, and normal-appearing WM (NAWM) to investigate the in vivo pathological and clinical relevance of neuroinflammation. Fifteen secondary-progressive MS (SPMS) patients, 12 relapsing-remitting MS (RRMS) patients, and 14 matched healthy controls underwent 11 C-PBR28 MR-PET. MS subjects underwent 7T T2*-weighted imaging for cortical lesion segmentation, and neurological and cognitive evaluation. 11 C-PBR28 binding was measured using normalized 60- to 90-minute standardized uptake values and volume of distribution ratios. Relative to controls, MS subjects exhibited abnormally high 11 C-PBR28 binding across the brain, the greatest increases being in cortex and cortical lesions, thalamus, hippocampus, and NAWM. MS WM lesions showed relatively modest TSPO increases. With the exception of cortical lesions, where TSPO expression was similar, 11 C-PBR28 uptake across the brain was greater in SPMS than in RRMS. In MS, increased 11 C-PBR28 binding in cortex, deep GM, and NAWM correlated with neurological disability and impaired cognitive performance; cortical thinning correlated with increased thalamic TSPO levels. In MS, neuroinflammation is present in the cortex, cortical lesions, deep GM, and NAWM, is closely linked to poor clinical outcome, and is at least partly linked to neurodegeneration. Distinct inflammatory-mediated factors may underlie accumulation of cortical and WM lesions. Quantification of TSPO levels in MS could prove to be a sensitive tool for evaluating in vivo the inflammatory component of GM pathology, particularly in cortical lesions. Ann Neurol 2016;80:776-790. © 2016 American Neurological Association.

PET Using a GRPR Antagonist 68Ga-RM26 in Healthy Volunteers and Prostate Cancer Patients.

PubMed

Zhang, Jingjing; Niu, Gang; Fan, Xinrong; Lang, Lixin; Hou, Guozhu; Chen, Libo; Wu, Huanwen; Zhu, Zhaohui; Li, Fang; Chen, Xiaoyuan

2018-06-01

This study was designed to analyze the safety, biodistribution, and radiation dosimetry of a gastrin-releasing peptide receptor (GRPR) antagonist PET tracer, 68 Ga-RM26; to assess its clinical diagnostic value in prostate cancer patients; and to perform a direct comparison between GRPR antagonist 68 Ga-RM26 and agonist 68 Ga-BBN. Methods: Five healthy volunteers were enrolled to validate the safety of 68 Ga-RM26 and calculate dosimetry. A total of 28 patients with prostate cancer (17 newly diagnosed and 11 posttherapy) were recruited and provided written informed consent. All the cancer patients underwent PET/CT at 15-30 min after intravenous injection of 1.85 MBq (0.05 mCi) per kilogram of body weight of 68 Ga-RM26. Among them, 22 patients (11 newly diagnosed and 11 posttherapy) underwent 68 Ga-BBN PET/CT for comparison within 1 wk. 99m Tc-MDP (methylene diphosphonate) bone scans were obtained within 2 wk for comparison. GRPR immunohistochemical staining of tumor samples was performed. Results: The administration of 68 Ga-M26 was well tolerated by all subjects, with no adverse symptoms being noticed or reported during the procedure and at 2-wk follow-up. The total effective dose equivalent and effective dose were 0.0912 ± 0.0140 and 0.0657 ± 0.0124 mSv/MBq, respectively. In the 17 patients with newly diagnosed prostate cancer, 68 Ga-RM26 PET/CT showed positive prostate-confined findings in 15 tumors with an SUV max of 6.49 ± 2.37. In the 11 patients who underwent prostatectomy or brachytherapy with or without androgen deprivation therapy, 68 Ga-RM26 PET/CT detected 8 metastatic lymph nodes in 3 patients with an SUV max of 4.28 ± 1.25 and 21 bone lesions in 8 patients with an SUV max of 3.90 ± 3.07. Compared with 68 Ga-RM26 PET/CT, GRPR agonist 68 Ga-BBN PET/CT detected fewer primary lesions and lymph node metastases as well as demonstrated lower tracer accumulation. There was a significant positive correlation between SUV derived from 68 Ga-RM26 PET and the expression level of GRPR ( P < 0.001). Conclusion: This study indicates the safety and significant efficiency of GRPR antagonist 68 Ga-RM26. 68 Ga-RM26 PET/CT would have remarkable value in detecting both primary prostate cancer and metastasis. 68 Ga-RM26 is also expected to be better than GRPR agonist as an imaging marker to evaluate GRPR expression in prostate cancer. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung

2011-07-01

Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including humanmore » specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.« less

Expression and Bioinformatics Analysis of Pectate Lyase Gene from Bacillus subtilis521

NASA Astrophysics Data System (ADS)

Xiao, Jing; Lu, Fu-Ping; Li, Yu; Li, Jin-Ting

In order to exploit new genetic resources, Pectate lyase(PEL) gene was amplified by PCR using the genome DNA from an alkaline Bacillus subtilis521. The PCR product was inserted into pET22b(+) vector. The recombinant plasmids were cloned in E.coli DH5α and then expressed in E.coli BL21. When cultured in the optimized medium, the positive clones E.coli BL21(pET22b(+)pel)showed intracellular pectate lyase activity of 90.0 U/mL. It was indicated that we had obtained the correct PEL gene. The pel has an open reading frame of 1263 nucleotides and codes for a product of 420 amino acids with a calculated molecular mass of 45.5 kD. Based on computer assisted analysis, a signal peptides and two conserved domains were revealed. The sequence analysis for PEL showed that it shares 26-82% homology with other strains in GenBank. In addition, the advanced structure of PEL were also predicted and analysed. This study will help to the experimental design of PEL fermentation and production purification and enzyme evolution.

Cloning and construction of recombinant palI gene from Klebsiella oxytoca on pET-32b into E. coli BL21 (DE3) pLysS for production of isomaltulose, a new generation of sugar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moeis, Maelita R., E-mail: sony@sith.itb.ac.id; Berlian, Liska, E-mail: sony@sith.itb.ac.id; Suhandono, Sony, E-mail: sony@sith.itb.ac.id

Klebsiella oxytoca produces sucrose isomerase which catalyses the conversion of sucrose to isomaltulose, a new generation of sugar. From the previous study, palI gene from Klebsiella oxytoca was succesfully isolated from sapodilla fruit (Manilkara zapota). The full-length palI gene sequence of Klebsiella oxytoca was cloned in E. coli DH5α. The deduced amino acid sequence shows 498 residues which includes conserved motif for sucrose isomerisation {sup 325}RLDRD{sup 329} and 97% identical to palI gene from Klebsiella sp. LX3 (GenBank:AAK82938.1). This fragment was succesfullly ligated into the expression vector pET-32b using overlap-extension PCR and cloned in Escherichia coli BL21 (DE3) pLysS. DNAmore » sequencing result shows that palI gene of Klebsiella oxytoca was inserted in-frame in pET-32b. This is the first report on cloning of palI gene from Klebsiella oxytoca.« less

[Characterization of a malic enzyme isoform V from Mucor circinelloides].

PubMed

Zhang, Yingtong; Chen, Haiqin; Song, Yuanda; Zhang, Hao; Chen, Yongquan; Chen, Wei

2016-02-04

We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

Novel method for hit-position reconstruction using voltage signals in plastic scintillators and its application to Positron Emission Tomography

NASA Astrophysics Data System (ADS)

Raczyński, L.; Moskal, P.; Kowalski, P.; Wiślicki, W.; Bednarski, T.; Białas, P.; Czerwiński, E.; Kapłon, Ł.; Kochanowski, A.; Korcyl, G.; Kowal, J.; Kozik, T.; Krzemień, W.; Kubicz, E.; Molenda, M.; Moskal, I.; Niedźwiecki, Sz.; Pałka, M.; Pawlik-Niedźwiecka, M.; Rudy, Z.; Salabura, P.; Sharma, N. G.; Silarski, M.; Słomski, A.; Smyrski, J.; Strzelecki, A.; Wieczorek, A.; Zieliński, M.; Zoń, N.

2014-11-01

Currently inorganic scintillator detectors are used in all commercial Time of Flight Positron Emission Tomograph (TOF-PET) devices. The J-PET collaboration investigates a possibility of construction of a PET scanner from plastic scintillators which would allow for single bed imaging of the whole human body. This paper describes a novel method of hit-position reconstruction based on sampled signals and an example of an application of the method for a single module with a 30 cm long plastic strip, read out on both ends by Hamamatsu R4998 photomultipliers. The sampling scheme to generate a vector with samples of a PET event waveform with respect to four user-defined amplitudes is introduced. The experimental setup provides irradiation of a chosen position in the plastic scintillator strip with an annihilation gamma quanta of energy 511 keV. The statistical test for a multivariate normal (MVN) distribution of measured vectors at a given position is developed, and it is shown that signals sampled at four thresholds in a voltage domain are approximately normally distributed variables. With the presented method of a vector analysis made out of waveform samples acquired with four thresholds, we obtain a spatial resolution of about 1 cm and a timing resolution of about 80 ps (σ).

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV.

PubMed

Wang, Jian; Guo, Hong-yan; Jia, Rui; Xu, Xuan; Tan, Juan; Geng, Yun-qi; Qiao, Wen-tao

2010-04-01

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Detection of increased 64Cu uptake by human copper transporter 1 gene overexpression using PET with 64CuCl2 in human breast cancer xenograft model.

PubMed

Kim, Kwang Il; Jang, Su Jin; Park, Ju Hui; Lee, Yong Jin; Lee, Tae Sup; Woo, Kwang Sun; Park, Hyun; Choe, Jae Gol; An, Gwang Il; Kang, Joo Hyun

2014-10-01

Copper is an essential cofactor for a variety of biochemical processes including oxidative phosphorylation, cellular antioxidant activity, and elimination of free radicals. The copper transporter 1 is known to be involved in cellular uptake of copper ions. In this study, we evaluated the utility of human copper transporter 1 (hCTR1) gene as a new reporter gene for (64)Cu PET imaging. Human breast cancer cells (MDA-MB-231) were infected with a lentiviral vector constitutively expressing the hCTR1 gene under super cytomegalovirus promoter, and positive clones (MDA-MB-231-hCTR1) were selected. The expression of hCTR1 gene in MDA-MB-231-hCTR1 cells was measured by reverse transcription polymerase chain reaction, Western blot, and (64)Cu uptake assay. To evaluate the cytotoxic effects induced by hCTR1 expression, the dose-dependent cell survival rate after treatment with cisplatin (Cis-diaminedichloroplatinum (II) [CDDP]) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion. Small-animal PET images were acquired in tumor-bearing mice from 2 to 48 h after an intravenous injection of (64)Cu. The hCTR1 gene expression in MDA-MB-231-hCTR1 cells was confirmed at the RNA and protein expression and the cellular (64)Cu uptake level. MTT assay and trypan blue dye exclusion showed that the cell viability of MDA-MB-231-hCTR1 cells decreased more rapidly than that of MDA-MB-231 cells after treatment with CDDP for 96 or 72 h, respectively. Small-animal PET imaging revealed a higher accumulation of (64)Cu in MDA-MB-231-hCTR1 tumors than in MDA-MB-231 tumors. With respect to the biodistribution data, the percentage injected dose per gram of (64)Cu in the MDA-MB-231 tumors and MDA-MB-231-hCTR1 tumors at 48 h after (64)Cu injection was 2.581 ± 0.254 and 5.373 ± 1.098, respectively. An increase in (64)Cu uptake induced by the expression of hCTR1 gene was demonstrated in vivo and in vitro, suggesting the potential use of hCTR1 gene as a new imaging reporter gene for PET with (64)CuCl2. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

PubMed

Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun

2014-01-01

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

NASA Astrophysics Data System (ADS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Commissioning of the J-PET Detector for Studies of Decays of Positronium Atoms

NASA Astrophysics Data System (ADS)

Czerwiński, E.; Dulski, K.; Białas, P.; Curceanu, C.; Gajos, A.; Głowacz, B.; Gorgol, M.; Hiesmayr, B. C.; Jasińska, B.; Kisielewska, D.; Korcyl, G.; Kowalski, P.; Kozik, T.; Krawczyk, N.; Krzemień, W.; Kubicz, E.; Mohammed, M.; Niedźwiecki, Sz.; Pałka, M.; Pawlik-Niedźwiecka, M.; Raczyński, L.; Rudy, Z.; Sharma, N. G.; Sharma, S.; Shopa, R. Y.; Silarski, M.; Skurzok, M.; Wieczorek, A.; Wiślicki, W.; Zgardzińska, B.; Zieliński, M.; Moskal, P.

The Jagiellonian Positron Emission Tomograph (J-PET) is a detector for medical imaging of the whole human body as well as for physics studies involving detection of electron-positron annihilation into photons. J-PET has high angular and time resolution and allows for measurement of spin of the positronium and the momenta and polarization vectors of annihilation quanta. In this article, we present the potential of the J-PET system for background rejection in the decays of positronium atoms.

Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli.

PubMed

Cheng, Zhuan; Jiang, Jiaqi; Wu, Hui; Li, Zhimin; Ye, Qin

2016-01-01

In this study, production of 3-HP via malonyl-CoA was investigated by using metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase (Acc) from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. Three engineered E. coli strains with different host-vector systems were constructed and investigated. The results indicated that the combination of E. coli BL21(DE3) and pET28a was the most efficient host-vector system for 3-HP production, and the highest concentration of 3-HP attained in shake flask cultivation reached 1.80g/L by the strain BE-MDA with induction at 0.25mM IPTG and 25°C, and supplementation of NaHCO3 and biotin. In fed-batch fermentation performed in a 5-L reactor, the concentration of 3-HP achieved 10.08g/L in 36h. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression and purification of lacticin Q by small ubiquitin-related modifier fusion in Escherichia coli.

PubMed

Ma, Qingshan; Yu, Zhanqiao; Han, Bing; Wang, Qing; Zhang, Rijun

2012-04-01

Lacticin Q is a broad-spectrum class II bacteriocin with potential as an alternative to conventional antibiotics. The objective of this study was to produce recombinant lacticin Q using a small ubiquitin-related modifier (SUMO) fusion protein expression system. The 168-bp lacticin Q gene was cloned into the expression vector pET SUMO and transformed into Escherichia coli BL21(DE3). The soluble fusion protein was recovered with a Ni-NTA Sepharose column (95% purity); 130 mg protein was obtained per liter of fermentation culture. The SUMO tag was then proteolytically cleaved from the protein, which was re-applied to the column. Finally, about 32 mg lacticin Q (≥96% purity) was obtained. The recombinant protein exhibited antimicrobial properties similar to that of the native protein, demonstrating that lacticin Q had been successfully expressed by the SUMO fusion system.

Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

PubMed

Padaria, Jasdeep Chatrath; Vishwakarma, Harinder; Biswas, Koushik; Jasrotia, Rahul Singh; Singh, Gyanendra Pratap

2014-10-10

Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India. qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting. TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

Clinical Utility of Amyloid PET Imaging in the Differential Diagnosis of Atypical Dementias and Its Impact on Caregivers.

PubMed

Bensaïdane, Mohamed Reda; Beauregard, Jean-Mathieu; Poulin, Stéphane; Buteau, François-Alexandre; Guimond, Jean; Bergeron, David; Verret, Louis; Fortin, Marie-Pierre; Houde, Michèle; Bouchard, Rémi W; Soucy, Jean-Paul; Laforce, Robert

2016-04-18

Recent studies have supported a role for amyloid positron emission tomography (PET) imaging in distinguishing Alzheimer's disease (AD) pathology from other pathological protein accumulations leading to dementia. We investigated the clinical utility of amyloid PET in the differential diagnosis of atypical dementia cases and its impact on caregivers. Using the amyloid tracer 18F-NAV4694, we prospectively scanned 28 patients (mean age 59.3 y, s.d. 5.8; mean MMSE 21.4, s.d. 6.0) with an atypical dementia syndrome. Following a comprehensive diagnostic workup (i.e., history taking, neurological examination, blood tests, neuropsychological evaluation, MRI, and FDG-PET), no certain diagnosis could be arrived at. Amyloid PET was then conducted and classified as positive or negative. Attending physicians were asked to evaluate whether this result led to a change in diagnosis or altered management. They also reported their degree of confidence in the diagnosis. Caregivers were met after disclosure of amyloid PET results and completed a questionnaire/interview to assess the impact of the scan. Our cohort was evenly divided between positive (14/28) and negative (14/28) 18F-NAV4694 cases. Amyloid PET resulted in a diagnostic change in 9/28 cases (32.1%: 17.8% changed from AD to non-AD, 14.3% from non-AD to AD). There was a 44% increase in diagnostic confidence. Altered management occurred in 71.4% (20/28) of cases. Knowledge of amyloid status improved caregivers' outcomes in all domains (anxiety, depression, disease perception, future anticipation, and quality of life). This study suggests a useful additive role for amyloid PET in atypical cases with an unclear diagnosis beyond the extensive workup of a tertiary memory clinic. Amyloid PET increased diagnostic confidence and led to clinically significant alterations in management. The information gained from that test was well received by caregivers and encouraged spending quality time with their loved ones.

The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia.

PubMed

Lee, Young Mok; Pan, Chi-Jiunn; Koeberl, Dwight D; Mansfield, Brian C; Chou, Janice Y

2013-11-01

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia. © 2013.

[Prokaryotic expression of recombinant prochymosin gene and its antiserum preparation].

PubMed

Li, Xin-ping; Liu, Huan-huan; Pu, Yan; Zhang, Fu-chun; Li, Yi-jie

2012-07-01

To optimize the prochymosin (pCHY) gene codons and express the gene in Escherichia coli (E.coli), and to prepare its antiserum and detect chymosin protein specifically. According to codon usage bias of E.coli, prochymosin gene sequence was synthesized based on the conserved sequences of prochymosin gene from bovine, lamb and camel, and then cloned into the plasmid pET-30a and pcDNA3-AAT-COMP-C3d3 (pcD-ACC), respectively. pET-30a-pCHY was expressed, as the detected antigen, in E.coli BL21(DE3) after IPTG induction. RT-PCR was used to detect prochymosin mRNA expression in liver from the mice injected pcDNA3-AAT-COMP-pCHY-C3d3(pACCC) by hydrodynamics-based transfection method. To prepare the antiserum of prochymosin, pACCC and GST-pCHY proteins were used to immunize New Zealand rabbits in accordance with DNA prime-protein boost strategy. Antibody levels were tested by ELISA. Western blotting showed the molecular weight of His-pCHY protein was about 55 000, similar to the expected molecular size. ELISA demonstrated that the titer level of prochymosin antiserum was high. Based on the codon optimization, we have obtained high-titer prochymosin antiserum through DNA vaccine vector pcD-ACC combined with DNA prime-protein boost strategy, similar to that by protein vaccine.

Respiratory motion correction in 4D-PET by simultaneous motion estimation and image reconstruction (SMEIR)

PubMed Central

Kalantari, Faraz; Li, Tianfang; Jin, Mingwu; Wang, Jing

2016-01-01

In conventional 4D positron emission tomography (4D-PET), images from different frames are reconstructed individually and aligned by registration methods. Two issues that arise with this approach are as follows: 1) the reconstruction algorithms do not make full use of projection statistics; and 2) the registration between noisy images can result in poor alignment. In this study, we investigated the use of simultaneous motion estimation and image reconstruction (SMEIR) methods for motion estimation/correction in 4D-PET. A modified ordered-subset expectation maximization algorithm coupled with total variation minimization (OSEM-TV) was used to obtain a primary motion-compensated PET (pmc-PET) from all projection data, using Demons derived deformation vector fields (DVFs) as initial motion vectors. A motion model update was performed to obtain an optimal set of DVFs in the pmc-PET and other phases, by matching the forward projection of the deformed pmc-PET with measured projections from other phases. The OSEM-TV image reconstruction was repeated using updated DVFs, and new DVFs were estimated based on updated images. A 4D-XCAT phantom with typical FDG biodistribution was generated to evaluate the performance of the SMEIR algorithm in lung and liver tumors with different contrasts and different diameters (10 to 40 mm). The image quality of the 4D-PET was greatly improved by the SMEIR algorithm. When all projections were used to reconstruct 3D-PET without motion compensation, motion blurring artifacts were present, leading up to 150% tumor size overestimation and significant quantitative errors, including 50% underestimation of tumor contrast and 59% underestimation of tumor uptake. Errors were reduced to less than 10% in most images by using the SMEIR algorithm, showing its potential in motion estimation/correction in 4D-PET. PMID:27385378

Respiratory motion correction in 4D-PET by simultaneous motion estimation and image reconstruction (SMEIR)

NASA Astrophysics Data System (ADS)

Kalantari, Faraz; Li, Tianfang; Jin, Mingwu; Wang, Jing

2016-08-01

In conventional 4D positron emission tomography (4D-PET), images from different frames are reconstructed individually and aligned by registration methods. Two issues that arise with this approach are as follows: (1) the reconstruction algorithms do not make full use of projection statistics; and (2) the registration between noisy images can result in poor alignment. In this study, we investigated the use of simultaneous motion estimation and image reconstruction (SMEIR) methods for motion estimation/correction in 4D-PET. A modified ordered-subset expectation maximization algorithm coupled with total variation minimization (OSEM-TV) was used to obtain a primary motion-compensated PET (pmc-PET) from all projection data, using Demons derived deformation vector fields (DVFs) as initial motion vectors. A motion model update was performed to obtain an optimal set of DVFs in the pmc-PET and other phases, by matching the forward projection of the deformed pmc-PET with measured projections from other phases. The OSEM-TV image reconstruction was repeated using updated DVFs, and new DVFs were estimated based on updated images. A 4D-XCAT phantom with typical FDG biodistribution was generated to evaluate the performance of the SMEIR algorithm in lung and liver tumors with different contrasts and different diameters (10-40 mm). The image quality of the 4D-PET was greatly improved by the SMEIR algorithm. When all projections were used to reconstruct 3D-PET without motion compensation, motion blurring artifacts were present, leading up to 150% tumor size overestimation and significant quantitative errors, including 50% underestimation of tumor contrast and 59% underestimation of tumor uptake. Errors were reduced to less than 10% in most images by using the SMEIR algorithm, showing its potential in motion estimation/correction in 4D-PET.

Quantitative assessment of Zirconium-89 labeled cetuximab using PET/CT imaging in patients with advanced head and neck cancer: a theragnostic approach.

PubMed

Even, Aniek J G; Hamming-Vrieze, Olga; van Elmpt, Wouter; Winnepenninckx, Véronique J L; Heukelom, Jolien; Tesselaar, Margot E T; Vogel, Wouter V; Hoeben, Ann; Zegers, Catharina M L; Vugts, Daniëlle J; van Dongen, Guus A M S; Bartelink, Harry; Mottaghy, Felix M; Hoebers, Frank; Lambin, Philippe

2017-01-17

Biomarkers predicting treatment response to the monoclonal antibody cetuximab in locally advanced head and neck squamous cell carcinomas (LAHNSCC) are lacking. We hypothesize that tumor accessibility is an important factor in treatment success of the EGFR targeting drug. We quantified uptake of cetuximab labeled with Zirconium-89 (89Zr) using PET/CT imaging.Seventeen patients with stage III-IV LAHNSCC received a loading dose unlabeled cetuximab, followed by 10 mg 54.5±9.6 MBq 89Zr-cetuximab. PET/CT images were acquired either 3 and 6 or 4 and 7 days post-injection. 89Zr-cetuximab uptake was quantified using standardized uptake value (SUV) and tumor-to-background ratio (TBR), and correlated to EGFR immunohistochemistry. TBR was compared between scan days to determine optimal timing.Uptake of 89Zr-cetuximab varied between patients (day 6-7: SUVpeak range 2.5-6.2). TBR increased significantly (49±28%, p < 0.01) between first (1.1±0.3) and second scan (1.7±0.6). Between groups with a low and high EGFR expression a significant difference in SUVmean (2.1 versus 3.0) and SUVpeak (3.2 versus 4.7) was found, however, not in TBR. Data is available at www.cancerdata.org (DOI: 10.17195/candat.2016.11.1).In conclusion, 89Zr-cetuximab PET imaging shows large inter-patient variety in LAHNSCC and provides additional information over FDG-PET and EGFR expression. Validation of the predictive value is recommended with scans acquired 6-7 days post-injection.

Quantitative assessment of Zirconium-89 labeled cetuximab using PET/CT imaging in patients with advanced head and neck cancer: a theragnostic approach

PubMed Central

van Elmpt, Wouter; Winnepenninckx, Véronique J.L.; Heukelom, Jolien; Tesselaar, Margot E.T.; Vogel, Wouter V.; Hoeben, Ann; Zegers, Catharina M.L.; Vugts, Daniëlle J.; van Dongen, Guus A.M.S.; Bartelink, Harry; Mottaghy, Felix M.; Hoebers, Frank; Lambin, Philippe

2017-01-01

Biomarkers predicting treatment response to the monoclonal antibody cetuximab in locally advanced head and neck squamous cell carcinomas (LAHNSCC) are lacking. We hypothesize that tumor accessibility is an important factor in treatment success of the EGFR targeting drug. We quantified uptake of cetuximab labeled with Zirconium-89 (89Zr) using PET/CT imaging. Seventeen patients with stage III-IV LAHNSCC received a loading dose unlabeled cetuximab, followed by 10 mg 54.5±9.6 MBq 89Zr-cetuximab. PET/CT images were acquired either 3 and 6 or 4 and 7 days post-injection. 89Zr-cetuximab uptake was quantified using standardized uptake value (SUV) and tumor-to-background ratio (TBR), and correlated to EGFR immunohistochemistry. TBR was compared between scan days to determine optimal timing. Uptake of 89Zr-cetuximab varied between patients (day 6-7: SUVpeak range 2.5-6.2). TBR increased significantly (49±28%, p < 0.01) between first (1.1±0.3) and second scan (1.7±0.6). Between groups with a low and high EGFR expression a significant difference in SUVmean (2.1 versus 3.0) and SUVpeak (3.2 versus 4.7) was found, however, not in TBR. Data is available at www.cancerdata.org (DOI: 10.17195/candat.2016.11.1). In conclusion, 89Zr-cetuximab PET imaging shows large inter-patient variety in LAHNSCC and provides additional information over FDG-PET and EGFR expression. Validation of the predictive value is recommended with scans acquired 6-7 days post-injection. PMID:27965472

Evaluating [11C]PBR28 PET for Monitoring Gut and Brain Inflammation in a Rat Model of Chemically Induced Colitis.

PubMed

Kurtys, E; Doorduin, J; Eisel, U L M; Dierckx, R A J O; de Vries, E F J

2017-02-01

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon that affects an increasing number of patients. High comorbidity is observed between UC and other diseases in which inflammation may be involved, including brain diseases such as cognitive impairment, mental disorders, anxiety, and depression. To investigate the increased occurrence of these brain diseases in patients with UC, non-invasive methods for monitoring peripheral and central inflammation could be applied. Therefore, the goal of this study is to assess the feasibility of monitoring gut and brain inflammation in a rat model of chemically induced colitis by positron emission tomography (PET) with [ 11 C]PBR28, a tracer targeting the translocator protein (TSPO), which is upregulated when microglia and macrophages are activated. Colitis was induced in rats by intra-rectal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Rats with colitis and healthy control animals were subjected to [ 11 C]PBR28 PET of the abdomen followed by ex vivo biodistribution in order to assess whether inflammation in the gut could be detected. Another group of rats with colitis underwent repetitive [ 11 C]PBR28 PET imaging of the brain to investigate the development of neuroinflammation. Eleven days after TNBS injection, ex vivo biodistribution studies demonstrated increased [ 11 C]PBR28 uptake in the inflamed cecum and colon of rats with colitis as compared to healthy controls, whereas PET imaging did not show any difference between groups at any time. Similarly, repetitive PET imaging of the brain did not reveal any neuroinflammation induced by the TNBS administration in the colon. In contrast, significantly increased [ 11 C]PBR28 uptake in cerebellum could be detected in ex vivo biodistribution studies on day 11. Inflammation in both the gut and the brain of rats with chemically induced colitis was observed by ex vivo biodistribution. However, these effects could not be detected by [ 11 C]PBR28 PET imaging in our colitis model, which is likely due to spill-over effects and insufficient resolution of the PET camera.

Expression of Momordica charantia MAP30 and its antitumor effect on bladder cancer cells.

PubMed

Hlin, Hao; Zhi-Guo, Zhang; Cong-Hui, Han; Yan, Zhao; Qing, Liang; Bo, Jiang; Hou-Guang, He; Jun-Jie, Zhang; Pei-Ying, Zhang

2016-06-01

Momordica charantia (MC) is an edible medicinal plant that is known for its diversified biological functions. Momordica Antiviral Protein 30kD (MAP30) is a type I single chain ribosome-inactivating protein (RIP) isolated from the mature fruit and seeds of MC. Since MAP30 content in MC is limited, the study aim was to generate the recombinant MAP30 protein using prokaryotic expression system and determine its apoptotic/growth inhibitory effects on bladder cancer 5637 cells. MAP30 gene was amplified by PCR from MC genomic DNA and identified by sequencing. The target gene was inserted into pET-28a (+) vector and transformed into E. coli BL21 (DE3) cells. Positive clones were selected by PCR. Recombinant protein was efficiently expressed under induction with 1.0 mM Isopropylthio-β-D-galactoside (IPTG) at 30° C for 4 hours. Cytotoxicity studies were performed using MTT assay by treating 5637 bladder cancer cells with 100 µg/mL, 200 µg/mL, and 400 µg/mL concentrations of MAP30 for 24 hours and 48 hours, respectively. Flow cytometry was used to measure the apoptosis of MAP30-treatedcells in time course experiments. Full-length MAP30 gene was successfully expressed in Escherichia coli (E. coli) BL21 strain and MAP30 recombinant protein inhibited the growth of bladder cancer 5637 cells at 200 µg/mL and 400 µg/mL concentrations by inducing apoptosis of target cells in a dose- and time-dependent manner. It was, therefore, concluded that the MAP30 recombinant protein displayed potent antitumor activity in vitro.

Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.

PubMed

Dormiani, Kianoush; Mir Mohammad Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Forouzanfar, Mahboobeh; Baharvand, Hossein; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2017-01-01

Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

Utilization of a hybrid finite-element based registration method to quantify heterogeneous tumor response for adaptive treatment for lung cancer patients

NASA Astrophysics Data System (ADS)

Sharifi, Hoda; Zhang, Hong; Bagher-Ebadian, Hassan; Lu, Wei; Ajlouni, Munther I.; Jin, Jian-Yue; (Spring Kong, Feng-Ming; Chetty, Indrin J.; Zhong, Hualiang

2018-03-01

Tumor response to radiation treatment (RT) can be evaluated from changes in metabolic activity between two positron emission tomography (PET) images. Activity changes at individual voxels in pre-treatment PET images (PET1), however, cannot be derived until their associated PET-CT (CT1) images are appropriately registered to during-treatment PET-CT (CT2) images. This study aimed to investigate the feasibility of using deformable image registration (DIR) techniques to quantify radiation-induced metabolic changes on PET images. Five patients with non-small-cell lung cancer (NSCLC) treated with adaptive radiotherapy were considered. PET-CTs were acquired two weeks before RT and 18 fractions after the start of RT. DIR was performed from CT1 to CT2 using B-Spline and diffeomorphic Demons algorithms. The resultant displacements in the tumor region were then corrected using a hybrid finite element method (FEM). Bitmap masks generated from gross tumor volumes (GTVs) in PET1 were deformed using the four different displacement vector fields (DVFs). The conservation of total lesion glycolysis (TLG) in GTVs was used as a criterion to evaluate the quality of these registrations. The deformed masks were united to form a large mask which was then partitioned into multiple layers from center to border. The averages of SUV changes over all the layers were 1.0  ±  1.3, 1.0  ±  1.2, 0.8  ±  1.3, 1.1  ±  1.5 for the B-Spline, B-Spline  +  FEM, Demons and Demons  +  FEM algorithms, respectively. TLG changes before and after mapping using B-Spline, Demons, hybrid-B-Spline, and hybrid-Demons registrations were 20.2%, 28.3%, 8.7%, and 2.2% on average, respectively. Compared to image intensity-based DIR algorithms, the hybrid FEM modeling technique is better in preserving TLG and could be useful for evaluation of tumor response for patients with regressing tumors.

[Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

PubMed

Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

2003-02-01

Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

[Pet ownership and health status of pets from immunocompromised children, with emphasis in zoonotic diseases].

PubMed

Abarca V, Katia; López Del P, Javier; Peña D, Anamaría; López G, J Carlos

2011-06-01

To characterize pet ownership and pet health status in families of immunocompromised (IS) children, with emphasis in zoonotic diseases. Families of IS children from two hospitals in Santiago, Chile, were interviewed and their pets were evaluated by veterinary examination, coproparasitologic and skin dermatophytes test. In specific cases, other laboratory tests were performed in IS children or their relatives. 47 out of 70 contacted families had pets, 42 participated in the study. Several risk factors for IS children were observed, as having a turtle as a pet and to clean cat or turtle faeces. Lack of adequate veterinary control, immunizations and deparasitation of pets were observed. Some animals showed zoonotic diseases or agents, as Brucella canis, Cryptosporidium sp, Giardia intestinalis, Toxocara canis and scabies. 44% of dogs had ticks and 37% had fleas, both potential vectors of infections. Our results suggest that policies to provide safer pet contact in IS children are needed.

Frequency of neurotic symptoms shortly after the death of a pet.

PubMed

Kimura, Yuya; Kawabata, Hidenobu; Maezawa, Masaji

2014-04-01

Some individuals manifest psychosomatic symptoms after the death of their pets. A survey was conducted at four public and commercial animal cremation service centers in Japan. In each center, a questionnaire was distributed to 100 individuals (400 in total). The questionnaire consisted of the 28-item version of the General Health Questionnaire (GHQ28), the social readjustment rating scale (SRRS) and a series of questions regarding demographic information and the circumstances of their pet's death. In total, 82 returned questionnaires were available for analysis. GHQ28 proved the existence of neurotic symptoms in 46 responses (56.1%; 95% confidence interval: 44.7%-67.0%). Analysis of the responses using the GHQ28 subscales with a Likert scoring system demonstrated more somatic dysfunction in females (GHQ-A: P=0.04). Furthermore, significant correlations were identified among the following factors: owner's age (GHQ-A: ρ=-0.60, P=0.01; GHQ-B: ρ=-0.29, P=0.01; GHQ-C: ρ=-0.32, P<0.01; GHQ-D: ρ=-0.42, P<0.01), SRRS score (GHQ-A: ρ=0.32, P<0.01; GHQ-B: ρ=0.25, P=0.02; GHQ-D: ρ=0.30, P=0.01) and animal's age (GHQ-D: ρ=-0.26, P=0.02). The death of indoor pets caused deeper depression (GHQ-D: P=0.01) than that of outdoor or visiting pets. The results revealed neurotic symptoms in almost half of the pet owners shortly after their pet's death.

Silk-based anisotropical 3D biotextiles for bone regeneration.

PubMed

Ribeiro, Viviana P; Silva-Correia, Joana; Nascimento, Ana I; da Silva Morais, Alain; Marques, Alexandra P; Ribeiro, Ana S; Silva, Carla J; Bonifácio, Graça; Sousa, Rui A; Oliveira, Joaquim M; Oliveira, Ana L; Reis, Rui L

2017-04-01

Bone loss in the craniofacial complex can been treated using several conventional therapeutic strategies that face many obstacles and limitations. In this work, novel three-dimensional (3D) biotextile architectures were developed as a possible strategy for flat bone regeneration applications. As a fully automated processing route, this strategy as potential to be easily industrialized. Silk fibroin (SF) yarns were processed into weft-knitted fabrics spaced by a monofilament of polyethylene terephthalate (PET). A comparative study with a similar 3D structure made entirely of PET was established. Highly porous scaffolds with homogeneous pore distribution were observed using micro-computed tomography analysis. The wet state dynamic mechanical analysis revealed a storage modulus In the frequency range tested, the storage modulus values obtained for SF-PET scaffolds were higher than for the PET scaffolds. Human adipose-derived stem cells (hASCs) cultured on the SF-PET spacer structures showed the typical pattern for ALP activity under osteogenic culture conditions. Osteogenic differentiation of hASCs on SF-PET and PET constructs was also observed by extracellular matrix mineralization and expression of osteogenic-related markers (osteocalcin, osteopontin and collagen type I) after 28 days of osteogenic culture, in comparison to the control basal medium. The quantification of convergent macroscopic blood vessels toward the scaffolds by a chick chorioallantoic membrane assay, showed higher angiogenic response induced by the SF-PET textile scaffolds than PET structures and gelatin sponge controls. Subcutaneous implantation in CD-1 mice revealed tissue ingrowth's accompanied by blood vessels infiltration in both spacer constructs. The structural adaptability of textile structures combined to the structural similarities of the 3D knitted spacer fabrics to craniofacial bone tissue and achieved biological performance, make these scaffolds a possible solution for tissue engineering approaches in this area. Copyright © 2017 Elsevier Ltd. All rights reserved.

The role of ¹⁸F-FDG PET/CT for the diagnosis of infections in patients with hematological malignancies and persistent febrile neutropenia.

PubMed

Gafter-Gvili, Anat; Paul, Mical; Bernstine, Hanna; Vidal, Liat; Ram, Ron; Raanani, Pia; Yeshurun, Moshe; Tadmor, Boaz; Leibovici, Leonard; Shpilberg, Ofer; Groshar, David

2013-09-01

We assessed the performance of PET/CT for diagnosis and management of infections in high-risk hematological cancer patients with persistent febrile neutropenia in a prospective study. (18)F-FDG PET/CT with contrast-enhanced CT was performed on day 5-7 of persistent fever. Between 2008 and 2011, 91 PET/CT examinations were performed for different episodes in 79 patients, resulting in 117 diagnoses. The sensitivity of the PET/CT was 79.8% (71/89) compared to 51.7% (46/89) with chest/sinus CT alone. Specificities were 32.14% (9/28) vs. 42.85% (12/28), respectively. PET/CT resulted in a change from the pre-test diagnosis in 63/91 (69%) of episodes and in modification of patients' management in 46/91 (55%). PET/CT was beneficial in diagnosing abdominal infections. PET/CT has a potential role in the diagnostic evaluation of patients with persistent febrile neutropenia. Copyright © 2013 Elsevier Ltd. All rights reserved.

PirAB protein from Xenorhabdus nematophila HB310 exhibits a binary toxin with insecticidal activity and cytotoxicity in Galleria mellonella.

PubMed

Yang, Qing; Zhang, Jie; Li, Tianhui; Liu, Shen; Song, Ping; Nangong, Ziyan; Wang, Qinying

2017-09-01

PirAB (Photorhabdus insect-related proteins, PirAB) toxin was initially found in the Photorhabdus luminescens TT01 strain and has been shown to be a binary toxin with high insecticidal activity. Based on GenBank data, this gene was also found in the Xenorhabdus nematophila genome sequence. The predicted amino acid sequence of pirA and pirB in the genome of X. nematophila showed 51% and 50% identity with those gene sequences from P. luminescens. The purpose of this experiment is to identify the relevant information for this toxin gene in X. nematophila. The pirA, pirB and pirAB genes of X. nematophila HB310 were cloned and expressed in Escherichia coli BL21 (DE3) using the pET-28a vector. A PirAB-fusion protein (PirAB-F) was constructed by linking the pirA and pirB genes with the flexible linker (Gly) 4 DNA encoding sequence and then efficiently expressed in E. coli. The hemocoel and oral insecticidal activities of the recombinant proteins were analyzed against the larvae of Galleria mellonella. The results show that PirA/B alone, PirA/B mixture, co-expressed PirAB protein, and PirAB-F all had no oral insecticidal activity against the second-instar larvae of G. mellonella. Only PirA/B mixture and co-expressed PirAB protein had hemocoel insecticidal activity against G. mellonella fifth-instar larvae, with an LD 50 of 2.718μg/larva or 1.566μg/larva, respectively. Therefore, we confirmed that PirAB protein of X. nematophila HB310 is a binary insecticidal toxin. The successful expression and purification of PirAB laid a foundation for further studies on the function, insecticidal mechanism and expression regulation of the binary toxin. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-Tmore » Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.« less

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PubMed

Blixt, Maria K E; Hallböök, Finn

2016-01-01

Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Cloning and Characterization of Cold-Adapted α-Amylase from Antarctic Arthrobacter agilis.

PubMed

Kim, Su-Mi; Park, Hyun; Choi, Jong-Il

2017-03-01

In this study, the gene encoding an α-amylase from a psychrophilic Arthrobacter agilis PAMC 27388 strain was cloned into a pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3). The recombinant α-amylase with a molecular mass of about 80 kDa was purified by using Ni 2+ -NTA affinity chromatography. This recombinant α-amylase exhibited optimal activity at pH 3.0 and 30 °C and was highly stable at varying temperatures (30-60 °C) and within the pH range of 4.0-8.0. Furthermore, α-amylase activity was enhanced in the presence of FeCl 3 (1 mM) and β-mercaptoethanol (5 mM), while CoCl 2 (1 mM), ammonium persulfate (5 mM), SDS (10 %), Triton X-100 (10 %), and urea (1 %) inhibited the enzymatic activity. Importantly, the presence of Ca 2+ ions and phenylmethylsulfonyl fluoride (PMSF) did not affect enzymatic activity. Thin layer chromatography (TLC) analysis showed that recombinant A. agilis α-amylase hydrolyzed starch, maltotetraose, and maltotriose, producing maltose as the major end product. These results make recombinant A. agilis α-amylase an attractive potential candidate for industrial applications in the textile, paper, detergent, and pharmaceutical industries.

Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01.

PubMed

Chae, J P; Valeriano, V D; Kim, G-B; Kang, D-K

2013-01-01

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l(-1) isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel-nitrilotriacetic acid (Ni(2+) -NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety. BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes. © 2012 The Society for Applied Microbiology.

Immunodetection of Canine Parvovirus (CPV) in clinical samples by polyclonal antisera against CPV-VP2 protein expressed in Esherichia coli as an antigen.

PubMed

Park, Jae Sung; Choi, Bong Kum; Vijayachandran, Lakshmi Sumitra; Ayyappan, Vasudevan; Chong, Chom-Kyu; Lee, Ki-Sung; Kim, Sei Chang; Choi, Chang Won

2007-12-01

The entire virion protein 2 (VP2) gene of Canine Parvovirus (CPV) was amplified by polymerase chain reaction (PCR) and engineered to be expressed by a bacterial expression vector pET-28a, under the control of the IPTG-inducible T7lac promoter. SDS-PAGE gel revealed that VP2 expressed as a 67kDa, and found mainly in the pellet of the bacterial lysates, suggesting that cytoplasmic expression is not preferred. The recombinant protein VP2 fused with His-tag was purified from Esherichia coli using Ni-NTA resin under denaturing conditions. SDS-PAGE analysis also showed the high expression of several lower molecular weight (LMW) bands. Western blot analysis showed that polyclonal antisera produced by rabbit against E. coli-VP2 protein reacted specifically with the purified VP2 protein as well as two other LMW bands. Some of the resulting LMW products failed to keep their antigenic site in the N-terminal region of the VP2. The degradation of recombinant VP2 protein in E. coli could be due to the action of host proteases. The immunodetection ability of the polyclonal antisera was compared with that of a commercial monoclonal antibody to test numerous clinical specimens by immuno-dot blot assays. There were distinctive differences in the degree of immunodetection ability of polyclonal antisera and monoclonal antibody to react with CPV antigens. The reaction time of polyclonal antisera was much faster in visual color appearance than that of monoclonal antibody during NBT/BCIP staining. The result from diagnostic PCR assay confirmed the presence of CPV in 44 out of 46 specimens collected, consistent with polyclonal antisera-positive result. Therefore, the polyclonal antisera can be used for CPV detection in the faeces of diarrhoeic dogs, which was found to be more rapid, sensitive, broad but less specific than the monoclonal antibody.

Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

PubMed Central

Matroudi, S.; Zamani, M.R.; Motallebi, M.

2008-01-01

In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli. PMID:24031242

Pose-variant facial expression recognition using an embedded image system

NASA Astrophysics Data System (ADS)

Song, Kai-Tai; Han, Meng-Ju; Chang, Shuo-Hung

2008-12-01

In recent years, one of the most attractive research areas in human-robot interaction is automated facial expression recognition. Through recognizing the facial expression, a pet robot can interact with human in a more natural manner. In this study, we focus on the facial pose-variant problem. A novel method is proposed in this paper to recognize pose-variant facial expressions. After locating the face position in an image frame, the active appearance model (AAM) is applied to track facial features. Fourteen feature points are extracted to represent the variation of facial expressions. The distance between feature points are defined as the feature values. These feature values are sent to a support vector machine (SVM) for facial expression determination. The pose-variant facial expression is classified into happiness, neutral, sadness, surprise or anger. Furthermore, in order to evaluate the performance for practical applications, this study also built a low resolution database (160x120 pixels) using a CMOS image sensor. Experimental results show that the recognition rate is 84% with the self-built database.

One Health in Practice: A Pilot Project for Integrated Care of Zoonotic Infections in Immunocompromised Children and Their Pets in Chile.

PubMed

Peña, A; Abarca, K; Weitzel, T; Gallegos, J; Cerda, J; García, P; López, J

2016-08-01

Although pets provide physiological and psychological benefits to their owners, they are a potential source of zoonotic infections, especially for vulnerable individuals such as immunocompromised patients. During 1 year, we therefore performed a pilot project, which included 32 immunocompromised Chilean children and their family pets (35 dogs and 9 cats) with the aim of detecting, treating and preventing zoonotic infections. Children were examined by Infectious Diseases paediatricians and demographical and clinical information related to zoonotic infections were recorded. Pets were examined and sampled by veterinarians, who also administered missing routine vaccines and anti-parasitics. During family visits, all members were informed and educated about zoonoses and a satisfaction survey was performed. Visits also included vector control and indoor residual spraying with pyrethroids. Children were re-examined and re-tested according to the findings of their pets, and all detected zoonotic infections were treated both in children and pets. Physical examination revealed abnormalities in 18 dogs (51.4%) and three cats (33.3%). Twenty-eight (63.6%) of the pets were diagnosed with a zoonotic pathogen, and seven (15.9%) with a facultative pathogen. Most zoonotic agents were isolated from the pet's external ear and intestine. Bacteria with the highest pathogenic potential were Campylobacter jejuni and Brucella canis. In two children and their respective pets, the same zoonotic diseases were diagnosed (toxocariasis and giardiasis). Arthropods serving as potential vectors of zoonotic infections were found in 49% of dogs and 44% of cats. The pilot project was positively evaluated by the participating families. Our pilot project confirmed that pets are reservoir for various zoonotic agents in Chile and that the implementation of an integrated multidisciplinary programme was a valuable tool to prevent, diagnose and treat such zoonotic infections in vulnerable patients such as immunocompromised children. © 2015 Blackwell Verlag GmbH.

Patch-based image reconstruction for PET using prior-image derived dictionaries

NASA Astrophysics Data System (ADS)

Tahaei, Marzieh S.; Reader, Andrew J.

2016-09-01

In PET image reconstruction, regularization is often needed to reduce the noise in the resulting images. Patch-based image processing techniques have recently been successfully used for regularization in medical image reconstruction through a penalized likelihood framework. Re-parameterization within reconstruction is another powerful regularization technique in which the object in the scanner is re-parameterized using coefficients for spatially-extensive basis vectors. In this work, a method for extracting patch-based basis vectors from the subject’s MR image is proposed. The coefficients for these basis vectors are then estimated using the conventional MLEM algorithm. Furthermore, using the alternating direction method of multipliers, an algorithm for optimizing the Poisson log-likelihood while imposing sparsity on the parameters is also proposed. This novel method is then utilized to find sparse coefficients for the patch-based basis vectors extracted from the MR image. The results indicate the superiority of the proposed methods to patch-based regularization using the penalized likelihood framework.

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli▿

PubMed Central

Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q. S.; Qiao, Chuanling

2007-01-01

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His6-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the Km and kcat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min−1, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling. PMID:17575004

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

PubMed Central

Latifi, Ali Mohammad; Khajeh, Khosro; Farnoosh, Gholamreza; Hassanpour, Kazem; Khodi, Samaneh

2015-01-01

Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process. Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b). Materials and Methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd. Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band. Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step. PMID:26870308

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

NASA Technical Reports Server (NTRS)

Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

1999-01-01

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

Phenotyping of tumor biology in patients by multimodality multiparametric imaging: relationship of microcirculation, alphavbeta3 expression, and glucose metabolism.

PubMed

Metz, Stephan; Ganter, Carl; Lorenzen, Sylvie; van Marwick, Sandra; Herrmann, Ken; Lordick, Florian; Nekolla, Stephan G; Rummeny, Ernst J; Wester, Hans-Jürgen; Brix, Gunnar; Schwaiger, Markus; Beer, Ambros J

2010-11-01

Both dynamic contrast-enhanced (DCE) MRI and PET provide quantitative information on tumor biology in living organisms. However, imaging biomarkers often neglect tissue heterogeneity by focusing on distributional summary statistics. We analyzed the spatial relationship of α(v)β(3) expression, glucose metabolism, and perfusion by PET and DCE MRI, focusing on tumor heterogeneity. Thirteen patients with primary or metastasized cancer (non-small cell lung cancer, n = 9; others, n = 4) were examined with DCE MRI and with PET using (18)F-galacto-RGD and (18)F-FDG. Twenty-three different regions of interest were defined by cluster analysis based on the heterogeneity of tracer uptake. In these regions, the initial area under the gadopentetate dimeglumine concentration-time curve (IAUGC), as well as the regional blood volume (rBV) and regional blood flow (rBF), were estimated from DCE MRI and correlated with standardized uptake values from PET. Regions with simultaneously high uptake of (18)F-galacto-RGD and (18)F-FDG showed higher functional MRI data (IAUGC, 0.35 ± 0.04 mM·s; rBF, 70.2 ± 12.7 mL/min/100 g; rBV, 23.3 ± 2.7 mL/100 g) than did areas with low uptake of both tracers (IAUGC, 0.15 ± 0.04 mM·s [P < 0.01]; rBF, 28.3 ± 10.8 mL/min/100 g; rBV, 9.9 ± 1.9 mL/100 g [P < 0.01]). There was a weak to moderate correlation between the functional MRI parameters and (18)F-galacto-RGD (r = 0.30-0.62) and also (18)F-FDG (r = 0.44-0.52); these correlations were significant (P < 0.05), except for (18)F-galacto-RGD versus rBF (P = 0.17). These data show that multiparametric assessment of tumor heterogeneity is feasible by combining PET and MRI. Perfusion is highest in tumor areas with simultaneously high α(v)β(3) expression and high glucose metabolism and restricted in areas with both low α(v)β(3) expression and low glucose metabolism. The current limitations resulting from imaging with separate scanners might be overcome by future hybrid PET/MRI scanners.

Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

PubMed

Kanao, Megumi; Kanda, Hirotsugu; Huang, Wan; Liu, Shue; Yi, Hyun; Candiotti, Keith A; Lubarsky, David A; Levitt, Roy C; Hao, Shuanglin

2015-06-01

Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection-related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of γ-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBPβ was tested using immunohistochemistry. In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time-effect curves in the HSV vector expressing GAD67 was increased compared with that in the control vectors (P = 0.0005). Intrathecal GABA-A/B agonists elevated mechanical threshold in the pain model. The HSV vectors expressing GAD67 reversed the lowered GABA immunoreactivity in the spinal dorsal horn in the neuropathic rats. HSV vectors expressing GAD67 in the neuropathic rats reversed the increased signals of mitochondrial superoxide in the spinal dorsal horn. The vectors expressing GAD67 reversed the upregulated immunoreactivity expression of pCREB and pC/EBPβ in the spinal dorsal horn in rats exhibiting NP. Based on our results, we suggest that GAD67 mediated by HSV vectors acting through the suppression of mitochondrial reactive oxygen species and transcriptional factors in the spinal cord decreases pain in the HIV-related neuropathic pain model, providing preclinical evidence for gene therapy applications in patients with HIV-related pain states.

Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines.

PubMed

Agheli, Reza; Emkanian, Bijan; Halabian, Raheleh; Fallah Mehrabadi, Jalil; Imani Fooladi, Abbas Ali

2017-02-01

About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. For the construction of pET-21a/ entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/ entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A to activate T cells in vitro. Lymphocyte cells derived from lymph node BALB/c mice were exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin (1.10, 102,103, and 104 ng/µL) in order to evaluate the magnitude of proliferation, activation, and apoptosis of lymphocyte cells based on MTT and apoptosis assays, respectively. Our investigation showed that the function of cloned staphylococcal enterotoxin A was same as standard staphylococcal enterotoxin A, and the optimal concentration for the activation of lymphocyte cells and induction of apoptosis was 100 ng/µL and 1000 ng/µL ( P < .05), respectively. Quantification of cytokines clearly showed that lymphocyte cells exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A significantly secreted higher interferon γ and tumor necrosis factor α compared to control. According to our results, the biological activity of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A is identical; therefore, these procedures may be approved as an efficient method to express and purify this protein in a large scale.

Prokaryotic Expression of Hepatitis C Virus-NS3 Protein and Preparation of a Monoclonal Antibody.

PubMed

Xi, Yun; Zhang, Yuming; Fang, Jianmin; Whittaker, Kelly; Luo, Shuhong; Huang, Ruo-Pan

2017-12-01

Hepatitis C virus (HCV) is a significant health threat that has been extensively investigated worldwide. Improving the sensitivity and specificity of laboratory tests for screening and early diagnosis of HCV in a relevant population is an effective measure to control the spread of HCV. To build a more reliable diagnostic method for HCV, we expressed gene fragments of HCV-NS3 linked to a carrier, pET28a, and then transformed this vector into Escherichia. coli. The produced recombinant NS3 protein with a molecular weight of 38 kDa, which was purified through Ni-chelating affinity chromatography, was used to immunize BALB/C mice, which generated a serum antibody titer of 1:160,000 against the immunogen. Three positive monoclonal isolates (2A5, 2A6, and 5B12) were screened and established. Western blot and enzyme-linked immunosorbent assay (ELISA) results of these monoclonal cells show that each could specifically recognize the recombinant protein. Antibodies 2A5 and 2A6 were developed into an ELISA sandwich antibody pair for the recombinant protein. The detection sensitivity of our developed ELISA was 1.6 ng/mL, with a linear range of 2.5-80 ng/mL (R 2  = 0.998). Serum NS3 ELISA results show that the average value in the healthy group, liver disease group, and hepatitis C group was 3.71, 7.28, and 13.11 ng/mL, respectively. The positive rates of HCV-NS3 protein in the liver disease group and hepatitis C group was 17.2% and 41.7%, respectively. Detection of HCV-NS3 antigen can be used as an auxiliary test for anti-HCV antibody detection, thus reducing leakage detection and providing a reliable basis for clinical practice.

Sequential PET estimation of cerebral oxygen metabolism with spontaneous respiration of 15O-gas in mice with bilateral common carotid artery stenosis.

PubMed

Temma, Takashi; Yamazaki, Makoto; Miyanohara, Jun; Shirakawa, Hisashi; Kondo, Naoya; Koshino, Kazuhiro; Kaneko, Shuji; Iida, Hidehiro

2017-10-01

Positron emission tomography with 15 O-labeled gases ( 15 O-PET) is important for in vivo measurement of cerebral oxygen metabolism both in clinical and basic settings. However, there are currently no reports concerning 15 O-PET in mice. Here, we developed an 15 O-PET method applicable to mice with spontaneous respiration of 15 O-gas without a tracheotomy catheter. Sequential 15 O-PET was also performed in a mouse model of chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) induced by placement of microcoils. 15 O-gas with isoflurane was supplied to the nose of mouse with evacuation of excess 15 O-gas surrounding the body. 15 O-PET was performed on days 3, 7, 14, 21, and 28 after surgery. Cerebral blood flow (CBF), cerebral blood volume, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO 2 ) were calculated in whole brains. A significant decrease in CBF and compensatory increase in OEF in the BCAS group produced CMRO 2 values comparable to that of the sham group at three days post-operation. Although CBF and OEF in the BCAS group gradually recovered over the first 28 days, the CMRO 2 showed a gradual decrease to 68% of sham values at 28 days post-operation. In conclusion, we successfully developed a noninvasive 15 O-PET method for mice.

miR-28 regulates the germinal center reaction and blocks tumor growth in preclinical models of non-Hodgkin lymphoma

PubMed Central

Bartolomé-Izquierdo, Nahikari; Mur, Sonia M.

2017-01-01

Non-Hodgkin lymphoma comprises a variety of neoplasms, many of which arise from germinal center (GC)-experienced B cells. microRNA-28 (miR-28) is a GC-specific miRNA whose expression is lost in numerous mature B-cell neoplasms. Here we show that miR-28 regulates the GC reaction in primary B cells by impairing class switch recombination and memory B and plasma cell differentiation. Deep quantitative proteomics combined with transcriptome analysis identified miR-28 targets involved in cell-cycle and B-cell receptor signaling. Accordingly, we found that miR-28 expression diminished proliferation in primary and lymphoma cells in vitro. Importantly, miR-28 reexpression in human Burkitt (BL) and diffuse large B-cell lymphoma (DLBCL) xenografts blocked tumor growth, both when delivered in viral vectors or as synthetic, clinically amenable, molecules. Further, the antitumoral effect of miR-28 is conserved in a primary murine in vivo model of BL. Thus, miR-28 replacement is uncovered as a novel therapeutic strategy for DLBCL and BL treatment. PMID:28188132

Effect of transient receptor potential vanilloid 6 gene silencing on the expression of calcium transport genes in chicken osteoblasts.

PubMed

Zhang, Jie; Deng, Yifeng; Ma, Huijie; Hou, Jiafa; Zhou, ZhenLei

2015-03-01

Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of transient receptor potential vanilloid 6 in chickens. In this study, we evaluated the effects of transient receptor potential vanilloid 6 gene interference on the expression of calbindin-D28K, Na+/Ca2+ exchangers, and plasma membrane Ca2+ ATPase 1b to investigate the mechanism underlying the regulation of transient receptor potential vanilloid 6. Three hairpin siRNA expression vectors targeting transient receptor potential vanilloid 6 (pSIREN- transient receptor potential vanilloid 6) and a negative control (pSIREN-control) were constructed and transfected into chicken osteoblasts. The mRNA and protein expression levels were evaluated by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. The mRNA expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 45.7% (P<0.01) and 27.9% (P<0.01), respectively, 48 h after transfection with one of the three constructs (pSIREN- transient receptor potential vanilloid 6-3) compared with the level obtained in the untreated group. There was no significant difference in the mRNA expression levels of Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPase 1b. The protein expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 40.2% (P<0.01) and 29.8% (P<0.01), respectively, 48 h after transfection with pSIREN-transient receptor potential vanilloid 6-3 compared with the level obtained in the untreated group. In conclusion, the vector-based transient receptor potential vanilloid 6-shRNA can efficiently suppress the mRNA and protein expression of transient receptor potential vanilloid 6 in chicken osteoblasts, and transient receptor potential vanilloid 6 regulates the expression of calbindin-D28K during Ca2+ transport. © 2015 Poultry Science Association Inc.

[Identification and detection of trag: a new infection-related gene expressed in vivo from isolates of Streptococcus suis].

PubMed

Zhu, Haodan; Gu, Hongwei; Lu, Chengping

2008-12-01

The trag (transfer gene G) was one of the novel infection-related factors identified by in vivo-induced antigen technology (IVIAT) from Streptococcus suis type 2 expression libraries with swine convalesecent sera in our former research. We detected the distribution of trag in different Streptococcus suis isolates and identify the differential expression of the new infection-related factor between in vivo and in vitro condition. According to the sequence of trag of North American strain 89/1591, a pair of primers were designed to detect the distribution of trag in total 43 SS isolates. Another pair of primers were designed to amplify the ORF of trag of 5 SS representive strains (ZY05719, HA9801, 98012, SH040805, SH040917). Partial gene of trag was cloned and inserted into expression vector pET28a(+), and induced by IPTG to express recombinant TRAG. The recombinant protein was probed with swine convalescent sera and immune sera respectively. The trag was detected in the most of SS2 isolates (30/32), in SS9 isolates (4/6), and 1 isolate of SS7, while it was not found in SS2 European strain ATCC43765, avirulent strain SS2 T15, 1 isolates of SS1, 1 isolates of SS1/2 and 2 isolates of group C streptococcal strains from pigs. Comparisons between the sequences of TRAG of 5 isolates with that of SS isolates, showed a high homology (>97%) with North American strain 89/1589 and China strains 98HAH33, 05ZYH33. The immunoreactivity was only presented with convalescent sera. The trag was detected from virulent SS isolates but not from avirulent strain, which suggested that this gene may be related to the pathogenicity of SS. The special reactivity was only present with convalescent sera, and it indicated that TRAG might play a role during SS2 invasive course.

[A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

PubMed

Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

2017-01-01

Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

Adenovirus-Associated Virus Vector–Mediated Gene Transfer in Hemophilia B

PubMed Central

Nathwani, Amit C.; Tuddenham, Edward G.D.; Rangarajan, Savita; Rosales, Cecilia; McIntosh, Jenny; Linch, David C.; Chowdary, Pratima; Riddell, Anne; Pie, Arnulfo Jaquilmac; Harrington, Chris; O’Beirne, James; Smith, Keith; Pasi, John; Glader, Bertil; Rustagi, Pradip; Ng, Catherine Y.C.; Kay, Mark A.; Zhou, Junfang; Spence, Yunyu; Morton, Christopher L.; Allay, James; Coleman, John; Sleep, Susan; Cunningham, John M.; Srivastava, Deokumar; Basner-Tschakarjan, Etiena; Mingozzi, Federico; High, Katherine A.; Gray, John T.; Reiss, Ulrike M.; Nienhuis, Arthur W.; Davidoff, Andrew M.

2012-01-01

BACKGROUND Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS We infused a single dose of a serotype-8–pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid–specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.) PMID:22149959

In Vivo Demonstration of PSMA Expression in Adenocarcinoma Urinary Bladder Using 68Ga-PSMA 11 PET/CT.

PubMed

Roy, Shambo Guha; Parida, Girish Kumar; Tripathy, Sarthak; Singhal, Abhinav; Tripathi, Madhavi; Bal, Chandrasekhar

2017-07-01

In vitro and in vivo studies have demonstrated prostate-specific membrane antigen (PSMA) expression in various malignant and benign tumors. Based on the recent immunohistochemical study showing PSMA expression in adenocarcinoma of urinary bladder, we hypothesized that PSMA expression in adenocarcinoma of urinary bladder can be demonstrated in vivo using Ga-PSMA 11 PET/CT. We present a man with exstrophy bladder, presenting with adenocarcinoma urinary bladder referred for staging PET/CT. Both F-FDG and Ga-PSMA-11 PET/CT were done, which showed PSMA expression in the primary tumor as well as metastatic lymph nodes.

Mesenchymal stem cells are efficiently transduced with adenoviruses bearing type 35-derived fibers and the transduced cells with the IL-28A gene produces cytotoxicity to lung carcinoma cells co-cultured.

PubMed

Suzuki, Takeo; Kawamura, Kiyoko; Li, Quanhai; Okamoto, Shinya; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Yamaguchi, Naoto; Tagawa, Masatoshi

2014-09-25

Transduction of human mesenchymal stem cells (MSCs) with type 5 adenoviruses (Ad5) is limited in the efficacy because of the poor expression level of the coxsackie adenovirus receptor (CAR) molecules. We examined a possible improvement of Ad-mediated gene transfer in MSCs by substituting the fiber region of type 5 Ad with that of type 35 Ad. Expression levels of CAR and CD46 molecules, which are the major receptors for type 5 and type 35 Ad, respectively, were assayed with flow cytometry. We constructed vectors expressing the green fluorescent protein gene with Ad5 or modified Ad5 bearing the type 35 fiber region (AdF35), and examined the infectivity to MSCs with flow cytometry. We investigated anti-tumor effects of MSCs transduced with interleukin (IL)-28A gene on human lung carcinoma cells with a colorimetric assay. Expression of IL-28A receptors was tested with the polymerase chain reaction. A promoter activity of transcriptional regulatory regions in MSCs was determined with a luciferase assay and a tumor growth-promoting ability of MSCs was tested with co-injection of human tumor cells in nude mice. MSCs expressed CD46 but scarcely CAR molecules, and subsequently were transduced with AdF35 but not with Ad5. Growth of MSCs transduced with the IL-28A gene remained the same as that of untransduced cells since MSCs were negative for the IL-28A receptors. The IL-28A-transduced MSCs however suppressed growth of lung carcinoma cells co-cultured, whereas MSCs transduced with AdF35 expressing the β-galactosidase gene did not. A regulatory region of the cyclooygenase-2 gene possessed transcriptional activities greater than other tumor promoters but less than the cytomegalovirus promoter, and MSCs themselves did not support tumor growth in vivo. AdF35 is a suitable vector to transduce MSCs that are resistant to Ad5-mediated gene transfer. MSCs infected with AdF35 that activate an exogenous gene by the cytomegalovirus promoter can be a vehicle to deliver the gene product to targeted cells.

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1.

Expression, purification and epitope analysis of Pla a 2 allergen from Platanus acerifolia pollen.

PubMed

Wang, De-Wang; Ni, Wei-Wei; Zhou, Yan-Jun; Huang, Wen; Cao, Meng-Da; Meng, Ling; Wei, Ji-Fu

2018-01-01

Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ‑3.0 and ‑2.2. In total, eight B cell epitopes (15‑24, 60‑66, 78‑86, 109‑124, 232‑240, 260‑269, 298‑306 and 315‑322) and five T cell epitopes (62‑67, 86‑91, 125‑132, 217‑222 and 343‑350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen‑associated allergic reactions.

Bio-inorganic synthesis of ZnO powders using recombinant His-tagged ZnO binding peptide as a promoter.

PubMed

Song, Lei; Liu, Yingying; Zhang, Zhifang; Wang, Xi; Chen, Jinchun

2010-10-01

Inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs), are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. Recent advances in nanotechnology and molecular biology allow the engineering of these peptides with specific affinity to inorganics, often used as molecular linkers or assemblers, to facilitate materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here we report a novel biosynthetic ZnO binding protein and its application in promoting bio-inorganic materials synthesis. In brief, the gene encoding a ZnO binding peptide(ZBP) was genetically fused with His(6)-tag and GST-tag using E.coli expression vector pET-28a (+) and pGEX-4T-3. The recombinant protein GST-His-ZBP was expressed, purified with Ni-NTA system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Affinity adsorption test demonstrated that the fusion protein had a specific avidity for ZnO nanoparticles (NPs). Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)(2) sol. X-ray diffraction (XRD) analysis on the final products after calcining the precursor powders showed that hexagonal wurtzite ZnO crystals were obtained. Our work suggested a novel approach to the application about the organic-inorganic interactions.

PetIGA: A framework for high-performance isogeometric analysis

DOE PAGES

Dalcin, Lisandro; Collier, Nathaniel; Vignal, Philippe; ...

2016-05-25

We present PetIGA, a code framework to approximate the solution of partial differential equations using isogeometric analysis. PetIGA can be used to assemble matrices and vectors which come from a Galerkin weak form, discretized with Non-Uniform Rational B-spline basis functions. We base our framework on PETSc, a high-performance library for the scalable solution of partial differential equations, which simplifies the development of large-scale scientific codes, provides a rich environment for prototyping, and separates parallelism from algorithm choice. We describe the implementation of PetIGA, and exemplify its use by solving a model nonlinear problem. To illustrate the robustness and flexibility ofmore » PetIGA, we solve some challenging nonlinear partial differential equations that include problems in both solid and fluid mechanics. Lastly, we show strong scaling results on up to 4096 cores, which confirm the suitability of PetIGA for large scale simulations.« less

"De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

PubMed

Yefremova, Yelena; Al-Majdoub, Mahmoud; Opuni, Kwabena F M; Koy, Cornelia; Cui, Weidong; Yan, Yuetian; Gross, Michael L; Glocker, Michael O

2015-03-01

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

Characterization of Truncated Tumor-Associated NADH Oxidase (ttNOX)

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Malone, Christine C.; Burk, Melissa; Moore, Blake P.; Achari, Aniruddha; Curreri, Peter A. (Technical Monitor)

2002-01-01

Bacterial, plant and animal cells possess novel surface proteins that exhibit both NADH oxidation (NOX) or hydroquinone and protein disulfide-thiol interchange. These enzymatic activities alternate to yield oscillating patterns wjth period lengths of approximately 24 minutes. The catalytic period of NOX proteins are temperature compensated and gravity responsive. We report the cloning, expression and characterization of truncated tumor-associated NADH oxidase (ttNOX), in which the membrane spanning region has been deleted. The cDNA (originated from HeLa cells) was cloned into pET-34b and pET-14b (Novagen) vectors for E. coli expression. Optimized expression and purification protocols yielded greater than 300mg per liter of culture with greater than 95% purity. Circular dichroism data was collected from a 2.7mg/ml solution in a 0.1mm cuvette with variable scanning using an Olis RSM CD spectrophotometer. The ellipticity values were scanned from 190 to 260nm. The spectra recorded have characteristics for alpha proteins with band maxima at 216nm and a possible shoulder at 212nm at 12OC and 250 C. Protein crystal screens are in progress and, to date, only small crystals have been observed. The regular periodic oscillatory change in the ttNOX protein is indicative of a possible time-keeping functional role. A single protein possessing alternating catalytic activities, with a potential biological clock function, is unprecedented and structural determination is paramount to understanding this role.

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.

PubMed

Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi

2012-12-01

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

Construction of a complementary DNA library for Parelaphostrongylus tenuis and identification of a potentially sero-diagnostic recombinant antigen.

PubMed

Ogunremi, Oladele; Benjamin, Jane; MacDonald, Lily; Schimpf, Robert

2008-12-01

Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms, and ligation with the vector lambda-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned and expressed using the pET expression vector to produce a glutathione S transferase (GST)-His-S.Tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An enzyme-linked immunosorbent assay utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, 10 out of 10) that had been inoculated with 6 - 150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.

Site Specific Discrete PEGylation of 124I-Labeled mCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice

PubMed Central

Ding, Haiming; Carlton, Michelle M.; Povoski, Stephen P.; Milum, Keisha; Kumar, Krishan; Kothandaraman, Shankaran; Hinkle, George H.; Colcher, David; Brody, Rich; Davis, Paul D.; Pokora, Alex; Phelps, Mitchell; Martin, Edward W.; Tweedle, Michael F.

2014-01-01

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2–4 kDa discrete, branched PEGylation reagents on mCC49 Fab′ (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab′ (Fab′-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 (124I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab′ with Mal-dPEG-A (Fab′-A) reduced the binding affinity of the non PEGylated Fab′ by 30%; however, in vivo, Fab′-A significantly lengthened the blood retention vs Fab′-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab′-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A. PMID:24175669

PET image reconstruction using multi-parametric anato-functional priors

NASA Astrophysics Data System (ADS)

Mehranian, Abolfazl; Belzunce, Martin A.; Niccolini, Flavia; Politis, Marios; Prieto, Claudia; Turkheimer, Federico; Hammers, Alexander; Reader, Andrew J.

2017-08-01

In this study, we investigate the application of multi-parametric anato-functional (MR-PET) priors for the maximum a posteriori (MAP) reconstruction of brain PET data in order to address the limitations of the conventional anatomical priors in the presence of PET-MR mismatches. In addition to partial volume correction benefits, the suitability of these priors for reconstruction of low-count PET data is also introduced and demonstrated, comparing to standard maximum-likelihood (ML) reconstruction of high-count data. The conventional local Tikhonov and total variation (TV) priors and current state-of-the-art anatomical priors including the Kaipio, non-local Tikhonov prior with Bowsher and Gaussian similarity kernels are investigated and presented in a unified framework. The Gaussian kernels are calculated using both voxel- and patch-based feature vectors. To cope with PET and MR mismatches, the Bowsher and Gaussian priors are extended to multi-parametric priors. In addition, we propose a modified joint Burg entropy prior that by definition exploits all parametric information in the MAP reconstruction of PET data. The performance of the priors was extensively evaluated using 3D simulations and two clinical brain datasets of [18F]florbetaben and [18F]FDG radiotracers. For simulations, several anato-functional mismatches were intentionally introduced between the PET and MR images, and furthermore, for the FDG clinical dataset, two PET-unique active tumours were embedded in the PET data. Our simulation results showed that the joint Burg entropy prior far outperformed the conventional anatomical priors in terms of preserving PET unique lesions, while still reconstructing functional boundaries with corresponding MR boundaries. In addition, the multi-parametric extension of the Gaussian and Bowsher priors led to enhanced preservation of edge and PET unique features and also an improved bias-variance performance. In agreement with the simulation results, the clinical results also showed that the Gaussian prior with voxel-based feature vectors, the Bowsher and the joint Burg entropy priors were the best performing priors. However, for the FDG dataset with simulated tumours, the TV and proposed priors were capable of preserving the PET-unique tumours. Finally, an important outcome was the demonstration that the MAP reconstruction of a low-count FDG PET dataset using the proposed joint entropy prior can lead to comparable image quality to a conventional ML reconstruction with up to 5 times more counts. In conclusion, multi-parametric anato-functional priors provide a solution to address the pitfalls of the conventional priors and are therefore likely to increase the diagnostic confidence in MR-guided PET image reconstructions.

Improved characterization of molecular phenotypes in breast lesions using 18F-FDG PET image homogeneity

NASA Astrophysics Data System (ADS)

Cao, Kunlin; Bhagalia, Roshni; Sood, Anup; Brogi, Edi; Mellinghoff, Ingo K.; Larson, Steven M.

2015-03-01

Positron emission tomography (PET) using uorodeoxyglucose (18F-FDG) is commonly used in the assessment of breast lesions by computing voxel-wise standardized uptake value (SUV) maps. Simple metrics derived from ensemble properties of SUVs within each identified breast lesion are routinely used for disease diagnosis. The maximum SUV within the lesion (SUVmax) is the most popular of these metrics. However these simple metrics are known to be error-prone and are susceptible to image noise. Finding reliable SUV map-based features that correlate to established molecular phenotypes of breast cancer (viz. estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression) will enable non-invasive disease management. This study investigated 36 SUV features based on first and second order statistics, local histograms and texture of segmented lesions to predict ER and PR expression in 51 breast cancer patients. True ER and PR expression was obtained via immunohistochemistry (IHC) of tissue samples from each lesion. A supervised learning, adaptive boosting-support vector machine (AdaBoost-SVM), framework was used to select a subset of features to classify breast lesions into distinct phenotypes. Performance of the trained multi-feature classifier was compared against the baseline single-feature SUVmax classifier using receiver operating characteristic (ROC) curves. Results show that texture features encoding local lesion homogeneity extracted from gray-level co-occurrence matrices are the strongest discriminator of lesion ER expression. In particular, classifiers including these features increased prediction accuracy from 0.75 (baseline) to 0.82 and the area under the ROC curve from 0.64 (baseline) to 0.75.

Identification and characterization of a chitin deacetylase from a metagenomic library of deep-sea sediments of the Arctic Ocean.

PubMed

Liu, Jinlin; Jia, Zhijuan; Li, Sha; Li, Yan; You, Qiang; Zhang, Chunyan; Zheng, Xiaotong; Xiong, Guomei; Zhao, Jin; Qi, Chao; Yang, Jihong

2016-09-15

The chemical and biological compositions of deep-sea sediments are interesting because of the underexplored diversity when it comes to bioprospecting. The special geographical location and climates make Arctic Ocean a unique ocean area containing an abundance of microbial resources. A metagenomic library was constructed based on the deep-sea sediments of Arctic Ocean. Part of insertion fragments of this library were sequenced. A chitin deacetylase gene, cdaYJ, was identified and characterized. A metagenomic library with 2750 clones was obtained and ten clones were sequenced. Results revealed several interesting genes, including a chitin deacetylase coding sequence, cdaYJ. The CdaYJ is homologous to some known chitin deacetylases and contains conserved chitin deacetylase active sites. CdaYJ protein exhibits a long N-terminal and a relative short C-terminal. Phylogenetic analysis revealed that CdaYJ showed highest homology to CDAs from Alphaproteobacteria. The cdaYJ gene was subcloned into the pET-28a vector and the recombinant CdaYJ (rCdaYJ) was expressed in Escherichia coli BL21 (DE3). rCdaYJ showed a molecular weight of 43kDa, and exhibited deacetylation activity by using p-nitroacetanilide as substrate. The optimal pH and temperature of rCdaYJ were tested as pH7.4 and 28°C, respectively. The construction of metagenomic library of the Arctic deep-sea sediments provides us an opportunity to look into the microbial communities and exploiting valuable gene resources. A chitin deacetylase CdaYJ was identified from the library. It showed highest deacetylation activity under slight alkaline and low temperature conditions. CdaYJ might be a candidate chitin deacetylase that possesses industrial and pharmaceutical potentials. Copyright © 2016 Elsevier B.V. All rights reserved.

(18)F-FDG PET-CT simulation for non-small-cell lung cancer: effect in patients already staged by PET-CT.

PubMed

Hanna, Gerard G; McAleese, Jonathan; Carson, Kathryn J; Stewart, David P; Cosgrove, Vivian P; Eakin, Ruth L; Zatari, Ashraf; Lynch, Tom; Jarritt, Peter H; Young, V A Linda; O'Sullivan, Joe M; Hounsell, Alan R

2010-05-01

Positron emission tomography (PET), in addition to computed tomography (CT), has an effect in target volume definition for radical radiotherapy (RT) for non-small-cell lung cancer (NSCLC). In previously PET-CT staged patients with NSCLC, we assessed the effect of using an additional planning PET-CT scan for gross tumor volume (GTV) definition. A total of 28 patients with Stage IA-IIIB NSCLC were enrolled. All patients had undergone staging PET-CT to ensure suitability for radical RT. Of the 28 patients, 14 received induction chemotherapy. In place of a RT planning CT scan, patients underwent scanning on a PET-CT scanner. In a virtual planning study, four oncologists independently delineated the GTV on the CT scan alone and then on the PET-CT scan. Intraobserver and interobserver variability were assessed using the concordance index (CI), and the results were compared using the Wilcoxon signed ranks test. PET-CT improved the CI between observers when defining the GTV using the PET-CT images compared with using CT alone for matched cases (median CI, 0.57 for CT and 0.64 for PET-CT, p = .032). The median of the mean percentage of volume change from GTV(CT) to GTV(FUSED) was -5.21% for the induction chemotherapy group and 18.88% for the RT-alone group. Using the Mann-Whitney U test, this was significantly different (p = .001). PET-CT RT planning scan, in addition to a staging PET-CT scan, reduces interobserver variability in GTV definition for NSCLC. The GTV size with PET-CT compared with CT in the RT-alone group increased and was reduced in the induction chemotherapy group.

Canine Comfort: Pet Affinity Buffers the Negative Impact of Ambivalence over Emotional Expression on Perceived Social Support.

PubMed

Bryan, Jennifer L; Quist, Michelle C; Young, Chelsie M; Steers, Mai-Ly N; Foster, Dawn W; Lu, Qian

2014-10-01

This study evaluated pet affinity as a buffer between ambivalence over emotional expression (AEE) and social support. AEE occurs when one desires to express emotions but is reluctant to do so and is related to negative psychological outcomes. Individuals high in AEE may have difficulty receiving social support and thus may not gain accompanying benefits. Social support has been associated with positive health outcomes, and pet support is positively associated with human social support. The present study explores the potential protective effect of pet affinity. One hundred ninety-eight undergraduate dog owners completed measures assessing perceived social support, pet affinity, and AEE. AEE was expected to be negatively associated with social support, and pet affinity was expected to buffer the negative effects of AEE on social support. We found that AEE was negatively associated with perceived social support. An interaction between pet affinity and AEE emerged such that the negative association between AEE and social support was weaker among those higher in pet affinity. Thus, at high levels of AEE, those who felt a close connection with their pets reported more perceived social support than those less connected with their pets. Overall, these findings emphasize the potential benefits of pet affinity.

Associations Between PET Textural Features and GLUT1 Expression, and the Prognostic Significance of Textural Features in Lung Adenocarcinoma.

PubMed

Koh, Young Wha; Park, Seong Yong; Hyun, Seung Hyup; Lee, Su Jin

2018-02-01

We evaluated the association between positron emission tomography (PET) textural features and glucose transporter 1 (GLUT1) expression level and further investigated the prognostic significance of textural features in lung adenocarcinoma. We evaluated 105 adenocarcinoma patients. We extracted texture-based PET parameters of primary tumors. Conventional PET parameters were also measured. The relationships between PET parameters and GLUT1 expression levels were evaluated. The association between PET parameters and overall survival (OS) was assessed using Cox's proportional hazard regression models. In terms of PET textural features, tumors expressing high levels of GLUT1 exhibited significantly lower coarseness, contrast, complexity, and strength, but significantly higher busyness. On univariate analysis, the metabolic tumor volume, total lesion glycolysis, contrast, busyness, complexity, and strength were significant predictors of OS. Multivariate analysis showed that lower complexity (HR=2.017, 95%CI=1.032-3.942, p=0.040) was independently associated with poorer survival. PET textural features may aid risk stratification in lung adenocarcinoma patients. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A method to rapidly and accurately compare relative efficacies of non-invasive imaging reporter genes in a mouse model, and its application to luciferase reporters

PubMed Central

Gil, Jose S.; Machado, Hidevaldo B.; Herschman, Harvey R.

2013-01-01

Purpose Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure, and compare the luciferase reporter efficacies. Procedures Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. Results We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed a greater that 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc, and a greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. Conclusions Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes, and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET). PMID:21850545

Chromium resistance characteristics of Cr(VI) resistance genes ChrA and ChrB in Serratia sp. S2.

PubMed

He, Yuan; Dong, Lanlan; Zhou, Simin; Jia, Yan; Gu, Ruijia; Bai, Qunhua; Gao, Jieying; Li, Yingli; Xiao, Hong

2018-08-15

To find an efficient chromium (VI) resistance system, with a highly efficient, economical, safe, and environmentally friendly chromium-removing strain, ChrA, ChrB, and ChrAB fragments of the chromium (VI) resistance gene in Serratia sp. S2 were cloned, and their prokaryotic expression vectors were constructed and transformed into E. coli BL21. The anti-chromium (VI) capacity and characteristics of engineered bacteria, role of ChrA and ChrB genes in the anti-chromium (VI) processes, and the mechanism of chromium metabolism, were explored. The PCR technique was used to amplify ChrA, ChrB, and ChrAB genes from the Serratia sp. S2 genome. ChrA, ChrB, and ChrAB genes were connected to the prokaryotic expression vector pET-28a and transferred into E. coli BL21 for prokaryotic expression. Cr-absorption and Cr-efflux ability of the engineered strains were determined. The effects of respiratory inhibitors and oxygenated anions on Cr-efflux of ChrA and ChrB engineered strains were explored. ChrA, ChrB, and ChrAB engineered strains were constructed successfully; there was no significant difference between the control strain and the ChrB engineered strain for Cr-metabolism (P > 0.05). Cr-absorption and Cr-efflux of ChrA and ChrAB engineered strains were significantly stronger than the control strain (P < 0.05). Oxyanions (sulfate and molybdate) and inhibitors (valinomycin and CN - ) could significantly inhibit the Cr-efflux capacities of ChrA and ChrAB engineered strains (P < 0.05), while NADPH could significantly promote such capacities (P < 0.05). The Cr-transporter, encoded by ChrA gene, confer the ability to pump out intracellular Cr on ChrA and ChrAB engineered strains. The ChrB gene plays a positive regulatory role in ChrA gene regulation. The Cr-metabolism ability of the ChrAB engineered strain is stronger than the ChrA engineered strain. ChrA and ChrAB genes in the Cr-resistance system may involve a variety of mechanisms, such as sulfate ion channel and respiratory chain electron transfer. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum.

PubMed

Rajulapati, Vikky; Goyal, Arun

2017-05-01

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0-9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca 2+ or Mg 2+ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca 2+ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.

Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio).

PubMed

Ken, C F; Lin, C T; Wu, J L; Shaw, J F

2000-06-01

A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.

18F-Fluoromisonidazole positron emission tomography (FMISO-PET) may reflect hypoxia and cell proliferation activity in oral squamous cell carcinoma.

PubMed

Sato, Jun; Kitagawa, Yoshimasa; Watanabe, Shiro; Asaka, Takuya; Ohga, Noritaka; Hirata, Kenji; Okamoto, Shozo; Shiga, Tohru; Shindoh, Masanobu; Kuge, Yuji; Tamaki, Nagara

2017-09-01

Hypoxia is a common feature and prognostic factor in cancer. 18 F-fluoromisonidazole (FMISO) positron emission tomography (PET) can detect tumor hypoxia noninvasively. The aim of this study was to assess the correlations between FMISO-PET and 18 F-fluorodexyglucose (FDG)-PET parameters with cell proliferation and hypoxia in patients with oral squamous cell carcinoma (OSCC). Twenty-three preoperative patients with OSCC were included. The tumor/muscle ratio (TMR) of FMISO-PET, the maximum standardized uptake values (SUV max ) of FDG-PET, metabolic tumor volume, and total lesion glycolysis were measured. Ki-67 and hypoxia-inducible factor-1α (HIF-1α) expression was immunohistochemically evaluated. FMISO TMR (P = .003) and FDG SUV max (P = .04) were significantly higher in patients with high expression of Ki-67 compared with those with low expression of Ki-67. FMISO TMR (P = .006) and FDG SUV max (P = .01) were also significantly higher in patients with HIF-1α expression than in those without HIF-1α expression. Metabolic tumor volume was not significantly related to either Ki-67 or HIF-1α expression. Multivariate analysis showed that FMISO TMR was independently predictive of Ki-67 (P = .002; odds ratio 31.1) and HIF-1α (P = .049; odds ratio 10.5) expression. FMISO-PET showed significant relationships with Ki-67 and HIF-1α expression, which are key features of cell proliferation and hypoxia in OSCC. Copyright © 2017 Elsevier Inc. All rights reserved.

LIN28A enhances the therapeutic potential of cultured neural stem cells in a Parkinson's disease model.

PubMed

Rhee, Yong-Hee; Kim, Tae-Ho; Jo, A-Young; Chang, Mi-Yoon; Park, Chang-Hwan; Kim, Sang-Mi; Song, Jae-Jin; Oh, Sang-Min; Yi, Sang-Hoon; Kim, Hyeon Ho; You, Bo-Hyun; Nam, Jin-Wu; Lee, Sang-Hun

2016-10-01

The original properties of tissue-specific stem cells, regardless of their tissue origins, are inevitably altered during in vitro culturing, lessening the clinical and research utility of stem cell cultures. Specifically, neural stem cells derived from the ventral midbrain lose their dopamine neurogenic potential, ventral midbrain-specific phenotypes, and repair capacity during in vitro cell expansion, all of which are critical concerns in using the cultured neural stem cells in therapeutic approaches for Parkinson's disease. In this study, we observed that the culture-dependent changes of neural stem cells derived from the ventral midbrain coincided with loss of RNA-binding protein LIN28A expression. When LIN28A expression was forced and sustained during neural stem cell expansion using an inducible expression-vector system, loss of dopamine neurogenic potential and midbrain phenotypes after long-term culturing was blocked. Furthermore, dopamine neurons that differentiated from neural stem cells exhibited remarkable survival and resistance against toxic insults. The observed effects were not due to a direct action of LIN28A on the differentiated dopamine neurons, but rather its action on precursor neural stem cells as exogene expression was switched off in the differentiating/differentiated cultures. Remarkable and reproducible behavioural recovery was shown in all Parkinson's disease rats grafted with neural stem cells expanded with LIN28A expression, along with extensive engraftment of dopamine neurons expressing mature neuronal and midbrain-specific markers. These findings suggest that LIN28A expression during stem cell expansion could be used to prepare therapeutically competent donor cells. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy.

PubMed

Wang, Xiaolong; Zhou, PeiHua; Sun, XueJun; Wei, GuangBing; Zhang, Li; Wang, Hui; Yao, JianFeng; Jia, PengBo; Zheng, JianBao

2016-05-01

One of the current challenges facing cancer gene therapy is the tumour-specific targeting of therapeutic genes. Effective targeting in gene therapy requires accurate spatial and temporal control of gene expression. To develop a sufficient and accurate tumour-targeting method for cancer gene therapy, we have investigated the use of hyperthermia to control the expression of a transgene under the control of the human telomerase reverse transcriptase (hTERT) promoter and eight heat shock elements (8HSEs). Luciferase reporters were constructed by inserting eight HSEs and the hTERT promoter (8HSEs-hTERTp) upstream of the pGL4.20 vector luciferase gene. The luciferase activity of the hTERT promoter and 8HSEs-hTERT promoter were then compared in the presence and absence of heat. The differences in luciferase activity were analysed using dual luciferase assays in SW480 (high hTERT expression), MKN28 and MRC-5 cells (low hTERT expression). The luciferase activity of the Hsp70B promoter was also compared to the 8HSEs-hTERT promoter in the above listed cell lines. Lentiviral vector and heat-induced expression of EGFP expression under the control of the 8HSEs-hTERT promoter in cultured cells and mouse tumour xenografts was measured by reverse transcription polymerase (RT-PCR), Western blot and immunofluorescence assays. hTERT promoter activity was higher in SW480 cells than in MKN28 or MRC-5 cells. At 43 °C, the luciferase activity of the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN28 or MRC-5 cells. Importantly, the differences in luciferase activity were much more obvious in both high (SW480) and low (MKN28 and MRC-5) hTERT expressing cells when the activity of the 8HSEs-hTERT promoter was compared to the Hsp70B promoter. Moreover, under the control of 8HSEs-hTERT promoter in vitro and in vivo, EGFP expression was obviously increased by heat treatment in SW480 cells but not in MKN28 or MRC-5 cells, nor was expression increased under normal temperature conditions. The hTERT promoter is a potentially powerful tumour-specific promoter and gene therapy tool for cancer treatment. Incorporating heat-inducible therapeutic elements (8HSEs) into the hTERT promoter may enhance the efficiency and specificity of cancer targeting gene therapy under hyperthermic clinical conditions.

Integrated MRI and [11 C]-PBR28 PET Imaging in Amyotrophic Lateral sclerosis.

PubMed

Alshikho, Mohamad J; Zürcher, Nicole R; Loggia, Marco L; Cernasov, Paul; Reynolds, Beverly; Pijanowski, Olivia; Chonde, Daniel B; Izquierdo Garcia, David; Mainero, Caterina; Catana, Ciprian; Chan, James; Babu, Suma; Paganoni, Sabrina; Hooker, Jacob M; Atassi, Nazem

2018-05-08

To characterize [ 11 C]-PBR28 brain uptake using positron emission tomography (PET) in people with amyotrophic lateral sclerosis (ALS), and primary lateral sclerosis (PLS). We have previously shown increased [ 11 C]-PBR28 uptake in the precentral gyrus in a small group of ALS patients. Herein, we confirm our initial finding, study the longitudinal changes, and characterize the gray vs. white matter distribution of [ 11 C]-PBR28 uptake in a larger cohort of patients with ALS and PLS. Eighty-five participants including 53 ALS, 11 PLS and 21 healthy controls underwent integrated [ 11 C]-PBR28 PET-MR brain imaging. Patients were clinically assessed using the upper motor neuron burden (UMNB), and the revised ALS functional rating scale (ALSFRS-R). [ 11 C]-PBR28 uptake was quantified as standardized uptake value ratio (SUVR), and compared between groups. Cortical thickness, and fractional anisotropy were compared between groups and correlated with SUVR and the clinical data. [ 11 C]-PBR28 uptake and ALSFRS-R were compared longitudinally over six-month in ten ALS individuals. Whole brain voxel-wise, surface-based and region of interest analyses revealed increased [ 11 C]-PBR28 uptake in the precentral and paracentral gyri in ALS, and in the sub-cortical white matter for the same regions in PLS, compared to controls. The increase in [ 11 C]-PBR28 uptake co-localized and correlated with cortical thinning, reduced fractional anisotropy, increased mean diffusivity, and correlated with higher UMNB score. No significant changes were detected in [ 11 C]-PBR28 uptake over six-month despite clinical progression. Glial activation measured by in vivo [ 11 C]-PBR28 PET is increased in pathologically relevant regions in people with ALS and correlates with clinical measures. This article is protected by copyright. All rights reserved. © 2018 American Neurological Association.

Signal detection using support vector machines in the presence of ultrasonic speckle

NASA Astrophysics Data System (ADS)

Kotropoulos, Constantine L.; Pitas, Ioannis

2002-04-01

Support Vector Machines are a general algorithm based on guaranteed risk bounds of statistical learning theory. They have found numerous applications, such as in classification of brain PET images, optical character recognition, object detection, face verification, text categorization and so on. In this paper we propose the use of support vector machines to segment lesions in ultrasound images and we assess thoroughly their lesion detection ability. We demonstrate that trained support vector machines with a Radial Basis Function kernel segment satisfactorily (unseen) ultrasound B-mode images as well as clinical ultrasonic images.

Optimizing modelling in iterative image reconstruction for preclinical pinhole PET

NASA Astrophysics Data System (ADS)

Goorden, Marlies C.; van Roosmalen, Jarno; van der Have, Frans; Beekman, Freek J.

2016-05-01

The recently developed versatile emission computed tomography (VECTor) technology enables high-energy SPECT and simultaneous SPECT and PET of small animals at sub-mm resolutions. VECTor uses dedicated clustered pinhole collimators mounted in a scanner with three stationary large-area NaI(Tl) gamma detectors. Here, we develop and validate dedicated image reconstruction methods that compensate for image degradation by incorporating accurate models for the transport of high-energy annihilation gamma photons. Ray tracing software was used to calculate photon transport through the collimator structures and into the gamma detector. Input to this code are several geometric parameters estimated from system calibration with a scanning 99mTc point source. Effects on reconstructed images of (i) modelling variable depth-of-interaction (DOI) in the detector, (ii) incorporating photon paths that go through multiple pinholes (‘multiple-pinhole paths’ (MPP)), and (iii) including various amounts of point spread function (PSF) tail were evaluated. Imaging 18F in resolution and uniformity phantoms showed that including large parts of PSFs is essential to obtain good contrast-noise characteristics and that DOI modelling is highly effective in removing deformations of small structures, together leading to 0.75 mm resolution PET images of a hot-rod Derenzo phantom. Moreover, MPP modelling reduced the level of background noise. These improvements were also clearly visible in mouse images. Performance of VECTor can thus be significantly improved by accurately modelling annihilation gamma photon transport.

PET/CT alignment calibration with a non-radioactive phantom and the intrinsic 176Lu radiation of PET detector

NASA Astrophysics Data System (ADS)

Wei, Qingyang; Ma, Tianyu; Wang, Shi; Liu, Yaqiang; Gu, Yu; Dai, Tiantian

2016-11-01

Positron emission tomography/computed tomography (PET/CT) is an important tool for clinical studies and pre-clinical researches which provides both functional and anatomical images. To achieve high quality co-registered PET/CT images, alignment calibration of PET and CT scanner is a critical procedure. The existing methods reported use positron source phantoms imaged both by PET and CT scanner and then derive the transformation matrix from the reconstructed images of the two modalities. In this paper, a novel PET/CT alignment calibration method with a non-radioactive phantom and the intrinsic 176Lu radiation of the PET detector was developed. Firstly, a multi-tungsten-alloy-sphere phantom without positron source was designed and imaged by CT and the PET scanner using intrinsic 176Lu radiation included in LYSO. Secondly, the centroids of the spheres were derived and matched by an automatic program. Lastly, the rotation matrix and the translation vector were calculated by least-square fitting of the centroid data. The proposed method was employed in an animal PET/CT system (InliView-3000) developed in our lab. Experimental results showed that the proposed method achieves high accuracy and is feasible to replace the conventional positron source based methods.

Synthesis and biological evaluation of copper-64 radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a novel bivalent targeting vector having affinity for two distinct biomarkers (GRPr/PSMA) of prostate cancer.

PubMed

Bandari, Rajendra Prasad; Jiang, Zongrun; Reynolds, Tamila Stott; Bernskoetter, Nicole E; Szczodroski, Ashley F; Bassuner, Kurt J; Kirkpatrick, Daniel L; Rold, Tammy L; Sieckman, Gary L; Hoffman, Timothy J; Connors, James P; Smith, Charles J

2014-04-01

Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] radioligand for prostate cancer imaging, where DUPA = (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH2 = bombesin, a GRPr-specific peptide targeting probe. The PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH2], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with (64)CuCl2 and (nat)CuCl2. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.). Competitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-((nat)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18h p.i. with collateral, background radiation also being observed in non-target tissue. DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular of imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2 in tumor-bearing mice indicate that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues. Published by Elsevier Inc.

Synthesis and Biological Evaluation of Copper-64 Radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a Novel Bivalent Targeting Vector Having Affinity for Two Distinct Biomarkers (GRPr/PSMA) of Prostate Cancer

PubMed Central

Bandari, Rajendra Prasad; Jiang, Zongrun; Reynolds, Tamila Stott; Bernskoetter, Nicole E.; Szczodroski, Ashley F.; Bassuner, Kurt J.; Kirkpatrick, Daniel L.; Rold, Tammy L.; Sieckman, Gary L.; Hoffman, Timothy J.; Connors, James P.; Smith, Charles J.

2014-01-01

Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-(64Cu-NODAGA)-5-Ava-BBN(7-14)NH2] radioligand for prostate cancer imaging, where DUPA = 2-[3-(1,3-Bis-tertbutoxycarbonylpropyl)-ureido]pentanedioic acid, a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH2 = bombesin or BBN, a GRPr-specific peptide targeting probe. Methods The PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH2], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with 64CuCl2 and natCuCl2. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.). Results Competitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-(natCu-NODAGA)-5-Ava-BBN(7-14)NH2] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18 h p.i. with collateral, background radiation also being observed in non-target tissue. Conclusions [DUPA-6-Ahx-(64Cu-NODAGA)-5-Ava-BBN(7-14)NH2] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-(64Cu-NODAGA)-5-Ava-BBN(7-14)NH2] in tumor-bearing mice indicates that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues. PMID:24508213

Heterogeneity of (18)F-FDG PET combined with expression of EGFR may improve the prognostic stratification of advanced oropharyngeal carcinoma.

PubMed

Wang, Hung-Ming; Cheng, Nai-Ming; Lee, Li-Yu; Fang, Yu-Hua Dean; Chang, Joseph Tung-Chieh; Tsan, Din-Li; Ng, Shu-Hang; Liao, Chun-Ta; Yang, Lan-Yan; Yen, Tzu-Chen

2016-02-01

The Ang's risk profile (based on p16, smoking and cancer stage) is a well-known prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). Whether heterogeneity in (18)F-fluorodeoxyglucose (FDG) positron emission tomographic (PET) images and epidermal growth factor receptor (EGFR) expression could provide additional information on clinical outcomes in advanced-stage OPSCC was investigated. Patients with stage III-IV OPSCC who completed primary therapy were eligible. Zone-size nonuniformity (ZSNU) extracted from pretreatment FDG PET scans was used as an index of image heterogeneity. EGFR and p16 expression were examined by immunohistochemistry. Disease-specific survival (DSS) and overall survival (OS) served as outcome measures. Kaplan-Meier estimates and Cox proportional hazards regression models were used for survival analysis. A bootstrap resampling technique was applied to investigate the stability of outcomes. Finally, a recursive partitioning analysis (RPA)-based model was constructed. A total of 113 patients were included, of which 28 were p16-positive. Multivariate analysis identified the Ang's profile, EGFR and ZSNU as independent predictors of both DSS and OS. Using RPA, the three risk factors were used to devise a prognostic scoring system that successfully predicted DSS in both p16-positive and -negative cases. The c-statistic of the prognostic index for DSS was 0.81, a value which was significantly superior to both AJCC stage (0.60) and the Ang's risk profile (0.68). In patients showing an Ang's high-risk profile (N = 77), the use of our scoring system clearly identified three distinct prognostic subgroups. It was concluded that a novel index may improve the prognostic stratification of patients with advanced-stage OPSCC. © 2015 UICC.

Pet-1 Switches Transcriptional Targets Postnatally to Regulate Maturation of Serotonin Neuron Excitability.

PubMed

Wyler, Steven C; Spencer, W Clay; Green, Noah H; Rood, Benjamin D; Crawford, LaTasha; Craige, Caryne; Gresch, Paul; McMahon, Douglas G; Beck, Sheryl G; Deneris, Evan

2016-02-03

Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1(-/-) neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. Five-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. The regulatory mechanisms that control functional maturation of neurons are poorly understood. We show that in addition to inducing brain serotonin (5-HT) synthesis and reuptake, the Pet-1 ETS (E26 transformation-specific) factor subsequently globally coordinates postmitotic expression trajectories of genes necessary for maturation of 5-HT neuron excitability. Further, Pet-1 switches its transcriptional targets as 5-HT neurons mature from 5-HT synthesis genes to G-protein-coupled receptors, which are necessary for afferent synaptic modulation of 5-HT neuron excitability. Our findings uncover gene-specific switching of downstream targets as a previously unrecognized regulatory strategy through which continuously expressed transcription factors control acquisition of neuronal identity at different stages of development. Copyright © 2016 the authors 0270-6474/16/361758-17$15.00/0.

Infectious microorganisms in mice (Mus musculus) purchased from commercial pet shops in Germany.

PubMed

Dammann, P; Hilken, G; Hueber, B; Köhl, W; Bappert, M T; Mähler, M

2011-10-01

In this study, we investigated the prevalence of infectious microorganisms (viruses, bacteria, fungi and eukaryotic parasites) in mice from different pet shops in Germany; such animals may compromise the hygienic integrity of laboratory animal vivaria if private pet holders act as unintended vectors of infections carried by them. House mice sold as pets or feed specimens were purchased from different pet shops and tested for a comprehensive panel of unwanted microorganisms. We found a number of microorganisms in these pet shop mice, the most prevalent of which were Helicobacter species (92.9%), mouse parvovirus (89.3%), mouse hepatitis virus (82.7%), Pasteurella pneumotropica (71.4%) and Syphacia species (57.1%). Several microorganisms (e.g. mouse parvovirus, Theiler's murine encephalomyelitis virus, pneumonia virus of mice, Encephalitozoon cuniculi, Clostridium piliforme) had considerably higher prevalences than those reported in similar studies on wild mice from North America, Europe or Australia. Our study shows that direct contact with pet shop mice may constitute a risk for laboratory animal vivaria if hygienic precautions are not taken. However, even relatively simple precautions seem effective enough to hold the risk at bay.

Parametric mapping using spectral analysis for 11C-PBR28 PET reveals neuroinflammation in mild cognitive impairment subjects.

PubMed

Fan, Zhen; Dani, Melanie; Femminella, Grazia D; Wood, Melanie; Calsolaro, Valeria; Veronese, Mattia; Turkheimer, Federico; Gentleman, Steve; Brooks, David J; Hinz, Rainer; Edison, Paul

2018-07-01

Neuroinflammation and microglial activation play an important role in amnestic mild cognitive impairment (MCI) and Alzheimer's disease. In this study, we investigated the spatial distribution of neuroinflammation in MCI subjects, using spectral analysis (SA) to generate parametric maps and quantify 11 C-PBR28 PET, and compared these with compartmental and other kinetic models of quantification. Thirteen MCI and nine healthy controls were enrolled in this study. Subjects underwent 11 C-PBR28 PET scans with arterial cannulation. Spectral analysis with an arterial plasma input function was used to generate 11 C-PBR28 parametric maps. These maps were then compared with regional 11 C-PBR28 V T (volume of distribution) using a two-tissue compartment model and Logan graphic analysis. Amyloid load was also assessed with 18 F-Flutemetamol PET. With SA, three component peaks were identified in addition to blood volume. The 11 C-PBR28 impulse response function (IRF) at 90 min produced the lowest coefficient of variation. Single-subject analysis using this IRF demonstrated microglial activation in five out of seven amyloid-positive MCI subjects. IRF parametric maps of 11 C-PBR28 uptake revealed a group-wise significant increase in neuroinflammation in amyloid-positive MCI subjects versus HC in multiple cortical association areas, and particularly in the temporal lobe. Interestingly, compartmental analysis detected group-wise increase in 11 C-PBR28 binding in the thalamus of amyloid-positive MCI subjects, while Logan parametric maps did not perform well. This study demonstrates for the first time that spectral analysis can be used to generate parametric maps of 11 C-PBR28 uptake, and is able to detect microglial activation in amyloid-positive MCI subjects. IRF parametric maps of 11 C-PBR28 uptake allow voxel-wise single-subject analysis and could be used to evaluate microglial activation in individual subjects.

Functional imaging of human epidermal growth factor receptor 2-positive metastatic breast cancer using (64)Cu-DOTA-trastuzumab PET.

PubMed

Mortimer, Joanne E; Bading, James R; Colcher, David M; Conti, Peter S; Frankel, Paul H; Carroll, Mary I; Tong, Shan; Poku, Erasmus; Miles, Joshua K; Shively, John E; Raubitschek, Andrew A

2014-01-01

Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of (64)Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including (18)F-FDG PET/CT. For 6 of the 8 patients, (64)Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21-25 (day 1) and 47-49 (day 2) h after (64)Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of (18)F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). Liver uptake of (64)Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and (18)F-FDG, respectively. On average, tumor uptake was similar for (64)Cu-DOTA-trastuzumab and (18)F-FDG (SUVmax and range, 8.1 and 3.0-22.5 for day 1 [n = 48]; 8.9 and 0.9-28.9 for day 2 [n = 38]; 9.7 and 3.3-25.4 for (18)F-FDG [n = 56]), but same-lesion SUVmax was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from (64)Cu-DOTA-trastuzumab was similar to (18)F-FDG. (64)Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a (64)Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. (64)Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.

Functional characterization of GH7 endo-1,4-β-glucanase from Aspergillus fumigatus and its potential industrial application.

PubMed

Bernardi, Aline Vianna; de Gouvêa, Paula Fagundes; Gerolamo, Luis Eduardo; Yonamine, Deborah Kimie; de Lourdes de Lima Balico, Laís; Uyemura, Sergio Akira; Dinamarco, Taisa Magnani

2018-04-30

A gene encoding an endo-1,4-β-glucanase (Afu6g01800) from A. fumigatus was cloned into the vector pET-28a(+) and expressed in the E. coli strain RosettaTM (DE3) pLysS. Sequence analysis indicated that the enzyme Af-EGL7 belonged to the GH7 family. The gene Af-egl7 encoded a protein comprising 460 amino acids, with a CBM1 domain at residues 424-460 and molecular mass of 52 kDa, as estimated by SDS-PAGE. This enzyme was optimally active at pH and temperatures ranging from 4.5 to 5.5 and from 40 to 60 °C, respectively. Mn 2+ addition significantly enhanced the Af-EGL7 cellulase activity by 233%, whereas SDS addition fully inhibited this activity. Higher activity was observed toward β-glucan than toward xyloglucan and CM-Cellulose, suggesting that the enzyme corresponds to a β-1,3-1,4-glucanase. qRT-PCR in different culture media helped to establish the time-course expression profile. Different polysaccharides induced the gene Af-egl7 in a time-dependent manner; in the particular case of the substrate sugarcane exploded bagasse (SEB), Af-egl7 was induced 2500-fold. Upon addition to a commercial cellulase cocktail, Af-EGL7 significantly improved SEB saccharification, which suggested that the enzyme Af-EGL7 had great potential to hydrolyze complex biomass. From a biotechnological point of view, A. fumigatus Af-EGL7 is a promising candidate to enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing. Copyright © 2018 Elsevier Inc. All rights reserved.

Frequency of Neurotic Symptoms Shortly after the Death of a Pet

PubMed Central

KIMURA, Yuya; KAWABATA, Hidenobu; MAEZAWA, Masaji

2013-01-01

ABSTRACT Some individuals manifest psychosomatic symptoms after the death of their pets. A survey was conducted at four public and commercial animal cremation service centers in Japan. In each center, a questionnaire was distributed to 100 individuals (400 in total). The questionnaire consisted of the 28-item version of the General Health Questionnaire (GHQ28), the social readjustment rating scale (SRRS) and a series of questions regarding demographic information and the circumstances of their pet’s death. In total, 82 returned questionnaires were available for analysis. GHQ28 proved the existence of neurotic symptoms in 46 responses (56.1%; 95% confidence interval: 44.7%–67.0%). Analysis of the responses using the GHQ28 subscales with a Likert scoring system demonstrated more somatic dysfunction in females (GHQ-A: P=0.04). Furthermore, significant correlations were identified among the following factors: owner’s age (GHQ-A: ρ=−0.60, P=0.01; GHQ-B: ρ=−0.29, P=0.01; GHQ-C: ρ=−0.32, P<0.01; GHQ-D: ρ=−0.42, P<0.01), SRRS score (GHQ-A: ρ=0.32, P<0.01; GHQ-B: ρ=0.25, P=0.02; GHQ-D: ρ=0.30, P=0.01) and animal’s age (GHQ-D: ρ=−0.26, P=0.02). The death of indoor pets caused deeper depression (GHQ-D: P=0.01) than that of outdoor or visiting pets. The results revealed neurotic symptoms in almost half of the pet owners shortly after their pet’s death. PMID:24334827

Simulation study comparing the helmet-chin PET with a cylindrical PET of the same number of detectors

NASA Astrophysics Data System (ADS)

Ahmed, Abdella M.; Tashima, Hideaki; Yoshida, Eiji; Nishikido, Fumihiko; Yamaya, Taiga

2017-06-01

There is a growing interest in developing brain PET scanners with high sensitivity and high spatial resolution for early diagnosis of neurodegenerative diseases and studies of brain functions. Sensitivity of the PET scanner can be improved by increasing the solid angle. However, conventional PET scanners are designed based on a cylindrical geometry, which may not be the most efficient design for brain imaging in terms of the balance between sensitivity and cost. We proposed a dedicated brain PET scanner based on a hemispheric shape detector and a chin detector (referred to as the helmet-chin PET), which is designed to maximize the solid angle by increasing the number of lines-of-response in the hemisphere. The parallax error, which PET scanners with a large solid angle tend to have, can be suppressed by the use of depth-of-interaction detectors. In this study, we carry out a realistic evaluation of the helmet-chin PET using Monte Carlo simulation based on the 4-layer GSO detector which consists of a 16  ×  16  ×  4 array of crystals with dimensions of 2.8  ×  2.8  ×  7.5 mm3. The purpose of this simulation is to show the gain in imaging performance of the helmet-chin PET compared with the cylindrical PET using the same number of detectors in each configuration. The sensitivity of the helmet-chin PET evaluated with a cylindrical phantom has a significant increase, especially at the top of the (field-of-view) FOV. The peak-NECR of the helmet-chin PET is 1.4 times higher compared to the cylindrical PET. The helmet-chin PET provides relatively low noise images throughout the FOV compared to the cylindrical PET which exhibits enhanced noise at the peripheral regions. The results show the helmet-chin PET can significantly improve the sensitivity and reduce the noise in the reconstructed images.

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buarque, Diego S.; Spindola, Leticia M.N.; Martins, Rafael M.

2011-09-23

Highlights: {yields} Tigutcystatin inhibits Trypanosoma cruzi cysteine proteases with high specificity. {yields} Tigutcystatin expression is up-regulated in response to T. cruzi infection. {yields} It is the first cysteine proteases inhibitor characterized from a triatomine insect. -- Abstract: The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatinmore » was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K{sub i} = 3.29 nM) and human cathepsin L (K{sub i} = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.« less

The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

PubMed

Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

2017-12-01

To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.

PubMed

Mason, J B; Gurda, B L; Van Wettere, A; Engiles, J B; Wilson, J M; Richardson, D W

2017-01-01

Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. Randomised in vivo experiment. We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken β-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the distribution pattern of transduction. © 2015 EVJ Ltd.

Cloning and heterologous expression of plnE, -F, -J and -K genes derived from soil metagenome and purification of active plantaricin peptides.

PubMed

Pal, Gargi; Srivastava, Sheela

2014-02-01

Plantaricin gene-specific primers were used to obtain plnE, -F, -J and -K structural gene amplicons from soil metagenome. These amplicons were cloned and expressed in pET32a (+) vector in Escherichia coli BL21 (DE3). PlnE, -F, -J and -K peptides were expressed as His-tagged-fusion proteins and were separated by Ni(2+) -chelating affinity chromatography. The peptides were released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. The cleaved peptides were further analysed for antimicrobial activity and found to be active against Listeria innocua NRRL B33314, Micrococcus luteus MTCC 106 and lactic acid bacteria, such as Enterococcus casseliflavus NRRL B3502, Lactococcus lactis lactis NRRL 1821, Lactobacillus curvatus NRRL B4562 and Lactobacillus plantarum NRRL B4496. E. coli has been successfully exploited as a host for heterologous expression with a significant yield of fused and cleaved peptides in the range of 8-12 and 1-1.5 mg/l of the culture, respectively. Heterologous expression, therefore, can be used to overcome the constraints of low yield often reported from a native strain.

Novel vector vaccine against Brucella abortus based on influenza A viruses expressing Brucella L7/L12 or Omp16 proteins: evaluation of protection in pregnant heifers.

PubMed

Tabynov, Kaissar; Yespembetov, Bolat; Sansyzbay, Abylai

2014-10-14

The present study provides the first information about the protection of a novel influenza viral vector vaccine expressing the Brucella proteins ribosomal L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with Brucella abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Via both the conjunctival or subcutaneous route, evaluation of protectiveness against abortion, effectiveness of vaccination and index of infection (in heifers and their fetuses or calves) demonstrated the vector vaccine provided good protection against B. abortus 544 infection compared to the negative control group (PBS+Montanide Gel01) and comparable protection to commercial vaccines B. abortus S19 or B. abortus RB51. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cloning and expression of an envelope gene of West Nile virus and evaluation of the protein for use in an IgM ELISA.

PubMed

Saxena, Divyasha; Parida, Manmohan; Rao, Putcha Venkata L; Kumar, Jyoti S

2013-04-01

West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and epidemiologic control in areas where multiple flaviviruses are endemic. The coexistence of WNV along with other members of flaviviruses like dengue and Japanese encephalitis in India has complicated the serodiagnosis due to cross-reactive antigens. In the present study, the development and evaluation of a highly sensitive and specific IgM enzyme-linked immunosorbent assay (ELISA) using the recombinant envelope protein (rWNV-Env) for rapid, early, and accurate diagnosis of WNV are reported. The gene coding for the envelope protein of WNV was cloned and expressed in pET 28a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rWNV-Env protein was optimized having no cross reactivity with healthy human serum. Furthermore, the specificity of this assay was confirmed by cross checking with serum samples obtained from patients with dengue and Japanese encephalitis viruses. The comparative evaluation of this rWNV-Env protein-specific IgM ELISA with plaque reduction neutralization test assay using 105 acute phase of clinical samples revealed 95% concordance with sensitivity and specificity of 92% and 97%, respectively. The positive and negative predictive values of recombinant-based Env ELISA were 94% and 96%, respectively. The recombinant envelope protein-based WNV-specific ELISA reported in this study will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks. Copyright © 2013 Elsevier Inc. All rights reserved.

Online survey as empathic bridging for the disenfranchised grief of pet loss.

PubMed

Packman, Wendy; Carmack, Betty J; Katz, Rachel; Carlos, France; Field, Nigel P; Landers, Craig

2014-01-01

The current cross-cultural study investigated grief reactions of bereaved individuals following the death of a pet. We used qualitative methodology to compare, analyze, and report responses of U.S. and French Canadian participants to the last open-ended question on our online pet loss survey. We explored the degree to which our data illustrated pet loss as disenfranchised grief and asked whether there are differences and commonalities in the expression of grief between the two samples. Four major themes emerged: lack of validation and support; intensity of loss; nature of the human pet relationship; and continuing bonds. Findings confirm that, for both the U.S. and French Canadian participants, pet loss is often disenfranchised grief and there are ways to facilitate expressions of grief. Many participants wrote that the survey was therapeutic. Our survey allowed participants to express their grief in an anonymous, safe way by serving as empathic bridging and a willingness to help others.

Multiple human exposures to a rabid bear cub at a petting zoo and barnwarming--Iowa, August 1999.

PubMed

1999-09-03

On August 27, 1999, a black bear cub, approximately 5-6 months old, died after several hours of acute central nervous system symptoms; preliminary test results available on August 28 indicated the bear had rabies. The bear was part of the Swenson's Wild Midwest Exotic Petting Zoo in Clermont, Iowa (northeastern Iowa). At the petting zoo, visitors fed, wrestled, and may have been nipped by the bear. The bear also was taken to an August 14 barnwarming at the Tharp barn in Holy Cross, Iowa (eastern Iowa), where it reportedly nipped people. An estimated 400 people from 10 states (Arizona, California, Florida, Illinois, Iowa, Minnesota, New Mexico, New York, Ohio, and Wisconsin) and Australia had contact with the bear cub at either the petting zoo or the barnwarming during the 28 days before its death, during which the bear may have transmitted rabies virus.

Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications

PubMed Central

Lange, Sara E.S.; Zheleznyak, Alex; Studer, Matthew; O'Shannessy, Daniel J.; Lapi, Suzanne E.; Van Tine, Brian A.

2016-01-01

Purpose The complexity of sarcoma has led to the need for patient selection via in vivo biomarkers. Tumor endothelial marker-1 (TEM-1) is a cell surface marker expressed by the tumor microenvironment. Currently MORAb-004 (Ontuxizumab), an anti-TEM-1 humanized monoclonal antibody, is in sarcoma clinical trials. Development of positron emission tomography (PET) for in vivo TEM-1 expression may allow for stratification of patients, potentially enhancing clinical outcomes seen with Ontuxizumab. Results Characterization of cell lines revealed clear differences in TEM-1 expression. One high expressing (RD-ES) and one low expressing (LUPI) cell line were xenografted, and mice were injected with 89Zr-Ontuxizumab. PET imaging post-injection revealed that TEM-1 was highly expressed and readily detectable in vivo only in RD-ES. In vivo biodistribution studies confirmed high radiopharmaceutical uptake in tumor relative to normal organs. Experimental Design Sarcoma cell lines were characterized for TEM-1 expression. Ontuxizumab was labeled with 89Zr and evaluated for immunoreactivity preservation. 89Zr-Ontuxizumab was injected into mice with high or null expressing TEM-1 xenografts. In vivo PET imaging experiments were performed. Conclusion 89Zr-Ontuxizumab can be used in vivo to determine high versus low TEM-1 expression. Reliable PET imaging of TEM-1 in sarcoma patients may allow for identification of patients that will attain the greatest benefit from anti-TEM-1 therapy. PMID:26909615

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves.

PubMed

Liu, Zhen; Chen, Zhe; Hong, Jian; Wang, Xuefeng; Zhou, Changyong; Zhou, Xueping; Wu, Jianxiang

2016-08-01

Citrus tristeza virus (CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein (CP) gene of CTV was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-28a and purified through Ni+-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines (14B10, 14H11, 20D5, and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10(-6) to 10(-7) in indirect enzyme-linked immunosorbent assay (ELISA). Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich (TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and 1:10, 240 (w/v, g/mL), respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.

Positron Emission Tomography studies with [11C]PBR28 in the Healthy Rodent Brain: Validating SUV as an Outcome Measure of Neuroinflammation.

PubMed

Tóth, Miklós; Doorduin, Janine; Häggkvist, Jenny; Varrone, Andrea; Amini, Nahid; Halldin, Christer; Gulyás, Balázs

2015-01-01

Molecular imaging of the 18 kD Translocator protein (TSPO) with positron emission tomography (PET) is of great value for studying neuroinflammation in rodents longitudinally. Quantification of the TSPO in rodents is, however, quite challenging. There is no suitable reference region and the use of plasma-derived input is not an option for longitudinal studies. The aim of this study was therefore to evaluate the use of the standardized uptake value (SUV) as an outcome measure for TSPO imaging in rodent brain PET studies, using [11C]PBR28. In the first part of the study, healthy male Wistar rats (n = 4) were used to determine the correlation between the distribution volume (VT, calculated with Logan graphical analysis) and the SUV. In the second part, healthy male Wistar rats (n = 4) and healthy male C57BL/6J mice (n = 4), were used to determine the test-retest variability of the SUV, with a 7-day interval between measurements. Dynamic PET scans of 63 minutes were acquired with a nanoScan PET/MRI and nanoScan PET/CT. An MRI scan was made for anatomical reference with each measurement. The whole brain VT of [11C]PBR28 in rats was 42.9 ± 1.7. A statistically significant correlation (r2 = 0.96; p < 0.01) was found between the VT and the SUV. The test-retest variability in 8 brain region ranged from 8 to 20% in rats and from 7 to 23% in mice. The interclass correlation coefficient (ICC) was acceptable to excellent for rats, but poor to acceptable for mice. The SUV of [11C]PBR28 showed a high correlation with VT as well as good test-retest variability. For future longitudinal small animal PET studies the SUV can thus be used to describe [11C]PBR28 uptake in healthy brain tissue. Based on the present observations, further studies are needed to explore the applicability of this approach in small animal disease models, with special regard to neuroinflammatory models.

Evidence of Prostate-Specific Membrane Antigen Expression in Metastatic Differentiated Thyroid Cancer Using 68Ga-PSMA-HBED-CC PET/CT.

PubMed

Verma, Priyanka; Malhotra, Gaurav; Agrawal, Ritesh; Sonavane, Sunita; Meshram, Vilas; Asopa, Ramesh V

2018-06-12

Prostate-specific membrane antigen (PSMA) overexpression is not restricted to prostate cancer, but it has also been demonstrated in gliomas, lung cancer, and in tumor neovasculature. Systematic studies exploring PSMA uptake in thyroid tumors are lacking. The aim of this pilot study was to assess PSMA expression in patients with metastatic differentiated thyroid cancer (mDTC). Ten patients of mDTC harboring 32 lesions (5 men; age range, 38-65 years; mean age, 50 years) underwent prospective evaluation with radioiodine (I), F-FDG PET, and Ga-PSMA-HBED-CC PET scans as per the institution protocol. PSMA expression (SUVmax) was compared with F-FDG and I scan findings in all patients. Lesions were radioiodine avid in 8 patients, whereas 2 were classified as thyroglobulin elevation with negative iodide scintigraphy (TENIS) patients. All patients with iodine-avid metastatic disease showed substantial PSMA uptake. PSMA PET detected 30/32 total lesions (93.75%; SUVmax ranging from 4.86 to 101.81 with median SUVmax of 31.35), whereas FDG PET/CT was positive in 23/32 lesions (81.85%). Twenty-one (70%) of 30 lesions that showed PSMA expression were localized to the bones. PSMA localized a lesion in each of the 2 TENIS patients similar to FDG PET scan. Ga-PSMA-HBED-CC PET/CT is a potentially useful imaging modality in patients of mDTC with most (70%) of PSMA expressing metastasis being localized to the bones. PSMA PET/CT could be useful for identifying patients with limited therapeutic options (eg, TENIS) who might benefit from PSMA-targeted radionuclide therapy.

Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells.

PubMed

Kamath, Anant; Ternes, Sara; McGowan, Stephen; English, Anthony; Mallampalli, Rama; Moy, Alan B

2017-08-01

Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28 . We describe a robust Myc and Lin28 -free IPSC reprogramming approach using reprogramming molecules. IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28 -free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28 . This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.

Functional Imaging of HER2-Positive Metastatic Breast Cancer Using 64Cu-DOTA-Trastuzumab Positron Emission Tomography

PubMed Central

Mortimer, Joanne E.; Bading, James R.; Colcher, David M.; Conti, Peter S.; Frankel, Paul H.; Carroll, Mary I.; Tong, Shan; Poku, Erasmus; Miles, Joshua K.; Shively, John E.; Raubitschek, Andrew A.

2014-01-01

Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion which may not be representative of the larger tumor mass or other sites of disease. Our long-range goal is to develop positron emission tomography (PET) of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET-CT of 64Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Methods Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for ≥ 4 mo underwent complete staging, including 18F-fluorodeoxyglucose (FDG)/PET-CT. For 6 of the 8 patients, 64Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg trastuzumab) was preceded by trastuzumab infusion (45 mg). PET-CT (PET scan duration 1 h) was performed 21-25 (“Day 1”) and 47-49 (“Day 2”) h after 64Cu-DOTA-trastuzumab injection. Scan fields of view were chosen based on 18F-FDG/PET-CT. Lesions visualized relative to adjacent tissue on PET were considered PET-positive; analysis was limited to lesions identifiable on CT. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). Results Liver uptake of 64Cu was reduced approximately 75% with the 45 mg trastuzumab pre-dose, without significant effect on tumor uptake. The study included 89 CT-positive lesions; detection sensitivity was 77, 89 and 93% for Day 1, Day 2 and 18F-FDG, respectively. On average, tumor uptake was similar for 64Cu-DOTA-trastuzumab and 18F-FDG [SUVmax (mean, range): Day 1 (8.1, 3.0-22.5, n=48); Day 2 (8.9, 0.9-28.9, n=38); 18F-FDG (9.7, 3.3-25.4, n=56)], but the extent of same-lesion uptake was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from 64Cu-DOTA-trastuzumab was similar to 18F-FDG. Conclusion 64Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity, and is effective in surveying disseminated disease. A 45 mg trastuzumab pre-dose provides a 64Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. 64Cu-DOTA-trastuzumab/PET-CT warrants further evaluation for assessing tumor HER2 expression and measuring delivery of trastuzumab-based therapy. PMID:24337604

2-Methyl-3-buten-2-ol (MBO) synthase expression in Nostoc punctiforme leads to over production of phytols.

PubMed

Gupta, Dinesh; Ip, Tina; Summers, Michael L; Basu, Chhandak

2015-01-01

Phytol is a diterpene alcohol of medicinal importance and it also has potential to be used as biofuel. We found over production of phytol in Nostoc punctiforme by expressing a 2-Methyl-3-buten-2-ol (MBO) synthase gene. MBO synthase catalyzes the conversion of dimethylallyl pyrophosphate (DMAPP) into MBO, a volatile hemiterpene alcohol, in Pinus sabiniana. The result of enhanced phytol production in N. punctiforme, instead of MBO, could be explained by one of the 2 models: either the presence of a native prenyltransferase enzyme with a broad substrate specificity, or appropriation of a MBO synthase metabolic intermediate by a native geranyl diphosphate (GDP) synthase. In this work, an expression vector with an indigenous petE promoter for gene expression in the cyanobacterium N. punctiforme was constructed and MBO synthase gene expression was successfully shown using reverse transcriptase (RT)-PCR and SDS-PAGE. Gas chromatography--mass spectrophotometry (GC-MS) was performed to confirm phytol production from the transgenic N. punctiforme strains. We conclude that the expression of MBO synthase in N. punctiforme leads to overproduction of an economically important compound, phytol. This study provides insights about metabolic channeling of isoprenoids in cyanobacteria and also illustrates the challenges of bioengineering non-native hosts to produce economically important compounds.

Noninvasive imaging of islet grafts using positron-emission tomography

NASA Astrophysics Data System (ADS)

Lu, Yuxin; Dang, Hoa; Middleton, Blake; Zhang, Zesong; Washburn, Lorraine; Stout, David B.; Campbell-Thompson, Martha; Atkinson, Mark A.; Phelps, Michael; Gambhir, Sanjiv Sam; Tian, Jide; Kaufman, Daniel L.

2006-07-01

Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. However, islet transplantation is not routinely successful because most islet recipients gradually lose graft function. Furthermore, serological markers of islet function are insensitive to islet loss until the latter stages of islet graft rejection. A noninvasive method of monitoring islet grafts would aid in the assessment of islet graft survival and the evaluation of interventions designed to prolong graft survival. Here, we show that recombinant adenovirus can engineer isolated islets to express a positron-emission tomography (PET) reporter gene and that these islets can be repeatedly imaged by using microPET after transplantation into mice. The magnitude of signal from engineered islets implanted into the axillary cavity was directly related to the implanted islet mass. PET signals attenuated over the following weeks because of the transient nature of adenovirus-mediated gene expression. Because the liver is the preferred site for islet implantation in humans, we also tested whether islets could be imaged after transfusion into the mouse liver. Control studies revealed that both intrahepatic islet transplantation and hyperglycemia altered the biodistribution kinetics of the PET probe systemically. Although transplanted islets were dispersed throughout the liver, clear signals from the liver region of mice receiving PET reporter-expressing islets were detectable for several weeks. Viral transduction, PET reporter expression, and repeated microPET imaging had no apparent deleterious effects on islet function after implantation. These studies lay a foundation for noninvasive quantitative assessments of islet graft survival using PET. diabetes | transplantation

Applications of immunoPET: Using 124I-anti-PSCA A11 minibody for imaging disease progression and response to therapy in mouse xenograft models of prostate cancer

DOE PAGES

Knowles, Scott M.; Tavare, Richard; Zettlitz, Kirstin A.; ...

2014-10-17

Here, prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate cancer bone metastases and its expression correlates with androgen receptor activation and a poor prognosis. Here in this study, we investigate the potential clinical applications of immunoPET with the anti-PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11 minibody immunoPET to 18F-Fluoride PET bone scans for detecting prostate cancer bone tumors and evaluate the ability of the A11 minibody to image tumor response to androgen deprivation. Osteoblastic, PSCA expressing, LAPC-9 intratibial xenografts were imaged with serial 124I-anti-PSCA A11more » minibody immunoPET and 18F-Fluoride bone scans. Mice bearing LAPC-9 subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody immunoPET/CT scans pre- and post-treatment. Ex vivo flow cytometry measured the change in PSCA expression in response to androgen deprivation. A11 minibody demonstrated improved sensitivity and specificity over 18F-Fluoride bone scans for detecting LAPC-9 intratibial xenografts at all time points. Finally, LAPC-9 subcutaneous xenografts showed downregulation of PSCA when treated with MDV-3100 which A11 minibody immunoPET was able to detect in vivo.« less

Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies.

PubMed

Karray, Aida; Amara, Sawsan; Carrière, Frédéric; Gargouri, Youssef; Bezzine, Sofiane

2014-06-01

The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group IIA sPLA2, has been amplified from chicken intestine. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg/l of pure refolded fully active enzyme to be obtained. Recombinant expression of chicken intestinal sPLA2-IIA (ChPLA2-IIA) in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 10-fold preference. Indeed, we report in this work, a comparative kinetic study between the wild type and the recombinant ChPLA2-IIA, on zwitterionic head group phospholipids (DDPC) and negatively charged phospholipids (POPG) using the monomolecular film technique. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

Constructing Chimeric Antigen for Precise Screening of HTLV-I Infection.

PubMed

Heydari Zarnagh, Hafez; Hassanpour, Kazem; Rasaee, Mohammad Javad

2015-08-01

Individual preparation of two human T-cell lymphotropic virus type I (HTLV-I) diagnostic GST fused peptides (MTA-1 and GD21) is time-consuming and expensive. The aim of this study was to design a novel single chimeric antigen (SCA) to obviate separate expression of proteins and reduce the cost of reagent preparation. Structural protein fragments, including immunodominant B cell linear epitopes, were selected and different SCAs were designed. Tertiary structure, epitope exposure, solubility and stability were calculated for each SCA and compared with each other. The synthetic DNA encoding the interested SCA was sub-cloned into pET32a expression vector, expressed as a soluble form in Escherichia coli BL21 (DE3) cells and purified under native condition using affinity chromatography. The SDS-PAGE results indicated that thioredoxin-fused SCA was successfully expressed as a soluble form in E. coli BL21 (DE3) cells. The results of ELISA confirmed that SCA reacted with anti-HTLV-I antibodies in a concentration-dependent manner. Our results indicated that the designed SCA may be a good candidate for the screening of HTLV-I carriers with antigen-antibody-based tests.

Microbiological survey of mice (Mus musculus) purchased from commercial pet shops in Kanagawa and Tokyo, Japan.

PubMed

Hayashimoto, Nobuhito; Morita, Hanako; Ishida, Tomoko; Uchida, Ritsuki; Tanaka, Mai; Ozawa, Midori; Yasuda, Masahiko; Itoh, Toshio

2015-01-01

Information regarding the prevalence of infectious agents in mice in pet shops in Japan is scarce. This information is particularly useful for minimizing the risk of potential transmission of infections to laboratory mice. Therefore, we surveyed infectious agents in mice from pet shops in Kanagawa and Tokyo, Japan. The survey was conducted in 28 mice from 5 pet shops to screen for 47 items (17 viruses, 22 bacteria and fungi, 10 parasites) using culture tests, serology, PCR, and microscopy. The most common viral agent detected was murine norovirus (17 mice; 60.7%), followed by Theiler's murine encephalomyelitis virus (13 mice; 46.4%), and mouse hepatitis virus (12 mice; 42.8%). The most common agent amongst the bacteria and fungi was Pasteurella pneumotropica (10 mice; 35.7%), followed by Helicobacter ganmani and Pneumocystis murina (8 mice; 28.5%, for both). Tritrichomonas muris was the most common parasite (19 mice; 67.8%), followed by Spironucleus muris (13 mice; 46.4%), Aspiculuris tetraptera, and Syphacia obvelata (8 mice each; 28.5%). Remarkably, a zoonotic agent, Hymenolepis nana, was found in 7 mice (25%). Given these results, we suggest that the workers in laboratory animal facilities should recognize again the potential risks of mice outside of the laboratory animal facilities as an infectious source, and avoid keeping mice as pets or as feed for carnivorous reptiles as much as possible for risk management.

Expression of Caenorhabditis elegans antimicrobial peptide NLP-31 in Escherichia coli

NASA Astrophysics Data System (ADS)

Lim, Mei-Perng; Nathan, Sheila

2014-09-01

Burkholderia pseudomallei is the causative agent of melioidosis, a fulminant disease endemic in Southeast Asia and Northern Australia. The standardized form of therapy is antibiotics treatment; however, the bacterium has become increasingly resistant to these antibiotics. This has spurred the need to search for alternative therapeutic agents. Antimicrobial peptides (AMPs) are small proteins that possess broad-spectrum antimicrobial activity. In a previous study, the nematode Caenorhabditis elegans was infected by B. pseudomallei and a whole animal transcriptome analysis identified a number of AMP-encoded genes which were induced significantly in the infected worms. One of the AMPs identified is NLP-31 and to date, there are no reports of anti-B. pseudomallei activity demonstrated by NLP-31. To produce NLP-31 protein for future studies, the gene encoding for NLP-31 was cloned into the pET32b expression vector and transformed into Escherichia coli BL21(DE3). Protein expression was induced with 1 mM IPTG for 20 hours at 20°C and recombinant NLP-31 was detected in the soluble fraction. Taken together, a simple optimized heterologous production of AMPs in an E. coli expression system has been successfully developed.

Borrelia miyamotoi, Other Vector-Borne Agents in Cat Blood and Ticks in Eastern Maryland.

PubMed

Shannon, Avery B; Rucinsky, Renee; Gaff, Holly D; Brinkerhoff, R Jory

2017-12-01

We collected blood and tick samples in eastern Maryland to quantify vector-borne pathogen exposure and infection in healthy cats and to assess occupational disease risk to veterinary professionals and others who regularly interact with household pets. Thirty-six percent of healthy cats parasitized by ticks at time of examination (9/25) were exposed to, and 14% of bloods (7/49) tested PCR-positive for, at least one vector-borne pathogen including several bloods and ticks with Borrelia miyamotoi, a recently recognized tick-borne zoonotic bacterium. There was no indication that high tick burdens were associated with exposure to vector-borne pathogens. Our results underscore the potential importance of cats to human vector-borne disease risk.

Quantitative PET imaging of Met-expressing human cancer xenografts with 89Zr-labelled monoclonal antibody DN30.

PubMed

Perk, Lars R; Stigter-van Walsum, Marijke; Visser, Gerard W M; Kloet, Reina W; Vosjan, Maria J W D; Leemans, C René; Giaccone, Giuseppe; Albano, Raffaella; Comoglio, Paolo M; van Dongen, Guus A M S

2008-10-01

Targeting the c-Met receptor with monoclonal antibodies (MAbs) is an appealing approach for cancer diagnosis and treatment because this receptor plays a prominent role in tumour invasion and metastasis. Positron emission tomography (PET) might be a powerful tool for guidance of therapy with anti-Met MAbs like the recently described MAb DN30 because it allows accurate quantitative imaging of tumour targeting (immuno-PET). We considered the potential of PET with either (89)Zr-labelled (residualising radionuclide) or (124)I-labelled (non-residualising radionuclide) DN30 for imaging of Met-expressing tumours. The biodistribution of co-injected (89)Zr-DN30 and iodine-labelled DN30 was compared in nude mice bearing either the human gastric cancer line GLT-16 (high Met expression) or the head-and-neck cancer line FaDu (low Met expression). PET images were acquired in both xenograft models up to 4 days post-injection (p.i.) and used for quantification of tumour uptake. Biodistribution studies in GTL-16-tumour-bearing mice revealed that (89)Zr-DN30 achieved much higher tumour uptake levels than iodine-labelled DN30 (e.g. 19.6%ID/g vs 5.3%ID/g, 5 days p.i.), while blood levels were similar, indicating internalisation of DN30. Therefore, (89)Zr-DN30 was selected for PET imaging of GLT-16-bearing mice. Tumours as small as 11 mg were readily visualised with immuno-PET. A distinctive lower (89)Zr uptake was observed in FaDu compared to GTL-16 xenografts (e.g. 7.8%ID/g vs 18.1%ID/g, 3 days p.i.). Nevertheless, FaDu xenografts were also clearly visualised with (89)Zr-DN30 immuno-PET. An excellent correlation was found between PET-image-derived (89)Zr tumour uptake and ex-vivo-assessed (89)Zr tumour uptake (R(2)=0.98). The long-lived positron emitter (89)Zr seems attractive for PET-guided development of therapeutic anti-c-Met MAbs.

A PET imaging agent for evaluating PARP-1 expression in ovarian cancer.

PubMed

Makvandi, Mehran; Pantel, Austin; Schwartz, Lauren; Schubert, Erin; Xu, Kuiying; Hsieh, Chia-Ju; Hou, Catherine; Kim, Hyoung; Weng, Chi-Chang; Winters, Harrison; Doot, Robert; Farwell, Michael D; Pryma, Daniel A; Greenberg, Roger A; Mankoff, David A; Simpkins, Fiona; Mach, Robert H; Lin, Lilie L

2018-05-01

Poly(ADP-ribose) polymerase (PARP) inhibitors are effective in a broad population of patients with ovarian cancer; however, resistance caused by low enzyme expression of the drug target PARP-1 remains to be clinically evaluated in this context. We hypothesize that PARP-1 expression is variable in ovarian cancer and can be quantified in primary and metastatic disease using a novel PET imaging agent. We used a translational approach to describe the significance of PET imaging of PARP-1 in ovarian cancer. First, we produced PARP1-KO ovarian cancer cell lines using CRISPR/Cas9 gene editing to test the loss of PARP-1 as a resistance mechanism to all clinically used PARP inhibitors. Next, we performed preclinical microPET imaging studies using ovarian cancer patient-derived xenografts in mouse models. Finally, in a phase I PET imaging clinical trial we explored PET imaging as a regional marker of PARP-1 expression in primary and metastatic disease through correlative tissue histology. We found that deletion of PARP1 causes resistance to all PARP inhibitors in vitro, and microPET imaging provides proof of concept as an approach to quantify PARP-1 in vivo. Clinically, we observed a spectrum of standard uptake values (SUVs) ranging from 2-12 for PARP-1 in tumors. In addition, we found a positive correlation between PET SUVs and fluorescent immunohistochemistry for PARP-1 (r2 = 0.60). This work confirms the translational potential of a PARP-1 PET imaging agent and supports future clinical trials to test PARP-1 expression as a method to stratify patients for PARP inhibitor therapy. Clinicaltrials.gov NCT02637934. Research reported in this publication was supported by the Department of Defense OC160269, a Basser Center team science grant, NIH National Cancer Institute R01CA174904, a Department of Energy training grant DE-SC0012476, Abramson Cancer Center Radiation Oncology pilot grants, the Marsha Rivkin Foundation, Kaleidoscope of Hope Foundation, and Paul Calabresi K12 Career Development Award 5K12CA076931.

In vitro synthesis of 9,10-dihydroxyhexadecanoic acid using recombinant Escherichia coli.

PubMed

Kaprakkaden, Anees; Srivastava, Preeti; Bisaria, Virendra Swarup

2017-05-18

Hydroxy fatty acids are widely used in food, chemical and cosmetic industries. A variety of dihydroxy fatty acids have been synthesized so far; however, no studies have been done on the synthesis of 9,10-dihydroxyhexadecanoic acid. In the present study recombinant E. coli has been used for the heterologous expression of fatty acid hydroxylating enzymes and the whole cell lysate of the induced culture was used for in vitro production of 9,10-dihydroxyhexadecanoic acid. A first of its kind proof of principle has been successfully demonstrated for the production of 9,10-dihydroxyhexadecanoic acid using three different enzymes viz. fatty acid desaturase (FAD) from Saccharomyces cerevisiae, epoxide hydrolase (EH) from Caenorhabditis elegance and epoxygenase (EPOX) from Stokasia laevis. The genes for these proteins were codon-optimised, synthesised and cloned in pET 28a (+) vector. The culture conditions for induction of these three proteins in E. coli were optimised in shake flask. The induced cell lysates were used both singly and in combination along with the trans-supply of hexadecanoic acid and 9-hexadecenoic acid, followed by product profiling by GC-MS. Formation of 9,10-dihydroxyhexadecanoic acid was successfully achieved when combination of induced cell lysates of recombinant E. coli containing FAD, EH, and EPOX were incubated with 9-hexadecenoic acid. The in vitro production of 9,10-dihydroxyhexadecanoic acid synthesis using three fatty acid modification genes from different sources has been successfully demonstrated. The strategy adopted can be used for the production of similar compounds.

Gene therapy using self-complementary Y733F capsid mutant AAV2/8 restores vision in a model of early onset Leber congenital amaurosis.

PubMed

Ku, Cristy A; Chiodo, Vince A; Boye, Sanford L; Goldberg, Andrew F X; Li, Tiansen; Hauswirth, William W; Ramamurthy, Visvanathan

2011-12-01

Defects in the photoreceptor-specific gene aryl hydrocarbon receptor interacting protein-like 1 (Aipl1) are associated with Leber congenital amaurosis (LCA), a childhood blinding disease with early-onset retinal degeneration and vision loss. Furthermore, Aipl1 defects are characterized at the most severe end of the LCA spectrum. The rapid photoreceptor degeneration and vision loss observed in the LCA patient population are mimicked in a mouse model lacking AIPL1. Using this model, we evaluated if gene replacement therapy using recent advancements in adeno-associated viral vectors (AAV) provides advantages in preventing rapid retinal degeneration. Specifically, we demonstrated that the novel self-complementary Y733F capsid mutant AAV2/8 (sc-Y733F-AAV) provided greater preservation of photoreceptors and functional vision in Aipl1 null mice compared with single-stranded AAV2/8. The benefits of sc-Y733F-AAV were evident following viral administration during the active phase of retinal degeneration, where only sc-Y733F-AAV treatment achieved functional vision rescue. This result was likely due to higher and earlier onset of Aipl1 expression. Based on our studies, we conclude that the sc-Y733F-AAV2/8 viral vector, to date, achieves the best rescue for rapid retinal degeneration in Aipl1 null mice. Our results provide important considerations for viral vectors to be used in future gene therapy clinical trials targeting a wider severity spectrum of inherited retinal dystrophies.

68Ga-PSMA PET/CT in prostate cancer.

PubMed

García Garzón, J R; de Arcocha Torres, M; Delgado-Bolton, R; Ceci, F; Alvarez Ruiz, S; Orcajo Rincón, J; Caresia Aróztegui, A P; García Velloso, M J; García Vicente, A M

Positron emission tomography/computed tomography (PET/CT) with 68 Ga-PSMA is a non-invasive diagnostic technique to image prostate cancer with increased prostate-specific membrane antigen (PSMA) expression. PSMA is a transmembrane protein present in all prostatic tissues. Increased PSMA expression is seen in several malignancies, although prostate cancer is the tumour where it presents higher concentrations. Almost all prostate adenocarcinomas show PSMA expression in most of lesions, primary and metastatic. Immunohistochemistry has demonstrated that the expression of PSMA increases in patients with de-differentiated, metastatic or hormone-refractory tumours. Moreover, the expression level of PSMA has a prognostic value for disease outcome. PET measures the three-dimensional distribution of 68 Ga-PSMA, producing semi-quantitative images that allow for non-invasive assessment of PSMA expression. Copyright © 2017 Elsevier España, S.L.U. y SEMNIM. All rights reserved.

Seroprevalences of antibodies against Bartonella henselae and Toxoplasma gondii and fecal shedding of Cryptosporidium spp, Giardia spp, and Toxocara cati in feral and pet domestic cats.

PubMed

Nutter, Felicia B; Dubey, J P; Levine, Jay F; Breitschwerdt, Edward B; Ford, Richard B; Stoskopf, Michael K

2004-11-01

To compare seroprevalences of antibodies against Bartonella henselae and Toxoplasma gondii and fecal shedding of Cryptosporidium spp, Giardia spp, and Toxocara cati in feral and pet domestic cats. Prospective cross-sectional serologic and coprologic survey. 100 feral cats and 76 pet domestic cats from Randolph County, NC. Blood and fecal samples were collected and tested. Percentages of feral cats seropositive for antibodies against B. henselae and T. gondii (93% and 63%, respectively) were significantly higher than percentages of pet cats (75% and 34%). Percentages of feral and pet cats with Cryptosporidium spp (7% of feral cats; 6% of pet cats), Giardia spp (6% of feral cats; 5% of pet cats), and T. cati ova (21% of feral cats; 18% of pet cats) in their feces were not significantly different between populations. Results of CBCs and serum biochemical analyses were not significantly different between feral and pet cats, except that feral cats had a significantly lower median PCV and significantly higher median neutrophil count. Results suggested that feral and pet cats had similar baseline health status, as reflected by results of hematologic and serum biochemical testing and similar prevalences of infection with Cryptosporidium spp, Giardia spp, and T. cati. Feral cats did have higher seroprevalences of antibodies against B. henselae and T. gondii than did pet cats, but this likely was related to greater exposure to vectors of these organisms.

68Ga-PSMA PET-CT Imaging of Metastatic Adenoid Cystic Carcinoma.

PubMed

de Keizer, Bart; Krijger, Gerard C; Ververs, F Tessa; van Es, Robert J J; de Bree, Remco; Willems, Stefan

2017-12-01

A patient with a history of adenoid cystic carcinoma of the nasal cavity presented himself with bone pain and an elevated PSA level. On suspicion of metastatic prostate cancer a 68 Ga-PSMA PET-CT was performed. The PET-CT showed numerous lung and non-sclerotic bone metastasis. Biopsy of a bone metastasis was performed and pathology showed adenoid cystic carcinoma instead of prostate cancer. Immunohistochemical PSMA staining of the primary tumour showed intense PSMA expression in adenoid cystic carcinoma tumour cells. Because of the high PSMA expression of adenoid cystic carcinoma, 68 Ga-PSMA PET-CT might be a promising imaging modality for this malignancy.

Polymeric nanoparticle PET/MR imaging allows macrophage detection in atherosclerotic plaques

PubMed Central

Majmudar, Maulik D.; Yoo, Jeongsoo; Keliher, Edmund J.; Truelove, Jessica; Iwamoto, Yoshiko; Sena, Brena; Dutta, Partha; Borodovsky, Anna; Fitzgerald, Kevin; Di Carli, Marcelo; Libby, Peter; Anderson, Daniel G.; Swirski, Filip K.; Weissleder, Ralph; Nahrendorf, Matthias

2013-01-01

Rationale Myeloid cell content in atherosclerotic plaques associates with rupture and thrombosis. Thus, imaging of lesional monocyte and macrophages (Mo/Mϕ) could serve as a biomarker of disease progression and therapeutic intervention. Objective To noninvasively assess plaque inflammation with dextran nanoparticle-facilitated hybrid PET/MR imaging. Methods and Results Using clinically approved building blocks, we systematically developed 13nm polymeric nanoparticles consisting of crosslinked short chain dextrans which were modified with desferoxamine for zirconium-89 radiolabeling (89Zr-DNP) and a near infrared fluorochrome (VT680) for microscopic and cellular validation. Flow cytometry of cells isolated from excised aortas showed DNP uptake predominantly in Mo/Mϕ (76.7%) and lower signal originating from other leukocytes such as neutrophils and lymphocytes (11.8% and 0.7%, p<0.05 versus Mo/Mϕ). DNP colocalized with the myeloid cell marker CD11b on immunohistochemistry. PET/MRI revealed high uptake of 89Zr-DNP in the aortic root of ApoE−/− mice (standard uptake value, ApoE−/− mice versus wild type controls, 1.9±0.28 versus 1.3±0.03, p<0.05), corroborated by ex vivo scintillation counting and autoradiography. Therapeutic silencing of the monocyte-recruiting receptor CCR2 with siRNA decreased 89Zr-DNP plaque signal (p<0.05) and inflammatory gene expression (p<0.05). Conclusions Hybrid PET/MR imaging with a 13nm DNP enables noninvasive assessment of inflammation in experimental atherosclerotic plaques and reports on therapeutic efficacy of anti-inflammatory therapy. PMID:23300273

Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.

PubMed

Li, Bao-Cun; Zhang, Shuang-Quan; Dan, Wen-Bing; Chen, Yu-Qing; Cao, Peng

2007-07-01

The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

The Bartonella quintana Extracytoplasmic Function Sigma Factor RpoE Has a Role in Bacterial Adaptation to the Arthropod Vector Environment

PubMed Central

Abromaitis, Stephanie

2013-01-01

Bartonella quintana is a vector-borne bacterial pathogen that causes fatal disease in humans. During the infectious cycle, B. quintana transitions from the hemin-restricted human bloodstream to the hemin-rich body louse vector. Because extracytoplasmic function (ECF) sigma factors often regulate adaptation to environmental changes, we hypothesized that a previously unstudied B. quintana ECF sigma factor, RpoE, is involved in the transition from the human host to the body louse vector. The genomic context of B. quintana rpoE identified it as a member of the ECF15 family of sigma factors found only in alphaproteobacteria. ECF15 sigma factors are believed to be the master regulators of the general stress response in alphaproteobacteria. In this study, we examined the B. quintana RpoE response to two stressors that are encountered in the body louse vector environment, a decreased temperature and an increased hemin concentration. We determined that the expression of rpoE is significantly upregulated at the body louse (28°C) versus the human host (37°C) temperature. rpoE expression also was upregulated when B. quintana was exposed to high hemin concentrations. In vitro and in vivo analyses demonstrated that RpoE function is regulated by a mechanism involving the anti-sigma factor NepR and the response regulator PhyR. The ΔrpoE ΔnepR mutant strain of B. quintana established that RpoE-mediated transcription is important in mediating the tolerance of B. quintana to high hemin concentrations. We present the first analysis of an ECF15 sigma factor in a vector-borne human pathogen and conclude that RpoE has a role in the adaptation of B. quintana to the hemin-rich arthropod vector environment. PMID:23564167

Therapeutic implications of continuing bonds expressions following the death of a pet.

PubMed

Packman, Wendy; Carmack, Betty J; Ronen, Rama

Through the exploration of 12 continuing bonds expressions (CBE), this current study investigated the grief reaction and continuing impact of the death of a pet. Thirty-three individuals were interviewed to determine the degree of connection maintained with the deceased pet and how that affects their coping. Findings emphasize that the majority of respondents frequently maintain ongoing meaningful ties with their deceased pet through the use of CBE such as fond memories, rituals, dreams. The findings suggest that it is not the number of CBE but the degree of adaptability that is significant. The importance of recognizing the unique, total experience of those grieving the death of a pet is addressed. Implications for those working with and supporting those in grief are included. Future directions for research are described.

Development of novel types of plastid transformation vectors and evaluation of factors controlling expression.

PubMed

Herz, Stefan; Füssl, Monika; Steiger, Sandra; Koop, Hans-Ulrich

2005-12-01

Two new vector types for plastid transformation were developed and uidA reporter gene expression was compared to standard transformation vectors. The first vector type does not contain any plastid promoter, instead it relies on extension of existing plastid operons and was therefore named "operon-extension" vector. When a strongly expressed plastid operon like psbA was extended by the reporter gene with this vector type, the expression level was superior to that of a standard vector under control of the 16S rRNA promoter. Different insertion sites, promoters and 5'-UTRs were analysed for their effect on reporter gene expression with standard and operon-extension vectors. The 5'-UTR of phage 7 gene 10 in combination with a modified N-terminus was found to yield the highest expression levels. Expression levels were also strongly dependent on external factors like plant or leaf age or light intensity. In the second vector type, named "split" plastid transformation vector, modules of the expression cassette were distributed on two separate vectors. Upon co-transformation of plastids with these vectors, the complete expression cassette became inserted into the plastome. This result can be explained by successive co-integration of the split vectors and final loop-out recombination of the duplicated sequences. The split vector concept was validated with different vector pairs.

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants.

PubMed

Nizampatnam, Narasimha Rao; Doodhi, Harinath; Kalinati Narasimhan, Yamini; Mulpuri, Sujatha; Viswanathaswamy, Dinesh Kumar

2009-03-01

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

PubMed

Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

2012-04-01

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

[Microeconomics of introduction of a PET system based on the revised Japanese National Insurance reimbursement system].

PubMed

Abe, Katsumi; Kosuda, Shigeru; Kusano, Shoichi; Nagata, Masayoshi

2003-11-01

It is crucial to evaluate an annual balance before-hand when an institution installs a PET system because the revised Japanese national insurance reimbursement system set the cost of a FDG PET study as 75,000 yen. A break-even point was calculated in an 8-hour or a 24-hour operation of a PET system, based on the total costs reported. The break-even points were as follows: 13.4, 17.7, 22.1 studies per day for the 1 cyclotron-1 PET camera, 1 cyclotron-2 PET cameras, 1 cyclotron-3 PET cameras system, respectively, in an ordinary PET system operation of 8 hours. The break-even points were 19.9, 25.5, 31.2 studies per day for the 1 cyclotron-1 PET camera, 1 cyclotron-2 PET cameras, 1 cyclotron-3 PET cameras system, respectively, in a full PET system operation of 24 hours. The results indicate no profit would accrue in an ordinary PET system operation of 8 hours. The annual profit and break-even point for the total cost including the initial investment would be respectively 530 million yen and 2.8 years in a 24-hour operation with 1 cyclotron-3 PET cameras system.

Imaging Transgene Expression with Radionuclide Imaging Technologies1

PubMed Central

Gambhir, SS; Herschman, HR; Cherry, SR; Barrio, JR; Satyamurthy, N; Toyokuni, T; Phelps, ME; Larson, SM; Balaton, J; Finn, R; Sadelain, M; Tjuvajev, J

2000-01-01

Abstract A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (micro PET) designed specifically for studies of small animals. We review “marker/reporter gene” imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications. PMID:10933072

Impact of Endoscopic Ultrasonography on 18F-FDG-PET/CT Upfront Towards Patient Specific Esophageal Cancer Treatment.

PubMed

Hulshoff, J B; Mul, V E M; de Boer, H E M; Noordzij, W; Korteweg, T; van Dullemen, H M; Nagengast, W B; Oppedijk, V; Pierie, J P E N; Plukker, John Th M

2017-07-01

In patients with potentially resectable esophageal cancer (EC), the value of endoscopic ultrasonography (EUS) after fluorine-18 labeled fluorodeoxyglucose positron emission tomography with computed tomography ( 18 F-FDG-PET/CT) is questionable. Retrospectively, we assessed the impact of EUS after PET/CT on the given treatment in EC patients. During the period 2009-2015, 318 EC patients were staged as T1-4aN0-3M0 with hybrid 18 F-FDG-PET/CT or 18 F-FDG-PET with CT and EUS if applicable in a nonspecific order. We determined the impact of EUS on the given treatment in 279 patients who also were staged with EUS. EUS had clinical consequences if it changed curability, extent of radiation fields or lymph node resection (AJCC stations 2-5), and when the performed fine-needle aspiration (FNA) provided conclusive information of suspicious lymph node. EUS had an impact in 80 (28.7%) patients; it changed the radiation field in 63 (22.6%), curability in 5 (1.8%), lymphadenectomy in 48 (17.2%), and FNA was additional in 21 (7.5%). In patients treated with nCRT (n = 194), EUS influenced treatment in 53 (27.3%) patients; in 38 (19.6%) the radiation field changed, in 3 (1.5%) the curability, in 35 (18.0%) the lymphadenectomy, and in 17 (8.8%) FNA was additional. EUS influenced both the extent of radiation field and nodal resection in 31 (16.0%) nCRT patients. EUS had an impact on the given treatment in approximately 29%. In most patients, the magnitude of EUS found expression in the extent of radiotherapy target volume delineation to upper/high mediastinal lymph nodes.

Comparison of hybrid (68)Ga-PSMA PET/MRI and (68)Ga-PSMA PET/CT in the evaluation of lymph node and bone metastases of prostate cancer.

PubMed

Freitag, Martin T; Radtke, Jan P; Hadaschik, Boris A; Kopp-Schneider, A; Eder, Matthias; Kopka, Klaus; Haberkorn, Uwe; Roethke, Matthias; Schlemmer, Heinz-Peter; Afshar-Oromieh, Ali

2016-01-01

To evaluate the reproducibility of the combination of hybrid PET/MRI and the (68)Ga-PSMA-11 tracer in depicting lymph node (LN) and bone metastases of prostate cancer (PC) in comparison with that of PET/CT. A retrospective analysis of 26 patients who were subjected to (68)Ga-PSMA PET/CTlow-dose (1 h after injection) followed by PET/MRI (3 h after injection) was performed. MRI sequences included T1-w native, T1-w contrast-enhanced, T2-w fat-saturated and diffusion-weighted sequences (DWIb800). Discordant PET-positive and morphological findings were evaluated. Standardized uptake values (SUV) of PET-positive LNs and bone lesions were quantified and their morphological size and conspicuity determined. Comparing the PET components, the proportion of discordant PSMA-positive suspicious findings was very low (98.5 % of 64 LNs concordant, 100 % of 28 bone lesions concordant). Two PET-positive bone metastases could not be confirmed morphologically using CTlow-dose, but could be confirmed using MRI. In 12 of 20 patients, 47 PET-positive LNs (71.9 %) were smaller than 1 cm in short axis diameter. There were significant linear correlations between PET/MRI SUVs and PET/CT SUVs in the 64 LN metastases (p < 0.0001) and in the 28 osseous metastases (p < 0.0001) for SUVmean and SUVmax, respectively. The LN SUVs were significantly higher on PET/MRI than on PET/CT (p SUVmax < 0.0001; p SUVmean < 0.0001) but there was no significant difference between the bone lesion SUVs (p SUVmax = 0.495; p SUVmean = 0.381). Visibility of LNs was significantly higher on MRI using the T1-w contrast-enhanced fat-saturated sequence (p = 0.013), the T2-w fat-saturated sequence (p < 0.0001) and the DWI sequence (p < 0.0001) compared with CTlow-dose. For bone lesions, only the overall conspicuity was higher on MRI compared with CTlow-dose (p < 0.006). Nodal and osseous metastases of PC are accurately and reliably depicted by hybrid PET/MRI using (68)Ga-PSMA-11 with very low discordance compared with PET/CT including PET-positive LNs of normal size. The correlation between PET/MRI SUVs and PET/CT SUVs was linear in LN and bone metastases but was significantly lower in control (non-metastatic) tissue.

Can Imaging Parameters Provide Information Regarding Histopathology in Head and Neck Squamous Cell Carcinoma? A Meta-Analysis.

PubMed

Surov, Alexey; Meyer, Hans Jonas; Wienke, Andreas

2018-04-01

Our purpose was to provide data regarding relationships between different imaging and histopathological parameters in HNSCC. MEDLINE library was screened for associations between different imaging parameters and histopathological features in HNSCC up to December 2017. Only papers containing correlation coefficients between different imaging parameters and histopathological findings were acquired for the analysis. Associations between 18 F-FDG positron emission tomography (PET) and KI 67 were reported in 8 studies (236 patients). The pooled correlation coefficient was 0.20 (95% CI = [-0.04; 0.44]). Furthermore, in 4 studies (64 patients), associations between 18 F-fluorothymidine PET and KI 67 were analyzed. The pooled correlation coefficient between SUV max and KI 67 was 0.28 (95% CI = [-0.06; 0.94]). In 2 studies (23 patients), relationships between KI 67 and dynamic contrast-enhanced magnetic resonance imaging were reported. The pooled correlation coefficient between K trans and KI 67 was -0.68 (95% CI = [-0.91; -0.44]). Two studies (31 patients) investigated correlation between apparent diffusion coefficient (ADC) and KI 67. The pooled correlation coefficient was -0.61 (95% CI = [-0.84; -0.38]). In 2 studies (117 patients), relationships between 18 F-FDG PET and p53 were analyzed. The pooled correlation coefficient was 0.0 (95% CI = [-0.87; 0.88]). There were 3 studies (48 patients) that investigated associations between ADC and tumor cell count in HNSCC. The pooled correlation coefficient was -0.53 (95% CI = [-0.74; -0.32]). Associations between 18 F-FDG PET and HIF-1α were investigated in 3 studies (72 patients). The pooled correlation coefficient was 0.44 (95% CI = [-0.20; 1.08]). ADC may predict cell count and proliferation activity, and SUV max may predict expression of HIF-1α in HNSCC. SUV max cannot be used as surrogate marker for expression of KI 67 and p53. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Radiomic biomarkers from PET/CT multi-modality fusion images for the prediction of immunotherapy response in advanced non-small cell lung cancer patients

NASA Astrophysics Data System (ADS)

Mu, Wei; Qi, Jin; Lu, Hong; Schabath, Matthew; Balagurunathan, Yoganand; Tunali, Ilke; Gillies, Robert James

2018-02-01

Purpose: Investigate the ability of using complementary information provided by the fusion of PET/CT images to predict immunotherapy response in non-small cell lung cancer (NSCLC) patients. Materials and methods: We collected 64 patients diagnosed with primary NSCLC treated with anti PD-1 checkpoint blockade. Using PET/CT images, fused images were created following multiple methodologies, resulting in up to 7 different images for the tumor region. Quantitative image features were extracted from the primary image (PET/CT) and the fused images, which included 195 from primary images and 1235 features from the fusion images. Three clinical characteristics were also analyzed. We then used support vector machine (SVM) classification models to identify discriminant features that predict immunotherapy response at baseline. Results: A SVM built with 87 fusion features and 13 primary PET/CT features on validation dataset had an accuracy and area under the ROC curve (AUROC) of 87.5% and 0.82, respectively, compared to a model built with 113 original PET/CT features on validation dataset 78.12% and 0.68. Conclusion: The fusion features shows better ability to predict immunotherapy response prediction compared to individual image features.

Correlation between PET/CT parameters and KRAS expression in colorectal cancer.

PubMed

Chen, Shang-Wen; Chiang, Hua-Che; Chen, William Tzu-Liang; Hsieh, Te-Chun; Yen, Kuo-Yang; Chiang, Shu-Fen; Kao, Chia-Hung

2014-08-01

The objective of this study was to correlate the association between mutated KRAS and wild-type colorectal cancer (CRC) by using various F-FDG PET-related parameters. One hundred twenty-one CRC patients who had undergone preoperative PET/CT were included in this study. Several PET/CT-related parameters, including SUVmax and various thresholds of metabolic tumor volume, total lesion glycolysis, and PET/CT-based tumor width, were measured. Tumor- and PET/CT-related parameters were correlated with genomic expression between KRAS mutant and wild-type groups, using a Mann-Whitney U test and logistic regression analysis. Colorectal cancer tumors with a mutated KRAS exhibited higher SUVmax and an increased accumulation of FDG among several threshold methods. Multivariate analysis showed that SUVmax and using a 40% threshold level for maximal uptake of TW (TW40%) were the 2 predictors of KRAS mutations. The odds ratio was 1.23 for SUVmax (P = 0.02; 95% confidence interval, 1.01-1.52) and 1.15 for TW40% (P = 0.02; 95% confidence interval, 1.02-1.30). The accuracy of SUVmax for predicting mutated KRAS was higher in patients with colon or sigmoid colon cancers, whereas it was TW40% in those with rectal cancers. SUVmax and TW40% were associated in CRC with KRAS mutations. PET/CT parameters can supplement genomic analysis to determine KRAS expression in CRC.

Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.

PubMed

Hadj Sassi, Azza; Trigui-Lahiani, Hèla; Abdeljalil, Salma; Gargouri, Ali

2017-02-01

Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form. Copyright © 2016 Elsevier B.V. All rights reserved.

Inter-crystal scatter identification for a depth-sensitive detector using support vector machine for small animal positron emission tomography

NASA Astrophysics Data System (ADS)

Yoshida, Eiji; Kitamura, Keishi; Kimura, Yuichi; Nishikido, Fumihiko; Shibuya, Kengo; Yamaya, Taiga; Murayama, Hideo

2007-02-01

In a conventional positron emission tomography (PET) detector, detected events are projected onto a 2D position histogram by an Anger calculation for crystal identification. However, the measured histogram is affected by inter-crystal scatterings (ICS) which occur in the entire detector. Peaks which are projected for each crystal in the histogram are blurred, and this causes ICS mispositioning. A depth-of-interaction (DOI) detector has been developed for the small animal PET scanner jPET-RD. This DOI detector uses 32×32 crystals with four layers and a 256-channel multi-anode flat panel photomultiplier tube (FP-PMT) which was developed by Hamamatsu Photonics K.K. Each crystal element is 1.45×1.45×4.5 mm 3. The FP-PMT has a large detective area (49×49 mm 2) and a small anode pitch (3.04 mm). Therefore, the FP-PMT can extensively trace the behavior of incident γ rays in the crystals including ICS event. We, therefore, propose a novel method for ICS estimation using a statistical pattern recognition algorithm based on a support vector machine (SVM). In this study, we applied the SVM for discriminating photoelectric events from ICS events generated from multiple-anode outputs. The SVM was trained by uniform irradiation events generated from a detector simulator using a Monte Carlo calculation. The success rate for ICS event identification is about 78% for non-training data. The SVM can achieve a true subtraction of ICS events from measured events, and it is also useful for random correction in PET.

MAX Mutations in Endometrial Cancer: Clinicopathologic Associations and Recurrent MAX p.His28Arg Functional Characterization.

PubMed

Walker, Christopher J; Rush, Craig M; Dama, Paola; O'Hern, Matthew J; Cosgrove, Casey M; Gillespie, Jessica L; Zingarelli, Roman A; Smith, Blair; Stein, Maggie E; Mutch, David G; Shakya, Reena; Chang, Chia-Wen; Selvendiran, Karuppaiyah; Song, Jonathan W; Cohn, David E; Goodfellow, Paul J

2018-05-01

Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes. MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine pro-angiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided. Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio = 4.00, 95% confidence interval = 1.15 to 13.91, P = .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P = .003 and P = .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P = .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs. These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.

Reproducibility of functional volume and activity concentration in 18F-FDG PET/CT of liver metastases in colorectal cancer.

PubMed

Heijmen, Linda; de Geus-Oei, Lioe-Fee; de Wilt, Johannes H W; Visvikis, Dimitris; Hatt, Mathieu; Visser, Eric P; Bussink, Johan; Punt, Cornelis J A; Oyen, Wim J G; van Laarhoven, Hanneke W M

2012-12-01

Several studies showed potential for monitoring response to systemic therapy in metastatic colorectal cancer patients with (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Before (18)F-FDG PET can be implemented for response evaluation the repeatability should be known. This study was performed to assess the magnitude of the changes in standardized uptake value (SUV), volume and total lesion glycolysis (TLG) in colorectal liver metastases and validate the biological basis of (18)F-FDG PET in colorectal liver metastases. Twenty patients scheduled for liver metastasectomy underwent two (18)F-FDG PET scans within 1 week. Bland-Altman analysis was performed to assess repeatability of SUV(max), SUV(mean), volume and TLG. Tumours were delineated using an adaptive threshold method (PET(SBR)) and a semiautomatic fuzzy locally adaptive Bayesian (FLAB) delineation method. Coefficient of repeatability of SUV(max) and SUV(mean) were ∼39 and ∼31 %, respectively, independent of the delineation method used and image reconstruction parameters. However, repeatability was worse in recently treated patients. The FLAB delineation method improved the repeatability of the volume and TLG measurements compared to PET(SBR), from coefficients of repeatability of over 85 % to 45 % and 57 % for volume and TLG, respectively. Glucose transporter 1 (GLUT1) expression correlated to the SUV(mean). Vascularity (CD34 expression) and tumour hypoxia (carbonic anhydrase IX expression) did not correlate with (18)F-FDG PET parameters. In conclusion, repeatability of SUV(mean) and SUV(max) was mainly affected by preceding systemic therapy. The repeatability of tumour volume and TLG could be improved using more advanced and robust delineation approaches such as FLAB, which is recommended when (18)F-FDG PET is utilized for volume or TLG measurements. Improvement of repeatability of PET measurements, for instance by dynamic PET scanning protocols, is probably necessary to effectively use PET for early response monitoring.

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

PubMed Central

Hsu, Cary; Jones, Stephanie A.; Cohen, Cyrille J.; Zheng, Zhili; Kerstann, Keith; Zhou, Juhua; Robbins, Paul F.; Peng, Peter D.; Shen, Xinglei; Gomes, Theotonius J.; Dunbar, Cynthia E.; Munroe, David J.; Stewart, Claudia; Cornetta, Kenneth; Wangsa, Danny; Ried, Thomas; Rosenberg, Steven A.

2007-01-01

Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8+ T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus–based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28−, CD45RA−, CD45RO+, and CD62L−, a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen–specific T-cell receptors, the clone secreted IFN-γ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15Rα expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation. PMID:17353346

Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.

PubMed

Chen, Gen-Hung; Yin, Li-Jung; Chiang, I-Hua; Jiang, Shann-Tzong

2008-12-01

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

Stress-altered synaptic plasticity and DAMP signaling in the hippocampus-PFC axis; elucidating the significance of IGF-1/IGF-1R/CaMKIIα expression in neural changes associated with a prolonged exposure therapy.

PubMed

Ogundele, Olalekan M; Ebenezer, Philip J; Lee, Charles C; Francis, Joseph

2017-06-14

Traumatic stress patients showed significant improvement in behavior after a prolonged exposure to an unrelated stimulus. This treatment method attempts to promote extinction of the fear memory associated with the initial traumatic experience. However, the subsequent prolonged exposure to such stimulus creates an additional layer of neural stress. Although the mechanism remains unclear, prolonged exposure therapy (PET) likely involves changes in synaptic plasticity, neurotransmitter function and inflammation; especially in parts of the brain concerned with the formation and retrieval of fear memory (Hippocampus and Prefrontal Cortex: PFC). Since certain synaptic proteins are also involved in danger-associated molecular pattern signaling (DAMP), we identified the significance of IGF-1/IGF-1R/CaMKIIα expression as a potential link between the concurrent progression of synaptic and inflammatory changes in stress. Thus, a comparison between IGF-1/IGF-1R/CaMKIIα, synaptic and DAMP proteins in stress and PET may highlight the significance of PET on synaptic morphology and neuronal inflammatory response. In behaviorally characterized Sprague-Dawley rats, there was a significant decline in neural IGF-1 (p<0.001), hippocampal (p<0.001) and cortical (p<0.05) IGF-1R expression. These animals showed a significant loss of presynaptic markers (synaptophysin; p<0.001), and changes in neurotransmitters (VGLUT2, Tyrosine hydroxylase, GABA, ChAT). Furthermore, naïve stressed rats recorded a significant decrease in post-synaptic marker (PSD-95; p<0.01) and synaptic regulator (CaMKIIα; p<0.001). As part of the synaptic response to a decrease in brain CaMKIIα, small ion conductance channel (KCa2.2) was upregulated in the brain of naïve stressed rats (p<0.01). After a PET, an increase in IGF-1 (p<0.05) and IGF-1R was recorded in the Stress-PET group (p<0.001). As such, hippocampal (p<0.001), but not cortical (ns) synaptophysin expression increased in Stress-PET. Although PSD-95 was relatively unchanged in the hippocampus and PFC, CaMKIIα (p<0.001) and KCa2.2 (p<0.01) were upregulated in Stress-PET, and may be involved in extinction of fear memory-related synaptic potentials. These changes were also associated with a normalized neurotransmitter function, and a significant reduction in open space avoidance; when the animals were assessed in elevated plus maze (EPM). In addition to a decrease in IGF-1/IGF-1R, an increase in activated hippocampal and cortical microglia was seen in stress (p<0.05) and after a PET (Stress-PET; p<0.001). Furthermore, this was linked with a significant increase in HMGB1 (Hippocampus: p<0.001, PFC: p<0.05) and TLR4 expression (Hippocampus: p<0.01; PFC: ns) in the neurons. Taken together, this study showed that traumatic stress and subsequent PET involves an event-dependent alteration of IGF1/IGF-1R/CaMKIIα. Firstly, we showed a direct relationship between IGF-1/IGF-1R expression, presynaptic function (synaptophysin) and neurotransmitter activity in stress and PET. Secondly, we identified the possible role of CaMKIIα in post-synaptic function and regulation of small ion conductance channels. Lastly, we highlighted some of the possible links between IGF1/IGF-1R/CaMKIIα, the expression of DAMP proteins, Microglia activation, and its implication on synaptic plasticity during stress and PET. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Using CT Data to Improve the Quantitative Analysis of 18F-FBB PET Neuroimages

PubMed Central

Segovia, Fermín; Sánchez-Vañó, Raquel; Górriz, Juan M.; Ramírez, Javier; Sopena-Novales, Pablo; Testart Dardel, Nathalie; Rodríguez-Fernández, Antonio; Gómez-Río, Manuel

2018-01-01

18F-FBB PET is a neuroimaging modality that is been increasingly used to assess brain amyloid deposits in potential patients with Alzheimer's disease (AD). In this work, we analyze the usefulness of these data to distinguish between AD and non-AD patients. A dataset with 18F-FBB PET brain images from 94 subjects diagnosed with AD and other disorders was evaluated by means of multiple analyses based on t-test, ANOVA, Fisher Discriminant Analysis and Support Vector Machine (SVM) classification. In addition, we propose to calculate amyloid standardized uptake values (SUVs) using only gray-matter voxels, which can be estimated using Computed Tomography (CT) images. This approach allows assessing potential brain amyloid deposits along with the gray matter loss and takes advantage of the structural information provided by most of the scanners used for PET examination, which allow simultaneous PET and CT data acquisition. The results obtained in this work suggest that SUVs calculated according to the proposed method allow AD and non-AD subjects to be more accurately differentiated than using SUVs calculated with standard approaches. PMID:29930505

(124)I-iodopyridopyrimidinone for PET of Abl kinase-expressing tumors in vivo.

PubMed

Doubrovin, Mikhail; Kochetkova, Tatiana; Santos, Elmer; Veach, Darren R; Smith-Jones, Peter; Pillarsetty, Nagavarakishore; Balatoni, Julius; Bornmann, William; Gelovani, Juri; Larson, Steven M

2010-01-01

Because of the recent development of an iodopyridopyrimidinone Abl protein kinase inhibitor (PKI), (124)I-SKI-212230 ((124)I-SKI230), we investigated the feasibility of a PET-based molecular imaging method for the direct visualization of Abl kinase expression and PKI treatment. In vitro pharmacokinetic properties, including specific and nonspecific binding of (124)I-SKI230 to its Abl kinase target and interaction with other PKIs, were assessed in cell-free medium and chronic myelogenous leukemia (CML) cells overexpressing BCR-Abl (K562), in comparison with BT-474 cells that are low in Abl expression. In a xenograft tumor model, we assessed the in vivo pharmacokinetics of (124)I-SKI230 using PET and postmortem tissue sampling. We also tested a paradigm of (124)I-SKI230 PET after treatment of the animal with a dose of Abl-specific PKI for the monitoring of the tumor response. In vitro studies confirmed that SKI230 binds to Abl kinase with nanomolar affinity, that selective uptake occurs in cell lines known to express Abl kinase, that RNAi knock-down supports specificity of cellular uptake due to Abl kinase, and that imatinib, an archetype Abl PKI, completely displaces SKI230. With SKI230, we obtained successful in vivo PET of Abl-expressing human tumors in a nude rat. We were also able to demonstrate evidence of substrate inhibition of in vivo radiotracer uptake in the xenograft tumor after treatment of the animal as a model of PKI treatment monitoring. These results support the hypothesis that molecular imaging using PET will be useful for the study of in vivo pharmacodynamics of Abl PKI molecular therapy in humans.

[Prokaryotic Expression and Purification of the Notch Ligand and Its Effect on Hematopoiesis after Carbon Tetrachloride Damage].

PubMed

Chen, Juan-Juan; Huang, Si-Yong; Ma, Peng-Fei; Wu, Bi-Jia; Zhou, Si-Lang; Zhao, Yong-Xing; Gong, Jun-Mei; Liang, Ying-Min

2018-04-01

To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni 2+ -beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The Lin - Scal-1 + c-Kit + cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10. The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury. A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.

Prostate-Specific Membrane Antigen Expression in Distal Radius Fracture.

PubMed

Hoberück, Sebastian; Michler, Enrico; Kaiser, Daniel; Röhnert, Anne; Zöphel, Klaus; Kotzerke, Jörg

2018-06-12

A 79-year old man with prostate cancer under active surveillance for 5 years was referred for a PSMA-PET/MRI for re-evaluation because of a rising prostate-specific antigen value. PET/MRI revealed a ribbonlike tracer accumulation in a healing fracture of the distal radius. This case illustrates that PSMA expression may occur in healing bone fractures in the distal radius. It can be assumed that benign causes of tracer accumulations in the upper extremities are missed in PET/CT due to elevated position of the arms during image acquisition.

Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.).

PubMed

Elhag, Osama; Zhou, Dingzhong; Song, Qi; Soomro, Abdul Aziz; Cai, Minmin; Zheng, Longyu; Yu, Ziniu; Zhang, Jibin

2017-01-01

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.)

PubMed Central

Elhag, Osama; Zhou, Dingzhong; Song, Qi; Soomro, Abdul Aziz; Cai, Minmin; Zheng, Longyu; Yu, Ziniu; Zhang, Jibin

2017-01-01

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens. PMID:28056070

In vitro evaluation of the monoclonal antibody 64Cu-IgG M75 against human carbonic anhydrase IX and its in vivo imaging.

PubMed

Čepa, Adam; Ráliš, Jan; Král, Vlastimil; Paurová, Monika; Kučka, Jan; Humajová, Jana; Lázníček, Milan; Lebeda, Ondřej

2018-03-01

Specific oncology diagnostics requires new types of the selective radiopharmaceuticals, particularly those suitable for the molecular PET imaging. The aim of this work is to present a new, specific PET-immunodiagnostic radiopharmaceutical based on the monoclonal antibody IgG M75 targeting human carbonic anhydrase IX labelled with 64 Cu (T ½ = 12.70h) and its in vitro and in vivo evaluation. The antibody IgG M75 was conjugated with a non-commercial copper-specific chelator "phosphinate" and then labelled with the positron emitter 64 Cu. Stability of the labelled conjugated was tested in human serum. The immunoreactivity of the labelled conjugate was evaluated in vitro on a suitable cell cultures of the colorectal carcinoma (HT-29) and its imaging properties were estimated in vivo on a mouse model with inoculated colorectal carcinoma HT-29 imaged on a µPET/CT. The tested radioimmunoconjugate was obtained in a specific activity of 0.25-0.5 MBq/µg. In vitro uptake experiments revealed specific binding to the HT-29 cells (45 ± 2.8% of the total added activity) and the measured K D value was found to be 9.2nM. Imaging clearly demonstrated significant uptake of the labelled monoclonal antibody in the tumour at 18h post administration. The radioimmunoconjugate 64 Cu-PS-IgG M75 seems to be a suitable candidate for PET diagnostics of hypoxic tumours expressing human carbonic anhydrase IX. Copyright © 2017 Elsevier Ltd. All rights reserved.

Attenuation correction in 4D-PET using a single-phase attenuation map and rigidity-adaptive deformable registration

PubMed Central

Kalantari, Faraz; Wang, Jing

2017-01-01

Purpose Four-dimensional positron emission tomography (4D-PET) imaging is a potential solution to the respiratory motion effect in the thoracic region. Computed tomography (CT)-based attenuation correction (AC) is an essential step toward quantitative imaging for PET. However, due to the temporal difference between 4D-PET and a single attenuation map from CT, typically available in routine clinical scanning, motion artifacts are observed in the attenuation-corrected PET images, leading to errors in tumor shape and uptake. We introduced a practical method to align single-phase CT with all other 4D-PET phases for AC. Methods A penalized non-rigid Demons registration between individual 4D-PET frames without AC provides the motion vectors to be used for warping single-phase attenuation map. The non-rigid Demons registration was used to derive deformation vector fields (DVFs) between PET matched with the CT phase and other 4D-PET images. While attenuated PET images provide useful data for organ borders such as those of the lung and the liver, tumors cannot be distinguished from the background due to loss of contrast. To preserve the tumor shape in different phases, an ROI-covering tumor was excluded from non-rigid transformation. Instead the mean DVF of the central region of the tumor was assigned to all voxels in the ROI. This process mimics a rigid transformation of the tumor along with a non-rigid transformation of other organs. A 4D-XCAT phantom with spherical lung tumors, with diameters ranging from 10 to 40 mm, was used to evaluate the algorithm. The performance of the proposed hybrid method for attenuation map estimation was compared to 1) the Demons non-rigid registration only and 2) a single attenuation map based on quantitative parameters in individual PET frames. Results Motion-related artifacts were significantly reduced in the attenuation-corrected 4D-PET images. When a single attenuation map was used for all individual PET frames, the normalized root mean square error (NRMSE) values in tumor region were 49.3% (STD: 8.3%), 50.5% (STD: 9.3%), 51.8% (STD: 10.8%) and 51.5% (STD: 12.1%) for 10-mm, 20-mm, 30-mm and 40-mm tumors respectively. These errors were reduced to 11.9% (STD: 2.9%), 13.6% (STD: 3.9%), 13.8% (STD: 4.8%), and 16.7% (STD: 9.3%) by our proposed method for deforming the attenuation map. The relative errors in total lesion glycolysis (TLG) values were −0.25% (STD: 2.87%) and 3.19% (STD: 2.35%) for 30-mm and 40-mm tumors respectively in proposed method. The corresponding values for Demons method were 25.22% (STD: 14.79%) and 18.42% (STD: 7.06%). Our proposed hybrid method outperforms the Demons method especially for larger tumors. For tumors smaller than 20 mm, non-rigid transformation could also provide quantitative results. Conclusion Although non-AC 4D-PET frames include insignificant anatomical information, they are still useful to estimate the DVFs to align the attenuation map for accurate AC. The proposed hybrid method can recover the AC-related artifacts and provide quantitative AC-PET images. PMID:27987223

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria.

PubMed

Klocke, Michael; Mundt, Kerstin; Idler, Frank; Jung, Sabrina; Backhausen, Jan E

2005-06-01

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.

Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants.

PubMed

Xu, Li; Ye, Rongjian; Zheng, Yusheng; Wang, Zhekui; Zhou, Peng; Lin, Yongjun; Li, Dongdong

2010-09-01

As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5'-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the -817 bp and -453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli.

PubMed

Cao, Wei; Zhou, Yuxun; Ma, Yushu; Luo, Qingping; Wei, Dongzhi

2005-04-01

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.

[Production of recombinant human interleukin-38 and its inhibitory effect on the expression of proinflammatory cytokines in THP-1 cells].

PubMed

Yuan, X L; Li, Y; Pan, X H; Zhou, M; Gao, Q Y; Li, M C

2016-01-01

Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-β-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/μg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1β, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical applications.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed Central

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors. Images PMID:2404285

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Investigation of spatial resolution improvement by use of a mouth-insert detector in the helmet PET scanner.

PubMed

Ahmed, Abdella M; Tashima, Hideaki; Yamaya, Taiga

2018-03-01

The dominant factor limiting the intrinsic spatial resolution of a positron emission tomography (PET) system is the size of the crystal elements in the detector. To increase sensitivity and achieve high spatial resolution, it is essential to use advanced depth-of-interaction (DOI) detectors and arrange them close to the subject. The DOI detectors help maintain high spatial resolution by mitigating the parallax error caused by the thickness of the scintillator near the peripheral regions of the field-of-view. As an optimal geometry for a brain PET scanner, with high sensitivity and spatial resolution, we proposed and developed the helmet-chin PET scanner using 54 four-layered DOI detectors consisting of a 16 × 16 × 4 array of GSOZ scintillator crystals with dimensions of 2.8 × 2.8 × 7.5 mm 3 . All the detectors used in the helmet-chin PET scanner had the same spatial resolution. In this study, we conducted a feasibility study of a new add-on detector arrangement for the helmet PET scanner by replacing the chin detector with a segmented crystal cube, having high spatial resolution in all directions, which can be placed inside the mouth. The crystal cube (which we have named the mouth-insert detector) has an array of 20 × 20 × 20 LYSO crystal segments with dimensions of 1 × 1 × 1 mm 3 . Thus, the scanner is formed by the combination of the helmet and mouth-insert detectors, and is referred to as the helmet-mouth-insert PET scanner. The results show that the helmet-mouth-insert PET scanner has comparable sensitivity and improved spatial resolution near the center of the hemisphere, compared to the helmet-chin PET scanner.

Optimization of a Pretargeted Strategy for the PET Imaging of Colorectal Carcinoma via the Modulation of Radioligand Pharmacokinetics

PubMed Central

Zeglis, Brian M.; Brand, Christian; Abdel-Atti, Dalya; Carnazza, Kathryn E.; Cook, Brendon E.; Carlin, Sean; Reiner, Thomas; Lewis, Jason S.

2015-01-01

Pretargeted PET imaging has emerged as an effective strategy for merging the exquisite selectivity of antibody-based targeting vectors with the rapid pharmacokinetics of radiolabeled small molecules. We previously reported the development of a strategy for the pretargeted PET imaging of colorectal cancer based on the bioorthogonal inverse electron demand Diels–Alder reaction between a tetrazine-bearing radioligand and a transcyclooctene-modified huA33 immunoconjugate. Although this method effectively delineated tumor tissue, its clinical potential was limited by the somewhat sluggish clearance of the radioligand through the gastrointestinal tract. Herein, we report the development and in vivo validation of a pretargeted strategy for the PET imaging of colorectal carcinoma with dramatically improved pharmacokinetics. Two novel tetrazine constructs, Tz-PEG7-NOTA and Tz-SarAr, were synthesized, characterized, and radiolabeled with 64Cu in high yield (>90%) and radiochemical purity (>99%). PET imaging and biodistribution experiments in healthy mice revealed that although 64Cu-Tz-PEG7-NOTA is cleared via both the gastrointestinal and urinary tracts, 64Cu-Tz-SarAr is rapidly excreted by the renal system alone. On this basis, 64Cu-Tz-SarAr was selected for further in vivo evaluation. To this end, mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts were administered huA33-TCO, and the immunoconjugate was given 24 h to accumulate at the tumor and clear from the blood, after which 64Cu-Tz-SarAr was administered via intravenous tail vein injection. PET imaging and biodistribution experiments revealed specific uptake of the radiotracer in the tumor at early time points (5.6 ± 0.7 %ID/g at 1 h p.i.), high tumor-to-background activity ratios, and rapid elimination of unclicked radioligand. Importantly, experiments with longer antibody accumulation intervals (48 and 120 h) yielded slight decreases in tumoral uptake but also concomitant increases in tumor-to-blood activity concentration ratios. This new strategy offers dosimetric benefits as well, yielding a total effective dose of 0.041 rem/mCi, far below the doses produced by directly labeled 64Cu-NOTA-huA33 (0.133 rem/mCi) and 89Zr-DFO-huA33 (1.54 rem/mCi). Ultimately, this pretargeted PET imaging strategy boasts a dramatically improved pharmacokinetic profile compared to our first generation system and is capable of clearly delineating tumor tissue with high image contrast at only a fraction of the radiation dose created by directly labeled radioimmunoconjugates. PMID:26287993

Optimization of a Pretargeted Strategy for the PET Imaging of Colorectal Carcinoma via the Modulation of Radioligand Pharmacokinetics.

PubMed

Zeglis, Brian M; Brand, Christian; Abdel-Atti, Dalya; Carnazza, Kathryn E; Cook, Brendon E; Carlin, Sean; Reiner, Thomas; Lewis, Jason S

2015-10-05

Pretargeted PET imaging has emerged as an effective strategy for merging the exquisite selectivity of antibody-based targeting vectors with the rapid pharmacokinetics of radiolabeled small molecules. We previously reported the development of a strategy for the pretargeted PET imaging of colorectal cancer based on the bioorthogonal inverse electron demand Diels-Alder reaction between a tetrazine-bearing radioligand and a transcyclooctene-modified huA33 immunoconjugate. Although this method effectively delineated tumor tissue, its clinical potential was limited by the somewhat sluggish clearance of the radioligand through the gastrointestinal tract. Herein, we report the development and in vivo validation of a pretargeted strategy for the PET imaging of colorectal carcinoma with dramatically improved pharmacokinetics. Two novel tetrazine constructs, Tz-PEG7-NOTA and Tz-SarAr, were synthesized, characterized, and radiolabeled with (64)Cu in high yield (>90%) and radiochemical purity (>99%). PET imaging and biodistribution experiments in healthy mice revealed that although (64)Cu-Tz-PEG7-NOTA is cleared via both the gastrointestinal and urinary tracts, (64)Cu-Tz-SarAr is rapidly excreted by the renal system alone. On this basis, (64)Cu-Tz-SarAr was selected for further in vivo evaluation. To this end, mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts were administered huA33-TCO, and the immunoconjugate was given 24 h to accumulate at the tumor and clear from the blood, after which (64)Cu-Tz-SarAr was administered via intravenous tail vein injection. PET imaging and biodistribution experiments revealed specific uptake of the radiotracer in the tumor at early time points (5.6 ± 0.7 %ID/g at 1 h p.i.), high tumor-to-background activity ratios, and rapid elimination of unclicked radioligand. Importantly, experiments with longer antibody accumulation intervals (48 and 120 h) yielded slight decreases in tumoral uptake but also concomitant increases in tumor-to-blood activity concentration ratios. This new strategy offers dosimetric benefits as well, yielding a total effective dose of 0.041 rem/mCi, far below the doses produced by directly labeled (64)Cu-NOTA-huA33 (0.133 rem/mCi) and (89)Zr-DFO-huA33 (1.54 rem/mCi). Ultimately, this pretargeted PET imaging strategy boasts a dramatically improved pharmacokinetic profile compared to our first generation system and is capable of clearly delineating tumor tissue with high image contrast at only a fraction of the radiation dose created by directly labeled radioimmunoconjugates.

Comparative Analysis of the Magnitude, Quality, Phenotype and Protective Capacity of SIV Gag-Specific CD8+ T Cells Following Human-, Simian- and Chimpanzee-Derived Recombinant Adenoviral Vector Immunisation

PubMed Central

Quinn, Kylie M.; Costa, Andreia Da; Yamamoto, Ayako; Berry, Dana; Lindsay, Ross W.B.; Darrah, Patricia A.; Wang, Lingshu; Cheng, Cheng; Kong, Wing-Pui; Gall, Jason G.D.; Nicosia, Alfredo; Folgori, Antonella; Colloca, Stefano; Cortese, Riccardo; Gostick, Emma; Price, David A.; Gomez, Carmen E.; Esteban, Mariano; Wyatt, Linda S.; Moss, Bernard; Morgan, Cecilia; Roederer, Mario; Bailer, Robert T.; Nabel, Gary J.; Koup, Richard A.; Seder, Robert A.

2013-01-01

Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. Here we show low seroreactivity in humans against simian- (sAd11, sAd16), or chimpanzee-derived (chAd3, chAd63) compared to human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107 to 109 PU), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFNγ+TNFα+IL-2+ and KLRG1+CD127- CD8+ T cells, but strikingly ~30–80% of memory CD8+ T cells co-expressed CD127 and KLRG1. To further optimise CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ~60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared to prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively. PMID:23390298

Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

PubMed

Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

2014-11-01

Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates heterologous expression of large gene clusters for drug discovery. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression, purification and characterisation of recombinant Escherichia coli derived chicken interleukin-12.

PubMed

Thomas, Jesse D; Morris, Kirsten R; Godfrey, Dale I; Lowenthal, John W; Bean, Andrew G D

2008-12-15

Zoonotic viruses, such as H5N1 Avian Influenza, pose major threats to both animals and humans, and with this in mind there is a need for the development of new anti-viral strategies. The cytokine interleukin-12 (IL-12) is known to play a pivotal regulatory role in the anti-viral response due to its role in the induction of the key anti-viral cytokine IFN-gamma. Therefore, strategies which provide a means for the production of therapeutic quantities of IL-12 may be of major benefit. Here we describe the development of biologically active Escherichia coli (E. coli) derived chicken IL-12 (ChIL-12). The single chain ChIL-12 gene was cloned into the pET32b expression vector, transformed into the BL-21 E. coli strain and expression induced with IPTG. Over expressed protein was solubilised with zwittergent detergent and isolated utilising Nickel ion affinity chromatography. Biological activity was determined as ChIL-12 stimulated proliferation of pre-treated T-cells in vitro. This study is the first example of a biologically active E. coli derived IL-12 from a non-mammalian vertebrate subsequently providing a means for testing the anti-viral therapeutic potential of ChIL-12 in an in vivo model.

Efficient production of mutant phytase (phyA-7) derived from Selenomonas ruminantium using recombinant Escherichia coli in pilot scale.

PubMed

Chi-Wei Lan, John; Chang, Chih-Kai; Wu, Ho-Shing

2014-09-01

A mutant gene of rumen phytase (phyA-7) was cloned into pET23b(+) vector and expressed in the Escherichia coli BL21 under the control of the T7 promoter. The study of fermentation conditions includes the temperature impacts of mutant phytase expression, the effect of carbon supplements over induction stage, the inferences of acetic acid accumulation upon enzyme expression and the comparison of one-stage and two-stage operations in batch mode. The maximum value of phytase activity was reached 107.0 U mL(-1) at induction temperature of 30°C. Yeast extract supplement demonstrated a significant increase on both protein concentration and phytase activity. The acetic acid (2 g L(-1)) presented in the modified synthetic medium demonstrated a significant decrease on expressed phytase activity. A two-stage batch operation enhanced the level of phytase activity from 306 to 1204 U mL(-1) in the 20 L of fermentation scale. An overall 3.7-fold improvement in phytase yield (35,375.72-1,31,617.50 U g(-1) DCW) was achieved in the two-stage operation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

PubMed

Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

2010-09-01

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

Influence of alcoholism and cholesterol on TSPO binding in brain: PET [11C]PBR28 studies in humans and rodents.

PubMed

Kim, Sung Won; Wiers, Corinde E; Tyler, Ryan; Shokri-Kojori, Ehsan; Jang, Yeon Joo; Zehra, Amna; Freeman, Clara; Ramirez, Veronica; Lindgren, Elsa; Miller, Gregg; Cabrera, Elizabeth A; Stodden, Tyler; Guo, Min; Demiral, Şükrü B; Diazgranados, Nancy; Park, Luke; Liow, Jeih-San; Pike, Victor; Morse, Cheryl; Vendruscolo, Leandro F; Innis, Robert B; Koob, George F; Tomasi, Dardo; Wang, Gene-Jack; Volkow, Nora D

2018-05-03

Neuroinflammation appears to contribute to neurotoxicity observed with heavy alcohol consumption. To assess whether chronic alcohol results in neuroinflammation we used PET and [ 11 C]PBR28, a ligand that binds to the 18-kDa translocator protein (TSPO), to compare participants with an alcohol use disorder (AUD: n = 19) with healthy controls (HC: n = 17), and alcohol-dependent (n = 9) with -nondependent rats (n = 10). Because TSPO is implicated in cholesterol's transport for steroidogenesis, we investigated whether plasma cholesterol levels influenced [ 11 C]PBR28 binding. [ 11 C]PBR28 binding did not differ between AUD and HC. However, when separating by TSPO genotype rs6971, we showed that medium-affinity binders AUD participants showed lower [ 11 C]PBR28 binding than HC in regions of interest (whole brain, gray and white matter, hippocampus, and thalamus), but no group differences were observed in high-affinity binders. Cholesterol levels inversely correlated with brain [ 11 C]PBR28 binding in combined groups, due to a correlation in AUD participants. In rodents, we observed no differences in brain [ 11 C]PBR28 uptake between alcohol-dependent and -nondependent rats. These findings, which are consistent with two previous [ 11 C]PBR28 PET studies, may indicate lower activation of microglia in AUD, whereas failure to observe alcohol effects in the rodent model indicate that species differences do not explain the discrepancy with prior rodent autoradiographic studies reporting increases in TSPO binding with chronic alcohol. However, reduced binding in AUD participants could also reflect competition from endogenous TSPO ligands such as cholesterol; and since the rs6971 polymorphism affects the cholesterol-binding domain of TSPO this could explain why differences were observed only in medium-affinity binders.

ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice

PubMed Central

Mak, Kai Y; Chin, Ruth; Cunningham, Sharon C; Habib, Miriam R; Torresi, Joseph; Sharland, Alexandra F; Alexander, Ian E; Angus, Peter W; Herath, Chandana B

2015-01-01

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1–7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1–7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects. PMID:25997428

Adenocarcinoma Prostate With Neuroendocrine Differentiation: Potential Utility of 18F-FDG PET/CT and 68Ga-DOTANOC PET/CT Over 68Ga-PSMA PET/CT.

PubMed

Parida, Girish Kumar; Tripathy, Sarthak; Datta Gupta, Shreya; Singhal, Abhinav; Kumar, Rakesh; Bal, Chandrasekhar; Shamim, Shamim Ahmed

2018-04-01

Ga-PSMA PET/CT is the upcoming imaging modality for staging, restaging and response assessment of prostate cancer. However, due to neuroendocrine differentiation in some of patients with prostate cancer, they express somatostatin receptors instead of prostate specific membrane antigen. This can be exploited and other modalities like Ga-DOTANOC PET/CT and F-FDG PET/CT should be used in such cases for guiding management. We hereby discuss a similar case of 67-year-old man of adenocarcinoma prostate with neuroendocrine differentiation, which shows the potential pitfall of Ga-PSMA PET/CT imaging and benefit of Ga-DOTANOC PET/CT and F-FDG PET/CT in such cases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of amore » large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

Use of [18F]FDG PET to Monitor The Development of Cardiac Allograft Rejection

PubMed Central

Daly, Kevin P.; Dearling, Jason L. J.; Seto, Tatsuichiro; Dunning, Patricia; Fahey, Frederic; Packard, Alan B.; Briscoe, David M.

2014-01-01

Background Positron Emission Tomography (PET) has the potential to be a specific, sensitive and quantitative diagnostic test for transplant rejection. To test this hypothesis, we evaluated 18F-labeled fluorodeoxyglucose ([18F]FDG) and 13N-labeled ammonia ([13N]NH3) small animal PET imaging in a well-established murine cardiac rejection model. Methods Heterotopic transplants were performed using minor MHC mismatched B6.C-H2bm12 donor hearts in C57BL/6(H-2b) recipients. C57BL/6 donor hearts into C57BL/6 recipients served as isograft controls. [18F]FDG PET imaging was performed weekly between post-transplant days 7 and 42 and the percent injected dose was computed for each graft. [13N]NH3 imaging was performed to evaluate myocardial perfusion. Results There was a significant increase in [18F]FDG uptake in allografts from day 14 to day 21 (1.6% to 5.2%; P<0.001) and uptake in allografts was significantly increased on post-transplant days 21 (5.2% vs. 0.9%; P=0.005) and 28 (4.8% vs. 0.9%; P=0.006) compared to isograft controls. Furthermore, [18F]FDG uptake correlated with an increase in rejection within allografts between days 14 and 28 post-transplant. Finally, the uptake of [13N]NH3 was significantly lower relative to the native heart in allografts with chronic vasculopathy compared to isograft controls on day 28 (P=0.01). Conclusions PET imaging with [18F]FDG can be used following transplantation to monitor the evolution of rejection. In addition, decreased uptake of [13N]NH3 in rejecting allografts may be reflective of decreased myocardial blood flow. These data suggest that combined [18F]FDG and [13N]NH3 PET imaging could be used as a non-invasive, quantitative technique for serial monitoring of allograft rejection and has potential application in human transplant recipients. PMID:25675207

Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors.

PubMed

Kyostio-Moore, Sirkka; Berthelette, Patricia; Cornell, Cathleen Sookdeo; Nambiar, Bindu; Figueiredo, Monica Dias

2018-05-01

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction

PubMed Central

Huang, Mei; Chen, ZhiYing; Hu, Shijun; Jia, Fangjun; Li, Zongjin; Hoyt, Grant; Robbins, Robert C.; Kay, Mark A.; Wu, Joseph C.

2011-01-01

Background Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel non-viral vector which robustly and persistently expresses the hypoxia inducible factor-1 alpha (HIF-1α) therapeutic gene in the heart, leading to functional benefits following myocardial infarction (MI). Methods and Results We first created minicircles carrying double fusion (MC-DF) reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging (BLI) showed stable minicircle gene expression in the heart for >12 weeks and the activity level was 5.6±1.2 fold stronger than regular plasmid at day 4 (P<0.01). Next, we created minicircles carrying hypoxia inducible factor-1 alpha (MC-HIF-1α) therapeutic gene for treatment of MI. Adult FVB mice underwent LAD ligation and were injected intramyocardially with (1) MC-HIF-1α, (2) regular plasmid carrying HIF-1α (PL-HIF-1α) as positive control, and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction (LVEF) in the minicircle group (51.3%±3.6%) compared to regular plasmid group (42.3%±4.1%) and saline group (30.5%±2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed minicircles express >50% higher HIF-1α level than regular plasmid. Conclusion Taken together, this is the first study to demonstrate that minicircles can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel non-viral vector can be of great value for cardiac gene therapy protocols. PMID:19752373

Subacute cardiac rubidium-82 positron emission tomography (82Rb-PET) to assess myocardial area at risk, final infarct size, and myocardial salvage after STEMI.

PubMed

Ghotbi, Adam Ali; Kjaer, Andreas; Nepper-Christensen, Lars; Ahtarovski, Kiril Aleksov; Lønborg, Jacob Thomsen; Vejlstrup, Niels; Kyhl, Kasper; Christensen, Thomas Emil; Engstrøm, Thomas; Kelbæk, Henning; Holmvang, Lene; Bang, Lia E; Ripa, Rasmus Sejersten; Hasbak, Philip

2018-06-01

Determining infarct size and myocardial salvage in patients with ST-segment elevation myocardial infarction (STEMI) is important when assessing the efficacy of new reperfusion strategies. We investigated whether rest 82 Rb-PET myocardial perfusion imaging can estimate area at risk, final infarct size, and myocardial salvage index when compared to cardiac SPECT and magnetic resonance (CMR). Twelve STEMI patients were injected with 99m Tc-Sestamibi intravenously immediate prior to reperfusion. SPECT, 82 Rb-PET, and CMR imaging were performed post-reperfusion and at a 3-month follow-up. An automated algorithm determined area at risk, final infarct size, and hence myocardial salvage index. SPECT, CMR, and PET were performed 2.2 ± 0.5, 34 ± 8.5, and 32 ± 24.4 h after reperfusion, respectively. Mean (± SD) area at risk were 35.2 ± 16.6%, 34.7 ± 11.3%, and 28.1 ± 16.1% of the left ventricle (LV) in SPECT, CMR, and PET, respectively, P = 0.04 for difference. Mean final infarct size estimates were 12.3 ± 15.4%, 13.7 ± 10.4%, and 11.9 ± 14.6% of the LV in SPECT, CMR, and PET imaging, respectively, P = .72. Myocardial salvage indices were 0.64 ± 0.33 (SPECT), 0.65 ± 0.20 (CMR), and 0.63 ± 0.28 (PET), (P = .78). 82 Rb-PET underestimates area at risk in patients with STEMI when compared to SPECT and CMR. However, our findings suggest that PET imaging seems feasible when assessing the clinical important parameters of final infarct size and myocardial salvage index, although with great variability, in a selected STEMI population with large infarcts. These findings should be confirmed in a larger population.

(68)Ga-DOTATATE PET in juvenile angiofibroma.

PubMed

Gronkiewicz, Zuzanna; Kukwa, Wojciech; Krolicki, Leszek; Cyran-Chlebicka, Agata; Pawlak, Dariusz; Stankiewicz, Czeslaw; Krzeski, Antoni; Górnicka, Barbara; Wolosz, Dominika; Kunikowska, Jolanta

2016-06-01

As somatostatin receptors (SSTRs) may be overexpressed in rapidly growing vessels, the aim of this study was the analysis of in vivo and in vitro SSTR2A expression in juvenile angiofibroma (JA). A group of six male adolescents with a diagnosis of primary, recurrent/residual JA was enrolled in the study. All patients underwent (68)Ga-DOTATATE PET/computed tomography (CT) followed by immunohistochemical staining for SSTR expression. (68)Ga-DOTATATE PET/CT showed accumulation in areas matching the pathologic tissue in the nasopharynx of all patients studied with SUVmax of 5.1 ± 0.9 (ranging from 3.6 to 6.4). In all cases, the immunohistochemical examination showed a presence of SSTR2A with a high staining index. In vitro SSTR2A cytoplasm expression was found to be high in all tumor specimens. However, the uptake of (68)Ga-DOTATATE was weak in the PET/CT studies. We postulate that the intracellular localization of the SSTR2A in JA may cause this discrepancy.

Comparison of Coregistration Accuracy of Pelvic Structures Between Sequential and Simultaneous Imaging During Hybrid PET/MRI in Patients with Bladder Cancer.

PubMed

Rosenkrantz, Andrew B; Balar, Arjun V; Huang, William C; Jackson, Kimberly; Friedman, Kent P

2015-08-01

The aim of this study was to compare coregistration of the bladder wall, bladder masses, and pelvic lymph nodes between sequential and simultaneous PET and MRI acquisitions obtained during hybrid (18)F-FDG PET/MRI performed using a diuresis protocol in bladder cancer patients. Six bladder cancer patients underwent (18)F-FDG hybrid PET/MRI, including IV Lasix administration and oral hydration, before imaging to achieve bladder clearance. Axial T2-weighted imaging (T2WI) was obtained approximately 40 minutes before PET ("sequential") and concurrently with PET ("simultaneous"). Three-dimensional spatial coordinates of the bladder wall, bladder masses, and pelvic lymph nodes were recorded for PET and T2WI. Distances between these locations on PET and T2WI sequences were computed and used to compare in-plane (x-y plane) and through-plane (z-axis) misregistration relative to PET between T2WI acquisitions. The bladder increased in volume between T2WI acquisitions (sequential, 176 [139] mL; simultaneous, 255 [146] mL). Four patients exhibited a bladder mass, all with increased activity (SUV, 9.5-38.4). Seven pelvic lymph nodes in 4 patients showed increased activity (SUV, 2.2-9.9). The bladder wall exhibited substantially less misregistration relative to PET for simultaneous, compared with sequential, acquisitions in in-plane (2.8 [3.1] mm vs 7.4 [9.1] mm) and through-plane (1.7 [2.2] mm vs 5.7 [9.6] mm) dimensions. Bladder masses exhibited slightly decreased misregistration for simultaneous, compared with sequential, acquisitions in in-plane (2.2 [1.4] mm vs 2.6 [1.9] mm) and through-plane (0.0 [0.0] mm vs 0.3 [0.8] mm) dimensions. FDG-avid lymph nodes exhibited slightly decreased in-plane misregistration (1.1 [0.8] mm vs 2.5 [0.6] mm), although identical through-plane misregistration (4.0 [1.9] mm vs 4.0 [2.8] mm). Using hybrid PET/MRI, simultaneous imaging substantially improved bladder wall coregistration and slightly improved coregistration of bladder masses and pelvic lymph nodes.

Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic Antivector Immune Responses

PubMed Central

ABORDO-ADESIDA, EVELYN; FOLLENZI, ANTONIA; BARCIA, CARLOS; SCIASCIA, SANDRA; CASTRO, MARIA G.; NALDINI, LUIGI; LOWENSTEIN, PEDRO R.

2009-01-01

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose—response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 102, and maximum expression at 106, transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS. PMID:15960605

Analysis of polyethylene terephthalate PET plastic bottle jointing system using finite element method (FEM)

NASA Astrophysics Data System (ADS)

Zaidi, N. A.; Rosli, Muhamad Farizuan; Effendi, M. S. M.; Abdullah, Mohamad Hariri

2017-09-01

For almost all injection molding applications of Polyethylene Terephthalate (PET) plastic was analyzed the strength, durability and stiffness of properties by using Finite Element Method (FEM) for jointing system of wood furniture. The FEM was utilized for analyzing the PET jointing system for Oak and Pine as wood based material of furniture. The difference pattern design of PET as wood jointing furniture gives the difference value of strength furniture itself. The results show the wood specimen with grooves and eclipse pattern design PET jointing give lower global estimated error is 28.90%, compare to the rectangular and non-grooves wood specimen of global estimated error is 63.21%.

Evaluation of Multimodal Imaging Biomarkers of Prostate Cancer

DTIC Science & Technology

2014-09-01

scan duration ~ 21 min). PET imaging was performed on a Concorde Microsystems microPET Focus 220. Approximately 120 uCi of tracer was administered... PET tracer targeting translocator protein expression (TSPO), using 18F-VUIIS1008 (a probe developed in-house), and hypoxia, using 18F...manuscripts describing these efforts. First, we plan to submit a manuscript validating the use of the TSPO PET tracer developed in house in the Pten/p53

Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae.

PubMed

Rund, Samuel S C; Hou, Tim Y; Ward, Sarah M; Collins, Frank H; Duffield, Giles E

2011-08-09

Anopheles gambiae, the primary African vector of malaria parasites, exhibits numerous rhythmic behaviors including flight activity, swarming, mating, host seeking, egg laying, and sugar feeding. However, little work has been performed to elucidate the molecular basis for these daily rhythms. To study how gene expression is regulated globally by diel and circadian mechanisms, we have undertaken a DNA microarray analysis of An. gambiae under light/dark cycle (LD) and constant dark (DD) conditions. Adult mated, non-blood-fed female mosquitoes were collected every 4 h for 48 h, and samples were processed with DNA microarrays. Using a cosine wave-fitting algorithm, we identified 1,293 and 600 rhythmic genes with a period length of 20-28 h in the head and body, respectively, under LD conditions, representing 9.7 and 4.5% of the An. gambiae gene set. A majority of these genes was specific to heads or bodies. Examination of mosquitoes under DD conditions revealed that rhythmic programming of the transcriptome is dependent on an interaction between the endogenous clock and extrinsic regulation by the LD cycle. A subset of genes, including the canonical clock components, was expressed rhythmically under both environmental conditions. A majority of genes had peak expression clustered around the day/night transitions, anticipating dawn and dusk. Genes cover diverse biological processes such as transcription/translation, metabolism, detoxification, olfaction, vision, cuticle regulation, and immunity, and include rate-limiting steps in the pathways. This study highlights the fundamental roles that both the circadian clock and light play in the physiology of this important insect vector and suggests targets for intervention.

[Effect of white spot syndrome virus envelope protein Vp28 expressed in silkworm (Bombyx mori) pupae on disease resistence in Procambarus clarkii].

PubMed

Wei, Ke Qiang; Xu, Zi Rong

2005-06-01

The vaccine made of recombinant envelope protein (rVp28) of white spot syndrome virus (WSSV) expressed in silkworm (Bombyx mori) pupae using a baculovirus vector was used to investigate the efficacy of oral administration on WSSV disease resistance of Procambarus clarkii. Vaccine was mixed with diet at a ratio of 2% (w/w), and Procambarus clarkii were orally administered throughout 75 days. Vaccination with rVP28 showed the significantly higher cumulative survival compared with positive and negative control (P < 0.05) following an oral challenge on the 35th day post-vaccination (dpv), with PRP values 54.16% and 59.26%, respectively. rVP28 induced higher resistance via IM (intramuscular) injection challenge with WSSV stock, with PRP value of 46.12% and 49.99%, respectively. The survivors were subsequently re-challenged on the 55th dpv. rVP28 induced the significantly higher resistance to oral re-challenge (P < 0.05), with both PRP values 55.80% and 63.16%, respectively. rVP28 induced higher resistance to IM injection re-challenge, with both PRP values 31.25%. A DIG labeled WSSV DNA probe was used to detect WSSV by in situ hybridization. The positive cells were observed in epithelial cells of stomach, hepatopancreas and gut of the infected control crayfish, while negative reaction were observed in the tissues of survivors-vaccinated. These results indicated that vaccination of crayfish with recombinant protein had significant effect on oral infection, and had higher resistance against intramuscular injection challenge. This suggested the protection against WSSV could be induced in crayfish by recombinant protein rVp28 expressed in silkworm pupae.

High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad Javad; Forouzandeh-Moghadam, Mehdi; Allameh, Abdol-Amir

2005-11-01

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

Activity of Tachykinin1-Expressing Pet1 Raphe Neurons Modulates the Respiratory Chemoreflex

PubMed Central

Corcoran, Andrea E.; Brust, Rachael D.; Chang, YoonJeung; Nattie, Eugene E.

2017-01-01

Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 (Tac1) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1, referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine-N-oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2. Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a Pet1 neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei. SIGNIFICANCE STATEMENT Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using genetic tools, we characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 (Tac1-Pet1 neurons), mapping soma localization to the caudal medulla primarily and axonal projections to brainstem motor nuclei most prominently, and, when silenced, observed blunting of the ventilatory response to inhaled CO2. Tac1-Pet1 neurons thus appear distinct from and contrast previously described Egr2-Pet1 neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive. PMID:28073937

Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca).

PubMed

Su, Xiu-Lan; Hou, Yi-Ling; Yan, Xiang-Hui; Ding, Xiang; Hou, Wan-Ru; Sun, Bing; Zhang, Si-Nan

2012-09-01

Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 μg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.

Screening of cancer tissue arrays identifies CXCR4 on adrenocortical carcinoma: correlates with expression and quantification on metastases using 64Cu-plerixafor PET.

PubMed

Weiss, Ido D; Huff, Lyn M; Evbuomwan, Moses O; Xu, Xin; Dang, Hong Duc; Velez, Daniel S; Singh, Satya P; Zhang, Hongwei H; Gardina, Paul J; Lee, Jae-Ho; Lindenberg, Liza; Myers, Timothy G; Paik, Chang H; Schrump, David S; Pittaluga, Stefania; Choyke, Peter L; Fojo, Tito; Farber, Joshua M

2017-09-26

Expression of the chemokine receptor CXCR4 by many cancers correlates with aggressive clinical behavior. As part of the initial studies in a project whose goal was to quantify CXCR4 expression on cancers non-invasively, we examined CXCR4 expression in cancer samples by immunohistochemistry using a validated anti-CXCR4 antibody. Among solid tumors, we found expression of CXCR4 on significant percentages of major types of kidney, lung, and pancreatic adenocarcinomas, and, notably, on metastases of clear cell renal cell carcinoma and squamous cell carcinoma of the lung. We found particularly high expression of CXCR4 on adrenocortical cancer (ACC) metastases. Microarrays of ACC metastases revealed correlations between expression of CXCR4 and other chemokine system genes, particularly CXCR7/ACKR3 , which encodes an atypical chemokine receptor that shares a ligand, CXCL12, with CXCR4. A first-in-human study using 64 Cu-plerixafor for PET in an ACC patient prior to resection of metastases showed heterogeneity among metastatic nodules and good correlations among PET SUVs, CXCR4 staining, and CXCR4 mRNA. Additionally, we were able to show that CXCR4 expression correlated with the rates of growth of the pulmonary lesions in this patient. Further studies are needed to understand better the role of CXCR4 in ACC and whether targeting it may be beneficial. In this regard, non-invasive methods for assessing CXCR4 expression, such as PET using 64 Cu-plerixafor, should be important investigative tools.

[18F]tetrafluoroborate-PET/CT enables sensitive tumor and metastasis in vivo imaging in a sodium iodide symporter-expressing tumor model.

PubMed

Diocou, S; Volpe, A; Jauregui-Osoro, M; Boudjemeline, M; Chuamsaamarkkee, K; Man, F; Blower, P J; Ng, T; Mullen, G E D; Fruhwirth, G O

2017-04-19

Cancer cell metastasis is responsible for most cancer deaths. Non-invasive in vivo cancer cell tracking in spontaneously metastasizing tumor models still poses a challenge requiring highest sensitivity and excellent contrast. The goal of this study was to evaluate if the recently introduced PET radiotracer [ 18 F]tetrafluoroborate ([ 18 F]BF 4 - ) is useful for sensitive and specific metastasis detection in an orthotopic xenograft breast cancer model expressing the human sodium iodide symporter (NIS) as a reporter. In vivo imaging was complemented by ex vivo fluorescence microscopy and γ-counting of harvested tissues. Radionuclide imaging with [ 18 F]BF 4 - (PET/CT) was compared to the conventional tracer [ 123 I]iodide (sequential SPECT/CT). We found that [ 18 F]BF 4 - was superior due to better pharmacokinetics, i.e. faster tumor uptake and faster and more complete clearance from circulation. [ 18 F]BF 4 - -PET was also highly specific as in all detected tissues cancer cell presence was confirmed microscopically. Undetected comparable tissues were similarly found to be free of metastasis. Metastasis detection by routine metabolic imaging with [ 18 F]FDG-PET failed due to low standard uptake values and low contrast caused by adjacent metabolically active organs in this model. [ 18 F]BF 4 - -PET combined with NIS expressing disease models is particularly useful whenever preclinical in vivo cell tracking is of interest.

Targeting CXCR4 with [68Ga]Pentixafor: a suitable theranostic approach in pleural mesothelioma?

PubMed

Lapa, Constantin; Kircher, Stefan; Schirbel, Andreas; Rosenwald, Andreas; Kropf, Saskia; Pelzer, Theo; Walles, Thorsten; Buck, Andreas K; Weber, Wolfgang A; Wester, Hans-Juergen; Herrmann, Ken; Lückerath, Katharina

2017-11-14

C-X-C motif chemokine receptor 4 (CXCR4) is a key factor for tumor growth and metastasis in several types of human cancer. This study investigated the feasibility of CXCR4-directed imaging with positron emission tomography/computed tomography (PET/CT) using [ 68 Ga]Pentixafor in malignant pleural mesothelioma. Six patients with pleural mesothelioma underwent [ 68 Ga]Pentixafor-PET/CT. 2'-[ 18 F]fluoro-2'-deoxy-D-glucose ([ 18 F]FDG)-PET/CT (4/6 patients) and immunohistochemistry obtained from biopsy or surgery (all) served as standards of reference. Additionally, 9 surgical mesothelioma samples were available for histological work-up. Whereas [ 18 F]FDG-PET depicted active lesions in all patients, [ 68 Ga]Pentixafor-PET/CT recorded physiologic tracer distribution and none of the 6 patients presented [ 68 Ga]Pentixafor-positive lesions. This finding paralleled results of immunohistochemistry which also could not identify relevant CXCR4 surface expression in the samples analyzed. In contrast to past reports, our data suggest widely absence of CXCR4 expression in pleural mesothelioma. Hence, robust cell surface expression should be confirmed prior to targeting this chemokine receptor for diagnosis and/or therapy.

Noninvasive Interrogation of DLL3 Expression in Metastatic Small Cell Lung Cancer.

PubMed

Sharma, Sai Kiran; Pourat, Jacob; Abdel-Atti, Dalya; Carlin, Sean D; Piersigilli, Alessandra; Bankovich, Alexander J; Gardner, Eric E; Hamdy, Omar; Isse, Kumiko; Bheddah, Sheila; Sandoval, Joseph; Cunanan, Kristen M; Johansen, Eric B; Allaj, Viola; Sisodiya, Vikram; Liu, David; Zeglis, Brian M; Rudin, Charles M; Dylla, Scott J; Poirier, John T; Lewis, Jason S

2017-07-15

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody-drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed 89 Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the in vivo status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with 89 Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient-derived xenograft models. Cancer Res; 77(14); 3931-41. ©2017 AACR . ©2017 American Association for Cancer Research.

Recombinant production of the insecticidal scorpion toxin BjαIT in Escherichia coli.

PubMed

Li, Hongbo; Xia, Yuxian

2018-02-01

Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl β-d-1-thiogalactopyranoside, and it was purified by Ni 2+ -nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust (Locusta migratoria) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests. Copyright © 2017 Elsevier Inc. All rights reserved.

Matching the decay half-life with the biological half-life: ImmunoPET imaging with 44 Sc-labeled Cetuximab Fab fragment

DOE PAGES

Chakravarty, Rubel; Goel, Shreya; Valdovinos, Hector F.; ...

2014-11-11

Scandium-44 (t 1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-( para-isothiocyanato-phenyl)propyl]- trans-(S,S)-cyclohexane-1,2-diamine- N,N,N',N'',N''-pentaacetic acid (CHX-A"-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastomamore » (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ~12% ID/g at 4 h postinjection) of 44Sc–CHX-A"-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. As a result, this successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-''-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.« less

Therapeutic Benefits and Adverse Effects of Combined Proangiogenic Gene Therapy in Mouse Critical Leg Ischemia.

PubMed

Lebas, Benoît; Galley, Julien; Renaud-Gabardos, Edith; Pujol, Françoise; Lenfant, Françoise; Garmy-Susini, Barbara; Chaufour, Xavier; Prats, Anne-Catherine

2017-04-01

Critical leg ischemia (CLI) represents the ultimate stage of peripheral arterial disease. Despite current surgery advances, patients with CLI have limited therapeutic options. Therapeutic angiogenesis thus appears as a powerful approach, aiming to stimulate vessel formation by angiogenic molecules administration. In this context, combined gene therapy has been proved to be the most efficient. The present study aims to compare, in a preclinical mouse model, the therapeutic benefit of a combination of 2 angiogenic factors fibroblast growth factor 2 (FGF2) and Cyr61 using plasmid and viral vectors, able to generate short- or long-term transgene expression in the leg, respectively. Two therapeutic genes, FGF2 and Cyr61, were introduced into internal ribosome entry site-based expression vectors (FGFiCyr) allowing co-expression of the 2 transgenes. The proangiogenic plasmid pC-FGFiCyr was assessed by intramuscular administration followed by electrotransfer into ischemic legs. To generate long-term transgene expression, the FGFiCyr bicistronic cassette was introduced into an adenoassociated virus-derived vector (rAAV). The rAAV treatment was performed either before or immediately after surgery. Therapeutic effects were analyzed by laser Doppler imaging, clinical score, and angiography. The plasmid pC-FGFiCyr improved revascularization, reperfusion, and clinical score. Surprisingly, when AAV-FGFiCyr was injected 21 or 28 days before surgery, the proangiogenic rAAV was drastically deleterious on all measured parameters. In contrast, when administrated shortly after surgery, AAV-FGFiCyr generated therapeutic benefits, with a significantly better clinical score than after treatment with the plasmid. Therapeutic effects of the angiogenic combination FGF2-Cyr61 is observed with short-term transgene expression, but the treatment is significantly more efficient when a long-term expression viral vector is used. However, the rAAV-FGFiCyr generated therapeutic benefit only when injected in an ischemic leg, whereas the same dose of rAAV exhibited deleterious effects when administrated to healthy animals. These data may contribute to the understanding of the moderate success of proangiogenic treatments in CLI gene therapy clinical assays. Copyright © 2016 Elsevier Inc. All rights reserved.

PET Imaging of Tissue Factor in Pancreatic Cancer Using 64Cu-Labeled Active Site-Inhibited Factor VII.

PubMed

Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Lotte K; Jensen, Mette M; El Ali, Henrik H; Madsen, Jacob; Wiinberg, Bo; Petersen, Lars C; Kjaer, Andreas

2016-07-01

Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%-89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site-inhibited FVII (FVIIai) labeled with (64)Cu for PET imaging of TF expression. FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu ((64)Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of (64)Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with (64)Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using (64)Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. Longitudinal PET imaging with (64)Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3, and 2.4 ± 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor-to-normal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of (64)Cu-NOTA-FVIIai was colocalized with viable tumor tissue. (64)Cu-NOTA-FVIIai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

PET imaging of Hsp90 expression in pancreatic cancer using a new 64Cu-labeled dimeric Sansalvamide A decapeptide.

PubMed

Wang, Xiaohui; Zhang, Jun; Wu, Hubing; Li, Yumin; Conti, Peter S; Chen, Kai

2018-04-24

Heat shock protein 90 (Hsp90) plays a vital role in the progress of malignant disease and elevated Hsp90 expression has been reported in pancreatic cancer. In this study, we radiolabeled a dimeric Sansalvamide A derivative (Di-San A1) with 64 Cu, and evaluated the feasibility of using 64 Cu-Di-San A1 for PET imaging of Hsp90 expression in a mouse model of pancreatic cancer. A macrocyclic chelator NOTA (1,4,7-triazacyclononane-1,4,7-trisacetic acid) was conjugated to Di-San A1. 64 Cu-Di-San A1 was successfully prepared in a radiochemical yield > 97% with a radiochemical purity > 98%. 64 Cu-Di-San A1 is stable in PBS and mouse serum with > 92% of parent probe intact after 4 h incubation. The cell binding and uptake revealed that 64 Cu-Di-San A1 binds to Hsp90-positive PL45 pancreatic cancer cells, and the binding can be effectively blocked by an Hsp90 inhibitor (17AAG). For microPET study, 64 Cu-Di-San A1 shows good in vivo performance in terms of tumor uptake in nude mice bearing PL45 tumors. The Hsp90-specific tumor activity accumulation of 64 Cu-Di-San A1 was further demonstrated by significant reduction of PL45 tumor uptake with a pre-injected blocking dose of 17AAG. The ex vivo PET imaging and biodistribution results were consistent with the quantitative analysis of PET imaging, demonstrating good tumor-to-muscle ratio (5.35 ± 0.46) of 64 Cu-Di-San A1 at 4 h post-injection in PL45 tumor mouse xenografts. 64 Cu-Di-San A1 allows PET imaging of Hsp90 expression in PL45 tumors, which may provide a non-invasive method to quantitatively characterize Hsp90 expression in pancreatic cancer.

Early identification of MCI converting to AD: a FDG PET study.

PubMed

Pagani, Marco; Nobili, Flavio; Morbelli, Silvia; Arnaldi, Dario; Giuliani, Alessandro; Öberg, Johanna; Girtler, Nicola; Brugnolo, Andrea; Picco, Agnese; Bauckneht, Matteo; Piva, Roberta; Chincarini, Andrea; Sambuceti, Gianmario; Jonsson, Cathrine; De Carli, Fabrizio

2017-11-01

Mild cognitive impairment (MCI) is a transitional pathological stage between normal ageing (NA) and Alzheimer's disease (AD). Although subjects with MCI show a decline at different rates, some individuals remain stable or even show an improvement in their cognitive level after some years. We assessed the accuracy of FDG PET in discriminating MCI patients who converted to AD from those who did not. FDG PET was performed in 42 NA subjects, 27 MCI patients who had not converted to AD at 5 years (nc-MCI; mean follow-up time 7.5 ± 1.5 years), and 95 MCI patients who converted to AD within 5 years (MCI-AD; mean conversion time 1.8 ± 1.1 years). Relative FDG uptake values in 26 meta-volumes of interest were submitted to ANCOVA and support vector machine analyses to evaluate regional differences and discrimination accuracy. The MCI-AD group showed significantly lower FDG uptake values in the temporoparietal cortex than the other two groups. FDG uptake values in the nc-MCI group were similar to those in the NA group. Support vector machine analysis discriminated nc-MCI from MCI-AD patients with an accuracy of 89% (AUC 0.91), correctly detecting 93% of the nc-MCI patients. In MCI patients not converting to AD within a minimum follow-up time of 5 years and MCI patients converting within 5 years, baseline FDG PET and volume-based analysis identified those who converted with an accuracy of 89%. However, further analysis is needed in patients with amnestic MCI who convert to a dementia other than AD.

Recombinant expression and characterization of L-asparaginase II from a moderately thermotolerant bacterial isolate.

PubMed

Vidya, Jalaja; Pandey, Ashok

2012-07-01

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 °C, was isolated from cow dung; L-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37 °C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 °C with a 33% and 28% of activity retention after 45 and 60 min. The kinetic parameters for the enzyme were calculated from Lineweaver-Burk plot, and K(m) and V(max) were 0.89 mM and 0.18 U/mg, respectively.

[Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide].

PubMed

Peng, Jing; Wang, Qiong; Cheng, Xiaoling; Liu, Mengwen; Wang, Mei; Xin, Huawei

2018-04-25

We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.

Developing Targeted Hybrid Imaging Probes by Chelator Scaffolding

PubMed Central

2017-01-01

Positron emission tomography (PET) as well as optical imaging (OI) with peptide receptor targeting probes have proven their value for oncological applications but also show restrictions depending on the clinical field of interest. Therefore, the combination of both methods, particularly in a single molecule, could improve versatility in clinical routine. This proof of principle study aims to show that a chelator, Fusarinine C (FSC), can be utilized as scaffold for novel dimeric dual-modality imaging agents. Two targeting vectors (a minigastrin analogue (MG11) targeting cholecystokinin-2 receptor overexpression (CCK2R) or integrin αVβ3 targeting cyclic pentapeptides (RGD)) and a near-infrared fluorophore (Sulfo-Cyanine7) were conjugated to FSC. The probes were efficiently labeled with gallium-68 and in vitro experiments including determination of logD, stability, protein binding, cell binding, internalization, and biodistribution studies as well as in vivo micro-PET/CT and optical imaging in U-87MG αVβ3- and A431-CCK2R expressing tumor xenografted mice were carried out. Novel bioconjugates showed high receptor affinity and highly specific targeting properties at both receptors. Ex vivo biodistribution and micro-PET/CT imaging studies revealed specific tumor uptake accompanied by slow blood clearance and retention in nontargeted tissues (spleen, liver, and kidneys) leading to visualization of tumors at early (30 to 120 min p.i.). Excellent contrast in corresponding optical imaging studies was achieved especially at delayed time points (24 to 72 h p.i.). Our findings show the proof of principle of chelator scaffolding for hybrid imaging agents and demonstrate FSC being a suitable bifunctional chelator for this approach. Improvements to fine-tune pharmacokinetics are needed to translate this into a clinical setting. PMID:28462989

Reactivity of IgE to the allergen hyaluronidase from Polybia paulista (Hymenoptera, Vespidae) venom.

PubMed

Justo Jacomini, Débora Laís; Gomes Moreira, Susana Margarida; Campos Pereira, Franco Dani; Zollner, Ricardo de Lima; Brochetto Braga, Márcia Regina

2014-05-01

To date, there are no allergenic extracts or components available in Brazil to diagnosis and treatment of patients with venom allergy from social wasp (Vespidae Family; Polistinae Subfamily) despite of the great number of existing species. We evaluated the immunogenic potential of the Hyal recombinant protein (Pp-Hyal-rec) which was expressed in an insoluble form in comparison with the allergenic native protein (Pp-Hyal-nat) for recognition of immunoglobulin E (IgE) in the serum of allergic patients to venom of the endemic social wasp Polybia paulista from São Paulo State, Brazil. Hyal cDNA from the venom of the social wasp P. paulista (Pp-Hyal) (GI: 302201582) was cloned into the expression vector pET-28a in Escherichia coli DE3 (BL21) cells. Solubilization and purification of Pp-Hyal-rec from inclusion bodies were performed using Ni(2+) affinity chromatography (Ni-NTA-Agarose) under denaturing conditions. Both the native (Pp-Hyal-nat) and the recombinant (Pp-Hyal-rec) purified allergens were used for Western blotting to assess the levels of Pp-Hyal-IgE specific in the serum of 10 patients exclusively reactive to the venom of the social wasp P. paulista. The immune sera specifically recognized the band corresponding to the Pp-Hyal-rec protein (40 kDa) at a higher intensity than the native allergen (39 kDa). The sera recognized other proteins in P. paulista crude venom extract to a lesser extent, likely corresponding to other venom allergens such as phospholipase (34 kDa), Antigen 5 (25 kDa), and proteases. The recognition pattern of the immune sera to the Pp-Hyal-rec allergen strongly suggests that this recombinant antigen could be used for developing a diagnostic allergy test as well as for specific immunotherapy (IT). Copyright © 2014 Elsevier Ltd. All rights reserved.

Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons

PubMed Central

Morral, Núria; O’Neal, Wanda; Rice, Karen; Leland, Michele; Kaplan, Johanne; Piedra, Pedro A.; Zhou, Heshan; Parks, Robin J.; Velji, Rizwan; Aguilar-Córdova, Estuardo; Wadsworth, Samuel; Graham, Frank L.; Kochanek, Stefan; Carey, K. Dee; Beaudet, Arthur L.

1999-01-01

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes. PMID:10536005

Coronin 3 promotes gastric cancer metastasis via the up-regulation of MMP-9 and cathepsin K.

PubMed

Ren, Gui; Tian, Qifei; An, Yanxin; Feng, Bin; Lu, Yuanyuan; Liang, Jie; Li, Kai; Shang, Yulong; Nie, Yongzhan; Wang, Xin; Fan, Daiming

2012-09-14

Coronins are a family of highly evolutionary conserved proteins reportedly involved in the regulation of actin cytoskeletal dynamics, although only coronin 3 has been shown to be related to cancer cell migration. In glioblastoma cells, the knockdown of coronin 3 inhibits cell proliferation and invasion. Coronin 3 is also associated with the aggression and metastasis of hepatocellular carcinoma. In this paper, we analyze the migration, invasion and metastasis abilities of gastric cancer cells after up- or down-regulation of coronin 3, and explore the mechanism of coronin 3 in the process of gastric cancer metastasis. The expression of coronin 3 was higher in the highly metastatic sub-cell line MKN28-M, which we established in our laboratory. We also demonstrated that the expression of coronin 3 was remarkably higher in lymph lode metastases than in primary gastric cancer tissues, and over-expression of coronin 3 was correlated with the increased clinical stage and lymph lode metastasis. Recombinant lentiviral vectors encoding shRNAs were designed to down-regulate coronin 3 expression in gastric cancer cell lines. Stable knockdown of coronin 3 by this lentiviral vector could efficiently inhibit the migration and invasion of MKN45 gastric cancer cells. In contrast, up-regulation of coronin 3 significantly enhanced migration and invasion of MKN28-NM cells. In addition, knockdown of coronin 3 significantly reduced liver metastasis in mice after tail vein injection of gastric cancer cells. The Human Tumor Metastasis PCR Array was used to screen the metastasis-associated genes identified by the down-regulation of coronin 3, and the results suggested that, following the knockdown of coronin 3, the tumor cell migration and invasion were inhibited by the reduced expression of MMP-9 and cathepsin K. Coronin 3 is highly expressed in gastric cancer metastases and can promote the metastatic behaviors of gastric cancer cells, including their migration and invasion.

Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen

2006-10-01

Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni{sup 2+}-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution.more » The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°.« less

Associations of Tumor PD-1 Ligands, Immunohistochemical Studies, and Textural Features in 18F-FDG PET in Squamous Cell Carcinoma of the Head and Neck.

PubMed

Chen, Rui-Yun; Lin, Ying-Chun; Shen, Wei-Chih; Hsieh, Te-Chun; Yen, Kuo-Yang; Chen, Shang-Wen; Kao, Chia-Hung

2018-01-08

To know tumor PD-L1 expression through IHC or the FDG-PET related radiomics, we investigated the association between programmed cell death protein 1 ligand (PD-L1) expression and immunohistochemical (IHC) biomarkers or textural features of 18F-fluoro-2-deoxdeoxyglucose positron emission tomography ( 18 F-FDG PET) in 53 oropharyngeal or hypopharyngeal cancer patients who were ready to undergo radiotherapy-based treatment. Differences in textural features or biomarkers between tumors with and without PD-L1 expression were tested using a Mann-Whitney U test. The predicted values for PD-L1 expression were examined using logistic regression analysis. The mean percentages of tumor PD-L1 expression were 6.2 ± 13.5. Eighteen tumors had PD-L1 expression ≥5%, whereas 30 tumors ≥1%. Using a 5% cutoff, the p16 staining percentage and the textural index of correlation were two factors associated with PD-L1 expression. The odds ratios (ORs) were 17.00 (p = 0.028) and 0.009 (p = 0.015), respectively. When dichotomizing PD-L1 at 1%, the p16 and Ki-67 staining percentages were two predictors for PD-L1 expression with ORs of 11.41 (p = 0.035) and 757.77 (p = 0.045). p16 and Ki-67 staining percentages and several PET/CT-derived textural features can provide supplemental information to determine tumor PD-L1 expression in HNCs.

[Expression of OPN gene during different lactation stages in mammary gland of dairy goat and its effect on growth of MCF-7 cell line].

PubMed

Sun, Jie; Luo, Jun; Liu, Jun-Xia; Li, Da-Quan

2009-08-01

To investigate the expression pattern and preliminary function of OPN gene in mammary gland of dairy goat during different lactation stages, using b-actin gene as the internal control, the SYBR Green quantitative real-time PCR (QPCR) analysis was conducted to determine the mRNA expression of OPN gene in mammary gland at the 28th, 60th, 100th, 190th, 270th and 330th day after kidding. Recombinant plasmid of pcDNA3.1-OPN was constructed by inserting the fragment of OPN gene into eukaryotic expression vector pcDNA3.1 and used to transfect the MCF-7 cell line following the restrictive endonuclease cleavage and sequence identification of the target gene segment, the effect of OPN gene on MCF-7 cell proliferation was assessed by MTT analysis. The results indicated that OPN gene exhibited the higher expression level in early (28 d) and late (190 d) lactation stages and the lowest level at dry stage (330 d), which demonstrated a high-low-high-low pattern. There was a significant difference (P < 0. 05) in the proliferation between OPN gene transfected and non-transfected MCF-7 cells, which suggested that the expression of OPN gene could stimulate the proliferation of MCF-7 cells.

Protected engagement time on older adult mental health wards: A thematic analysis of the views of patients, carers, and staff.

PubMed

Dodd, Emily; Cheston, Richard; Procter, Charlie; Heneker, Sarah; Gray, Richard; Fox, Chris; Nolan, Fiona

2018-04-01

During protected engagement time (PET), ward routines are adjusted so that staff can spend time together with patients without interruption. The aim of PET is to increase staff and patient interaction on wards, and ultimately patient well-being. Although PET has been implemented on inpatient wards within the UK, including older adult wards, there is no systematic evidence as to how PET is carried out or how it is experienced by staff, patients, and families. Semistructured interviews were conducted with 28 participants (8 patients, 10 family members, and 10 ward staff) from three different wards with PET, and transcriptions were analysed using thematic analysis. Three themes were identified: (i) the patient is at the heart of care; (ii) PET depends on staff; and (iii) tensions in how PET operates. There was support in our sample for the principles of PET and its potential for a positive impact on patient well-being. However, the implementation of PET was identified as challenging, highlighting an existing tension between an individual's needs and the wider needs of patients on the ward as a whole. The impact of PET was generally described as being dependent on how PET was organized and the level of staff commitment to PET. Participants emphasized that if PET is to be successful, then it should be a fluid process that fits in with the local context. © 2017 Australian College of Mental Health Nurses Inc.

Small animal simultaneous PET/MRI: initial experiences in a 9.4 T microMRI

NASA Astrophysics Data System (ADS)

Harsha Maramraju, Sri; Smith, S. David; Junnarkar, Sachin S.; Schulz, Daniela; Stoll, Sean; Ravindranath, Bosky; Purschke, Martin L.; Rescia, Sergio; Southekal, Sudeepti; Pratte, Jean-François; Vaska, Paul; Woody, Craig L.; Schlyer, David J.

2011-04-01

We developed a non-magnetic positron-emission tomography (PET) device based on the rat conscious animal PET that operates in a small-animal magnetic resonance imaging (MRI) scanner, thereby enabling us to carry out simultaneous PET/MRI studies. The PET detector comprises 12 detector blocks, each being a 4 × 8 array of lutetium oxyorthosilicate crystals (2.22 × 2.22 × 5 mm3) coupled to a matching non-magnetic avalanche photodiode array. The detector blocks, housed in a plastic case, form a 38 mm inner diameter ring with an 18 mm axial extent. Custom-built MRI coils fit inside the positron-emission tomography (PET) device, operating in transceiver mode. The PET insert is integrated with a Bruker 9.4 T 210 mm clear-bore diameter MRI scanner. We acquired simultaneous PET/MR images of phantoms, of in vivo rat brain, and of cardiac-gated mouse heart using [11C]raclopride and 2-deoxy-2-[18F]fluoro-d-glucose PET radiotracers. There was minor interference between the PET electronics and the MRI during simultaneous operation, and small effects on the signal-to-noise ratio in the MR images in the presence of the PET, but no noticeable visual artifacts. Gradient echo and high-duty-cycle spin echo radio frequency (RF) pulses resulted in a 7% and a 28% loss in PET counts, respectively, due to high PET counts during the RF pulses that had to be gated out. The calibration of the activity concentration of PET data during MR pulsing is reproducible within less than 6%. Our initial results demonstrate the feasibility of performing simultaneous PET and MRI studies in adult rats and mice using the same PET insert in a small-bore 9.4 T MRI.

And then the dog died.

PubMed

Kaufman, Kenneth R; Kaufman, Nathaniel D

2006-01-01

Childhood grief and mourning of family and friends may have immediate and long-lasting consequences including depression, anxiety, social withdrawal, behavioral disturbances, and school underachievement. Childhood pet bereavement is no less important, because the pet is often considered a member of the family by the child. However, society does not always acknowledge the significance of pet bereavement, which can result in unresolved grief. This article, a case analysis with literature review, addresses childhood pet bereavement in the context of multiple prior losses (K. R. Kaufman & N. D. Kaufman, 2005). This case mirrors both old and new findings in grief research and therapy: (a) beneficial response to emotional expression of grief in context of search for meaning; (b) beneficial response to cognitive approach toward grief with ability to prevent development of complicated grief even in the face of multiple losses; (c) beneficial effects associated with supportive family and with positive self-concept; (d) intensity of grief magnified by the child's degree of attachment to the pet, the suddenness of the pet's death, the multiple prior losses, and the role of the pet in the child's life; and (e) resiliency. This case further emphasizes the need for parents not to trivialize death of pets, to appreciate the role pets have in children's lives, and to assist the child in multiple approaches toward expression (be it verbal, written, or artistic). Finally, this case reinforces the ability of the child to assist in family bereavement and to serve as teacher.

Matching the Decay Half-Life with the Biological Half-Life: ImmunoPET Imaging with 44Sc-Labeled Cetuximab Fab Fragment

PubMed Central

2015-01-01

Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of 44Sc–CHX-A″-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-A″-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments. PMID:25389697

In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector.

PubMed

Läppchen, Tilman; Kiefer, Yvonne; Holland, Jason P; Bartholomä, Mark D

2018-05-01

We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for complexation of the PET nuclides gallium-68 and copper-64. The main objective of this study was to evaluate the stability and pharmacokinetics of 68 Ga- and 64 Cu-complexes of the bifunctional chelator NODIA-Me 1 covalently bound to a PSMA targeting vector in vivo. NODIA-Me 1 was conjugated to the PSMA targeting Glu-NH-CO-NH-Lys moiety to give the bioconjugate NODIA-Me-NaI-Ahx-PSMA 4. The stability of [ 68 Ga]4 and [ 64 Cu]4 was assessed in vitro by serum stability studies. The PSMA binding affinity was determined in competitive cell experiments in LNCaP cells using 68 Ga-PSMA-HBED-CC as radioligand. The stability and pharmacokinetics of [ 68 Ga]4 and [ 64 Cu]4 was evaluated by PET imaging and ex vivo biodistribution studies in mice bearing subcutaneous LNCaP tumors. In human serum, [ 68 Ga]4 and [ 64 Cu]4 remained intact to 85% (3 h) and 92% (24 h), respectively. Nature of the metal chelate influenced PSMA binding affinity with IC 50 of 233 ± 10 nM for uncomplexed 4, 681 ± 7 nM for Cu-4 and 176 ± 10 nM for Ga-4. In animal studies, [ 68 Ga]4 and [ 64 Cu]4 revealed low uptake (≤1% IA g -1 ) in the majority of organs. Kidney uptake at 1 h p.i. was 6.28 ± 0.92% IA g -1 and 4.96 ± 0.79% IA g -1 and specific tumor uptake was 1.33 ± 0.46% IA g -1 and 2.15 ± 0.38% IA g -1 for [ 68 Ga]4 and [ 64 Cu]4, respectively. The bifunctional chelator NODIA-Me 1 was successfully conjugated to a PSMA targeting moiety. In small-animal PET imaging and ex vivo biodistribution studies, 68 Ga- and 64 Cu-labelled conjugates specifically delineated PSMA-positive LNCaP tumors and exhibited rapid renal clearance from non-target tissues with no significant demetallation/transchelation in vivo. The results support further development of this novel chelating platform for production of 68 Ga- and 64 Cu-labelled radiopharmaceuticals. Copyright © 2017. Published by Elsevier Inc.

Infections from leisure-time activities.

PubMed

Schlossberg, D

2001-05-01

Leisure-time activities expose us to a variety of infections. The traveler confronts new pathogens and vectors. Camping, hiking and gardening have attendant risks, as does exposure to fresh and salt water. Adventuresome eating poses gastronomic threats, and pets, sexual exposure and organized sports each contribute distinctive infectious risks to participants.

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Deepti; Rawat, Surender; Waseem, Mohd

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K{sub m} values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative propertymore » against BSA and Cu{sup 2+}/H{sub 2}O{sub 2} model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.« less

Recombinant antigens for immunodiagnosis of cystic echinococcosis

PubMed Central

Li, Jun; Zhang, Wen-Bao

2004-01-01

Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections. PMID:15188015

Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

PubMed

Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

2009-08-15

To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

WE-AB-204-09: Respiratory Motion Correction in 4D-PET by Simultaneous Motion Estimation and Image Reconstruction (SMEIR)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalantari, F; Wang, J; Li, T

2015-06-15

Purpose: In conventional 4D-PET, images from different frames are reconstructed individually and aligned by registration methods. Two issues with these approaches are: 1) Reconstruction algorithms do not make full use of all projections statistics; and 2) Image registration between noisy images can Result in poor alignment. In this study we investigated the use of simultaneous motion estimation and image reconstruction (SMEIR) method for cone beam CT for motion estimation/correction in 4D-PET. Methods: Modified ordered-subset expectation maximization algorithm coupled with total variation minimization (OSEM- TV) is used to obtain a primary motion-compensated PET (pmc-PET) from all projection data using Demons derivedmore » deformation vector fields (DVFs) as initial. Motion model update is done to obtain an optimal set of DVFs between the pmc-PET and other phases by matching the forward projection of the deformed pmc-PET and measured projections of other phases. Using updated DVFs, OSEM- TV image reconstruction is repeated and new DVFs are estimated based on updated images. 4D XCAT phantom with typical FDG biodistribution and a 10mm diameter tumor was used to evaluate the performance of the SMEIR algorithm. Results: Image quality of 4D-PET is greatly improved by the SMEIR algorithm. When all projections are used to reconstruct a 3D-PET, motion blurring artifacts are present, leading to a more than 5 times overestimation of the tumor size and 54% tumor to lung contrast ratio underestimation. This error reduced to 37% and 20% for post reconstruction registration methods and SMEIR respectively. Conclusion: SMEIR method can be used for motion estimation/correction in 4D-PET. The statistics is greatly improved since all projection data are combined together to update the image. The performance of the SMEIR algorithm for 4D-PET is sensitive to smoothness control parameters in the DVF estimation step.« less

Injuries caused by pets in Asian urban households: a cross-sectional telephone survey

PubMed Central

Chan, Emily Y Y; Gao, Yang; Li, Liping; Lee, Po Yi

2017-01-01

Objectives Little is known about pet-related injuries in Asian populations. This study primarily aimed to investigate the incidence rate of pet-related household injuries in Hong Kong, an urban Chinese setting. Setting Cantonese-speaking non-institutionalised population of all ages in Hong Kong accessible by telephone land-line. Participants A total of 43 542 telephone numbers were dialled and 6570 residents successfully completed the interviews. Primary and secondary outcome measures Data of pet-related household injuries in the previous 12 months, pet ownership and socio-demographic characteristics were collected with a questionnaire. Direct standardisation of the incidence rates of pet-related household injuries by gender and age to the 2009 Hong Kong Population Census was estimated. Univariate and multivariate analyses were performed to estimate risks of socio-demographic factors and pet ownership for the injury. Results A total of 84 participants experienced pet-related household injuries in the past 12 months, with an overall person-based incidence rate of 1.28%. The majority of the victims were injured once (69.6%). Cats (51.6%) were the most common pets involved. Pet owners were at an extremely higher risk after controlling for other factors (adjusted OR: 52.0, 95% CI 22.1 to 98.7). Females, the unmarried, those with higher monthly household income and those living in lower-density housing were more likely to be injured by pets. Conclusions We project a pet-related household injury incidence rate of 1.24% in the general Hong Kong population, with 86 334 residents sustaining pet-related injuries every year. Pet ownership puts people at extremely high risk, especially the unmarried. Further studies should focus on educating pet owners to reduce pet-related injuries in urban Greater China. PMID:28110284

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

PubMed Central

Marshall, Kathryn E.; Robinson, Errol W.; Hengel, Shawna M.; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation. PMID:22470473

FRET imaging of diatoms expressing a biosilica-localized ribose sensor.

PubMed

Marshall, Kathryn E; Robinson, Errol W; Hengel, Shawna M; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.

Utility of 18F-fluoro-deoxyglucose emission tomography/computed tomography fusion imaging (18F-FDG PET/CT) in combination with ultrasonography for axillary staging in primary breast cancer.

PubMed

Ueda, Shigeto; Tsuda, Hitoshi; Asakawa, Hideki; Omata, Jiro; Fukatsu, Kazuhiko; Kondo, Nobuo; Kondo, Tadaharu; Hama, Yukihiro; Tamura, Katsumi; Ishida, Jiro; Abe, Yoshiyuki; Mochizuki, Hidetaka

2008-06-09

Accurate evaluation of axillary lymph node (ALN) involvement is mandatory before treatment of primary breast cancer. The aim of this study is to compare preoperative diagnostic accuracy between positron emission tomography/computed tomography with 18F-fluorodeoxyglucose (18F-FDG PET/CT) and axillary ultrasonography (AUS) for detecting ALN metastasis in patients having operable breast cancer, and to assess the clinical management of axillary 18F-FDG PET/CT for therapeutic indication of sentinel node biopsy (SNB) and preoperative systemic chemotherapy (PSC). One hundred eighty-three patients with primary operable breast cancer were recruited. All patients underwent 18F-FDG PET/CT and AUS followed by SNB and/or ALN dissection (ALND). Using 18F-FDG PET/CT, we studied both a visual assessment of 18F-FDG uptake and standardized uptake value (SUV) for axillary staging. In a visual assessment of 18F-FDG PET/CT, the diagnostic accuracy of ALN metastasis was 83% with 58% in sensitivity and 95% in specificity, and when cut-off point of SUV was set at 1.8, sensitivity, specificity, and accuracy were 36, 100, and 79%, respectively. On the other hand, the diagnostic accuracy of AUS was 85% with 54% in sensitivity and 99% in specificity. By the combination of 18F-FDG PET/CT and AUS to the axilla, the sensitivity, specificity, and accuracy were 64, 94, and 85%, respectively. If either 18F-FDG PET uptake or AUS was positive in allixa, the probability of axillary metastasis was high; 50% (6 of 12) in 18F-FDG PET uptake only, 80% (4 of 5) in AUS positive only, and 100% (28 of 28) in dual positive. By the combination of AUS and 18F-FDG PET/CT, candidates of SNB were more appropriately selected. The axillary 18F-FDG uptake was correlated with the maximum size and nuclear grade of metastatic foci (p = 0.006 and p = 0.03). The diagnostic accuracy of 18F-FDG PET/CT was shown to be nearly equal to ultrasound, and considering their limited sensitivities, the high radiation exposure by 18F-FDG PET/CT and also costs of the examination, it is likely that AUS will be more cost-effective in detecting massive axillary tumor burden. However, when we cannot judge the axillary staging using AUS alone, metabolic approach of 18F-FDG PET/CT for axillary staging would enable us a much more confident diagnosis.

Assessment of α-fetoprotein targeted HSV1-tk expression in hepatocellular carcinoma with in vivo imaging.

PubMed

Park, Ju Hui; Kim, Kwang Il; Lee, Kyo Chul; Lee, Yong Jin; Lee, Tae Sup; Chung, Wee Sup; Lim, Sang Moo; Kang, Joo Hyun

2015-02-01

Tumor-specific enhancer/promoter is applicable for targeting gene expression in tumors and helpful for tumor-targeting imaging and therapy. We aimed to acquire α-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) specific images using adenovirus containing HSV1-tk gene controlled by AFP enhancer/promoter and evaluate in vivo ganciclovir (GCV)-medicated therapeutic effects on AFP-targeted HSV1-tk expression with (18)F-FDG positron emission tomography (PET). Recombinant adenovirus expressing HSV1-tk under AFP enhancer/promoter was produced (AdAFP-TK) and the expression levels were evaluated by RT-PCR and (125)I-IVDU uptake. GCV-mediated HSV1-tk cytotoxicity was determined by MTT assay. After the mixture of AdAFP-fLuc and AdAFP-TK was administrated, bioluminescent images (BLIs) and (18)F-FHBG PET images were obtained in tumor-bearing mice. In vivo therapeutic effects of AdAFP-TK and GCV in the HuH-7 xenograft model were monitored by (18)F-FDG PET. When infected with AdAFP-TK, cell viability in HuH-7 was reduced, but those in HT-29 and SK-Hep-1 were not significantly decreased at any GCV concentration less than 100 μM. AFP-targeted fLuc and HSV1-tk expression were clearly visualized by BLI and (18)F-FHBG PET images in AFP-producing HCC, respectively. In vivo GCV-mediated tumor growth inhibition by AFP-targeted HSV1-tk expression was monitored by (18)F-FDG PET. Recombinant AdAFP-TK could be applied for AFP-targeted HCC gene therapy and imaging in AFP-producing HCC.

Preoperative Staging With 11C-Choline PET/CT Is Adequately Accurate in Patients With Very High-Risk Prostate Cancer.

PubMed

Schiavina, Riccardo; Bianchi, Lorenzo; Mineo Bianchi, Federico; Borghesi, Marco; Pultrone, Cristian Vincenzo; Dababneh, Hussam; Castellucci, Paolo; Ceci, Francesco; Nanni, Cristina; Gaudiano, Caterina; Fiorentino, Michelangelo; Porreca, Angelo; Chessa, Francesco; Minervini, Andrea; Fanti, Stefano; Brunocilla, Eugenio

2018-05-30

To evaluate the accuracy of 11 C-choline positron emission tomography (PET)/computed tomography (CT) for nodal staging of prostate cancer (PCa) in different populations of high-risk patients. We evaluated 262 individuals with intermediate- or high-risk PCa submitted to radical prostatectomy and extended pelvic lymph node dissection. Within men with high-risk disease, we identified a subgroup of individuals harboring very high-risk (VHR, n = 28) disease: clinical stage ≥ T2c and more than 5 cores with Gleason score 8-10; primary biopsy Gleason score of 5; 3 high-risk features; or prostate-specific antigen ≥ 30 ng/mL. The diagnostic accuracy of PET/CT and contrast-enhanced CT (CECT) was assessed after stratifying patients according to risk group classification on a patient- and anatomic region-based analysis. On patient-based analysis, considering high-risk patients (n = 155), 11 C-choline PET/CT versus CECT had sensitivity and specificity of 50% and 76% versus 21% and 92%, respectively. Considering VHR men as separate subgroups (n = 28), 11 C-choline PET/CT versus CECT had sensitivity and specificity of 71% and 93% versus 25% and 79%, respectively. Accordingly, in the VHR category, the area under the curve of 11 C-choline PET/CT versus CECT was 0.86 (95% confidence interval, 0.71-1.0) versus 0.69 (95% confidence interval, 0.52-0.86), respectively. On anatomic region-based analysis, considering the VHR group, 11 C-choline PET/CT versus CECT had sensitivity and specificity of 70.6% and 95.5% versus 35.3% and 98.5%, respectively. Patients with VHR characteristics could represent the ideal candidate to undergo disease staging with PET/CT before surgery with the highest cost efficacy. Copyright © 2018 Elsevier Inc. All rights reserved.

Thymidine Kinase PET Reporter Gene Imaging of Cancer Cells In Vivo.

PubMed

McCracken, Melissa N

2018-01-01

Positron emission tomography (PET) is a three dimensional imaging modality that detects the accumulation of radiolabeled isotopes in vivo. Ectopic expression of a thymidine kinase reporter gene allows for the specific detection of reporter cells in vivo by imaging with the reporter specific probe. PET reporter imaging is sensitive, quantitative and can be scaled into larger tumors or animals with little to no tissue diffraction. Here, we describe how thymidine kinase PET reporter genes can be used to noninvasively image cancer cells in vivo.

Pets, depression and long term survival in community living patients following myocardial infarction

PubMed Central

Friedmann, Erika; Thomas, Sue A.; Son, Heesook

2011-01-01

Evidence supports the contribution of depression, anxiety, and poor social support to mortality of hospitalized myocardial infarction (MI) patients. The contribution of depression to survival is independent of disease severity. Pet ownership, a non-human form of social support, has also been associated with one year survival of post-MI patients. The current study addresses whether pet ownership contributes independently to long term survival beyond the contributions of depression, anxiety, or low social support in post-MI patients who have already survived at least 6 months. Data from patients (N = 460) enrolled in the “Psychosocial Responses in the Home Automated External Defibrillator Trial (PR-HAT)”were used. Seventeen patients died during a median follow-up of 2.8 years. In Cox proportional hazards regression model that included depression, lack of pet ownership, and the interaction between depression and lack of pet ownership, not owning a pet was the only significant independent predictor of mortality (p = 0.036). The interaction between pet ownership and depression tended to be significant indicating that the effect of pet ownership on survival in this group of people who have supportive spouses/companions living with them may relate to depression. PMID:21857770

Molecular cloning, expression, and in silico structural analysis of guinea pig IL-17.

PubMed

Dirisala, Vijaya R; Jeevan, Amminikutty; Ramasamy, Suresh K; McMurray, David N

2013-11-01

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.

Prostate-specific membrane antigen PET/MRI validation of MR textural analysis for detection of transition zone prostate cancer.

PubMed

Bates, Anthony; Miles, Kenneth

2017-12-01

To validate MR textural analysis (MRTA) for detection of transition zone (TZ) prostate cancer through comparison with co-registered prostate-specific membrane antigen (PSMA) PET-MR. Retrospective analysis was performed for 30 men who underwent simultaneous PSMA PET-MR imaging for staging of prostate cancer. Thirty texture features were derived from each manually contoured T2-weighted, transaxial, prostatic TZ using texture analysis software that applies a spatial band-pass filter and quantifies texture through histogram analysis. Texture features of the TZ were compared to PSMA expression on the corresponding PET images. The Benjamini-Hochberg correction controlled the false discovery rate at <5%. Eighty-eight T2-weighted images in 18 patients demonstrated abnormal PSMA expression within the TZ on PET-MR. 123 images were PSMA negative. Based on the corrected p-value of 0.005, significant differences between PSMA positive and negative slices were found for 16 texture parameters: Standard deviation and mean of positive pixels for all spatial filters (p = <0.0001 for both at all spatial scaling factor (SSF) values) and mean intensity following filtration for SSF 3-6 mm (p = 0.0002-0.0018). Abnormal expression of PSMA within the TZ is associated with altered texture on T2-weighted MR, providing validation of MRTA for the detection of TZ prostate cancer. • Prostate transition zone (TZ) MR texture analysis may assist in prostate cancer detection. • Abnormal transition zone PSMA expression correlates with altered texture on T2-weighted MR. • TZ with abnormal PSMA expression demonstrates significantly reduced MI, SD and MPP.

Survey of intestinal helminths collected from pet rodents in México.

PubMed

Panti-May, Jesús Alonso; Caraveo-Centeno, Luis; Hernández-Betancourt, Silvia F; Robles, María Del Rosario; Machain-Williams, Carlos

2017-11-01

In this survey, intestinal helminths from pet rodents in Mérida, México, were analyzed. A total of 46 mice Mus musculus, 28 hamsters Mesocricetus auratus, 23 rats Rattus norvegicus, and 1 gerbil Meriones unguiculatus were purchased from six pet shops and one black market for wildlife in the city of Mérida. The overall prevalence of helminths in rodents was 61.2% (60/98). Six species of helminths were identified: the zoonotic cestode Rodentolepis nana, and the nematodes Aspiculuris tetraptera, Dentostomella translucida, Syphacia obvelata, Syphacia mesocriceti, and Syphacia muris. Of the 60 infected rodents, 25 (41.7%) harbored 2 or 3 species of helminths. Rodentolepis nana was found in 4.3% of mice and 17.9% of hamsters. This is the first report of infection with S. muris in pet rats. Considering the close physical contact between pet rodents and humans, the presence of R. nana in pets represents a potential risk of transmission, especially to children and immunocompromised individuals.

Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen.

PubMed

Nasiri, K; Nassiri, M R; Tahmoorespur, M; Haghparast, A; Zibaee, S

2016-01-01

Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus.

SU-F-I-58: Image Quality Comparisons of Different Motion Magnitudes and TR Values in MR-PET

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patrick, J; Thompson, R; Tavallaei, M

2016-06-15

Purpose: The aim of this work is to evaluate the accuracy and sensitivity of a respiratory-triggered MR-PET protocol in detecting four different sized lesions at two different magnitudes of motion, with two different TR values, using a novel PET-MR-CT compatible respiratory motion phantom. Methods: The eight-compartment torso phantom was setup adjacent to the motion stage, which moved four spherical compartments (28, 22, 17, 10 mm diameter) in two separate (1 and 2 cm) linear motion profiles, simulating a 3.5 second respiratory cycle. Scans were acquired on a 3T MR-PET system (Biograph mMR; Siemens Medical Solutions, Germany). MR measurements were takenmore » with: 1) Respiratory-triggered T2-weighted turbo spin echo (BLADE) sequence in coronal orientation, and 2) Real-time balanced steady-state gradient echo sequence (TrueFISP) in coronal and sagittal planes. PET was acquired simultaneously with MR. Sphere geometries and motion profiles were measured and compared with ground truths for T2 BLADE-TSE acquisitions and real time TrueFISP images. PET quantification and geometry measurements were taken using standardized uptake values, voxel intensity plots and were compared with known values, and examined alongside MR-based attenuation maps. Contrast and signal-to-noise ratios were also compared for each of the acquisitions as functions of motion range and TR. Results: Comparison of lesion diameters indicate the respiratory triggered T2 BLADE-TSE was able to maintain geometry within −2 mm for 1 cm motion for both TR values, and within −3.1 mm for TR = 2000 ms at 2 cm motion. Sphere measurements in respiratory triggered PET images were accurate within +/− 5 mm for both ranges of motion for 28, 22, and 17 mm diameter spheres. Conclusion: Hybrid MR-PET systems show promise in imaging lung cancer in non-compliant patients, with their ability to acquire both modalities simultaneously. However, MR-based attenuation maps are still susceptible to motion derived artifacts and pose the potential to affect PET accuracy.« less

Quantitative agreement between [(15)O]H2O PET and model free QUASAR MRI-derived cerebral blood flow and arterial blood volume.

PubMed

Heijtel, D F R; Petersen, E T; Mutsaerts, H J M M; Bakker, E; Schober, P; Stevens, M F; van Berckel, B N M; Majoie, C B L M; Booij, J; van Osch, M J P; van Bavel, E T; Boellaard, R; Lammertsma, A A; Nederveen, A J

2016-04-01

The purpose of this study was to assess whether there was an agreement between quantitative cerebral blood flow (CBF) and arterial cerebral blood volume (CBVA) measurements by [(15)O]H2O positron emission tomography (PET) and model-free QUASAR MRI. Twelve healthy subjects were scanned within a week in separate MRI and PET imaging sessions, after which quantitative and qualitative agreement between both modalities was assessed for gray matter, white matter and whole brain region of interests (ROI). The correlation between CBF measurements obtained with both modalities was moderate to high (r(2): 0.28-0.60, P < 0.05), although QUASAR significantly underestimated CBF by 30% (P < 0.001). CBVA was moderately correlated (r(2): 0.28-0.43, P < 0.05), with QUASAR yielding values that were only 27% of the [(15)O]H2O-derived values (P < 0.001). Group-wise voxel statistics identified minor areas with significant contrast differences between [(15)O]H2O PET and QUASAR MRI, indicating similar qualitative CBVA and CBF information by both modalities. In conclusion, the results of this study demonstrate that QUASAR MRI and [(15)O]H2O PET provide similar CBF and CBVA information, but with systematic quantitative discrepancies. Copyright © 2016 John Wiley & Sons, Ltd.

SU-E-I-81: Targeting of HER2-Expressing Tumors with Dual PET-MR Imaging Probes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, P; Peng, Y; Sun, M

2015-06-15

Purpose: The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Trastuzumab, effective in about 15 % of women with breast cancer, downregulates signalling through the Akt/PI3K and MAPK pathways.These pathways modulate metabolism which can be monitored by positron emission tomography (PET) and magnetic resonance imaging (MRI). Methods: The relationship between response of HER2 overexpressing tumours and changes in imaging PET or SPECT and MRI willmore » be examined by a integrated bimodal imaging probe.Small (7 kDa) high-affinity anti-HER2 Affibody molecules and KCCYSL targeting peptide may be suitable tracers for visualization of HER2-expressing tumors. Peptide-conjugated iron oxide nanoparticles (Fe3O4 NPs) as MRI imaging and CB-TE2A as PET imaging are integrated into a single synthetic molecule in the HER2 positive cancer. Results: One of targeted contrast bimodal imaging probe agents was synthesized and evaluated to target HER2-expressing tumors in a HER2 positive rat model. We will report the newest results regarding the development of bimodal imaging probes. Conclusion: The preliminary results of the bimodal imaging probe presents high correlation of MRI signal and PET imaging intensity in vivo. This unique feature can hardly be obtained by single model contrast agents. It is envisioned that this bimodal agents can hold great potential for accurate detection of HER2-expressing tumors which are critical for clinical management of the disease.« less

Molecular cloning and expression of PoIR2, a novel gene involved in immune response in Japanese flounder ( Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Li, Shuo; Li, Chunmei; Wang, Xubo; Wang, Yanan; Liu, Zhipeng; Zhai, Teng; Zhang, Quanqi

2010-03-01

A novel immune-related gene was expressed in Japanese flounder ( Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 ( P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Activity of Tachykinin1-Expressing Pet1 Raphe Neurons Modulates the Respiratory Chemoreflex.

PubMed

Hennessy, Morgan L; Corcoran, Andrea E; Brust, Rachael D; Chang, YoonJeung; Nattie, Eugene E; Dymecki, Susan M

2017-02-15

Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 ( Tac1 ) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1 , referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine -N- oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO 2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO 2 Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a Pet1 neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei. SIGNIFICANCE STATEMENT Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using genetic tools, we characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 ( Tac1-Pet1 neurons), mapping soma localization to the caudal medulla primarily and axonal projections to brainstem motor nuclei most prominently, and, when silenced, observed blunting of the ventilatory response to inhaled CO 2 Tac1-Pet1 neurons thus appear distinct from and contrast previously described Egr2-Pet1 neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive. Copyright © 2017 the authors 0270-6474/17/371807-13$15.00/0.

High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26

PubMed Central

Thisgaard, Helge; Rosenström, Ulrika; Dam, Johan Hygum; Larhed, Mats

2017-01-01

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors. PMID:29097932

Comparison of receptor affinity of natSc-DOTA-TATE versus natGa-DOTA-TATE.

PubMed

Koumarianou, Eftychia; Pawlak, Dariusz; Korsak, Agnieszka; Mikolajczak, Renata

2011-01-01

44Sc as a positron emitter can be an interesting alternative to 68Ga (T½=67.71 min) due to its longer half-life (T½=3.97 h). Moreover, the b-emitter 47Sc can be used for therapy when attached to the same biomolecule vectors. DOTA as a chelating agent has been proven suitable for the radiolabelling of peptides recognising tumour cell receptors in vivo with M3+ radiometals. DOTA-derivatized peptides have been successfully labelled with 90Y and 177Lu for therapy, and with 68Ga for PET imaging. However, published data on 44Sc-labelled DOTA-biomolecules as potential PET radiotracers are still very limited. The aim of this study was to compare the affinity of natGa- and natSc-labelled DOTA-TATE to somatostatin receptors subtype 2 expressed in rat pancreatic cancer cell line AR42J. The cold complexes of DOTA-TATE with natGa and natSc were synthesized and identified by HPLC and MS analysis and evaluated in vitro for competitive binding to cancer cell line AR42J expressing somatostatin receptors subtype 2 (sstr2). The IC50 values calculated from the displacement curve of {125I-Tyr11}-SST-14 were: 0.20±0.18, 0.70±0.20, 0.64±0.22 and 0.67±0.12 for natGa-DOTA-TATE, natSc-DOTA-TATE, DOTA-TATE, and {Tyr11}-SST-14 complexes, respectively, with the affinity lowering in the decreasing order: natGa-DOTA-TATE>DOTA-TATE>Tyr11-SST-14>natSc-DOTA-TATE. The binding affinity of natGa-DOTA-TATE appeared higher than that of natSc-DOTA-TATE. Further in vitro and in vivo studies are needed to verify the influence of the chelated metal on the affinity and uptake of the respective radiolabelled compounds. This information might be crucial when the in vivo applications of peptides labelled with 68Ga and 44Sc for PET, as well as the use of 47Sc for radiotherapy are considered.

The effect of 18F-FDG-PET image reconstruction algorithms on the expression of characteristic metabolic brain network in Parkinson's disease.

PubMed

Tomše, Petra; Jensterle, Luka; Rep, Sebastijan; Grmek, Marko; Zaletel, Katja; Eidelberg, David; Dhawan, Vijay; Ma, Yilong; Trošt, Maja

2017-09-01

To evaluate the reproducibility of the expression of Parkinson's Disease Related Pattern (PDRP) across multiple sets of 18F-FDG-PET brain images reconstructed with different reconstruction algorithms. 18F-FDG-PET brain imaging was performed in two independent cohorts of Parkinson's disease (PD) patients and normal controls (NC). Slovenian cohort (20 PD patients, 20 NC) was scanned with Siemens Biograph mCT camera and reconstructed using FBP, FBP+TOF, OSEM, OSEM+TOF, OSEM+PSF and OSEM+PSF+TOF. American Cohort (20 PD patients, 7 NC) was scanned with GE Advance camera and reconstructed using 3DRP, FORE-FBP and FORE-Iterative. Expressions of two previously-validated PDRP patterns (PDRP-Slovenia and PDRP-USA) were calculated. We compared the ability of PDRP to discriminate PD patients from NC, differences and correlation between the corresponding subject scores and ROC analysis results across the different reconstruction algorithms. The expression of PDRP-Slovenia and PDRP-USA networks was significantly elevated in PD patients compared to NC (p<0.0001), regardless of reconstruction algorithms. PDRP expression strongly correlated between all studied algorithms and the reference algorithm (r⩾0.993, p<0.0001). Average differences in the PDRP expression among different algorithms varied within 0.73 and 0.08 of the reference value for PDRP-Slovenia and PDRP-USA, respectively. ROC analysis confirmed high similarity in sensitivity, specificity and AUC among all studied reconstruction algorithms. These results show that the expression of PDRP is reproducible across a variety of reconstruction algorithms of 18F-FDG-PET brain images. PDRP is capable of providing a robust metabolic biomarker of PD for multicenter 18F-FDG-PET images acquired in the context of differential diagnosis or clinical trials. Copyright © 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

Human placental peroxisome proliferator-activated receptor delta and gamma expression in healthy pregnancy and in preeclampsia and intrauterine growth restriction.

PubMed

Rodie, Vanessa A; Young, Anne; Jordan, Fiona; Sattar, Naveed; Greer, Ian A; Freeman, D J

2005-07-01

Human and animal studies have demonstrated that peroxisome proliferator-activated receptors (PPARs) are important in placental development and play key roles in metabolism and inflammation. We studied placental PPARdelta, PPARgamma, and retinoid X receptor alpha (RXRalpha) expression in healthy pregnancy and in preeclampsia (PET) and intrauterine growth restriction (IUGR). Using immunocytochemistry, PPARdelta, PPARgamma, and RXRalpha were localized to the cyto- and syncytiotrophoblast and invading trophoblast columns in first and second trimester placentas. Third trimester placentas from healthy pregnancy, and in PET and IUGR, demonstrated PPARdelta, PPARgamma, and RXRalpha staining within the syncytium, and localization within isolated cells in the stroma. In uncomplicated pregnancies, PPARdelta mRNA expression (PPARdelta:18s ratio, third trimester median 0.43 [interquartile (IQ) range 0.26-0.52] vs first trimester 0.20 [0.00-0.26], P = .03) and PPARdelta protein expression (third trimester 3.94 [2.45-4.68] vs first trimester 1.29 [0.78-2.29] optical densitometry [OD] mm(2), P = .04) were higher in the third trimester than in the first trimester. There were no consistent differences in PPARdelta, PPARgamma, or RXRalpha mRNA and protein expression among PET or IUGR placentas and controls. PPARdelta expression is up-regulated between the first and third trimester, indicating a role for this nuclear receptor in placental function. We found no evidence that placental PPARdelta, PPARgamma, and RXRalpha expression is changed in PET or IUGR. This suggests that changes in total placental PPAR expression are not involved in the pathophysiology of these conditions.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

PubMed

Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

64Cu-Labeled multifunctional dendrimers for targeted tumor PET imaging.

PubMed

Ma, Wenhui; Fu, Fanfan; Zhu, Jingyi; Huang, Rui; Zhu, Yizhou; Liu, Zhenwei; Wang, Jing; Conti, Peter S; Shi, Xiangyang; Chen, Kai

2018-03-29

We report the use of multifunctional folic acid (FA)-modified dendrimers as a platform to radiolabel with 64Cu for PET imaging of folate receptor (FR)-expressing tumors. In this study, amine-terminated generation 5 (G5) poly(amidoamine) dendrimers were sequentially modified with fluorescein isothiocyanate (FI), FA, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), followed by acetylation of the remaining dendrimer terminal amines. The as-formed multifunctional DOTA-FA-FI-G5·NHAc dendrimers were then radiolabeled with 64Cu via the DOTA chelation. We show that the FA modification renders the dendrimers with targeting specificity to cancer cells overexpressing FR in vitro. Importantly, the radiolabeled 64Cu-DOTA-FA-FI-G5·NHAc dendrimers can be used as a nanoprobe for specific targeting of FR-overexpressing cancer cells in vitro and targeted microPET imaging of the FR-expressing xenografted tumor model in vivo. The developed 64Cu-labeled multifunctional dendrimeric nanoprobe may hold great promise to be used for targeted PET imaging of different types of FR-expressing cancer.

Definition of Contravariant Velocity Components

NASA Technical Reports Server (NTRS)

Hung, Ching-moa; Kwak, Dochan (Technical Monitor)

2002-01-01

In this paper we have reviewed the basics of tensor analysis in an attempt to clarify some misconceptions regarding contravariant and covariant vector components as used in fluid dynamics. We have indicated that contravariant components are components of a given vector expressed as a unique combination of the covariant base vector system and, vice versa, that the covariant components are components of a vector expressed with the contravariant base vector system. Mathematically, expressing a vector with a combination of base vector is a decomposition process for a specific base vector system. Hence, the contravariant velocity components are decomposed components of velocity vector along the directions of coordinate lines, with respect to the covariant base vector system. However, the contravariant (and covariant) components are not physical quantities. Their magnitudes and dimensions are controlled by their corresponding covariant (and contravariant) base vectors.

Differential Receptor Tyrosine Kinase PET Imaging for Therapeutic Guidance.

PubMed

Wehrenberg-Klee, Eric; Turker, N Selcan; Heidari, Pedram; Larimer, Benjamin; Juric, Dejan; Baselga, José; Scaltriti, Maurizio; Mahmood, Umar

2016-09-01

Inhibitors of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway hold promise for the treatment of breast cancer, but resistance to these treatments can arise via feedback loops that increase surface expression of the receptor tyrosine kinases (RTK) epidermal growth factor receptor 1 (EGFR) and human epidermal growth factor receptor 3 (HER3), leading to persistent growth pathway signaling. We developed PET probes that provide a method of imaging this response in vivo, determining which tumors may use this escape pathway while avoiding the need for repeated biopsies. Anti-EGFR-F(ab')2 and anti-HER3-F(ab')2 were generated from monoclonal antibodies by enzymatic digestion, conjugated to DOTA, and labeled with (64)Cu. A panel of breast cancer cell lines was treated with increasing concentrations of the AKT inhibitor GDC-0068 or the PI3K inhibitor GDC-0941. Pre- and posttreatment expression of EGFR and HER3 was compared using Western blot and correlated to probe accumulation with binding studies. Nude mice xenografts of HCC-70 or MDA-MB-468 were treated with either AKT inhibitor or PI3K inhibitor and imaged with either EGFR or HER3 PET probe. Changes in HER3 and EGFR PET probe accumulation correlate to RTK expression change as assessed by Western blot (R(2) of 0.85-0.98). EGFR PET probe PET/CT imaging of HCC70 tumors shows an SUV of 0.32 ± 0.03 for vehicle-, 0.50 ± 0.01 for GDC-0941-, and 0.62 ± 0.01 for GDC-0068-treated tumors, respectively (P < 0.01 for both comparisons to vehicle). HER3 PET probe PET/CT imaging of MDAMB468 tumors shows an SUV of 0.35 ± 0.02 for vehicle- and 0.73 ± 0.05 for GDC-0068-treated tumors (P < 0.01). Our imaging studies, using PET probes specific to EGFR and HER3, show that changes in RTK expression indicative of resistance to PI3K and AKT inhibitors can be seen within days of therapy initiation and are of sufficient magnitude as to allow reliable clinical interpretation. Noninvasive PET monitoring of these RTK feedback loops should help to rapidly assess resistance to PI3K and AKT inhibitors and guide selection of an appropriate combinatorial therapeutic regimen on an individual patient basis. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

PD-L1 is an activation-independent marker of brown adipocytes.

PubMed

Ingram, Jessica R; Dougan, Michael; Rashidian, Mohammad; Knoll, Marko; Keliher, Edmund J; Garrett, Sarah; Garforth, Scott; Blomberg, Olga S; Espinosa, Camilo; Bhan, Atul; Almo, Steven C; Weissleder, Ralph; Lodish, Harvey; Dougan, Stephanie K; Ploegh, Hidde L

2017-09-21

Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or β-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state.Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody.

Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

PubMed

Kaur, Gagandeep; Singh, Amninder; Sharma, Rohit; Sharma, Vinay; Verma, Swati; Sharma, Pushpender K

2016-06-01

In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg -1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0-14.0 pH and 30-80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

(68)Ga-DOTA (0)-Tyr (3)-octreotide positron emission tomography in nasopharyngeal carcinoma.

PubMed

Schartinger, Volker H; Dudás, József; Url, Christoph; Reinold, Susanne; Virgolini, Irene J; Kroiss, Alexander; Riechelmann, Herbert; Uprimny, Christian

2015-01-01

PET/CT with (68)Ga-labelled [DOTA(0),Tyr(3)]-octreotide ((68)Ga-DOTA-TOC PET/CT) is a routinely used imaging modality for neuroendocrine tumours expressing somatostatin receptors (SSTR). Recent studies have shown SSTR expression in head and neck squamous cell carcinoma, albeit lower than in highly differentiated neuroendocrine tumours. We sought to determine whether nasopharyngeal carcinoma (NPC) positive for Epstein-Barr virus (EBV), a rare subtype of head and neck cancer, shows increased (68)Ga-DOTA-TOC uptake indicating expression of SSTR. Five patients with untreated, histologically proven EBV-positive NPC were referred for (68)Ga-DOTA-TOC PET/CT. Tracer uptake in tumour lesions was assessed visually and semiquantitatively measuring maximum standardized uptake values (SUVmax) and tumour to background ratios. Increased tumour-specific uptake was detected in all five patients with a median SUVmax of 10.6 (range 3.6 - 17.1) in the primary tumour and 13.2 (range 6.1 - 14.5) in cervical lymph node metastases. (68)Ga-DOTA-TOC PET/CT demonstrated tracer uptake in EBV-positive NPC comparable to that in highly differentiated neuroendocrine tumours. This observation is consistent with increased SSTR expression in EBV-positive NPC and may open new diagnostic and therapeutic windows in NPC.

Lower levels of the glial cell marker TSPO in drug-naive first-episode psychosis patients as measured using PET and [11C]PBR28.

PubMed

Collste, K; Plavén-Sigray, P; Fatouros-Bergman, H; Victorsson, P; Schain, M; Forsberg, A; Amini, N; Aeinehband, S; Erhardt, S; Halldin, C; Flyckt, L; Farde, L; Cervenka, S

2017-06-01

Several lines of evidence are indicative of a role for immune activation in the pathophysiology of schizophrenia. Nevertheless, studies using positron emission tomography (PET) and radioligands for the translocator protein (TSPO), a marker for glial activation, have yielded inconsistent results. Whereas early studies using a radioligand with low signal-to-noise in small samples showed increases in patients, more recent studies with improved methodology have shown no differences or trend-level decreases. Importantly, all patients investigated thus far have been on antipsychotic medication, and as these compounds may dampen immune cell activity, this factor limits the conclusions that can be drawn. Here, we examined 16 drug-naive, first-episode psychosis patients and 16 healthy controls using PET and the TSPO radioligand [ 11 C]PBR28. Gray matter (GM) volume of distribution (V T ) derived from a two-tissue compartmental analysis with arterial input function was the main outcome measure. Statistical analyses were performed controlling for both TSPO genotype, which is known to affect [ 11 C]PBR28 binding, and gender. There was a significant reduction of [ 11 C]PBR28 V T in patients compared with healthy controls in GM as well as in secondary regions of interest. No correlation was observed between GM V T and clinical or cognitive measures after correction for multiple comparisons. The observed decrease in TSPO binding suggests reduced numbers or altered function of immune cells in brain in early-stage schizophrenia.

Novel Nonreplicating Vaccinia Virus Vector Enhances Expression of Heterologous Genes and Suppresses Synthesis of Endogenous Viral Proteins.

PubMed

Wyatt, Linda S; Xiao, Wei; Americo, Jeffrey L; Earl, Patricia L; Moss, Bernard

2017-06-06

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation. Copyright © 2017 Wyatt et al.

Stable expression of the hepatitis B virus surface antigen containing pre-S2 protein in mouse cells using a bovine papillomavirus vector.

PubMed

Yoneyama, T; Akatsuka, T; Miyamura, T

1988-08-01

The large BglII fragment (2.8 kilobases) of hepatitis B virus DNA including the transcription unit for the hepatitis B surface antigen (HBsAg) was inserted into a bovine papillomavirus vector containing the neomycin resistance gene. The recombinant DNA was transfected into mouse C127 cells. A stable transformed cell line (MS128) secreting a large amount of 22 nm HBsAg particles containing pre-S2 protein was established. The secreted HBsAg particles had the receptor for polymerized human serum albumin. Immunoprecipitation and Western blot analyses showed that HBsAg particles consisted of two major proteins of 22K and 26K encoded by the S gene and a minor protein of 35K encoded by the pre-S2 and S genes. Southern blot analysis revealed that the transfected plasmid was integrated into the host chromosomal DNA and that most of the plasmid sequences were present. These results suggest that the stable expression of the HBsAg in MS128 cells is related to the integrated state of the recombinant DNA.

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

PubMed

Ferreira, Joshua P; Peacock, Ryan W S; Lawhorn, Ingrid E B; Wang, Clifford L

2011-12-01

The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

PubMed Central

Carnes, Aaron E.; Luke, Jeremy M.; Vincent, Justin M.; Anderson, Sheryl; Schukar, Angela; Hodgson, Clague P.; Williams, James A.

2010-01-01

Background For safety considerations, regulatory agencies recommend elimination of antibiotic resistance markers and nonessential sequences from plasmid DNA-based gene medicines. In the present study we analyzed antibiotic-free (AF) vector design criteria impacting bacterial production and mammalian transgene expression. Methods Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed EGFP and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction (RT-PCR), respectively, after transfection of human HEK293 cells. Results Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1,000 mg/L plasmid DNA) and high level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1,740 mg/L was developed. Conclusions We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors. PMID:20806425

Overexpression, purification, molecular characterization and the effect on tumor growth of ribosomal protein L22 from the Giant Panda (Ailuropoda melanoleuca).

PubMed

Li, Jian; Hou, Yiling; Ding, Xiang; Hou, Wanru; Song, Bo; Zeng, Yichun

2014-05-01

The ribosomal protein L22 (RPL22) protein belongs to the L22E family of ribosomal proteins. It is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of RPL22 of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL22 was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL22 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. The result indicated that the length of the fragment cloned is 414 bp, and it contains an open-reading frame of 387 bp encoding 128 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL22 protein is 14.74 kDa with a theoretical pI 9.21. The RPL22 gene can be really expressed in E. coli and the RPL22 protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 20.1 kDa polypeptide. The data showed that the recombinant protein RPL22 had a time- and dose-dependency on the cell growth inhibition rate. The human laryngeal carcinoma Hep-2 cells treated with 0.05-6 μg/ml of RPL22 for 24 h displayed significant cell growth inhibition (p<0.05, n=8) in assayed using MTT compared to the control (untreated) cells. The data indicate that the effect at low concentrations is better than high concentrations, and the concentration of 1.5 μg/ml has the best rate of growth inhibition of 47.70%. The inhibitory rate in mice treated with 1.5 μg/ml RPL22 protein can reach 43.75%. Histology of tumor organs shows that the tissues arranged looser in RPL22 group than those in control group. Meanwhile, there is no obvious damage to other organs, such as heart, lung and kidney. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL22 responsible for its anticancer activity.

Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin.

PubMed

Guo, L; Yan, X; Qian, S; Meng, G

2000-02-01

Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed against L-asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage-display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase-scFv46 complex had been treated with trypsin for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET-21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase-scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 degrees C. The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

PubMed

Bandeira, Vanessa S; Tomás, Hélio A; Alici, Evren; Carrondo, Manuel J T; Coroadinha, Ana S

2017-04-01

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na + ,K + -ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 10 7 infectious particles·ml -1 ). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.

Clinical utility of flumazenil-PET versus [18F]fluorodeoxyglucose-PET and MRI in refractory partial epilepsy. A prospective study in 100 patients.

PubMed

Ryvlin, P; Bouvard, S; Le Bars, D; De Lamérie, G; Grégoire, M C; Kahane, P; Froment, J C; Mauguière, F

1998-11-01

We assessed the clinical utility of [11C]flumazenil-PET (FMZ-PET) prospectively in 100 epileptic patients undergoing a pre-surgical evaluation, and defined the specific contribution of this neuro-imaging technique with respect to those of MRI and [18F]fluorodeoxyglucose-PET (FDG-PET). All patients benefited from a long term video-EEG monitoring, whereas an intracranial EEG investigation was performed in 40 cases. Most of our patients (73%) demonstrated a FMZ-PET abnormality; this hit rate was significantly higher in temporal lobe epilepsy (94%) than in other types of epilepsy (50%) (P < 0.001). Most FMZ-PET findings coexisted with a MRI abnormality (81%), including hippocampal atrophy (35%) and focal hypometabolism on FDG-PET (89%). The area of decreased FMZ binding was often smaller than that of glucose hypometabolism (48%) or larger than that of the MRI abnormality (28%). FMZ-PET did not prove superior to FDG-PET in assessing the extent of the ictal onset zone, as defined by intracranial EEG recordings. However, it provided useful data which were complementary to those of MRI and FDG-PET in three situations: (i) in temporal lobe epilepsy associated with MRI signs of hippocampal sclerosis, FMZ-PET abnormalities delineated the site of seizure onset precisely, whenever they were coextensive with FDG-PET abnormalities; (ii) in bi-temporal epilepsy, FMZ-PET helped to confirm the bilateral origin of seizures by showing a specific pattern of decreased FMZ binding in both temporal lobes in 33% of cases; (iii) in patients with a unilateral cryptogenic frontal lobe epilepsy, FMZ-PET provided further evidence of the side and site of seizure onset in 55% of cases. Thus, FMZ-PET deserves to be included in the pre-surgical evaluation of these specific categories of epileptic patients, representing approximately half of the population considered for epilepsy surgery.

Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum.

PubMed

Barranco-Medina, Sergio; López-Jaramillo, Francisco Javier; Bernier-Villamor, Laura; Sevilla, Francisca; Lázaro, Juan José

2006-07-01

A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 angstroms from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 angstroms, alpha = 102.90, beta = 104.40, gamma = 99.07 degrees, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.

Effect of Peptide Receptor Radionuclide Therapy on Somatostatin Receptor Status and Glucose Metabolism in Neuroendocrine Tumors: Intraindividual Comparison of Ga-68 DOTANOC PET/CT and F-18 FDG PET/CT

PubMed Central

Oh, Sowon; Prasad, Vikas; Lee, Dong Soo; Baum, R. P.

2011-01-01

The heterogeneous nature of the neuroendocrine tumors (NET) makes it challenging to find one uniformly applicable management protocol which is especially true for diagnosis. The discovery of the overexpression of somatostatin receptors (SMS-R) on neuroendocrine tumor cells lead to the generalized and rapid acceptance of radiolabeled somatostatin receptor analogs for staging and restaging of NET as well as for Peptide Receptor Radionuclide Therapy (PRRNT) using Y-90 and Lu-177 DOTATATE/DOTATOC. In this present work we tried to look in to the effect of PRRNT on the glucose metabolism assessed by F-18 FDG PET/CT and SMS-R density assessed by Ga-68 DOTANOC PET/CT. We observed a complex relationship between the somatostatin receptor expression and glucose metabolism with only 56% (77/138) of the lesions showing match, while the others show mismatch between the receptor status and metabolism. The match between receptor expression and glucose metabolism increases with the grade of NET. In grade 3 NET, there is a concurrence between the changes in glucose metabolism and somatostatin receptor expression. PRRNT was found to be more effective in lesions with higher receptor expression. PMID:22121482

[68Ga]Pentixafor-PET/CT for imaging of chemokine receptor CXCR4 expression in multiple myeloma - Comparison to [18F]FDG and laboratory values.

PubMed

Lapa, Constantin; Schreder, Martin; Schirbel, Andreas; Samnick, Samuel; Kortüm, Klaus Martin; Herrmann, Ken; Kropf, Saskia; Einsele, Herrmann; Buck, Andreas K; Wester, Hans-Jürgen; Knop, Stefan; Lückerath, Katharina

2017-01-01

Chemokine (C-X-C motif) receptor 4 (CXCR4) is a key factor for tumor growth and metastasis in several types of human cancer including multiple myeloma (MM). Proof-of-concept of CXCR4-directed radionuclide therapy in MM has recently been reported. This study assessed the diagnostic performance of the CXCR4-directed radiotracer [ 68 Ga]Pentixafor in MM and a potential role for stratifying patients to CXCR4-directed therapies. Thirty-five patients with MM underwent [ 68 Ga]Pentixafor-PET/CT for evaluation of eligibility for endoradiotherapy. In 19/35 cases, [ 18 F]FDG-PET/CT for correlation was available. Scans were compared on a patient and on a lesion basis. Tracer uptake was correlated with standard clinical parameters of disease activity. [ 68 Ga]Pentixafor-PET detected CXCR4-positive disease in 23/35 subjects (66%). CXCR4-positivity at PET was independent from myeloma subtypes, cytogenetics or any serological parameters and turned out as a negative prognostic factor. In the 19 patients in whom a comparison to [ 18 F]FDG was available, [ 68 Ga]Pentixafor-PET detected more lesions in 4/19 (21%) subjects, [ 18 F]FDG proved superior in 7/19 (37%). In the remaining 8/19 (42%) patients, both tracers detected an equal number of lesions. [ 18 F]FDG-PET positivity correlated with [ 68 Ga]Pentixafor-PET positivity (p=0.018). [ 68 Ga]Pentixafor-PET provides further evidence that CXCR4 expression frequently occurs in advanced multiple myeloma, representing a negative prognostic factor and a potential target for myeloma specific treatment. However, selecting patients for CXCR4 directed therapies and prognostic stratification seem to be more relevant clinical applications for this novel imaging modality, rather than diagnostic imaging of myeloma.

Human T cells engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity.

PubMed

Ankri, Chen; Shamalov, Katerina; Horovitz-Fried, Miryam; Mauer, Shmuel; Cohen, Cyrille J

2013-10-15

Adoptive transfer of T cells genetically modified to express cancer-specific receptors can mediate impressive tumor regression in terminally ill patients. However, T cell function and persistence over time could be hampered by the activation of inhibitory costimulatory pathways, such as programmed death 1 (PD1)/programmed death ligand 1, leading to T cell exhaustion and providing tumor cells with an escape mechanism from immunosurveillance. In addition, the lack of positive costimulation at the tumor site can further dampen T cell response. Thus, as T cell genetic engineering has become clinically relevant, we aimed at enhancing T cell antitumor activity by genetically diverting T cell-negative costimulatory signals into positive ones using chimeric costimulatory retargeting molecules and which are composed of the PD1 extracellular domain fused to the signaling domains of positive costimulatory molecules such as CD28 and 4-1BB. After characterizing the optimal PD1 chimera, we designed and optimized a tripartite retroviral vector that enables the simultaneous expression of this chimeric molecule in conjunction with a cancer-specific TCR. Human T cells, transduced to express a PD1/28 chimeric molecule, exhibited enhanced cytokine secretion and upregulation of activation markers upon coculture with tumor cells. These engineered cells also proliferated better compared with control cells. Finally, we tested the function of these cells in two xenograft models of human melanoma tumors and show that PD1/28-engineered human T cells demonstrated superior antitumor function. Overall, we propose that engineering T cells with a costimulatory retargeting molecule can enhance their function, which bears important implications for the improvement of T cell immunotherapy.

Improved distribution of small molecules and viral vectors in the murine brain using a hollow fiber catheter

PubMed Central

Seunguk, Oh; Odland, Rick; Wilson, Scott R.; Kroeger, Kurt M.; Liu, Chunyan; Lowenstein, Pedro R.; Castro, Maria G.; Hall, Walter A.; Ohlfest, John R.

2008-01-01

Object A hollow fiber catheter was developed to improve the distribution of drugs administered via direct infusion into the central nervous system (CNS). It is a porous catheter that significantly increases the surface area of brain tissue into which a drug is infused. Methods Dye was infused into the mouse brain through convection-enhanced delivery (CED) using a 28-gauge needle compared with a 3-mm-long hollow fiber catheter. To determine whether a hollow fiber catheter could increase the distribution of gene therapy vectors, a recombinant adenovirus expressing the firefly luciferase reporter was injected into the mouse striatum. Gene expression was monitored using in vivo bioluminescent imaging. To assess the distribution of gene transfer, an adenovirus expressing green fluorescent protein was injected into the striatum using a hollow fiber catheter or a needle. Results Hollow fiber catheter—mediated infusion increased the volume of brain tissue labeled with dye by 2.7 times relative to needle-mediated infusion. In vivo imaging revealed that catheter-mediated infusion of adenovirus resulted in gene expression that was 10 times greater than that mediated by a needle. The catheter appreciably increased the area of brain transduced with adenovirus relative to a needle, affecting a significant portion of the injected hemisphere. Conclusions The miniature hollow fiber catheter used in this study significantly increased the distribution of dye and adenoviral-mediated gene transfer in the mouse brain compared with the levels reached using a 28-gauge needle. Compared with standard single-port clinical catheters, the hollow fiber catheter has the advantage of millions of nanoscale pores to increase surface area and bulk flow in the CNS. Extending the scale of the hollow fiber catheter for the large mammalian brain shows promise in increasing the distribution and efficacy of gene therapy and drug therapy using CED. PMID:17886557

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

PubMed

Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

Neodymium-140 DOTA-LM3: Evaluation of an In Vivo Generator for PET with a Non-Internalizing Vector.

PubMed

Severin, Gregory W; Kristensen, Lotte K; Nielsen, Carsten H; Fonslet, Jesper; Jensen, Andreas I; Frellsen, Anders F; Jensen, K M; Elema, Dennis R; Maecke, Helmut; Kjær, Andreas; Johnston, Karl; Köster, Ulli

2017-01-01

140 Nd ( t 1/2  = 3.4 days), owing to its short-lived positron emitting daughter 140 Pr ( t 1/2  = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140 Nd is chemically disruptive to macrocycle-based radiolabeling, meaning that an in vivo redistribution of the daughter 140 Pr is expected before positron emission. The purpose of this study was to determine how the delayed positron from the de-labeled 140 Pr affects preclinical imaging with 140 Nd. To explore the effect, 140 Nd was produced at CERN-ISOLDE, reacted with the somatostatin analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(d-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)d-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify the in vivo redistribution of 140 Pr following 140 Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140 Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% ( n  = 4, mean ± SD) of the in situ produced 140 Pr washed out of the pancreas before decay. Contrastingly, the liver, spleen, and lungs acted as strong sink organs for free 140 Pr 3+ . Based upon these results, we conclude that 140 Nd imaging with a non-internalizing vector convolutes the biodistribution of the tracer with the accumulation pattern of free 140 Pr. This redistribution phenomenon may show promise as a probe of the cellular interaction with the vector, such as in determining tissue dependent internalization behavior.

Neodymium-140 DOTA-LM3: Evaluation of an In Vivo Generator for PET with a Non-Internalizing Vector

PubMed Central

Severin, Gregory W.; Kristensen, Lotte K.; Nielsen, Carsten H.; Fonslet, Jesper; Jensen, Andreas I.; Frellsen, Anders F.; Jensen, K. M.; Elema, Dennis R.; Maecke, Helmut; Kjær, Andreas; Johnston, Karl; Köster, Ulli

2017-01-01

140Nd (t1/2 = 3.4 days), owing to its short-lived positron emitting daughter 140Pr (t1/2 = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140Nd is chemically disruptive to macrocycle-based radiolabeling, meaning that an in vivo redistribution of the daughter 140Pr is expected before positron emission. The purpose of this study was to determine how the delayed positron from the de-labeled 140Pr affects preclinical imaging with 140Nd. To explore the effect, 140Nd was produced at CERN-ISOLDE, reacted with the somatostatin analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(d-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)d-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify the in vivo redistribution of 140Pr following 140Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140Pr washed out of the pancreas before decay. Contrastingly, the liver, spleen, and lungs acted as strong sink organs for free 140Pr3+. Based upon these results, we conclude that 140Nd imaging with a non-internalizing vector convolutes the biodistribution of the tracer with the accumulation pattern of free 140Pr. This redistribution phenomenon may show promise as a probe of the cellular interaction with the vector, such as in determining tissue dependent internalization behavior. PMID:28748183

A small animal PET based on GAPDs and charge signal transmission approach for hybrid PET-MR imaging

NASA Astrophysics Data System (ADS)

Kang, Jihoon; Choi, Yong; Hong, Key Jo; Hu, Wei; Jung, Jin Ho; Huh, Yoonsuk; Kim, Byung-Tae

2011-08-01

Positron emission tomography (PET) employing Geiger-mode avalanche photodiodes (GAPDs) and charge signal transmission approach was developed for small animal imaging. Animal PET contained 16 LYSO and GAPD detector modules that were arranged in a 70 mm diameter ring with an axial field of view of 13 mm. The GAPDs charge output signals were transmitted to a preamplifier located remotely using 300 cm flexible flat cables. The position decoder circuits (PDCs) were used to multiplex the PET signals from 256 to 4 channels. The outputs of the PDCs were digitized and further-processed in the data acquisition unit. The cross-compatibilities of the PET detectors and MRI were assessed outside and inside the MRI. Experimental studies of the developed full ring PET were performed to examine the spatial resolution and sensitivity. Phantom and mouse images were acquired to examine the imaging performance. The mean energy and time resolution of the PET detector were 17.6% and 1.5 ns, respectively. No obvious degradation on PET and MRI was observed during simultaneous PET-MRI data acquisition. The measured spatial resolution and sensitivity at the CFOV were 2.8 mm and 0.7%, respectively. In addition, a 3 mm diameter line source was clearly resolved in the hot-sphere phantom images. The reconstructed transaxial PET images of the mouse brain and tumor displaying the glucose metabolism patterns were imaged well. These results demonstrate GAPD and the charge signal transmission approach can allow the development of high performance small animal PET with improved MR compatibility.

The value of FDG PET/CT for follow-up of patients with melanoma: a retrospective analysis

PubMed Central

Vensby, Philip H; Schmidt, Grethe; Kjær, Andreas; Fischer, Barbara M

2017-01-01

The incidence of melanoma (MM) is among the fastest rising cancers in the western countries. Positron Emission Tomography with Computed Tomography (PET/CT) is a valuable non-invasive tool for the diagnosis and staging of patients with MM. However, research on the value of PET/CT in follow-up of melanoma patients is limited. This study assesses the diagnostic value of PET/CT for follow-up after melanoma surgery. This retrospective study includes patients with MM who performed at least one PET/CT scan after initial surgery and staging. PET/CT findings were compared to histology, MRI or fine needle aspiration (FNA) to estimate the diagnostic accuracy. The diagnostic performance of PET/CT performed in patients with and without a clinical suspicion of relapse was compared. 238 patients (526 scans) were included. Of the 526 scans 130 (25%) scans were PET-positive, 365 (69%) PET-negative, and 28 (5%) had equivocal findings. Sensitivity was 89% [0.82-0.94], specificity 92% [0.89-0.95], positive and negative predictive values of 78% [0.70-0.84] and 97% [0.94-0.98] respectively. When stratified for reason of referral there was no statistical significant difference in the diagnostic accuracy of PET/CT between patients referred with or without a clinical suspicion of relapse. This study demonstrates that PET/CT despite a moderate sensitivity has a high negative predictive value in the follow-up of melanoma patients. Thus, a negative PET/CT-scan essentially rules out relapse. However, the frequency of false positive findings is relatively high, especially among patients undergoing a “routine” PET/CT with no clinical suspicion of relapse, potentially causing anxiety and leading to further diagnostic procedures. PMID:29348980

The value of FDG PET/CT for follow-up of patients with melanoma: a retrospective analysis.

PubMed

Vensby, Philip H; Schmidt, Grethe; Kjær, Andreas; Fischer, Barbara M

2017-01-01

The incidence of melanoma (MM) is among the fastest rising cancers in the western countries. Positron Emission Tomography with Computed Tomography (PET/CT) is a valuable non-invasive tool for the diagnosis and staging of patients with MM. However, research on the value of PET/CT in follow-up of melanoma patients is limited. This study assesses the diagnostic value of PET/CT for follow-up after melanoma surgery. This retrospective study includes patients with MM who performed at least one PET/CT scan after initial surgery and staging. PET/CT findings were compared to histology, MRI or fine needle aspiration (FNA) to estimate the diagnostic accuracy. The diagnostic performance of PET/CT performed in patients with and without a clinical suspicion of relapse was compared. 238 patients (526 scans) were included. Of the 526 scans 130 (25%) scans were PET-positive, 365 (69%) PET-negative, and 28 (5%) had equivocal findings. Sensitivity was 89% [0.82-0.94], specificity 92% [0.89-0.95], positive and negative predictive values of 78% [0.70-0.84] and 97% [0.94-0.98] respectively. When stratified for reason of referral there was no statistical significant difference in the diagnostic accuracy of PET/CT between patients referred with or without a clinical suspicion of relapse. This study demonstrates that PET/CT despite a moderate sensitivity has a high negative predictive value in the follow-up of melanoma patients. Thus, a negative PET/CT-scan essentially rules out relapse. However, the frequency of false positive findings is relatively high, especially among patients undergoing a "routine" PET/CT with no clinical suspicion of relapse, potentially causing anxiety and leading to further diagnostic procedures.

Metabolically active tumour volume segmentation from dynamic [(18)F]FLT PET studies in non-small cell lung cancer.

PubMed

Hoyng, Lieke L; Frings, Virginie; Hoekstra, Otto S; Kenny, Laura M; Aboagye, Eric O; Boellaard, Ronald

2015-01-01

Positron emission tomography (PET) with (18)F-3'-deoxy-3'-fluorothymidine ([(18)F]FLT) can be used to assess tumour proliferation. A kinetic-filtering (KF) classification algorithm has been suggested for segmentation of tumours in dynamic [(18)F]FLT PET data. The aim of the present study was to evaluate KF segmentation and its test-retest performance in [(18)F]FLT PET in non-small cell lung cancer (NSCLC) patients. Nine NSCLC patients underwent two 60-min dynamic [(18)F]FLT PET scans within 7 days prior to treatment. Dynamic scans were reconstructed with filtered back projection (FBP) as well as with ordered subsets expectation maximisation (OSEM). Twenty-eight lesions were identified by an experienced physician. Segmentation was performed using KF applied to the dynamic data set and a source-to-background corrected 50% threshold (A50%) was applied to the sum image of the last three frames (45- to 60-min p.i.). Furthermore, several adaptations of KF were tested. Both for KF and A50% test-retest (TRT) variability of metabolically active tumour volume and standard uptake value (SUV) were evaluated. KF performed better on OSEM- than on FBP-reconstructed PET images. The original KF implementation segmented 15 out of 28 lesions, whereas A50% segmented each lesion. Adapted KF versions, however, were able to segment 26 out of 28 lesions. In the best performing adapted versions, metabolically active tumour volume and SUV TRT variability was similar to those of A50%. KF misclassified certain tumour areas as vertebrae or liver tissue, which was shown to be related to heterogeneous [(18)F]FLT uptake areas within the tumour. For [(18)F]FLT PET studies in NSCLC patients, KF and A50% show comparable tumour volume segmentation performance. The KF method needs, however, a site-specific optimisation. The A50% is therefore a good alternative for tumour segmentation in NSCLC [(18)F]FLT PET studies in multicentre studies. Yet, it was observed that KF has the potential to subsegment lesions in high and low proliferative areas.

A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein.

PubMed

Dar, Saira; Shuja, Rukhsana N; Shakoori, Abdul Rauf

2013-02-01

Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

Inhalation of Nebulized Perfluorochemical Enhances Recombinant Adenovirus and Adeno-Associated Virus-Mediated Gene Expression in Lung Epithelium

PubMed Central

Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J.; Wang, Lili; Gao, Guang Ping; Kolls, Jay K.; Bohm, Rudolf; Liggitt, Denny

2012-01-01

Abstract Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (p<0.05). However, vector administration 1 hr, 1 day, or 3 days after perflubron exposure was not different from either nebulized saline with vector or vector alone and a 60-min exposure to nebulized perflubron is required. In parallel pilot studies in macaques, inhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression. PMID:22568624

The Effect of Pet Remedy on the Behaviour of the Domestic Dog (Canis familiaris).

PubMed

Taylor, Sienna; Madden, Joah

2016-10-25

Stress-affected behaviour in companion animals can have an adverse effect on animal health and welfare and their relationships with humans. This stress can be addressed using chemical treatments, often in conjunction with behavioural therapies. Here, we investigated the efficacy of one commercial pharmacological intervention, Pet Remedy, advertised as a natural stress relief product for mammals. We aimed to see whether the product lowered stress-affected behaviour in dogs placed in a non-familiar environment. Behavioural responses of 28 dogs were video recorded using a double-blind, placebo-controlled, and counterbalanced repeated measures design. Dogs were exposed to both a placebo and Pet Remedy plug-in diffuser for 30 min with an intervening period of approximately 7 days between conditions. Multivariate regression analysis identified no significant differences in behaviour in either the Pet Remedy or placebo condition. In conclusion, in the current study, Pet Remedy did not reduce behavioural indicators indicative of a stress response. To determine the effects of Pet Remedy, future research using a larger sample size and controlling for breed would be beneficial.

TU-C-12A-12: Differentiating Bone Lesions and Degenerative Joint Disease in NaF PET/CT Scans Using Machine Learning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perk, T; Bradshaw, T; Muzahir, S

2014-06-15

Purpose: [F-18]NaF PET can be used to image bone metastases; however, tracer uptake in degenerative joint disease (DJD) often appears similar to metastases. This study aims to develop and compare different machine learning algorithms to automatically identify regions of [F-18]NaF scans that correspond to DJD. Methods: 10 metastatic prostate cancer patients received whole body [F-18]NaF PET/CT scans prior to treatment. Image segmentation resulted in 852 ROIs, 69 of which were identified by a nuclear medicine physician as DJD. For all ROIs, various PET and CT textural features were computed. ROIs were divided into training and testing sets used to trainmore » eight different machine learning classifiers. Classifiers were evaluated based on receiver operating characteristics area under the curve (AUC), sensitivity, specificity, and positive predictive value (PPV). We also assessed the added value of including CT features in addition to PET features for training classifiers. Results: The training set consisted of 37 DJD ROIs with 475 non-DJD ROIs, and the testing set consisted of 32 DJD ROIs with 308 non-DJD ROIs. Of all classifiers, generalized linear models (GLM), decision forests (DF), and support vector machines (SVM) had the best performance. AUCs of GLM (0.929), DF (0.921), and SVM (0.889) were significantly higher than the other models (p<0.001). GLM and DF, overall, had the best sensitivity, specificity, and PPV, and gave a significantly better performance (p<0.01) than all other models. PET/CT GLM classifiers had higher AUC than just PET or just CT. GLMs built using PET/CT information had superior or comparable sensitivities, specificities and PPVs to just PET or just CT. Conclusion: Machine learning algorithms trained with PET/CT features were able to identify some cases of DJD. GLM outperformed the other classification algorithms. Using PET and CT information together was shown to be superior to using PET or CT features alone. Research supported by the Prostate Cancer Foundation.« less

[PET/CT and biochemical recurrence of prostate adenocarcinoma: Added value of 68Ga-PSMA-11 when 18F-fluorocholine is non-contributive].

PubMed

Gauthé, M; Belissant, O; Girard, A; Zhang Yin, J; Ohnona, J; Cottereau, A-S; Nataf, V; Balogova, S; Pontvert, D; Lebret, T; Guillonneau, B; Cussenot, O; Talbot, J-N

Since April 201, we have introduced PET/CT using a ligand of prostate-specific membrane antigen labeled with gallium-68 (PSMA-11). We aimed to evaluate its positivity rate and impact in patients presenting biochemical recurrence of prostate cancer whose 18 F-fluorocholine (FCH) PET/CT was non-contributive. Patients were prospectively included between April and December 2016. PET/CT was performed 60min after injection of 2MBq/kg of body mass of 68 Ga-PSMA-11. Three anatomical areas were considered: prostatic lodge, pelvic lymph nodes and distant locations. The impact of PSMA-11 PET/CT was assessed by comparing changes in therapeutic strategy decided during multidisciplinary meeting. Thirty-three patients were included. The mean PSA serum level measured on the month of the PSMA-11 PET/CT was 2,8ng/mL. Twenty-five (76%) PSMA-11 PET/CT were positive, 7 (21%) negative and 1 (3%) equivocal. Of 11 patients whose FCH PET/CT showed equivocal foci, PSMA-11 PET/CT confirmed those foci in 5 cases. Follow-up was available for 18 patients (55%). PSMA-11 PET/CT results led to a change in management in 12 patients (67%). 68 Ga-PSMA-11 PET/CT is useful in detecting recurrence of prostate cancer, by identifying residual disease which was not detected on other imaging modalities and by changing management of 2 patients out of 3. 5. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

PubMed

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

PubMed

Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

2011-06-16

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

Development of siRNA expression vector utilizing rock bream beta-actin promoter: a potential therapeutic tool against viral infection in fish.

PubMed

Zenke, Kosuke; Nam, Yoon Kwon; Kim, Ki Hong

2010-01-01

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Pet owners' attitudes and behaviours related to smoking and second-hand smoke: a pilot study.

PubMed

Milberger, S M; Davis, R M; Holm, A L

2009-04-01

Although research indicates that second-hand smoke (SHS) harms both human and animal health, data on the percentage of pet owners who smoke or allow smoking in their homes are not readily available. To investigate pet owners' smoking behaviour and policies on smoking in their homes, and the potential for educational interventions to motivate change in pet owners' smoking behaviour. A web-based survey was used with 3293 adult pet owners. The main outcome measures were smoking behaviour of pet owners and their cohabitants; policies on smoking in pet owners' homes; and impact of information about the dangers of pet exposure to SHS on pet owners' smoking intentions. Of respondents, 21% were current smokers and 27% of participants lived with at least one smoker. Pet owners who smoke reported that information on the dangers of pet exposure to SHS would motivate them to try to quit smoking (28.4%) and ask the people with whom they live to quit smoking (8.7%) or not to smoke indoors (14.2%). Moreover, non-smoking pet owners who live with smokers said that they would ask the people with whom they live to quit (16.4%) or not smoke indoors (24.2%) if given this information. About 40% of current smokers and 24% of non-smokers living with smokers indicated that they would be interested in receiving information on smoking, quitting, or SHS. Educational campaigns informing pet owners of the risks of SHS exposure for pets could motivate some owners to quit smoking. It could also motivate these owners and non-smoking owners who cohabit with smokers make their homes smoke-free.

Imaging of Prostate-Specific Membrane Antigen Expression in Metastatic Differentiated Thyroid Cancer Using 68Ga-HBED-CC-PSMA PET/CT.

PubMed

Lütje, Susanne; Gomez, Benedikt; Cohnen, Joseph; Umutlu, Lale; Gotthardt, Martin; Poeppel, Thorsten D; Bockisch, Andreas; Rosenbaum-Krumme, Sandra

2017-01-01

The prostate-specific membrane antigen (PSMA) was shown to be overexpressed on the neovasculature of several malignancies. Here, the role of Ga-HBED-CC-PSMA PET/CT for the detection of PSMA expression in patients with metastasized differentiated thyroid cancer (DTC) was evaluated. Six patients with iodine-negative and F-FDG-positive metastasized DTC (mean TG, 1616 ng/mL) received 71-93 MBq of the Ga-labeled PSMA ligand and underwent PET/CT at 62 ± 7 minutes p.i.. Tumor accumulation capacity of the tracer and the detection rate of local recurrences and metastases were compared with F-FDG. Tracer uptake was quantified in terms of the SUVmax. In 5 of 6 patients, sites of putative metastatic disease could be identified using Ga-HBED-CC-PSMA PET/CT. All lesions detected with Ga-HBED-CC-PSMA PET/CT (n = 42) were confirmed by F-FDG PET/CT or conventional CT imaging. Using Ga-HBED-CC-PSMA PET/CT, all tumor lesions identified with F-FDG PET/CT imaging could be visualized in 3 of 5 patients. In 2 patients, only the most prominent lesions detected with F-FDG PET/CT imaging were visualized by Ga-HBED-CC-PSMA PET/CT. Ga-HBED-CC-PSMA uptake ranged from low in 1 patient (mean SUVmax 3.3) to intermediate (1 patient; mean SUVmax, 6.1) to intense (3 patients; mean SUVmax, 12.8, 16.2, and 18.3). The highest SUVmax values were observed for a bone lesion, reaching 39.7. These preliminary results indicate that Ga-HBED-CC-PSMA PET/CT might be suitable for staging of patients with metastasized DTC. Ga-HBED-CC-PSMA PET/CT could be useful for the identification of patients who might qualify for PSMA-targeted radionuclide therapy because of high PSMA uptake.

Polyethylene terephthalate recycling for food-contact applications: testing, safety and technologies: a global perspective.

PubMed

Bayer, Forrest L

2002-01-01

Studies were undertaken to determine the composition of five different types of post-consumer polyethylene terephthalate (PET) feedstreams to ascertain the relative amounts of food containers and non-food containers. Deposit post-consumer PET feedstreams contained approximately 100% food containers, whereas curbside feedstreams contained from 0.04 to 6% non-food containers. Analysis of the PET containers from the different type feedstreams after the containers were subjected to a commercial PET wash system and after processing with a proprietary decontamination technology was accomplished to determine the levels of compounds in the post-consumer PET after the various stages of processing. Comprehensive thermal desorption/GC/MS, purge and trap GC/MS purge and trap GC quantitation, PET dissolution and extraction GC analysis and PET dissolution HPLC analysis established the types and concentrations of compounds that absorb in the PET from the various types of postconsumer feedstreams. A total of 121 compounds were identified in the five different feedstreams. The concentration of absorbed compounds remaining in the deposit material and the non-food applications material after the commercial wash was 28 and 39mgkg(-1) respectively. Analysis of the feedstreams after subjecting the material to a proprietary decontamination process demonstrated the ability of removing all the absorbed compounds to a level below the level of the threshold of regulation. The safety of sourcing of post-consumer PET from food use applications verses non-food use applications of PET has been established.

Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.

PubMed

Chung, Wen-Jen; Huang, Chi-Lung; Gong, Hong-Yi; Ou, Tsung-Yin; Hsu, Jue-Liang; Hu, Shao-Yang

2015-06-01

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC-MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay. Copyright © 2015 Elsevier Inc. All rights reserved.

Injuries caused by pets in Asian urban households: a cross-sectional telephone survey.

PubMed

Chan, Emily Y Y; Gao, Yang; Li, Liping; Lee, Po Yi

2017-01-20

Little is known about pet-related injuries in Asian populations. This study primarily aimed to investigate the incidence rate of pet-related household injuries in Hong Kong, an urban Chinese setting. Cantonese-speaking non-institutionalised population of all ages in Hong Kong accessible by telephone land-line. A total of 43 542 telephone numbers were dialled and 6570 residents successfully completed the interviews. Data of pet-related household injuries in the previous 12 months, pet ownership and socio-demographic characteristics were collected with a questionnaire. Direct standardisation of the incidence rates of pet-related household injuries by gender and age to the 2009 Hong Kong Population Census was estimated. Univariate and multivariate analyses were performed to estimate risks of socio-demographic factors and pet ownership for the injury. A total of 84 participants experienced pet-related household injuries in the past 12 months, with an overall person-based incidence rate of 1.28%. The majority of the victims were injured once (69.6%). Cats (51.6%) were the most common pets involved. Pet owners were at an extremely higher risk after controlling for other factors (adjusted OR: 52.0, 95% CI 22.1 to 98.7). Females, the unmarried, those with higher monthly household income and those living in lower-density housing were more likely to be injured by pets. We project a pet-related household injury incidence rate of 1.24% in the general Hong Kong population, with 86 334 residents sustaining pet-related injuries every year. Pet ownership puts people at extremely high risk, especially the unmarried. Further studies should focus on educating pet owners to reduce pet-related injuries in urban Greater China. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Synthesis and evaluation of 18F-labeled CJ-042794 for imaging prostanoid EP4 receptor expression in cancer with positron emission tomography.

PubMed

Zhang, Zhengxing; Lau, Joseph; Kuo, Hsiou-Ting; Zhang, Chengcheng; Colpo, Nadine; Bénard, François; Lin, Kuo-Shyan

2017-05-15

The potent and selective prostanoid EP4 receptor antagonist CJ-042794 was radiolabeled with 18 F, and evaluated for imaging EP4 receptor expression in cancer with positron emission tomography (PET). The fluorination precursor, arylboronic acid pinacol ester 4, was prepared in 4 steps with 42% overall yield. 18 F-CJ-042794 was synthesized via a copper-mediated 18 F-fluorination reaction followed by base hydrolysis, and was obtained in 1.5±1.1% (n=2) decay-corrected radiochemical yield. PET/CT imaging and biodistribution studies in mice showed that 18 F-CJ-042794 was excreted through both renal and hepatobiliary pathways with significant retention in blood. The EP4-receptor-expressing LNCaP prostate cancer xenografts were clearly visualized in PET images with 1.12±0.08%ID/g (n=5) uptake value and moderate tumour-to-muscle contrast ratio (2.73±0.22) at 1h post-injection. However, the tumour uptake was nonspecific as it could not be blocked by co-injection of cold standard, precluding the application of 18 F-CJ-042794 for PET imaging of EP4 receptor expression in cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression.

PubMed

Lausberg, Frank; Chattopadhyay, Ava Rebecca; Heyer, Antonia; Eggeling, Lothar; Freudl, Roland

2012-09-01

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition. Copyright © 2012 Elsevier Inc. All rights reserved.

Looking for a relevant potential evapotranspiration model at the watershed scale

NASA Astrophysics Data System (ADS)

Oudin, L.; Hervieu, F.; Michel, C.; Perrin, C.; Anctil, F.; Andréassian, V.

2003-04-01

In this paper, we try to identify the most relevant approach to calculate Potential Evapotranspiration (PET) for use in a daily watershed model, to try to bring an answer to the following question: "how can we use commonly available atmospheric parameters to represent the evaporative demand at the catchment scale?". Hydrologists generally see the Penman model as the ideal model regarding to its good adequacy with lysimeter measurements and its physically-based formulation. However, in real-world engineering situations, where meteorological stations are scarce, hydrologists are often constrained to use other PET formulae with less data requirements or/and long-term average of PET values (the rationale being that PET is an inherently conservative variable). We chose to test 28 commonly used PET models coupled with 4 different daily watershed models. For each test, we compare both PET input options: actual data and long-term average data. The comparison is made in terms of streamflow simulation efficiency, over a large sample of 308 watersheds. The watersheds are located in France, Australia and the United States of America and represent varied climates. Strikingly, we find no systematic improvements of the watershed model efficiencies when using actual PET series instead of long-term averages. This suggests either that watershed models may not conveniently use the climatic information contained in PET values or that formulae are only awkward indicators of the real PET which watershed models need.

A relevant IgE-reactive 28kDa protein identified from Salsola kali pollen extract by proteomics is a natural degradation product of an integral 47kDa polygalaturonase.

PubMed

Mas, Salvador; Oeo-Santos, Carmen; Cuesta-Herranz, Javier; Díaz-Perales, Araceli; Colás, Carlos; Fernández, Javier; Barber, Domingo; Rodríguez, Rosalía; de Los Ríos, Vivian; Barderas, Rodrigo; Villalba, Mayte

2017-08-01

A highly prevalent IgE-binding protein band of 28kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein. The far-UV CD spectrum determined that rSal k 6 was folded. Immunostaining of the S. kali pollen protein extract with a rSal k 6-specific pAb and LC-MS/MS proteomic analyses confirmed the co-existence of the 28kDa band together with an allergenic band of about 47kDa in the pollen extract. Therefore, the 28kDa was assigned as a natural degradation product of the 47kDa integral polygalacturonase. The IgE-binding inhibition to S. kali pollen extract using rSal k 6 as inhibitor showed that signals directed to both protein bands of 28 and 47kDa were completely abrogated. The average prevalence of rSal k 6 among the three populations analyzed was 30%, with values correlating well with the levels of grains/m 3 of Amaranthaceae pollen. Sal k 6 shares IgE epitopes with Oleaceae members (Fraxinus excelsior, Olea europaea and Syringa vulgaris), with IgE-inhibition values ranging from 20% to 60%, respectively. No IgE-inhibition was observed with plant-derived food extracts. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and expression of the tig gene coding for trigger factor from psychrophilic bacteria with no information of genome sequence available.

PubMed

Lee, Kyunghee; Choi, Hyojung; Im, Hana

2009-08-01

Trigger factor (TF) plays a key role as a molecular chaperone with a peptidyl-prolyl cis-trans isomerase (PPIase) activity by which cells promote folding of newly synthesized proteins coming out of ribosomes. Since psychrophilic bacteria grow at a quite low temperature, between 4 and 15 degrees C, TF from such bacteria was investigated and compared with that of mesophilic bacteria E. coli in order to offer an explanation of cold-adaptation at a molecular level. Using a combination of gradient PCRs with homologous primers and LA PCR in vitro cloning technology, the tig gene was fully identified from Psychromonas arctica, whose genome sequence is not yet available. The resulting amino acid sequence of the TF was compared with other homologous TFs using sequence alignments to search for common domains. In addition, we have developed a protein expression system, by which TF proteins from P. arctica (PaTF) were produced by IPTG induction upon cloning the tig gene on expression vectors, such as pAED4. We have further examined the role of expressed psychrophilic PaTF on survival against cold treatment at 4 degrees C. Finally, we have attempted the in vitro biochemical characterization of TF proteins with His-tags expressed in a pET system, such as the PPIase activity of PaTF protein. Our results demonstrate that the expressed PaTF proteins helped cells survive against cold environments in vivo and the purified PaTF in vitro display the functional PPIase activity in a concentration dependent manner.

Cloning and expression of cyclophilin from Platanus orientalis pollens in Escherichia coli

PubMed Central

Sankian, Mojtaba; Vahedi, Fatemeh; Pazouki, Nazanin; Moghadam, Malihe; Jabbari Azad, Farahzad; Varasteh, Abdol-Reza

2012-01-01

Background: Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions. Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis. Methods: RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method. Results: The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen. Conclusion: The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen. PMID:26989705

The development of a personalized patient education tool for decision making for postmenopausal women with osteoporosis.

PubMed

Hiligsmann, M; Ronda, G; van der Weijden, T; Boonen, A

2016-08-01

A personalized patient education tool for decision making (PET) for postmenopausal women with osteoporosis was developed by means of a systematic development approach. A prototype was constructed and refined by involving various professionals and patients. Professionals and patients expressed a positive attitude towards the use of the PET. The purpose was to systematically develop a paper-based personalized PET to assist postmenopausal women with osteoporosis in selecting a treatment in line with their personal values and preferences. The development of the PET was based on a systematic process including scope, design, development of a prototype, and alpha testing among professionals and patients by semi-structured interviews. The design and development resulted in a four-page PET prototype together with a one-page fact sheet of the different drug options. The prototype PET provided the personal risk factors, the estimated individualized risk for a future major osteoporotic fracture and potential reduction with drugs, and a summary of advantages and disadvantages whether or not to start drugs. The drug fact sheet presents five attributes of seven drugs in a tabular format. The alpha testing with professionals resulted in some adaptations, e.g., inclusion of the possibility to calculate fracture risk based on various individual risk scoring methods. Important results from the alpha testing with patients were differences in the fracture risk percentage which was seen as worthwhile to start drugs, the importance of an overview of side effects, and of the timing of the PET into the patient pathway. All women indicated that the PET could be helpful for their decision to select a treatment. Physicians and patients expressed a positive attitude towards the use of the proposed PET. Further research would be needed to test the effects of the PET on feasibility in clinical workflow and on patient outcomes.

PET Imaging of VEGFR-2 Expression in Lung Cancer with 64Cu-Labeled Ramucirumab.

PubMed

Luo, Haiming; England, Christopher G; Graves, Stephen A; Sun, Haiyan; Liu, Glenn; Nickles, Robert J; Cai, Weibo

2016-02-01

Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2-positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2-targeted therapies. Here, we report the synthesis and characterization of an antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo. Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results. Flow cytometry examination revealed the specific binding capacity of fluorescein isothiocyanate-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with (64)Cu, PET imaging revealed specific and prominent uptake of (64)Cu-NOTA-RamAb in VEGFR-2-positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2-negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of (64)Cu-NOTA-RamAb. This study provides initial evidence that (64)Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find potential applications in monitoring the treatment response of VEGFR-2-targeted cancer therapy. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Measurement and Evaluation of Quantitative Performance of PET/CT Images before a Multicenter Clinical Trial.

PubMed

Zhu, Yanjia; Geng, Caizheng; Huang, Jia; Liu, Juzhen; Wu, Ning; Xin, Jun; Xu, Hao; Yu, Lijuan; Geng, Jianhua

2018-06-13

To ensure the reliability of the planned multi-center clinical trial, we assessed the consistence and comparability of the quantitative parameters of the eight PET/CT units that will be used in this trial. PET/CT images were scanned using a PET NEMA image quality phantom (Biodex) on the eight units of Discovery PET/CT 690 from GE Healthcare. The scanning parameters were the same with the ones to be used in the planned trial. The 18 F-NaF concentration in the background was 5.3 kBq/ml, while the ones in the spheres of diameter 37 mm, 22 mm, 17 mm and 10 mm were 8:1 as to that of the background and the ones in the spheres of diameter 28 mm and 13 mm were 0 kBq/ml. The consistency of hot sphere recovery coefficient (HRC), cold sphere recovery coefficient (CRC), hot sphere contrast (Q H ) and cold sphere contrast (Q c ) among these 8 PET/CTs was analyzed. The variation of the main quantitative parameters of the eight PET/CT systems was within 10%, which is acceptable for the clinical trial.

Evaluation of chemotherapy response in patients with advanced head and neck cancer using [F-18]fluorodeoxyglucose positron emission tomography.

PubMed

Lowe, V J; Dunphy, F R; Varvares, M; Kim, H; Wittry, M; Dunphy, C H; Dunleavy, T; McDonough, E; Minster, J; Fletcher, J W; Boyd, J H

1997-12-01

[F-18]Fluorodeoxyglucose (FDG)-positron emission tomography (PET) can measure the metabolic activity of tissues; FDG-PET may be able to predict response to chemotherapy by identifying changes in tumor metabolism. Measurement of response to treatment may help improve survival in the management of advanced head and neck cancer. We evaluated this particular use of FDG-PET in patients participating in a neoadjuvant organ-preservation protocol using taxol and carboplatin and compared pathologic response after chemotherapy with changes in tumor metabolism measured by FDG-PET. Serial FDG-PET studies (n = 56) were performed in patients (n = 28) with stage III/IV head and neck cancer participating in a neoadjuvant organ-preservation protocol. The FDG-PET studies were performed before and after chemotherapy. All patients had tissue biopsies before and after chemotherapy. Patients were classified as pathologic complete response (PCR) or residual disease (RD) based on tissue biopsies. Visual analysis of PET scans was performed to identify patients with complete response by PET, and these findings were compared with pathology results. Metabolic changes were also evaluated using standardized uptake ratios (SUR) of FDG. The sensitivity and specificity of PET for residual cancer after therapy was 90% (19/21) and 83% (5/6), respectively. Two patients had initially negative biopsies and positive PET studies for persistent disease. Pathology review and rebiospy led to confirmation of the PET results in these cases, giving a sensitivity of 90% for initial tissue biopsy. In this preliminary analysis, FDG-PET was accurate in classifying response to chemotherapy in most patients. Fluorodeoxyglucose-PET may identify residual viable tumor when it is otherwise undetectable.

Immuno-PET Imaging and Radioimmunotherapy of 64Cu-/177Lu-Labeled Anti-EGFR Antibody in Esophageal Squamous Cell Carcinoma Model.

PubMed

Song, In Ho; Lee, Tae Sup; Park, Yong Serk; Lee, Jin Sook; Lee, Byung Chul; Moon, Byung Seok; An, Gwang Il; Lee, Hae Won; Kim, Kwang Il; Lee, Yong Jin; Kang, Joo Hyun; Lim, Sang Moo

2016-07-01

Immuno-PET provides valuable information about tumor location, phenotype, susceptibility to therapy, and treatment response, especially to targeted radioimmunotherapy. In this study, we prepared antiepidermal growth factor receptor (EGFR) antibody via identical chelator, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-trience-3,6,9,-triacetic acid (PCTA), labeled with (64)Cu or (177)Lu to evaluate the EGFR expression levels using immuno-PET and the feasibility of radioimmunotherapy in an esophageal squamous cell carcinoma (ESCC) model. Cetuximab was conjugated with p-SCN-Bn-PCTA and radiolabeled with (64)Cu or (177)Lu. In vitro EGFR expression levels were determined and compared using flow cytometry and cell binding assay. In vivo EGFR expression levels were evaluated via immuno-PET imaging of (64)Cu-cetuximab and biodistribution analysis. Micro-SPECT/CT imaging, biodistribution, and radioimmunotherapy studies of (177)Lu-cetuximab were performed in the ESCC model. Therapeutic responses were monitored using (18)F-FDG PET and immunohistochemical staining. (64)Cu- or (177)Lu-labeled antibodies showed high radiolabeling yield (>98%), stability (>90%), and favorable immunoreactivity. In vitro EGFR status measured by cell binding assay was correlated with the flow cytometry data. Immuno-PET, micro-SPECT/CT, and biodistribution demonstrated specific uptake in ESCC tumors depending on the EGFR expression levels. Tumor accumulation of (64)Cu- and (177)Lu-cetuximab was peaked at 48 and 120 h, respectively. Radioimmunotherapy with (177)Lu-cetuximab showed significant inhibition of tumor growth (P < 0.01) and marked reduction of (18)F-FDG SUV compared with that of control (P < 0.05). Terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity and Ki-67 staining indices increased and decreased, respectively, in the radioimmunotherapy group compared with other groups (P < 0.01). (64)Cu-cetuximab immuno-PET represented EGFR expression levels in ESCC tumors, and (177)Lu-cetuximab radioimmunotherapy effectively inhibited the tumor growth. The diagnostic and therapeutic convergence radiopharmaceutical (64)Cu-/(177)Lu-PCTA-cetuximab may be useful as a diagnostic tool in patient selection and a potent radioimmunotherapy agent in EGFR-positive ESCC tumors. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

PubMed

Nakashima, N; Tamura, T

2013-06-01

Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-β-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose. © 2013 The Society for Applied Microbiology.

ImmunoPET with Anti-Mesothelin Antibody in Patients with Pancreatic and Ovarian Cancer before Anti-Mesothelin Antibody-Drug Conjugate Treatment.

PubMed

Lamberts, Laetitia E; Menke-van der Houven van Oordt, Catharina W; ter Weele, Eva J; Bensch, Frederike; Smeenk, Michiel M; Voortman, Johannes; Hoekstra, Otto S; Williams, Simon P; Fine, Bernard M; Maslyar, Daniel; de Jong, Johan R; Gietema, Jourik A; Schröder, Carolien P; Bongaerts, Alphons H H; Lub-de Hooge, Marjolijn N; Verheul, Henk M W; Sanabria Bohorquez, Sandra M; Glaudemans, Andor W J M; de Vries, Elisabeth G E

2016-04-01

Mesothelin (MSLN) is frequently overexpressed in pancreatic and ovarian cancers, making it a potential drug target. We performed an (89)Zr-PET imaging study with MMOT0530A, a MSLN antibody, in conjunction with a phase I study with the antibody-drug conjugate DMOT4039A, containing MMOT0530A bound to MMAE. The aim was to study antibody tumor uptake, whole-body distribution, and relation between uptake, response to treatment, and MSLN expression. Before DMOT4039A treatment, patients received 37 MBq (89)Zr-MMOT0530A followed by PET/CT imaging 2, 4, and 7 days postinjection. Tracer uptake was expressed as standardized uptake value (SUV). MSLN expression was determined with immunohistochemistry (IHC) on archival tumor tissue. Eleven patients were included, 7 with pancreatic and 4 with ovarian cancer. IHC MSLN expression varied from absent to strong. Suitable tracer antibody dose was 10 mg MMOT0530A and optimal imaging time was 4 and 7 days postinjection. Tumor tracer uptake occurred in 37 lesions with mean SUVmax of 13.1 (±7.5) on PET 4 days postinjection, with 11.5 (±7.5) in (N= 17) pancreatic and 14.5 (±8.7) in (N= 20) ovarian cancer lesions. Within patients, a mean 2.4-fold (±1.10) difference in uptake between tumor lesions existed. Uptake in blood, liver, kidneys, spleen, and intestine reflected normal antibody distribution. Tracer tumor uptake was correlated to IHC. Best response to DMOT4039A was partial response in one patient. With (89)Zr-MMOT0530A-PET, pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized. This technique can potentially guide individualized antibody-based treatment. ©2015 American Association for Cancer Research.

PET staging of amyloidosis using striatum.

PubMed

Hanseeuw, Bernard J; Betensky, Rebecca A; Mormino, Elizabeth C; Schultz, Aaron P; Sepulcre, Jorge; Becker, John A; Jacobs, Heidi I L; Buckley, Rachel F; LaPoint, Molly R; Vanini, Patrizia; Donovan, Nancy J; Chhatwal, Jasmeer P; Marshall, Gad A; Papp, Kathryn V; Amariglio, Rebecca E; Rentz, Dorene M; Sperling, Reisa A; Johnson, Keith A

2018-05-21

Amyloid PET data are commonly expressed as binary measures of cortical deposition. However, not all individuals with high cortical amyloid will experience rapid cognitive decline. Motivated by postmortem data, we evaluated a three-stage PET classification: low cortical; high cortical, low striatal; and high cortical, high striatal amyloid; hypothesizing this model could better reflect Alzheimer's dementia progression than a model based only on cortical measures. We classified PET data from 1433 participants (646 normal, 574 mild cognitive impairment, and 213 AD), explored the successive involvement of cortex and striatum using 3-year follow-up PET data, and evaluated the associations between PET stages, hippocampal volumes, and cognition. Follow-up data indicated that PET detects amyloid first in cortex and then in striatum. Our three-category staging including striatum better predicted hippocampal volumes and subsequent cognition than a three-category staging including only cortical amyloid. PET can evaluate amyloid expansion from cortex to subcortex. Using striatal signal as a marker of advanced amyloidosis may increase predictive power in Alzheimer's dementia research. Copyright © 2018. Published by Elsevier Inc.

The quest for a non-vector psyllid: Natural variation in acquisition and transmission of the huanglongbing pathogen ‘Candidatus Liberibacter asiaticus’ by Asian citrus psyllid isofemale lines

PubMed Central

Ammar, El-Desouky; Hall, David G.; Hosseinzadeh, Saeed

2018-01-01

Genetic variability in insect vectors is valuable to study vector competence determinants and to select non-vector populations that may help reduce the spread of vector-borne pathogens. We collected and tested vector competency of 15 isofemale lines of Asian citrus psyllid, Diaphorina citri, vector of ‘Candidatus Liberibacter asiaticus’ (CLas). CLas is associated with huanglongbing (citrus greening), the most serious citrus disease worldwide. D. citri adults were collected from orange jasmine (Murraya paniculata) hedges in Florida, and individual pairs (females and males) were caged on healthy Murraya plants for egg laying. The progeny from each pair that tested CLas-negative by qPCR were maintained on Murraya plants and considered an isofemale line. Six acquisition tests on D. citri adults that were reared as nymphs on CLas-infected citrus, from various generations of each line, were conducted to assess their acquisition rates (percentage of qPCR-positive adults). Three lines with mean acquisition rates of 28 to 32%, were classified as ‘good’ acquirers and three other lines were classified as ‘poor’ acquirers, with only 5 to 8% acquisition rates. All lines were further tested for their ability to inoculate CLas by confining CLas-exposed psyllids for one week onto healthy citrus leaves (6–10 adults/leaf/week), and testing the leaves for CLas by qPCR. Mean inoculation rates were 19 to 28% for the three good acquirer lines and 0 to 3% for the three poor acquirer lines. Statistical analyses indicated positive correlations between CLas acquisition and inoculation rates, as well as between CLas titer in the psyllids and CLas acquisition or inoculation rates. Phenotypic and molecular characterization of one of the good and one of the poor acquirer lines revealed differences between them in color morphs and hemocyanin expression, but not the composition of bacterial endosymbionts. Understanding the genetic architecture of CLas transmission will enable the development of new tools for combating this devastating citrus disease. PMID:29652934

An experimental study on stabilization of Pekan clay using polyethylene and polypropylene

NASA Astrophysics Data System (ADS)

Zukri, Azhani; Nazir, Ramli; Mender, Fatin Nabilah

2017-10-01

Many countries are expressing concern over the growing issues of polyethylene terephthalate (PET) bottles and polypropylene (PP) products made by the household sector. The rapid increase in the generation of plastic waste all around the world is due to the economic development and population growth. PP is the world's second-most widely produced synthetic plastic, after polyethylene. Statistics show that nearly 50% of the municipal solid waste in Malaysia comes from the institutional, industrial, residential, and construction waste. This paper presents the results of an investigation on the utilisation of fibres as products of PET bottles and PP products in order to improve the engineering properties of clay soil in Pekan. The soil samples were taken from Kampung Tanjung Medang, Pekan, Pahang. The basic properties of the clay soil were determined as follows; optimum moisture content: 32.5%, maximum dry density: 13.43 kN/m3, specific gravity: 2.51, liquid limit: 74.67%, plastic limit: 45.98%, and plasticity index: 28.69%. This investigation concentrates on the shear strength of the reinforced clay soils with PET and PP in random orientation. The reinforced soil samples were subjected to unconfined compression test (UCT) to differentiate their shear strength with that of the unreinforced soil. The tests found that the waste fibres (PET and PP) improved the strength properties of the Pekan clayey soils. The unconfined compressive strength (UCS) value increased with the increasing percentage of PET fibre and reached the optimum content at 10% reinforcement, where it showed the highest improvement of 365 kN/m2 from 325 kN/m2 and depleted when the optimum content reached 20% reinforcement. For PP fibre, the reinforced soil showed the highest UCS at 20% reinforcement with the improvement of 367 kN/m2. The study concluded that the PET and PP fibres can be utilised successfully as reinforcement materials for the stabilisation of clayey soils. The use of these waste compounds as alternative materials for clay soil stabilisation is reasonable and cost effective since they are constantly available.

(11)C-Choline PET/CT as a guide to radiation treatment planning of lymph-node relapses in prostate cancer patients.

PubMed

Picchio, M; Berardi, G; Fodor, A; Busnardo, E; Crivellaro, C; Giovacchini, G; Fiorino, C; Kirienko, M; Incerti, E; Messa, C; Gianolli, L; Di Muzio, N

2014-07-01

To evaluate, in prostate cancer (PCa) patients the potential of (11)C-choline PET/CT as a guide to helical tomotherapy (HTT) of lymph-node (LN) relapses with simultaneous integrated boost (SIB). The efficacy and feasibility of HTT in terms of acute toxicity were assessed. We enrolled 83 PCa patients (mean age 68 years, range 51 - 82 years) with biochemical recurrence after radical primary treatment (mean serum PSA 7.61 ng/ml, range 0.37 - 187.00 ng/ml; PSA0) who showed pathological findings on (11)C-choline PET/CT only at the LN site. (11)C-Choline PET/CT was performed for restaging and then for radiation treatment planning (PET/CT0). Of the 83 patients, 8 experienced further LN relapse, of whom 5 were retreated once and 3 were retreated twice (total 94 radiotherapy treatments). All pelvic and/or abdominal LNs positive on PET/CT0 were treated with high doses using SIB. Doses were in the range 36 - 74 Gy administered in 28 fractions. After the end of HTT (mean 83 days, range 16 - 365 days), serum PSA was measured in all patients (PSA1) and compared with PSA0 to evaluate early biochemical response. In 47 patients PET/CT was repeated (PET/CT1) to assess metabolic responses at the treated areas. Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) were used to assess acute toxicity. PET/CT0 revealed pathological LNs in the pelvis in 49 patients, pathological LNs in the abdomen in 15 patients pathological LNs in both the pelvis and abdomen in 18 patients, and pathological LNs in the pelvis or abdomen and other sites in 12 patients. All these sites were treated with HTT. With respect to PSA0, PSA1 (mean 6.28 ng/ml, range 0.00 - 220.46 ng/ml) showed a complete biochemical response after 66 of the 94 HTT treatments, a partial response after 12 treatments, stable disease after 1 treatment and progression of disease after 15 treatments. Of the 47 patients receiving PET/CT1, 20 showed a complete metabolic response at the treated area, 22 a partial metabolic response, 3 progression of disease and 2 stable disease. HTT with SIB was well tolerated in all patients. Grade 3 acute toxicity in the genitourinary tract was observed in two patients. (11)C-Choline PET/CT is a valuable tool for planning and monitoring HTT in LN relapse after primary treatment. High-dose hypofractionated (11)C-choline PET/CT-guided HTT with SIB is well tolerated and is associated with a high early biochemical response rate.

Topical or oral treatment of peach flower extract attenuates UV-induced epidermal thickening, matrix metalloproteinase-13 expression and pro-inflammatory cytokine production in hairless mice skin

PubMed Central

Yang, Jiwon; Shin, Chang-Yup; Chung, Jin Ho

2018-01-01

BACKGROUND/OBJECTIVES Ultraviolet radiation (UV) is a major cause of skin photoaging. Previous studies reported that ethanol extract (PET) of Prunus persica (L.) Batsch flowers (PPF, peach flowers) and its subfractions, particularly the ethylacetate (PEA) and n-butanol extracts (PBT), have potent antioxidant activity and attenuate the UV-induced matrix metalloproteinase (MMP) expression in human skin cells. In this study, we investigated the protective activity of PPF extract against UV-induced photoaging in a mouse model. MATERIALS/METHODS Hairless mice were treated with PET or a mixture of PEA and PBT either topically or orally along with UV irradiation. Histological changes and biochemical alterations of mouse skin were examined. Major phenolic compounds in PPF extract were analyzed using an ACQUITY UPLC system. RESULTS The overall effects of topical and oral treatments with PPF extract on the UV-induced skin responses exhibited similar patterns. In both experiments, the mixture of PEA and PBT significantly inhibited the UV-induced skin and epidermal thickening, while PET inhibited only the UV-induced epidermal thickening. Treatment of PET or the mixture of PEA and PBT significantly inhibited the UV-induced MMP-13 expression, but not typeⅠ collagen expression. Topical treatment of the mixture of PEA and PBT with UV irradiation significantly elevated catalase, superoxide dismutase (SOD) and glutathione-peroxidase (GPx) activities in the skin compared to those in the UV irradiated control group, while oral treatment of the mixture of PEA and PBT or PET elevated only catalase and SOD activities, but not GPx. Thirteen phytochemical compounds including 4-O-caffeoylquinic acid, cimicifugic acid E and B, quercetin-3-O-rhamnoside and kaempferol glycoside derivatives were identified in the PPF extract. CONCLUSIONS These results demonstrate that treatment with PET or the mixture of PEA and PBT, both topically or orally, attenuates UV-induced photoaging via the cooperative interactions of phenolic components having anti-oxidative and collagen-protective activities. PMID:29399294

SU-F-J-95: Impact of Shape Complexity On the Accuracy of Gradient-Based PET Volume Delineation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dance, M; Wu, G; Gao, Y

2016-06-15

Purpose: Explore correlation of tumor complexity shape with PET target volume accuracy when delineated with gradient-based segmentation tool. Methods: A total of 24 clinically realistic digital PET Monte Carlo (MC) phantoms of NSCLC were used in the study. The phantom simulated 29 thoracic lesions (lung primary and mediastinal lymph nodes) of varying size, shape, location, and {sup 18}F-FDG activity. A program was developed to calculate a curvature vector along the outline and the standard deviation of this vector was used as a metric to quantify a shape’s “complexity score”. This complexity score was calculated for standard geometric shapes and MC-generatedmore » target volumes in PET phantom images. All lesions were contoured using a commercially available gradient-based segmentation tool and the differences in volume from the MC-generated volumes were calculated as the measure of the accuracy of segmentation. Results: The average absolute percent difference in volumes between the MC-volumes and gradient-based volumes was 11% (0.4%–48.4%). The complexity score showed strong correlation with standard geometric shapes. However, no relationship was found between the complexity score and the accuracy of segmentation by gradient-based tool on MC simulated tumors (R{sup 2} = 0.156). When the lesions were grouped into primary lung lesions and mediastinal/mediastinal adjacent lesions, the average absolute percent difference in volumes were 6% and 29%, respectively. The former group is more isolated and the latter is more surround by tissues with relatively high SUV background. Conclusion: The complexity shape of NSCLC lesions has little effect on the accuracy of the gradient-based segmentation method and thus is not a good predictor of uncertainty in target volume delineation. Location of lesion within a relatively high SUV background may play a more significant role in the accuracy of gradient-based segmentation.« less

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

PubMed

Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

2016-04-12

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

PubMed Central

Kim, Woosuk; Le, Thuc M.; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T.; Abt, Evan R.; Capri, Joseph R.; Austin, Wayne R.; Van Valkenburgh, Juno S.; Steele, Dalton; Gipson, Raymond M.; Slavik, Roger; Cabebe, Anthony E.; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S.; Lee, Jason T.; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F.; Witte, Owen N.; Donahue, Timothy R.; Phelps, Michael E.; Herschman, Harvey R.; Herrmann, Ken; Czernin, Johannes; Radu, Caius G.

2016-01-01

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds—[18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) and 2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-guanine ([18F]F-AraG)—for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [18F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [18F]F-AraG is a better substrate for dGK than for dCK. [18F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [18F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [18F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [18F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [18F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [18F]CFA PET as a new cancer biomarker for treatment stratification and monitoring. PMID:27035974

Baseline knowledge of potential pet toxins among the US general public.

PubMed

Young, Natalie; Royal, Kenneth; Lovee, Bryan; Davidson, Gigi

2018-05-16

In 2014, the American Society for the Prevention of Cruelty toAnimals Animal Poison Control Center fielded more than 167,000cases of potential nonhuman animal toxicosis. Concomitantly, thereremain limited free and reputable veterinary toxicology resourcesavailable for companion-animal (pet) caregivers (owners) seekingassistance and advice about potentially harmful exposures inanimals. The objective of this study was to assess pet toxicantknowledge among a representative sample of Americans andgauge the need for additional toxicology resources. The studyinvolved a survey designed to capture participants' ability to identifypotential animal toxicants and what resource they would use ifan accidental toxic ingestion occurred. Participants were ableto correctly identify 52% of potential pet toxins. Women, olderparticipants and participants from the South expressed moreconcern about each potential pet poison. Approximately halfof participants indicated they would consult a veterinarian andwhereas most others indicated they would search the Internet formore information about pet toxicology. The findings suggest moreveterinary poisoning education is needed for pet owners to be ableto accurately distinguish potential pet toxicants from nontoxicants.

Non-invasive assessment of inter-and intrapatient variability of integrin expression in metastasized prostate cancer by PET.

PubMed

Beer, Ambros J; Schwarzenböck, Sarah M; Zantl, Niko; Souvatzoglou, Michael; Maurer, Tobias; Watzlowik, Petra; Kessler, Horst; Wester, Hans-Jürgen; Schwaiger, Markus; Krause, Bernd Joachim

2016-05-10

Due to the high expression of the integrin αvβ3 not only on endothelial cells, but also on mature osteoclasts and prostate cancer cells, imaging of osseous metastases with αvβ3-targeted tracers seems promising. However, little is known about the patterns of αvβ3-expression in metastasized prostate cancer lesions in-vivo. Thus we evaluated the uptake of the αvβ3-specific PET tracer [18F]Galacto-RGD for assessment of bone metastases in prostate cancer patients. [18F]Galacto-RGD PET identified 58/74 bone-lesions (detection rate of 78.4%) and lymph node metastases in 2/5 patients. The SUVmean was 2.12+/-0.94 (range 0.70-4.38; tumor/blood 1.36+/-0.53; tumor/muscle 2.82+/-1.31) in bone-lesions and 2.21+/-1.18 (range 0.75-3.56) in lymph node metastases. Good visualization and detection of bone metastases was feasible due to a low background activity of the surrounding normal bone tissue. 12 patients with known metastasized prostate cancer according to conventional staging (including bone-scintigraphy and contrast-enhanced CT; median PSA 68.63 ng/ml, range 3.72-1935) were examined with PET after i.v.-injection of [18F]Galacto-RGD. Two blinded nuclear-medicine physicians evaluated the PET-scans in consensus concerning lesion detectability. Volumes-of-interest were drawn in the PET-scans over all metastases defined by conventional staging (maximum of 11 lesions/patient), over the left ventricle, liver and muscle and standardized-uptake-values (SUVs) were calculated. Our data show generally elevated uptake of [18F]Galacto-RGD in bone metastases from prostate cancer with a marked inter- and intrapatient variability. While [18F]Galacto-RGD PET is inferior to bone scintigraphy for detection of osseous metastases, it might be valuable in patient screening and monitoring of αvβ3-targeted therapies due to the high variability of αvβ3-expression.

Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

PubMed

Ruby; Santosh Kumar, R J; Vishwakarma, Rishi K; Singh, Somesh; Khan, Bashir M

2014-07-01

Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.

The impact of FDG-PET/CT in the management of patients with vulvar and vaginal cancer.

PubMed

Robertson, N L; Hricak, H; Sonoda, Y; Sosa, R E; Benz, M; Lyons, G; Abu-Rustum, N R; Sala, E; Vargas, H A

2016-03-01

To evaluate the changes in prognostic impression and patient management following PET/CT in patients with vulvar and vaginal carcinoma; and to compare PET/CT findings with those of conventional imaging modalities. We summarized prospectively and retrospectively collected data for 50 consecutive patients from our institution that enrolled in the National Oncologic PET Registry and underwent FDG-PET/CT for a suspected or known primary or recurrent vulvar/vaginal cancer. 54/83 (65%) studies included had a diagnosis of vulvar cancer, and the remaining 29/83 (35%), a diagnosis of vaginal cancer. Following FDG-PET/CT, the physician's prognostic impression changed in 51% of cases. A change in patient management, defined as a change to/from a non-interventional strategy (observation or additional imaging), to/from an interventional strategy (biopsy or treatment), was documented in 36% of studies. The electronic records demonstrated that 95% of the management strategies recorded in the physician questionnaires were implemented as planned. MRI and/or CT were performed within one month of the FDG-PET/CT in 20/83 (24%) and 28/83 (34%) cases, respectively. FDG-PET/CT detected nodes suspicious for metastases on 29/83 (35%) studies performed. MRI and CT detected positive nodes on 6 and 11 studies respectively. Distant metastases were identified in 10 cases imaged with FDG-PET and 5 cases that had additional conventional CT imaging. All suspicious lesions seen on CT were positively identified on PET/CT. In 4 cases, an abnormality identified on PET/CT, was not seen on diagnostic CT. FDG-PET/CT may play an important role in the management of vulvar and vaginal carcinoma. Copyright © 2015 Elsevier Inc. All rights reserved.

PET Study of Sphingosine-1-Phosphate Receptor 1 Expression in Response to Vascular Inflammation in a Rat Model of Carotid Injury

DOE PAGES

Liu, Hui; Jin, Hongjun; Yue, Xuyi; ...

2017-01-30

Sphingosine-1-phosphate receptor (S1PR) activation plays a key role in vascular inflammatory response. Here, we report in vivo validation of [ 11C]TZ3321, a potent S1PR1 radioligand, for imaging vascular inflammation in a rat model of carotid injury. The right common carotid artery of male adult Sprague-Dawley rats was injured by balloon overinflation that denuded the endothelium and distended the vessel wall. Animals received a 60-minute micro-positron emission tomography (micro PET) scan with [ 11C]TZ3321 at 72 hours after injury. Ex vivo autoradiography was also conducted. The expression and cellular location of S1PR1 were examined by immunohistological analysis. Three-dimensional (3D) reconstruction ofmore » the first 100-second microPET/computed tomography (CT) image indicated the location of bilateral common carotid arteries. [ 11C]TZ3321 displayed significantly higher accumulation (standardized uptake values: 0.93 ± 0.07 vs 0.78 ± 0.09, n = 6, P = .001) in the injured carotid artery than in the contralateral side. Increased tracer uptake in the injured artery was confirmed by autoradiography (photostimulated luminescence measures: 85.5 ± 0.93 vs 71.48 ± 6.22, n = 2). Concordantly, high S1PR1expression was observed in infiltrated inflammatory cells in the injured artery. Our studies demonstrate [ 11C]TZ3321 microPET is able to detect the acute upregulation of S1PR1 expression in inflamed carotid artery. Therefore, [ 11C]TZ3321 has potential to be a PET radiotracer for detecting early inflammatory response and monitoring therapeutic efficacy of vascular inflammation.« less

PET Study of Sphingosine-1-Phosphate Receptor 1 Expression in Response to Vascular Inflammation in a Rat Model of Carotid Injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Hui; Jin, Hongjun; Yue, Xuyi

Sphingosine-1-phosphate receptor (S1PR) activation plays a key role in vascular inflammatory response. Here, we report in vivo validation of [ 11C]TZ3321, a potent S1PR1 radioligand, for imaging vascular inflammation in a rat model of carotid injury. The right common carotid artery of male adult Sprague-Dawley rats was injured by balloon overinflation that denuded the endothelium and distended the vessel wall. Animals received a 60-minute micro-positron emission tomography (micro PET) scan with [ 11C]TZ3321 at 72 hours after injury. Ex vivo autoradiography was also conducted. The expression and cellular location of S1PR1 were examined by immunohistological analysis. Three-dimensional (3D) reconstruction ofmore » the first 100-second microPET/computed tomography (CT) image indicated the location of bilateral common carotid arteries. [ 11C]TZ3321 displayed significantly higher accumulation (standardized uptake values: 0.93 ± 0.07 vs 0.78 ± 0.09, n = 6, P = .001) in the injured carotid artery than in the contralateral side. Increased tracer uptake in the injured artery was confirmed by autoradiography (photostimulated luminescence measures: 85.5 ± 0.93 vs 71.48 ± 6.22, n = 2). Concordantly, high S1PR1expression was observed in infiltrated inflammatory cells in the injured artery. Our studies demonstrate [ 11C]TZ3321 microPET is able to detect the acute upregulation of S1PR1 expression in inflamed carotid artery. Therefore, [ 11C]TZ3321 has potential to be a PET radiotracer for detecting early inflammatory response and monitoring therapeutic efficacy of vascular inflammation.« less

Acariasis on pet Burmese python, Python molurus bivittatus in Malaysia.

PubMed

Mariana, A; Vellayan, S; Halimaton, I; Ho, T M

2011-03-01

To identify the acari present on pet Burmese pythons in Malaysia and to determine whether there is any potential public health risk related to handling of the snakes. Two sub-adult Burmese pythons kept as pets for a period of about 6 to 7 months by different owners, were brought to an exotic animal practice for treatment. On a complete medical examination, some ticks and mites (acari) were detected beneath the dorsal and ventral scales along body length of the snakes. Ticks were directly identified and mites were mounted prior to identification. A total of 12 ticks represented by 3 males, 2 females and 7 nymphal stages of Rhipicephalus sanguineus (R. sanguineus) were extracted from the first python while the other one was with 25 female Ophionyssus natricis (O. natricis) mesostigmatid mites. Only adult female mites were found. These mites are common ectoparasites of Burmese pythons. Both the acarine species found on the Burmese pythons are known vectors of pathogens. This is the first record that R. sanguineus has been reported from a pet Burmese python in Malaysia. Copyright © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Convergence of the Insulin and Serotonin Programs in the Pancreatic β-Cell

PubMed Central

Ohta, Yasuharu; Kosaka, Yasuhiro; Kishimoto, Nina; Wang, Juehu; Smith, Stuart B.; Honig, Gerard; Kim, Hail; Gasa, Rosa M.; Neubauer, Nicole; Liou, Angela; Tecott, Laurence H.; Deneris, Evan S.; German, Michael S.

2011-01-01

OBJECTIVE Despite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences. RESEARCH DESIGN AND METHODS We used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas. RESULTS We found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In β-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance. CONCLUSIONS These studies demonstrate that a common transcriptional cascade drives the differentiation of β-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of diabetes. PMID:22013016

Convergence of the insulin and serotonin programs in the pancreatic β-cell.

PubMed

Ohta, Yasuharu; Kosaka, Yasuhiro; Kishimoto, Nina; Wang, Juehu; Smith, Stuart B; Honig, Gerard; Kim, Hail; Gasa, Rosa M; Neubauer, Nicole; Liou, Angela; Tecott, Laurence H; Deneris, Evan S; German, Michael S

2011-12-01

Despite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences. We used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas. We found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In β-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance. These studies demonstrate that a common transcriptional cascade drives the differentiation of β-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of diabetes.

Detection of Atherosclerotic Inflammation by 68Ga-DOTATATE PET Compared to [18F]FDG PET Imaging.

PubMed

Tarkin, Jason M; Joshi, Francis R; Evans, Nicholas R; Chowdhury, Mohammed M; Figg, Nichola L; Shah, Aarti V; Starks, Lakshi T; Martin-Garrido, Abel; Manavaki, Roido; Yu, Emma; Kuc, Rhoda E; Grassi, Luigi; Kreuzhuber, Roman; Kostadima, Myrto A; Frontini, Mattia; Kirkpatrick, Peter J; Coughlin, Patrick A; Gopalan, Deepa; Fryer, Tim D; Buscombe, John R; Groves, Ashley M; Ouwehand, Willem H; Bennett, Martin R; Warburton, Elizabeth A; Davenport, Anthony P; Rudd, James H F

2017-04-11

Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([ 18 F]FDG PET), [ 18 F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover. This study tested the efficacy of gallium-68-labeled DOTATATE ( 68 Ga-DOTATATE), a somatostatin receptor subtype-2 (SST 2 )-binding PET tracer, for imaging atherosclerotic inflammation. We confirmed 68 Ga-DOTATATE binding in macrophages and excised carotid plaques. 68 Ga-DOTATATE PET imaging was compared to [ 18 F]FDG PET imaging in 42 patients with atherosclerosis. Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific 68 Ga-DOTATATE ligand binding to SST 2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo 68 Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). 68 Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBR max ) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). 68 Ga-DOTATATE mTBR max predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and [ 18 F]FDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). [ 18 F]FDG mTBR max differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial [ 18 F]FDG spillover rendered coronary [ 18 F]FDG scans uninterpretable in 27 patients (64%). Coronary 68 Ga-DOTATATE PET scans were readable in all patients. We validated 68 Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that 68 Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than [ 18 F]FDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography [VISION]; NCT02021188). Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Combined Modality Treatment for PET-Positive Non-Hodgkin Lymphoma: Favorable Outcomes of Combined Modality Treatment for Patients With Non-Hodgkin Lymphoma and Positive Interim or Postchemotherapy FDG-PET

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halasz, Lia M.; Jacene, Heather A.; Catalano, Paul J.

2012-08-01

Purpose: To evaluate outcomes of patients treated for aggressive non-Hodgkin lymphoma (NHL) with combined modality therapy based on [{sup 18}F]fluoro-2-deoxy-2-D-glucose positron emission tomography (FDG-PET) response. Methods and Materials: We studied 59 patients with aggressive NHL, who received chemotherapy and radiation therapy (RT) from 2001 to 2008. Among them, 83% of patients had stage I/II disease. Patients with B-cell lymphoma received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone)-based chemotherapy, and 1 patient with anaplastic lymphoma kinase-negative anaplastic T-cell lymphoma received CHOP therapy. Interim and postchemotherapy FDG-PET or FDG-PET/computed tomography (CT) scans were performed for restaging. All patients received consolidated involved-field RT.more » Median RT dose was 36 Gy (range, 28.8-50 Gy). Progression-free survival (PFS) and local control (LC) rates were calculated with and without a negative interim or postchemotherapy FDG-PET scan. Results: Median follow-up was 46.5 months. Thirty-nine patients had negative FDG-PET results by the end of chemotherapy, including 12 patients who had a negative interim FDG-PET scan and no postchemotherapy PET. Twenty patients were FDG-PET-positive, including 7 patients with positive interim FDG-PET and no postchemotherapy FDG-PET scans. The 3-year actuarial PFS rates for patients with negative versus positive FDG-PET scans were 97% and 90%, respectively. The 3-year actuarial LC rates for patients with negative versus positive FDG-PET scans were 100% and 90%, respectively. Conclusions: Patients who had a positive interim or postchemotherapy FDG-PET had a PFS rate of 90% at 3 years after combined modality treatment, suggesting that a large proportion of these patients can be cured with consolidated RT.« less

Preclinical Evaluation of an Anti-Nectin-4 ImmunoPET Reagent in Tumor-Bearing Mice and Biodistribution Studies in Cynomolgus Monkeys.

PubMed

Campbell, Dean O; Noda, Akihiro; Verlinsky, Alla; Snyder, Josh; Fujita, Yuji; Murakami, Yoshihiro; Fushiki, Hiroshi; Miyoshi, Sosuke; Lacayo, Sergio; Cabral, Edward; Yang, Peng; Stover, David R; Joseph, Ingrid B J K

2016-10-01

Nectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models. The biodistribution of [(89)Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [(18)F]AGS-22M6 in cynomolgus monkeys was evaluated. A positive correlation was demonstrated between tumor nectin-4 expression and [(89)Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [(18)F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues. [(89)Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.

TimesVector: a vectorized clustering approach to the analysis of time series transcriptome data from multiple phenotypes.

PubMed

Jung, Inuk; Jo, Kyuri; Kang, Hyejin; Ahn, Hongryul; Yu, Youngjae; Kim, Sun

2017-12-01

Identifying biologically meaningful gene expression patterns from time series gene expression data is important to understand the underlying biological mechanisms. To identify significantly perturbed gene sets between different phenotypes, analysis of time series transcriptome data requires consideration of time and sample dimensions. Thus, the analysis of such time series data seeks to search gene sets that exhibit similar or different expression patterns between two or more sample conditions, constituting the three-dimensional data, i.e. gene-time-condition. Computational complexity for analyzing such data is very high, compared to the already difficult NP-hard two dimensional biclustering algorithms. Because of this challenge, traditional time series clustering algorithms are designed to capture co-expressed genes with similar expression pattern in two sample conditions. We present a triclustering algorithm, TimesVector, specifically designed for clustering three-dimensional time series data to capture distinctively similar or different gene expression patterns between two or more sample conditions. TimesVector identifies clusters with distinctive expression patterns in three steps: (i) dimension reduction and clustering of time-condition concatenated vectors, (ii) post-processing clusters for detecting similar and distinct expression patterns and (iii) rescuing genes from unclassified clusters. Using four sets of time series gene expression data, generated by both microarray and high throughput sequencing platforms, we demonstrated that TimesVector successfully detected biologically meaningful clusters of high quality. TimesVector improved the clustering quality compared to existing triclustering tools and only TimesVector detected clusters with differential expression patterns across conditions successfully. The TimesVector software is available at http://biohealth.snu.ac.kr/software/TimesVector/. sunkim.bioinfo@snu.ac.kr. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Neuroendocrine tumor recurrence: diagnosis with 68Ga-DOTATATE PET/CT.

PubMed

Haug, Alexander R; Cindea-Drimus, Ramona; Auernhammer, Christoph J; Reincke, Martin; Beuschlein, Felix; Wängler, Björn; Uebleis, Christopher; Schmidt, Gerwin P; Spitzweg, Christine; Bartenstein, Peter; Hacker, Marcus

2014-02-01

To evaluate diagnostic performance of gallium 68-tetraazacyclododecane tetraacetic acid-octreotate ((68)Ga-DOTATATE) in detection of recurrent neuroendocrine tumors (NETs). Approval was waived by the local ethics committee for this retrospective study. Between 2007 and 2011, 63 patients (mean age, 58 years) were examined with (68)Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) after primary NET curative resection. Reasons for PET/CT were regular follow-up examinations (n = 30), increased plasma levels of tumor markers (n = 27), or clinical suspicion of recurrence (n = 6). Final diagnosis was determined with histopathologic verification (n = 25) or clinical follow-up (n = 38). PET/CT scans were evaluated in consensus by two readers without blinding to clinical information and independently by two readers with blinding. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Final diagnosis of NET recurrence was determined in 29 patients. In three other patients, tumors of nonneuroendocrine origin were diagnosed. (68)Ga-DOTATATE PET/CT helped identify NET recurrence in 26 of 29 patients (sensitivity, 90%) and exclude presence of recurrent NET in 28 of 34 patients (specificity, 82% ). PET/CT provided false-positive and false-negative results in six and three patients (PPV, 81% [26 of 32]; NPV, 90% [28 of 31]; accuracy, 86% [54 of 63]). In gastroenteropancreatic NET (n = 45), sensitivity was 94% (17 of 18); specificity was 89% (24 of 27); PPV was 85% (17 of 20); NPV was 96% (24 of 25); and accuracy was 91% (41 of 45). Two blinded readers achieved sensitivity of 79% (23 of 29) and 76% (22 of 29); specificity of 85% (29 of 34) and 94% (32 of 34) (κ = 0.80); and accuracy of 83% and 86%. (68)Ga-DOTATATE PET/CT is accurate in detection of recurrent NET. Blinded PET/CT review markedly decreased sensitivity, underlining importance of considering clinical parameters in NET recurrence. Present results must be further validated to substantiate use of (68)Ga-DOTATATE PET/CT in routine follow-up after curative resection of NET. © RSNA, 2013

Targeted PET imaging strategy to differentiate malignant from inflamed lymph nodes in diffuse large B-cell lymphoma

PubMed Central

Salloum, Darin; Carney, Brandon; Brand, Christian; Kossatz, Susanne; Sadique, Ahmad; Lewis, Jason S.; Weber, Wolfgang A.; Wendel, Hans-Guido; Reiner, Thomas

2017-01-01

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults. DLBCL exhibits highly aggressive and systemic progression into multiple tissues in patients, particularly in lymph nodes. Whole-body 18F-fluodeoxyglucose positron emission tomography ([18F]FDG-PET) imaging has an essential role in diagnosing DLBCL in the clinic; however, [18F]FDG-PET often faces difficulty in differentiating malignant tissues from certain nonmalignant tissues with high glucose uptake. We have developed a PET imaging strategy for DLBCL that targets poly[ADP ribose] polymerase 1 (PARP1), the expression of which has been found to be much higher in DLBCL than in healthy tissues. In a syngeneic DLBCL mouse model, this PARP1-targeted PET imaging approach allowed us to discriminate between malignant and inflamed lymph nodes, whereas [18F]FDG-PET failed to do so. Our PARP1-targeted PET imaging approach may be an attractive addition to the current PET imaging strategy to differentiate inflammation from malignancy in DLBCL. PMID:28827325

SPET/CT image co-registration in the abdomen with a simple and cost-effective tool.

PubMed

Förster, Gregor J; Laumann, Christina; Nickel, Otmar; Kann, Peter; Rieker, Olaf; Bartenstein, Peter

2003-01-01

Fusion of morphology and function has been shown to improve diagnostic accuracy in many clinical circumstances. Taking this into account, a number of instruments combining computed tomography (CT) with positron emission tomography (PET) or single-photon emission tomography (SPET) are appearing on the market. The aim of this study was to evaluate a simple and cost-effective approach to generate fusion images of similar quality. For the evaluation of the proposed approach, patients with neuroendocrine abdominal tumours with liver metastases were chosen, since the exact superimposition in the abdomen is more difficult than in other regions. Five hours following the injection of 110 MBq (111)In-DTPA-octreotide, patients were fixed in a vacuum cushion (MED-TEC, Vac-Loc) and investigated with helical CT in a mid-inspiration position ( n=14). Directly following the CT, a SPET study (SPET1) of the abdominal region was performed without changing the position of the patient. A second SPET study (SPET2), 24 h p.i., was acquired after repositioning the patient in his or her individually moulded vacuum cushion. A total of nine markers suitable for imaging with CT and SPET were fixed on the cushion. Datasets were fused by means of internal landmarks (e.g. metastases or margin of abdominal organs) or by the external markers. Image fusion using external markers was fast and easy to handle compared with the use of internal landmarks. Using this technique, all lesions detectable by SPET ( n=28) appeared exactly superpositioned on the respective CT morphology by visual inspection. Image fusion of CT/SPET1 and CT/SPET2 showed a mean deviation of the external markers that in the former case was smaller than the voxel size of 4.67 mm: 4.17+/-0.61 (CT/SPET1; +/-SD) and 5.52+/-1.56 mm (CT/SPET2), respectively. Using internal landmarks, the mean deviation of the chosen landmarks was 6.47+/-1.37 and 7.78+/-1.21 mm. Vector subtraction of corresponding anatomical points of the CT and the re-sampled SPET volume datasets resulted in a similar accuracy. Vector subtraction of the metastases showed a significantly less accurate superimposition when internal landmarks were used ( P<0.001). The vacuum cushion did not affect the image quality of CT and SPET. The proposed technique is a simple and cost-effective way to generate abdominal datasets suitable for image fusion. External markers positioned on the cushion allow for a rapid and robust overlay even if no readily identifiable internal landmarks are present. This technique is, in principle, also suitable for CT/PET fusion as well as for fusions of MRI data with PET or SPET.

Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

PubMed

Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

2016-07-01

Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production.

Whole-Body 68Ga-DOTANOC PET/MRI Versus 68Ga-DOTANOC PET/CT in Patients With Neuroendocrine Tumors

PubMed Central

Berzaczy, Dominik; Giraudo, Chiara; Haug, Alexander R.; Raderer, Markus; Senn, Daniela; Karanikas, Georgios; Weber, Michael; Mayerhoefer, Marius E.

2017-01-01

Purpose The aim of this study was to assess the diagnostic performance of simultaneous whole-body 68Ga-DOTANOC PET/MRI compared with 68Ga-DOTANOC PET/CT for detection of distant metastatic disease in patients with well-differentiated neuroendocrine tumors (NETs). Methods Patients with histologically proven, well-differentiated NET (G1 or G2) were included in this prospective, institutional review board–approved study. Patients underwent 68Ga-DOTANOC PET/CT and subsequent 68Ga-DOTANOC PET/MRI after a single tracer injection on the same day for staging or restaging purposes. Images were evaluated for the presence of NET lesions by 2 rater teams, each consisting of a nuclear medicine physician and a radiologist, in an observer-blinded fashion. Overall agreement, accuracy, sensitivity, and specificity, relative to a composite reference standard (consensus review including follow-up data), were calculated. Results Between July 2014 and June 2016, 28 patients were enrolled. Overall agreement and accuracy between the 2 rater teams were 91.7% (95% confidence interval [CI], 87.5%–95.9%) and 97% (95% CI, 94.4%–99.6%) for PET/MRI and 92.3% (95% CI, 88.3%–96.3%) and 94.6% (95% CI, 91.2%–98.1%) for PET/CT, respectively (P = 1.00). Overall, PET/MRI reached 89.8% sensitivity (95% CI, 77.8%–96.6%) and 100% specificity (95% CI, 97%–100%); PET/CT showed 81.6% sensitivity (95% CI, 68%–91.2%) and 100% specificity (95% CI, 97%–100%) for the detection of metastatic disease in NETs. Conclusions Whole-body 68Ga-DOTANOC PET/MRI appears to be comparable to 68Ga-DOTANOC PET/CT for lesion detection in patients with well-differentiated NETs. PMID:28682844

Computational and experimental analysis of TMS-induced electric field vectors critical to neuronal activation

NASA Astrophysics Data System (ADS)

Krieg, Todd D.; Salinas, Felipe S.; Narayana, Shalini; Fox, Peter T.; Mogul, David J.

2015-08-01

Objective. Transcranial magnetic stimulation (TMS) represents a powerful technique to noninvasively modulate cortical neurophysiology in the brain. However, the relationship between the magnetic fields created by TMS coils and neuronal activation in the cortex is still not well-understood, making predictable cortical activation by TMS difficult to achieve. Our goal in this study was to investigate the relationship between induced electric fields and cortical activation measured by blood flow response. Particularly, we sought to discover the E-field characteristics that lead to cortical activation. Approach. Subject-specific finite element models (FEMs) of the head and brain were constructed for each of six subjects using magnetic resonance image scans. Positron emission tomography (PET) measured each subject’s cortical response to image-guided robotically-positioned TMS to the primary motor cortex. FEM models that employed the given coil position, orientation, and stimulus intensity in experimental applications of TMS were used to calculate the electric field (E-field) vectors within a region of interest for each subject. TMS-induced E-fields were analyzed to better understand what vector components led to regional cerebral blood flow (CBF) responses recorded by PET. Main results. This study found that decomposing the E-field into orthogonal vector components based on the cortical surface geometry (and hence, cortical neuron directions) led to significant differences between the regions of cortex that were active and nonactive. Specifically, active regions had significantly higher E-field components in the normal inward direction (i.e., parallel to pyramidal neurons in the dendrite-to-axon orientation) and in the tangential direction (i.e., parallel to interneurons) at high gradient. In contrast, nonactive regions had higher E-field vectors in the outward normal direction suggesting inhibitory responses. Significance. These results provide critical new understanding of the factors by which TMS induces cortical activation necessary for predictive and repeatable use of this noninvasive stimulation modality.

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

Molecular Targets for PET Imaging of Activated Microglia: The Current Situation and Future Expectations.

PubMed

Tronel, Claire; Largeau, Bérenger; Santiago Ribeiro, Maria Joao; Guilloteau, Denis; Dupont, Anne-Claire; Arlicot, Nicolas

2017-04-11

Microglia, as cellular mediators of neuroinflammation, are implicated in the pathogenesis of a wide range of neurodegenerative diseases. Positron emission tomography (PET) imaging of microglia has matured over the last 20 years, through the development of radiopharmaceuticals targeting several molecular biomarkers of microglial activation and, among these, mainly the translocator protein-18 kDa (TSPO). Nevertheless, current limitations of TSPO as a PET microglial biomarker exist, such as low brain density, even in a neurodegenerative setting, expression by other cells than the microglia (astrocytes, peripheral macrophages in the case of blood brain barrier breakdown), genetic polymorphism, inducing a variation for most of TSPO PET radiopharmaceuticals' binding affinity, or similar expression in activated microglia regardless of its polarization (pro- or anti-inflammatory state), and these limitations narrow its potential interest. We overview alternative molecular targets, for which dedicated radiopharmaceuticals have been proposed, including receptors (purinergic receptors P2X7, cannabinoid receptors, α7 and α4β2 nicotinic acetylcholine receptors, adenosine 2A receptor, folate receptor β) and enzymes (cyclooxygenase, nitric oxide synthase, matrix metalloproteinase, β-glucuronidase, and enzymes of the kynurenine pathway), with a particular focus on their respective contribution for the understanding of microglial involvement in neurodegenerative diseases. We discuss opportunities for these potential molecular targets for PET imaging regarding their selectivity for microglia expression and polarization, in relation to the mechanisms by which microglia actively participate in both toxic and neuroprotective actions in brain diseases, and then take into account current clinicians' expectations.

Comparison of planar, PET and well-counter measurements of total tumor radioactivity in a mouse xenograft model.

PubMed

Green, Michael V; Seidel, Jurgen; Williams, Mark R; Wong, Karen J; Ton, Anita; Basuli, Falguni; Choyke, Peter L; Jagoda, Elaine M

2017-10-01

Quantitative small animal radionuclide imaging studies are often carried out with the intention of estimating the total radioactivity content of various tissues such as the radioactivity content of mouse xenograft tumors exposed to putative diagnostic or therapeutic agents. We show that for at least one specific application, positron projection imaging (PPI) and PET yield comparable estimates of absolute total tumor activity and that both of these estimates are highly correlated with direct well-counting of these same tumors. These findings further suggest that in this particular application, PPI is a far more efficient data acquisition and processing methodology than PET. Forty-one athymic mice were implanted with PC3 human prostate cancer cells transfected with prostate-specific membrane antigen (PSMA (+)) and one additional animal (for a total of 42) with a control blank vector (PSMA (-)). All animals were injected with [ 18 F] DCFPyl, a ligand for PSMA, and imaged for total tumor radioactivity with PET and PPI. The tumors were then removed, assayed by well counting for total radioactivity and the values between these methods intercompared. PET, PPI and well-counter estimates of total tumor radioactivity were highly correlated (R 2 >0.98) with regression line slopes near unity (0.95

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

PubMed Central

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency. PMID:20042112

Design, Synthesis, and Biological Evaluation of 68Ga-DOTA-PA1 for Lung Cancer: A Novel PET Tracer for Multiple Somatostatin Receptor Imaging.

PubMed

Liu, Fei; Liu, Teli; Xu, Xiaoxia; Guo, Xiaoyi; Li, Nan; Xiong, Chiyi; Li, Chun; Zhu, Hua; Yang, Zhi

2018-02-05

Most of the radiolabeled somatostatin analogues (SSAs) are specific for subtype somatostatin receptor 2 (SSTR 2 ). Lack of ligands targeting other subtypes of SSTRs, especially SSTR 1, SSTR 3 , and SSTR 5 , limited their applications in tumors of low SSTR 2 expression, including lung tumor. In this study, we aimed to design and synthesize a positron emission tomography (PET) radiotracer targeting multi-subtypes of SSTRs for PET imaging. PA1 peptide and its conjugate with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator or fluorescein isothiocyanate (FITC) at the N-terminal of the lysine position were synthesized. 68 Ga was chelated to DOTA-PA1 to obtain 68 Ga-DOTA-PA1 radiotracer. The stability, lipophilicity, binding affinity, and binding specificity of 68 Ga-DOTA-PA1 and FITC-PA1 were evaluated by various in vitro experiments. Micro-PET imaging of 68 Ga-DOTA-PA1 was performed in nude mice bearing A549 lung adenocarcinoma, as compared with 68 Ga-DOTA-(Tyr3)-octreotate ( 68 Ga-DOTA-TATE). Histological analysis of SSTR expression in A549 tumor tissues and human tumor tissues was conducted using immunofluorescence staining and immunohistochemical assay. 68 Ga-DOTA-PA1 had high radiochemical yield and radiochemical purity of over 95% and 99%, respectively. The radiotracer was stable in vitro in different buffers over a 2 h incubation period. Cell uptake of 68 Ga-DOTA-PA1 was 1.31-, 1.33-, and 1.90-fold that of 68 Ga-DOTA-TATE, which has high binding affinity only for SSTR 2 , after 2 h incubation in H520, PG, and A549 lung cancer cell lines, respectively. Micro-PET images of 68 Ga-DOTA-PA1 showed that the PET imaging signal correlated with the total expression of SSTRs, instead of SSTR 2 only, which was measured by Western blotting and immunofluorescence analysis in mice bearing A549 tumors. In summary, a novel PET radiotracer, 68 Ga-DOTA-PA1, targeting multi-subtypes of SSTRs, was successfully synthesized and was confirmed to be useful for PET imaging. It may have potential as a noninvasive PET radiotracer for imaging SSTR-positive tumors.

Large Multiple Transmembrane Domain Fragments of a G Protein-Coupled Receptor: Biosynthesis, Purification, and Biophysical Studies

PubMed Central

Potetinova, Zhanna; Tantry, Subramanyam; Cohen, Leah S.; Caroccia, Katrina E.; Arshava, Boris; Becker, Jeffrey M.; Naider, Fred

2013-01-01

To conduct biophyiscal analyses on large domains of GPCRs, multi-milligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of 4 transmembrane and 5 transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G31-A198] (4TMN), four C-terminal Ste2p[T155-L340] (4TMC) or five C-terminal Ste2p[I120-L340] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced Escherichia coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multi-milligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [15N]-labeled 5TMC in 50% trifluoroethanol-d2/H2O/0.05% trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains. PMID:23203693

Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus.

PubMed

Shahein, Yasser Ezzat; El Sayed El-Hakim, Amr; Abouelella, Amira Mohamed Kamal; Hamed, Ragaa Reda; Allam, Shaimaa Abdul-Moez; Farid, Nevin Mahmoud

2008-03-25

A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.

Zoonoses in pet birds: review and perspectives

PubMed Central

2013-01-01

Pet birds are a not-so-well known veterinarian’s clientship fraction. Bought individually or in couples, as families often do (which is a lucrative business for pet shops or local breeders) or traded (sometimes illegally) for their very high genetic or exotic value, these birds, commonly canaries, parakeets or parrots, are regularly sold at high prices. These animals, however, are potential carriers and/or transmitters of zoonotic diseases. Some of them could have an important impact on human health, like chlamydophilosis, salmonellosis or even highly pathogenic avian influenza A H5N1. This review paper, although non exhaustive, aims at enlightening, by the description of several cases of bird-human transmission, the risks encountered by bird owners, including children. Public health consequences will be discussed and emphasis will be made on some vector-borne diseases, known to be emergent or which are underestimated, like those transmitted by the red mite Dermanyssus gallinae. Finally, biosecurity and hygiene, as well as prevention guidelines will be developed and perspectives proposed. PMID:23687940

Positron emission tomography imaging of CD105 expression in a rat myocardial infarction model with (64)Cu-NOTA-TRC105.

PubMed

Orbay, Hakan; Zhang, Yin; Valdovinos, Hector F; Song, Guoqing; Hernandez, Reinier; Theuer, Charles P; Hacker, Timothy A; Nickles, Robert J; Cai, Weibo

2013-01-01

Biological changes following myocardial infarction (MI) lead to increased secretion of angiogenic factors that subsequently stimulate the formation of new blood vessels as a compensatory mechanism to reverse ischemia. The goal of this study was to assess the role of CD105 expression during MI-induced angiogenesis by positron emission tomography (PET) imaging using (64)Cu-labeled TRC105, an anti-CD105 monoclonal antibody. MI was induced by ligation of the left anterior descending (LAD) artery in female rats. Echocardiography and (18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) PET scans were performed on post-operative day 3 to confirm the presence of MI in the infarct group and intact heart in the sham group, respectively. Ischemia-induced angiogenesis was non-invasively monitored with (64)Cu-NOTA-TRC105 (an extensively validated PET tracer in our previous studies) PET on post-operative days 3, 10, and 17. Tracer uptake in the infarct zone was highest on day 3 following MI, which was significantly higher than that in the sham group (1.41 ± 0.45 %ID/g vs 0.57 ± 0.07 %ID/g; n=3, p<0.05). Subsequently, tracer uptake in the infarct zone decreased over time to the background level on day 17, whereas tracer uptake in the heart of sham rats remained low at all time points examined. Histopathology documented increased CD105 expression following MI, which corroborated in vivo findings. This study indicated that PET imaging of CD105 can be a useful tool for MI-related research, which can potentially improve MI patient management in the future upon clinical translation of the optimized PET tracers.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

PubMed

Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh; Mahboudi, Fereidoun

2017-01-01

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

PubMed Central

Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh

2017-01-01

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios. PMID:28662065

Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

PubMed

Kimura, Tetsuya; Nakao, Akihide; Murata, Sachiko; Kobayashi, Yasuyuki; Tanaka, Yuji; Shibahara, Kenta; Kawazu, Tetsu; Nakagawa, Tsuyoshi

2013-01-01

We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

18F-fluorodeoxyglucose positron emission tomography/computed tomography-based radiotherapy target volume definition in non-small-cell lung cancer: delineation by radiation oncologists vs. joint outlining with a PET radiologist?

PubMed

Hanna, Gerard G; Carson, Kathryn J; Lynch, Tom; McAleese, Jonathan; Cosgrove, Vivian P; Eakin, Ruth L; Stewart, David P; Zatari, Ashraf; O'Sullivan, Joe M; Hounsell, Alan R

2010-11-15

(18)F-Fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) has benefits in target volume (TV) definition in radiotherapy treatment planning (RTP) for non-small-cell lung cancer (NSCLC); however, an optimal protocol for TV delineation has not been determined. We investigate volumetric and positional variation in gross tumor volume (GTV) delineation using a planning PET/CT among three radiation oncologists and a PET radiologist. RTP PET/CT scans were performed on 28 NSCLC patients (Stage IA-IIIB) of which 14 patients received prior induction chemotherapy. Three radiation oncologists and one PET radiologist working with a fourth radiation oncologist independently delineated the GTV on CT alone (GTV(CT)) and on fused PET/CT images (GTV(PETCT)). The mean percentage volume change (PVC) between GTV(CT) and GTV(PETCT) for the radiation oncologists and the PVC between GTV(CT) and GTV(PETCT) for the PET radiologist were compared using the Wilcoxon signed-rank test. Concordance index (CI) was used to assess both positional and volume change between GTV(CT) and GTV(PETCT) in a single measurement. For all patients, a significant difference in PVC from GTV(CT) to GTV(PETCT) exists between the radiation oncologist (median, 5.9%), and the PET radiologist (median, -0.4%, p = 0.001). However, no significant difference in median concordance index (comparing GTV(CT) and GTV(FUSED) for individual cases) was observed (PET radiologist = 0.73; radiation oncologists = 0.66; p = 0.088). Percentage volume changes from GTV(CT) to GTV(PETCT) were lower for the PET radiologist than for the radiation oncologists, suggesting a lower impact of PET/CT in TV delineation for the PET radiologist than for the oncologists. Guidelines are needed to standardize the use of PET/CT for TV delineation in RTP. Copyright © 2010 Elsevier Inc. All rights reserved.

Construction of two vectors for gene expression in Trichoderma reesei.

PubMed

Lv, Dandan; Wang, Wei; Wei, Dongzhi

2012-01-01

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry. Copyright © 2011 Elsevier Inc. All rights reserved.

Molecular characterization of enolase gene from Taenia multiceps.

PubMed

Li, W H; Qu, Z G; Zhang, N Z; Yue, L; Jia, W Z; Luo, J X; Yin, H; Fu, B Q

2015-10-01

Taenia multiceps is a cestode parasite with its larval stage, known as Coenurus cerebralis, mainly encysts in the central nervous system of sheep and other livestocks. Enolase is a key glycolytic enzyme and represents multifunction in most organisms. In the present study, a 1617bp full-length cDNA encoding enolase was cloned from T. multiceps and designated as TmENO. A putative encoded protein of 433 amino acid residues that exhibited high similarity to helminth parasites. The recombinant TmENO protein (rTmENO) showed the catalytic and plasminogen-binding characteristics after the TmENO was subcloned and expressed in the pET30a(+) vector. The TmENO gene was transcribed during the adult and larval stages and was also identified in both cyst fluid and as a component of the adult worms and the metacestode by western blot analysis. Taken together, our results will facilitate further structural characterization for TmENO and new potential control strategies for T. multiceps. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells].

PubMed

Ma, Benjiang; Hang, Changshou; Zhao, Yun; Wang, Shiwen; Xie, Yanxiang

2002-09-01

To construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses. This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

Quantitative PET Imaging of Tissue Factor Expression Using 18F-Labeled Active Site-Inhibited Factor VII.

PubMed

Nielsen, Carsten H; Erlandsson, Maria; Jeppesen, Troels E; Jensen, Mette M; Kristensen, Lotte K; Madsen, Jacob; Petersen, Lars C; Kjaer, Andreas

2016-01-01

Tissue factor (TF) is upregulated in many solid tumors, and its expression is linked to tumor angiogenesis, invasion, metastasis, and prognosis. A noninvasive assessment of tumor TF expression status is therefore of obvious clinical relevance. Factor VII is the natural ligand to TF. Here we report the development of a new PET tracer for specific imaging of TF using an (18)F-labeled derivative of factor VII. Active site-inhibited factor VIIa (FVIIai) was obtained by inactivation with phenylalanine-phenylalanine-arginine-chloromethyl ketone. FVIIai was radiolabeled with N-succinimidyl 4-(18)F-fluorobenzoate and purified. The corresponding product, (18)F-FVIIai, was injected into nude mice with subcutaneous human pancreatic xenograft tumors (BxPC-3) and investigated using small-animal PET/CT imaging 1, 2, and 4 h after injection. Ex vivo biodistribution was performed after the last imaging session, and tumor tissue was preserved for molecular analysis. A blocking experiment was performed in a second set of mice. The expression pattern of TF in the tumors was visualized by immunohistochemistry and the amount of TF in tumor homogenates was measured by enzyme-linked immunosorbent assay and correlated with the uptake of (18)F-FVIIai in the tumors measured in vivo by PET imaging. The PET images showed high uptake of (18)F-FVIIai in the tumor regions, with a mean uptake of 2.5 ± 0.3 percentage injected dose per gram (%ID/g) (mean ± SEM) 4 h after injection of 7.3-9.3 MBq of (18)F-FVIIai and with an average maximum uptake in the tumors of 7.1 ± 0.7 %ID/g at 4 h. In comparison, the muscle uptake was 0.2 ± 0.01 %ID/g at 4 h. At 4 h, the tumors had the highest uptake of any organ. Blocking with FVIIai significantly reduced the uptake of (18)F-FVIIai from 2.9 ± 0.1 to 1.4 ± 0.1 %ID/g (P < 0.001). The uptake of (18)F-FVIIai measured in vivo by PET imaging correlated (r = 0.72, P < 0.02) with TF protein level measured ex vivo. (18)F-FVIIai is a promising PET tracer for specific and noninvasive imaging of tumor TF expression. The tracer merits further development and clinical translation, with potential to become a companion diagnostics for emerging TF-targeted therapies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

The Processing of Human Emotional Faces by Pet and Lab Dogs: Evidence for Lateralization and Experience Effects

PubMed Central

Barber, Anjuli L. A.; Randi, Dania; Müller, Corsin A.; Huber, Ludwig

2016-01-01

From all non-human animals dogs are very likely the best decoders of human behavior. In addition to a high sensitivity to human attentive status and to ostensive cues, they are able to distinguish between individual human faces and even between human facial expressions. However, so far little is known about how they process human faces and to what extent this is influenced by experience. Here we present an eye-tracking study with dogs emanating from two different living environments and varying experience with humans: pet and lab dogs. The dogs were shown pictures of familiar and unfamiliar human faces expressing four different emotions. The results, extracted from several different eye-tracking measurements, revealed pronounced differences in the face processing of pet and lab dogs, thus indicating an influence of the amount of exposure to humans. In addition, there was some evidence for the influences of both, the familiarity and the emotional expression of the face, and strong evidence for a left gaze bias. These findings, together with recent evidence for the dog's ability to discriminate human facial expressions, indicate that dogs are sensitive to some emotions expressed in human faces. PMID:27074009

AAVPG: A vigilant vector where transgene expression is induced by p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.

2013-12-15

Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 foldmore » increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.« less

SU-E-J-254: Evaluating the Role of Mid-Treatment and Post-Treatment FDG-PET/CT in Predicting Progression-Free Survival and Distant Metastasis of Anal Cancer Patients Treated with Chemoradiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, H; Wang, J; Chuong, M

2015-06-15

Purpose: To evaluate the role of mid-treatment and post-treatment FDG-PET/CT in predicting progression-free survival (PFS) and distant metastasis (DM) of anal cancer patients treated with chemoradiotherapy (CRT). Methods: 17 anal cancer patients treated with CRT were retrospectively studied. The median prescription dose was 56 Gy (range, 50–62.5 Gy). All patients underwent FDG-PET/CT scans before and after CRT. 16 of the 17 patients had an additional FDG-PET/CT image at 3–5 weeks into the treatment (denoted as mid-treatment FDG-PET/CT). 750 features were extracted from these three sets of scans, which included both traditional PET/CT measures (SUVmax, SUVpeak, tumor diameters, etc.) and spatialtemporalmore » PET/CT features (comprehensively quantify a tumor’s FDG uptake intensity and distribution, spatial variation (texture), geometric property and their temporal changes relative to baseline). 26 clinical parameters (age, gender, TNM stage, histology, GTV dose, etc.) were also analyzed. Advanced analytics including methods to select an optimal set of predictors and a model selection engine, which identifies the most accurate machine learning algorithm for predictive analysis was developed. Results: Comparing baseline + mid-treatment PET/CT set to baseline + posttreatment PET/CT set, 14 predictors were selected from each feature group. Same three clinical parameters (tumor size, T stage and whether 5-FU was held during any cycle of chemotherapy) and two traditional measures (pre- CRT SUVmin and SUVmedian) were selected by both predictor groups. Different mix of spatial-temporal PET/CT features was selected. Using the 14 predictors and Naive Bayes, mid-treatment PET/CT set achieved 87.5% accuracy (2 PFS patients misclassified, all local recurrence and DM patients correctly classified). Post-treatment PET/CT set achieved 94.0% accuracy (all PFS and DM patients correctly predicted, 1 local recurrence patient misclassified) with logistic regression, neural network or support vector machine model. Conclusion: Applying radiomics approach to either midtreatment or post-treatment PET/CT could achieve high accuracy in predicting anal cancer treatment outcomes. This work was supported in part by the National Cancer Institute Grant R01CA172638.« less

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Weihong; Wu Jianqing; Zhong Li

2006-09-30

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency ofmore » conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.« less

A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

PubMed Central

Kobayashi, Kazuko; Sasaki, Takanori; Takenaka, Fumiaki; Yakushiji, Hiromasa; Fujii, Yoshihiro; Kishi, Yoshiro; Kita, Shoichi; Shen, Lianhua; Kumon, Hiromi; Matsuura, Eiji

2015-01-01

Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions. PMID:25883990

64Cu-DOTATATE PET/MRI for Detection of Activated Macrophages in Carotid Atherosclerotic Plaques: Studies in Patients Undergoing Endarterectomy.

PubMed

Pedersen, Sune Folke; Sandholt, Benjamin Vikjær; Keller, Sune Høgild; Hansen, Adam Espe; Clemmensen, Andreas Ettrup; Sillesen, Henrik; Højgaard, Liselotte; Ripa, Rasmus Sejersten; Kjær, Andreas

2015-07-01

A feature of vulnerable atherosclerotic plaques of the carotid artery is high activity and abundance of lesion macrophages. There is consensus that this is of importance for plaque vulnerability, which may lead to clinical events, such as stroke and transient ischemic attack. We used positron emission tomography (PET) and the novel PET ligand [(64)Cu] [1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid]-d-Phe1,Tyr3-octreotate ((64)Cu-DOTATATE) to specifically target macrophages via the somatostatin receptor subtype-2 in vivo. Ten patients underwent simultaneous PET/MRI to measure (64)Cu-DOTATATE uptake in carotid artery plaques before carotid endarterectomy. (64)Cu-DOTATATE uptake was significantly higher in symptomatic plaque versus the contralateral carotid artery (P<0.001). Subsequently, a total of 62 plaque segments were assessed for gene expression of selected markers of plaque vulnerability using real-time quantitative polymerase chain reaction. These results were compared with in vivo (64)Cu-DOTATATE uptake calculated as the mean standardized uptake value. Univariate analysis of real-time quantitative polymerase chain reaction and PET showed that cluster of differentiation 163 (CD163) and CD68 gene expression correlated significantly but weakly with mean standardized uptake value in scans performed 85 minutes post injection (P<0.001 and P=0.015, respectively). Subsequent multivariate analysis showed that CD163 correlated independently with (64)Cu-DOTATATE uptake (P=0.031) whereas CD68 did not contribute significantly to the final model. The novel PET tracer (64)Cu-DOTATATE accumulates in atherosclerotic plaques of the carotid artery. CD163 gene expression correlated independently with (64)Cu-DOTATATE uptake measured by real-time quantitative polymerase chain reaction in the final multivariate model, indicating that (64)Cu-DOTATATE PET is detecting alternatively activated macrophages. This association could potentially improve noninvasive identification and characterization of vulnerable plaques. © 2015 The Authors.

Vector-Borne Infections in Tornado-Displaced and Owner-Relinquished Dogs in Oklahoma, USA.

PubMed

Barrett, Anne W; Little, Susan E

2016-06-01

To determine the prevalence of infection with vector-borne agents in a cross-section of dogs from Oklahoma, where canine vector-borne diseases are common, blood samples were evaluated through serology and molecular analysis. Antibodies reactive to Ehrlichia spp., Rickettsia rickettsii, R. montanensis, and "R. amblyommii" were detected in 10.5% (11/105), 74.3% (78/105), 58.1% (61/105), and 55.2% (58/105) of dogs, respectively. Presence of spotted fever group Rickettsia spp. DNA was identified in 13.1% (8/61) of shelter dogs but not in any pet dogs (0/44). DNA of "R. amblyommii" was confirmed by sequencing, constituting the first report of this agent in a naturally infected dog. Antigen of Dirofilaria immitis was detected in 10.5% (11/105) and 16.2% (17/105) of samples before and after heat treatment, respectively. In total, 87.6% (92/105) of the dogs had evidence of infection with at least one vector-borne disease agent, confirming high risk of exposure to multiple vector-borne disease agents, several of which are zoonotic.

Lymphoma as a second malignancy in a patient with neuroendocrine tumor: mimicking dedifferentiation on dual-tracer PET/CT with 68Ga-DOTANOC and 18F-FDG.

PubMed

Jain, Sachin; Sharma, Punit; Dhull, Varun Singh; Bal, Chandrasekhar; Kumar, Rakesh

2014-04-01

Neuroendocrine tumors (NETs) are rare tumors which express somatostatin receptors (SSTRs). We here present a case of a 50-year-old female patient with metastatic bronchial carcinoid. She underwent 68Ga-DOTANOC PET/CT and 18F-FDG PET/CT which suggested a diagnosis of poorly differentiated NET. Biopsy of the lesion, however, revealed a second malignancy in the form of diffuse large B-cell lymphoma. Thus, very rarely, other primary tumors can mimic NETs on dual-tracer PET/CT, and biopsy is advised in doubtful cases.

Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.

PubMed

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-03-06

Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

PubMed Central

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-01-01

Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics. PMID:16519801

Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

PubMed

Iyer, Meera; Berenji, Manijeh; Templeton, Nancy S; Gambhir, Sanjiv S

2002-10-01

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

Lentivirus Vectors Incorporating the Immunoglobulin Heavy Chain Enhancer and Matrix Attachment Regions Provide Position-Independent Expression in B Lymphocytes

PubMed Central

Lutzko, Carolyn; Senadheera, Dinithi; Skelton, Dianne; Petersen, Denise; Kohn, Donald B.

2003-01-01

In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (Eμ) with and without associated matrix attachment regions (EμMAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34+ progenitors in vitro transduced with Eμ- and EμMAR-containing lentivectors. Lastly, we evaluated the expression from the EμMAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the Eμ and EμMAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the EμMAR-containing vector and not other cells types or vectors. Proviral genomes with the EμMAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the EμMAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies. PMID:12805432

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

PubMed

Arazi, T; Slutsky, S G; Shiboleth, Y M; Wang, Y; Rubinstein, M; Barak, S; Yang, J; Gal-On, A

2001-04-27

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli.

PubMed

Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

2016-08-01

Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans.

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

PubMed Central

Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

2016-01-01

Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization.

PubMed

Manzine, Livia Regina; Cassago, Alexandre; da Silva, Marco Túlio Alves; Thiemann, Otavio Henrique

2013-03-01

Selenocysteine Synthase (SELA, E.C. 2.9.1.1) from Escherichia coli is a homodecamer pyridoxal-5'-phosphate containing enzyme responsible for the conversion of seryl-tRNA(sec) into selenocysteyl-tRNA(sec) in the biosynthesis of the 21th amino acid, selenocysteine (Sec or U). This paper describes the cloning of the E. coli selA gene into a modified pET29a(+) vector and its expression in E. coli strain WL81460, a crucial modification allowing SELA expression without bound endogenous tRNA(sec). This expression strategy enabled the purification and additional biochemical and biophysical characterization of the SELA decamer. The homogeneous SELA protein was obtained using three chromatographic steps. Size Exclusion Chromatography and Native Gel Electrophoresis showed that SELA maintains a decameric state with molecular mass of approximately 500 kDa with an isoelectric point of 6,03. A predominance of α-helix structures was detected by circular dichroism with thermal stability up to 45 °C. The oligomeric assemblage of SELA was investigated by glutaraldehyde crosslinking experiments indicate that SELA homodecameric structure is the result of a stepwise addition of intermediate oligomeric states and not a direct monomer to homodecamer transition. Our results have contributed to the establishment of a robust expression model for the enzyme free of bound RNA and are of general interest to be taken into consideration in all cases of heterologous/homologous expressions of RNA-binding proteins avoiding the carryover of endogenous RNAs, which may interfere with further biochemical characterizations. Copyright © 2012 Elsevier Inc. All rights reserved.

Hybrid FDG-PET/MR compared to FDG-PET/CT in adult lymphoma patients.

PubMed

Atkinson, Wendy; Catana, Ciprian; Abramson, Jeremy S; Arabasz, Grae; McDermott, Shanaugh; Catalano, Onofrio; Muse, Victorine; Blake, Michael A; Barnes, Jeffrey; Shelly, Martin; Hochberg, Ephraim; Rosen, Bruce R; Guimaraes, Alexander R

2016-07-01

The goal of this study is to evaluate the diagnostic performance of simultaneous FDG-PET/MR including diffusion compared to FDG-PET/CT in patients with lymphoma. Eighteen patients with a confirmed diagnosis of non-Hodgkin's (NHL) or Hodgkin's lymphoma (HL) underwent an IRB-approved, single-injection/dual-imaging protocol consisting of a clinical FDG-PET/CT and subsequent FDG-PET/MR scan. PET images from both modalities were reconstructed iteratively. Attenuation correction was performed using low-dose CT data for PET/CT and Dixon-MR sequences for PET/MR. Diffusion-weighted imaging was performed. SUVmax was measured and compared between modalities and the apparent diffusion coefficient (ADC) using ROI analysis by an experienced radiologist using OsiriX. Strength of correlation between variables was measured using the Pearson correlation coefficient (r p). Of the 18 patients included in this study, 5 had HL and 13 had NHL. The median age was 51 ± 14.8 years. Sixty-five FDG-avid lesions were identified. All FDG-avid lesions were visible with comparable contrast, and therefore initial and follow-up staging was identical between both examinations. SUVmax from FDG-PET/MR [(mean ± sem) (21.3 ± 2.07)] vs. FDG-PET/CT (mean 23.2 ± 2.8) demonstrated a strongly positive correlation [r s = 0.95 (0.94, 0.99); p < 0.0001]. There was no correlation found between ADCmin and SUVmax from FDG-PET/MR [r = 0.17(-0.07, 0.66); p = 0.09]. FDG-PET/MR offers an equivalent whole-body staging examination as compared with PET/CT with an improved radiation safety profile in lymphoma patients. Correlation of ADC to SUVmax was weak, understating their lack of equivalence, but not undermining their potential synergy and differing importance.

Molecular and immunological characterization of subtilisin like serine protease, a major allergen of Curvularia lunata.

PubMed

Tripathi, Prabhanshu; Nair, Smitha; Singh, B P; Arora, Naveen

2011-03-01

Serine protease from numerous sources have been identified and characterized as major allergens. The present study aimed to clone, express and characterize a serine protease from Curvularia lunata. cDNA library screening identified partial protease clones. A clone showed significant homology to subtilisin like serine proteases from Aspergillus and Penicillium species. Full length sequence was generated by RACE PCR, subcloned in pET vector, protein expressed in Escherichia coli and purified from inclusion bodies yielding 0.5 mg/L of culture. Bioinformatic analysis identified serine protease motifs of subtilase family, catalytic triad and N-glycosylation sites on the primary sequence. The protein resolved at 54-kDa on SDS-PAGE and was recognized as a major allergen on immunoblot with 13/16 C. lunata sensitive patients' sera in ELISA and immunoblot. Recombinant protein reacted with rabbit polyclonal antibodies against alkaline serine proteases from C. lunata. Recombinant protein required 50-56 ng of same protein for 50% inhibition of IgE binding in competitive ELISA. In addition, 13 of 16 patients' samples showed significant basophil histamine release upon stimulation with purified recombinant protein. In conclusion, a 54 kDa major allergen of C. lunata was cloned, expressed, characterized and showed biological activity. It has potential to be used in molecule based approach for allergy diagnosis and therapy. Copyright © 2010 Elsevier GmbH. All rights reserved.

Molecular cloning, characterization and differential expression of novel phytocystatin gene during tropospheric ozone stress in maize (Zea mays) leaves.

PubMed

Ahmad, Rafiq; Zuily-Fodil, Yasmine; Passaquet, Chantal; Ali Khan, Sabaz; Repellin, Anne

2015-03-01

In this study, a full-length cDNA encoding a novel phytocystatin gene, designated CC14, was identified in maize leaves. The CC14 gene sequence reported in this study has been deposited in the GenBank database (accession number JF290478). The CC14 gene was cloned into an expression vector pET30 EK/LIC and was then transformed into Escherichia coli strain BL21 (DE3) pLysS to produce a recombinant CC14 protein. The recombinant protein was purified by nickel nitrilotriacetic acid affinity chromatography after induction with 1 mM IPTG. The purified CC14 protein was electrophoresed on SDS-PAGE and a protein 25 kDa in size was observed. Antiprotease activities of the purified recombinant CC14 protein against cysteine proteases and commercially available papain were tested. The results showed that CC14 purified protein suppressed 100% activity of papain and 57-86% plant cysteine protease activity. Moreover, an upregulation of CC14 gene expression was observed after 20 days of ozone stress in maize leaves. Together, these observations concurred to conclude that CC14 gene could potentially be used as a basis for the development of transgenic crops and natural pesticides that resist biotic and abiotic stresses. Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

PubMed Central

Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

2012-01-01

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

Functional characterization of recombinant bromelain of Ananas comosus expressed in a prokaryotic system.

PubMed

George, Susan; Bhasker, Salini; Madhav, Harish; Nair, Archana; Chinnamma, Mohankumar

2014-02-01

Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

PubMed

Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

2016-06-01

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases

PubMed Central

Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T. Michael; Sheibani, Nader; Gurel, Zafer; Boye, Sanford L.; Peterson, James J.; Boye, Shannon E.; Hauswirth, William W.; Chulay, Jeffrey D.

2016-01-01

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia. PMID:26603570

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases.

PubMed

Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T Michael; Sheibani, Nader; Gurel, Zafer; Boye, Sanford L; Peterson, James J; Boye, Shannon E; Hauswirth, William W; Chulay, Jeffrey D

2016-01-01

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.

Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5.

PubMed

Singh, Ram Sarup; Yadav, Mukesh

2012-07-01

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

18F-EF5 PET-based Imageable Hypoxia Predicts Local Recurrence in Tumors Treated With Highly Conformal Radiation Therapy.

PubMed

Qian, Yushen; Von Eyben, Rie; Liu, Yufei; Chin, Frederick T; Miao, Zheng; Apte, Sandeep; Carter, Justin N; Binkley, Michael S; Pollom, Erqi L; Harris, Jeremy P; Prionas, Nicolas D; Kissel, Madelyn; Simmons, Amanda; Diehn, Maximilian; Shultz, David B; Brown, J Martin; Maxim, Peter G; Koong, Albert C; Graves, Edward E; Loo, Billy W

2018-04-18

Tumor hypoxia contributes to radiation resistance. A noninvasive assessment of tumor hypoxia would be valuable for prognostication and possibly selection for hypoxia-targeted therapies. 18 F-pentafluorinated etanidazole ( 18 F-EF5) is a nitroimidazole derivative that has demonstrated promise as a positron emission tomography (PET) hypoxia imaging agent in preclinical and clinical studies. However, correlation of imageable hypoxia by 18 F-EF5 PET with clinical outcomes after radiation therapy remains limited. Our study prospectively enrolled 28 patients undergoing radiation therapy for localized lung or other tumors to receive pretreatment 18 F-EF5 PET imaging. Depending on the level of 18 F-EF5 tumor uptake, patients underwent functional manipulation of tumor oxygenation with either carbogen breathing or oral dichloroacetate followed by repeated 18 F-EF5 PET. The hypoxic subvolume of tumor was defined as the proportion of tumor voxels exhibiting higher 18 F-EF5 uptake than the 95th percentile of 18 F-EF5 uptake in the blood pool. Tumors with a hypoxic subvolume ≥ 10% on baseline 18 F-EF5 PET imaging were classified as hypoxic by imaging. A Cox model was used to assess the correlation between imageable hypoxia and clinical outcomes after treatment. At baseline, imageable hypoxia was demonstrated in 43% of all patients (12 of 28), including 6 of 16 patients with early-stage non-small cell lung cancer treated with stereotactic ablative radiation therapy and 6 of 12 patients with other cancers. Carbogen breathing was significantly associated with decreased imageable hypoxia, while dichloroacetate did not result in a significant change under our protocol conditions. Tumors with imageable hypoxia had a higher incidence of local recurrence at 12 months (30%) than those without (0%) (P < .01). Noninvasive hypoxia imaging by 18 F-EF5 PET identified imageable hypoxia in about 40% of tumors in our study population. Local tumor recurrence after highly conformal radiation therapy was higher in tumors with imageable hypoxia. Copyright © 2018 Elsevier Inc. All rights reserved.

The use of longitudinal 18F-FET MicroPET imaging to evaluate response to irinotecan in orthotopic human glioblastoma multiforme xenografts.

PubMed

Nedergaard, Mette K; Kristoffersen, Karina; Michaelsen, Signe R; Madsen, Jacob; Poulsen, Hans S; Stockhausen, Marie-Thérése; Lassen, Ulrik; Kjaer, Andreas

2014-01-01

Brain tumor imaging is challenging. Although 18F-FET PET is widely used in the clinic, the value of 18F-FET MicroPET to evaluate brain tumors in xenograft has not been assessed to date. The aim of this study therefore was to evaluate the performance of in vivo 18F-FET MicroPET in detecting a treatment response in xenografts. In addition, the correlations between the 18F-FET tumor accumulation and the gene expression of Ki67 and the amino acid transporters LAT1 and LAT2 were investigated. Furthermore, Ki67, LAT1 and LAT2 gene expression in xenograft and archival patient tumors was compared. Human GBM cells were injected orthotopically in nude mice and 18F-FET uptake was followed by weekly MicroPET/CT. When tumor take was observed, mice were treated with CPT-11 or saline weekly. After two weeks of treatment the brain tumors were isolated and quantitative polymerase chain reaction were performed on the xenograft tumors and in parallel on archival patient tumor specimens. The relative tumor-to-brain (T/B) ratio of SUV max was significantly lower after one week (123 ± 6%, n = 7 vs. 147 ± 6%, n = 7; p = 0.018) and after two weeks (142 ± 8%, n = 5 vs. 204 ± 27%, n = 4; p = 0.047) in the CPT-11 group compared with the control group. Strong negative correlations between SUV max T/B ratio and LAT1 (r = -0.62, p = 0.04) and LAT2 (r = -0.67, p = 0.02) were observed. In addition, a strong positive correlation between LAT1 and Ki67 was detected in xenografts. Furthermore, a 1.6 fold higher expression of LAT1 and a 23 fold higher expression of LAT2 were observed in patient specimens compared to xenografts. 18F-FET MicroPET can be used to detect a treatment response to CPT-11 in GBM xenografts. The strong negative correlation between SUV max T/B ratio and LAT1/LAT2 indicates an export transport function. We suggest that 18F-FET PET may be used for detection of early treatment response in patients.

Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones.

PubMed

Kaplan, Ondřej; Zárubová, Jana; Mikulová, Barbora; Filová, Elena; Bártová, Jiřina; Bačáková, Lucie; Brynda, Eduard

2016-01-01

We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.

Adenoviral vector tethering to metal surfaces via hydrolysable cross-linkers for the modulation of vector release and transduction

PubMed Central

Fishbein, Ilia; Forbes, Scott P.; Chorny, Michael; Connolly, Jeanne M.; Adamo, Richard F.; Corrales, Ricardo; Alferiev, Ivan S.; Levy, Robert J.

2013-01-01

The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolysable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37°C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolysable cross-linkers with structure-specific release kinetics. PMID:23777912

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna.

PubMed

Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

2016-02-01

To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna *

PubMed Central

Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

2016-01-01

To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna. PMID:26834012

Role of Combined 68Ga-DOTATOC and 18F-FDG Positron Emission Tomography/Computed Tomography in the Diagnostic Workup of Pancreas Neuroendocrine Tumors: Implications for Managing Surgical Decisions.

PubMed

Cingarlini, Sara; Ortolani, Silvia; Salgarello, Matteo; Butturini, Giovanni; Malpaga, Anna; Malfatti, Veronica; DʼOnofrio, Mirko; Davì, Maria Vittoria; Vallerio, Paola; Ruzzenente, Andrea; Capelli, Paola; Citton, Elia; Grego, Elisabetta; Trentin, Chiara; De Robertis, Riccardo; Scarpa, Aldo; Bassi, Claudio; Tortora, Giampaolo

2017-01-01

Ga-DOTATOC (Ga) positron emission tomography (PET)/computed tomography (CT) is recommended in the workup of pancreas neuroendocrine tumors (PanNETs); evidence suggests that F-FDG (F) PET/CT can also provide prognostic information. Aims of this study were to assess the role of combined Ga- and F-PET/CT in the evaluation of grade (G) 1-2 PanNETs and to test the correlation between F-PET/CT positivity and tumor grade. Preoperative Ga- and F-PET/CT of 35 patients with surgically resected G1-2 PanNETs were evaluated. For grading, the 2010 World Health Organization Classification was used; an ancillary analysis with Ki67 cutoffs at 5% to 20% was conducted. Correlation between F-PET/CT positivity (SUVmax > 3.5) and grade was assessed. Of 35 PanNETs, 28.6% and 71.4% were G1 and G2 as per World Health Organization. Ga-PET/CT showed high sensitivity (94.3%) in detecting G1-2 PanNETs. F-PET/CT was positive in 20% and 76% G1 and G2 tumors (P = 0.002). F-PET/CT identified G2 PanNETs with high positive predictive value (PPV, 90.5%). F-PET/CT correlated with tumor grade also in the ancillary analysis (P = 0.009). The high sensitivity of Ga-PET/CT in NET detection is known. The high PPV of F-PET/CT in the identification of G2 forms suggests its potential role in PanNETs prognostication and risk stratification.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

PubMed Central

2011-01-01

Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection. PMID:21679423

Evaluation of 89Zr-rituximab tracer by Cerenkov luminescence imaging and correlation with PET in a humanized transgenic mouse model to image NHL.

PubMed

Natarajan, Arutselvan; Habte, Frezghi; Liu, Hongguang; Sathirachinda, Ataya; Hu, Xiang; Cheng, Zhen; Nagamine, Claude M; Gambhir, Sanjiv Sam

2013-08-01

This research aimed to study the use of Cerenkov luminescence imaging (CLI) for non-Hodgkin's lymphoma (NHL) using 89Zr-rituximab positron emission tomography (PET) tracer with a humanized transgenic mouse model that expresses human CD20 and the correlation of CLI with PET. Zr-rituximab (2.6 MBq) was tail vein-injected into transgenic mice that express the human CD20 on their B cells (huCD20TM). One group (n=3) received 2 mg/kg pre-dose (blocking) of cold rituximab 2 h prior to tracer; a second group (n=3) had no pre-dose (non-blocking). CLI was performed using a cooled charge-coupled device optical imager. We also performed PET imaging and ex vivo studies in order to confirm the in vivo CLI results. At each time point (4, 24, 48, 72, and 96 h), two groups of mice were imaged in vivo and ex vivo with CLI and PET, and at 96 h, organs were measured by gamma counter. huCD20 transgenic mice injected with 89Zr-rituximab demonstrated a high-contrast CLI image compared to mice blocked with a cold dose. At various time points of 4-96 h post-radiotracer injection, the in vivo CLI signal intensity showed specific uptake in the spleen where B cells reside and, hence, the huCD20 biomarker is present at very high levels. The time-activity curve of dose decay-corrected CLI intensity and percent injected dose per gram of tissue of PET uptake in the spleen were increased over the time period (4-96 h). At 96 h, the 89Zr-rituximab uptake ratio (non-blocking vs blocking) counted (mean±standard deviation) for the spleen was 1.5±0.6 for CLI and 1.9±0.3 for PET. Furthermore, spleen uptake measurements (non-blocking and blocking of all time points) of CLI vs PET showed good correlation (R2=0.85 and slope=0.576), which also confirmed the corresponding correlations parameter value (R2=0.834 and slope=0.47) obtained for ex vivo measurements. CLI and PET of huCD20 transgenic mice injected with 89Zr-rituximab demonstrated that the tracer was able to target huCD20-expressing B cells. The in vivo and ex vivo tracer uptake corresponding to the CLI radiance intensity from the spleen is in good agreement with PET. In this report, we have validated the use of CLI with PET for NHL imaging in huCD20TM.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

PubMed

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors. PMID:29091919

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

PubMed

El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

2000-02-15

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

68Ga-HBED-CC-PSMA PET/CT Versus Histopathology in Primary Localized Prostate Cancer: A Voxel-Wise Comparison

PubMed Central

Zamboglou, Constantinos; Schiller, Florian; Fechter, Tobias; Wieser, Gesche; Jilg, Cordula Annette; Chirindel, Alin; Salman, Nasr; Drendel, Vanessa; Werner, Martin; Mix, Michael; Meyer, Philipp Tobias; Grosu, Anca Ligia

2016-01-01

Purpose: We performed a voxel-wise comparison of 68Ga-HBED-CC-PSMA PET/CT with prostate histopathology to evaluate the performance of 68Ga-HBED-CC-PSMA for the detection and delineation of primary prostate cancer (PCa). Methodology: Nine patients with histopathological proven primary PCa underwent 68Ga-HBED-CC-PSMA PET/CT followed by radical prostatectomy. Resected prostates were scanned by ex-vivo CT in a special localizer and histopathologically prepared. Histopathological information was matched to ex-vivo CT. PCa volume (PCa-histo) and non-PCa tissue in the prostate (NPCa-histo) were processed to obtain a PCa-model, which was adjusted to PET-resolution (histo-PET). Each histo-PET was coregistered to in-vivo PSMA-PET/CT data. Results: Analysis of spatial overlap between histo-PET and PSMA PET revealed highly significant correlations (p < 10-5) in nine patients and moderate to high coefficients of determination (R²) from 42 to 82 % with an average of 60 ± 14 % in eight patients (in one patient R2 = 7 %). Mean SUVmean in PCa-histo and NPCa-histo was 5.6 ± 6.1 and 3.3 ± 2.5 (p = 0.012). Voxel-wise receiver-operating characteristic (ROC) analyses comparing the prediction by PSMA-PET with the non-smoothed tumor distribution from histopathology yielded an average area under the curve of 0.83 ± 0.12. Absolute and relative SUV (normalized to SUVmax) thresholds for achieving at least 90 % sensitivity were 3.19 ± 3.35 and 0.28 ± 0.09, respectively. Conclusions: Voxel-wise analyses revealed good correlations of 68Ga-HBED-CC-PSMA PET/CT and histopathology in eight out of nine patients. Thus, PSMA-PET allows a reliable detection and delineation of PCa as basis for PET-guided focal therapies. PMID:27446496

SU-C-9A-06: The Impact of CT Image Used for Attenuation Correction in 4D-PET

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Y; Bowsher, J; Yan, S

2014-06-01

Purpose: To evaluate the appropriateness of using 3D non-gated CT image for attenuation correction (AC) in a 4D-PET (gated PET) imaging protocol used in radiotherapy treatment planning simulation. Methods: The 4D-PET imaging protocol in a Siemens PET/CT simulator (Biograph mCT, Siemens Medical Solutions, Hoffman Estates, IL) was evaluated. CIRS Dynamic Thorax Phantom (CIRS Inc., Norfolk, VA) with a moving glass sphere (8 mL) in the middle of its thorax portion was used in the experiments. The glass was filled with {sup 18}F-FDG and was in a longitudinal motion derived from a real patient breathing pattern. Varian RPM system (Varian Medicalmore » Systems, Palo Alto, CA) was used for respiratory gating. Both phase-gating and amplitude-gating methods were tested. The clinical imaging protocol was modified to use three different CT images for AC in 4D-PET reconstruction: first is to use a single-phase CT image to mimic actual clinical protocol (single-CT-PET); second is to use the average intensity projection CT (AveIP-CT) derived from 4D-CT scanning (AveIP-CT-PET); third is to use 4D-CT image to do the phase-matched AC (phase-matching- PET). Maximum SUV (SUVmax) and volume of the moving target (glass sphere) with threshold of 40% SUVmax were calculated for comparison between 4D-PET images derived with different AC methods. Results: The SUVmax varied 7.3%±6.9% over the breathing cycle in single-CT-PET, compared to 2.5%±2.8% in AveIP-CT-PET and 1.3%±1.2% in phasematching PET. The SUVmax in single-CT-PET differed by up to 15% from those in phase-matching-PET. The target volumes measured from single- CT-PET images also presented variations up to 10% among different phases of 4D PET in both phase-gating and amplitude-gating experiments. Conclusion: Attenuation correction using non-gated CT in 4D-PET imaging is not optimal process for quantitative analysis. Clinical 4D-PET imaging protocols should consider phase-matched 4D-CT image if available to achieve better accuracy.« less

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

PubMed

Casales, Erkuden; Aranda, Alejandro; Quetglas, Jose I; Ruiz-Guillen, Marta; Rodriguez-Madoz, Juan R; Prieto, Jesus; Smerdou, Cristian

2010-05-31

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells. 2010 Elsevier B.V. All rights reserved.

TU-AB-BRA-04: Quantitative Radiomics: Sensitivity of PET Textural Features to Image Acquisition and Reconstruction Parameters Implies the Need for Standards

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nyflot, MJ; Yang, F; Byrd, D

Purpose: Despite increased use of heterogeneity metrics for PET imaging, standards for metrics such as textural features have yet to be developed. We evaluated the quantitative variability caused by image acquisition and reconstruction parameters on PET textural features. Methods: PET images of the NEMA IQ phantom were simulated with realistic image acquisition noise. 35 features based on intensity histograms (IH), co-occurrence matrices (COM), neighborhood-difference matrices (NDM), and zone-size matrices (ZSM) were evaluated within lesions (13, 17, 22, 28, 33 mm diameter). Variability in metrics across 50 independent images was evaluated as percent difference from mean for three phantom girths (850,more » 1030, 1200 mm) and two OSEM reconstructions (2 iterations, 28 subsets, 5 mm FWHM filtration vs 6 iterations, 28 subsets, 8.6 mm FWHM filtration). Also, patient sample size to detect a clinical effect of 30% with Bonferroni-corrected α=0.001 and 95% power was estimated. Results: As a class, NDM features demonstrated greatest sensitivity in means (5–50% difference for medium girth and reconstruction comparisons and 10–100% for large girth comparisons). Some IH features (standard deviation, energy, entropy) had variability below 10% for all sensitivity studies, while others (kurtosis, skewness) had variability above 30%. COM and ZSM features had complex sensitivities; correlation, energy, entropy (COM) and zone percentage, short-zone emphasis, zone-size non-uniformity (ZSM) had variability less than 5% while other metrics had differences up to 30%. Trends were similar for sample size estimation; for example, coarseness, contrast, and strength required 12, 38, and 52 patients to detect a 30% effect for the small girth case but 38, 88, and 128 patients in the large girth case. Conclusion: The sensitivity of PET textural features to image acquisition and reconstruction parameters is large and feature-dependent. Standards are needed to ensure that prospective trials which incorporate textural features are properly designed to detect clinical endpoints. Supported by NIH grants R01 CA169072, U01 CA148131, NCI Contract (SAIC-Frederick) 24XS036-004, and a research contract from GE Healthcare.« less

Monitoring contact sensitization to p-phenylenediamine (PPD) by patch testing with PPD 0.3% in petrolatum.

PubMed

Geier, Johannes; Ballmer-Weber, Barbara K; Dickel, Heinrich; Frosch, Peter J; Bircher, Andreas; Weisshaar, Elke; Hillen, Uwe

2013-07-01

Being a contact allergen of general relevance, p-phenylenediamine (PPD) is patch tested in the baseline series. However, PPD 1% in petrolatum may actively sensitize. Patch testing with PPD at 0.35% pet. proved to be safe, as far as active sensitization is concerned. To determine whether PPD 0.3% pet. reliably detects PPD sensitization. Patch testing with PPD 0.3% pet. and 1% pet. synchronously was performed in consecutive patients in a multicentre study within the Information Network of Departments of Dermatology. Altogether, 2042 patients were patch tested. PPD 1% pet. yielded 6.0% positive reactions (n = 123), and PPD 0.3% pet. yielded 4.7% (n = 95). The synchronous reproducibility of PPD reactions was similar as known from parallel patch tests with identical PPD concentrations. The diagnostic properties of PPD 0.3% pet. expressed as reaction index and positivity ratio were good. Of the 123 patients reacting to PPD 1% pet., 32 (26%) had no positive reaction to PPD 0.3% pet. In 22 of these 32 patients (69%), no clinical relevance could be found. As patch testing with PPD 0.3% pet. is reliable according to our results, we recommend replacing PPD 1% pet. in the baseline series with PPD 0.3% pet. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of N-[(11)C]methyl-AMD3465 as a PET tracer for imaging of CXCR4 receptor expression in a C6 glioma tumor model.

PubMed

Hartimath, S V; van Waarde, A; Dierckx, R A J O; de Vries, E F J

2014-11-03

The chemokine receptor CXCR4 and its ligand CXCL12 play an important role in tumor progression and metastasis. CXCR4 receptors are expressed by many cancer types and provide a potential target for treatment. Noninvasive detection of CXCR4 may aid diagnosis and improve therapy selection. It has been demonstrated in preclinical studies that positron emission tomography (PET) with a radiolabeled small molecule could enable noninvasive monitoring of CXCR4 expression. Here, we prepared N-[(11)C]methyl-AMD3465 as a new PET tracer for CXCR4. N-[(11)C]Methyl-AMD3465 was readily prepared by N-methylation with [(11)C]CH3OTf. The tracer was obtained in a 60 ± 2% yield (decay corrected), the purity of the tracer was >99%, and specific activity was 47 ± 14 GBq/μmol. Tracer stability was tested in vitro using liver microsomes and rat plasma; excellent stability was observed. The tracer was evaluated in rat C6 glioma and human PC-3 cell lines. In vitro cellular uptake of N-[(11)C]methyl-AMD3465 was receptor mediated. The effect of transition metal ions (Cu(2+), Ni(2+), and Zn(2+)) on cellular binding was examined in C6 cells, and the presence of these ions increased the cellular binding of the tracer 9-, 7-, and 3-fold, respectively. Ex vivo biodistribution and PET imaging of N-[(11)C]methyl-AMD3465 were performed in rats with C6 tumor xenografts. Both PET and biodistribution studies demonstrated specific accumulation of the tracer in the tumor (SUV 0.6 ± 0.2) and other CXCR4 expressing organs, such as lymph node (1.5 ± 0.2), liver (8.9 ± 1.0), bone marrow (1.0 ± 0.3), and spleen (1.0 ± 0.1). Tumor uptake was significantly reduced (66%, p < 0.01) after pretreatment with Plerixafor (AMD3100). Biodistribution data indicates a tumor-to-muscle ratio of 7.85 and tumor-to-plasma ratio of 1.14, at 60 min after tracer injection. Our data demonstrated that N-[(11)C]methyl-AMD3465 is capable of detecting physiologic CXCR4 expression in tumors and other CXCR4 expressing tissues. These results warrant further evaluation of N-[(11)C]methyl-AMD3465 as a potential PET tracer for CXCR4 receptor imaging.

Alpharetroviral Self-inactivating Vectors: Long-term Transgene Expression in Murine Hematopoietic Cells and Low Genotoxicity

PubMed Central

Suerth, Julia D; Maetzig, Tobias; Brugman, Martijn H; Heinz, Niels; Appelt, Jens-Uwe; Kaufmann, Kerstin B; Schmidt, Manfred; Grez, Manuel; Modlich, Ute; Baum, Christopher; Schambach, Axel

2012-01-01

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively “extragenic” alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs. PMID:22334016

Covariance PET patterns in early Alzheimer's disease and subjects with cognitive impairment but no dementia: utility in group discrimination and correlations with functional performance

PubMed Central

Scarmeas, Nikolaos; Habeck, Christian G.; Zarahn, Eric; Anderson, Karen E.; Park, Aileen; Hilton, John; Pelton, Gregory H.; Tabert, Matthias H.; Honig, Lawrence S.; Moeller, James R.; Devanand, Davangere P.; Stern, Yaakov

2011-01-01

Although multivariate analytic techniques might identify diagnostic patterns that are not captured by univariate methods, they have rarely been used to study the neural correlates of Alzheimer's disease (AD) or cognitive impairment. Nonquantitative H215O PET scans were acquired during rest in 17 probable AD subjects selected for mild severity [mean-modified Mini Mental Status Examination (mMMS) 46/57; SD 5.1], 16 control subjects (mMMS 54; SD 2.5) and 23 subjects with minimal to mild cognitive impairment but no dementia (mMMS 53; SD 2.8). Expert clinical reading had low success in discriminating AD and controls. There were no significant mean flow differences among groups in traditional univariate SPM Voxel-wise analyses or region of interest (ROI) analyses. A covariance pattern was identified whose mean expression was significantly higher in the AD as compared to controls (P = 0.03; sensitivity 76–94%; specificity 63–81%). Sites of increased concomitant flow included insula, cuneus, pulvinar, lingual, fusiform, superior occipital and parahippocampal gyri, whereas decreased concomitant flow was found in cingulate, inferior parietal lobule, middle and inferior frontal, supramarginal and precentral gyri. The covariance analysis-derived pattern was then prospectively applied to the cognitively impaired subjects: as compared to subjects with Clinical Dementia Rating (CDR) = 0, subjects with CDR = 0.5 had significantly higher mean covariance pattern expression (P = 0.009). Expression of this pattern correlated inversely with Selective Reminding Test total recall (r = −0.401, P = 0.002), delayed recall (r = −0.351, P = 0.008) and mMMS scores (r = −0.401, P = 0.002) in all three groups combined. We conclude that patients with AD may differentially express resting cerebral blood flow covariance patterns even at very early disease stages. Significant alterations in expression of resting flow covariance patterns occur even for subjects with cognitive impairment. Expression of covariance patterns correlates with cognitive and functional performance measures, holding promise for meaningful associations with underlying biopathological processes. PMID:15325350

Quantitative characterisation of clinically significant intra-prostatic cancer by prostate-specific membrane antigen (PSMA) expression and cell density on PSMA-11.

PubMed

Domachevsky, Liran; Goldberg, Natalia; Bernstine, Hanna; Nidam, Meital; Groshar, David

2018-05-30

To quantitatively characterize clinically significant intra-prostatic cancer (IPC) by prostate-specific membrane antigen (PSMA) expression and cell density on PSMA-11 positron emission tomography/magnetic resonance (PET/MR). Retrospective study approved by the institutional review board with informed written consent obtained. Patients with a solitary, biopsy-proven prostate cancer, Gleason score (GS) ≥7, presenting for initial evaluation by PET/computerised tomography (PET/CT), underwent early prostate PET/MR immediately after PSMA-11 tracer injection. PET/MR [MRI-based attenuation correction (MRAC)] and PET/CT [CT-based AC (CTAC)] maximal standardised uptake value (SUVmax) and minimal and mean apparent diffusion coefficient (ADCmin, ADCmean; respectively) in normal prostatic tissue (NPT) were compared to IPC area. The relationship between SUVmax, ADCmin and ADCmean measurements was obtained. Twenty-two patients (mean age 69.5±5.0 years) were included in the analysis. Forty-four prostate areas were evaluated (22 IPC and 22 NPT). Median MRAC SUVmax of NPT was significantly lower than median MRAC SUVmax of IPC (p < 0.0001). Median ADCmin and ADCmean of NPT was significantly higher than median ADCmin and ADCmean of IPC (p < 0.0001). A very good correlation was found between MRAC SUVmax with CTAC SUVmax (rho = -0.843, p < 0.0001). A good inverse relationship was found between MRAC SUVmax and CTAC SUVmax with ADCmin (rho = -0.717, p < 0.0001 and -0.740, p < 0.0001; respectively; Z = 0.22, p = 0.82, NS) and with MRAC SUVmax and ADCmean (rho = -0.737, p < 0.0001). PET/MR SUVmax, ADCmin and ADCmean are distinct biomarkers able to differentiate between IPC and NPT in naïve prostate cancer patients with GS ≥ 7. • PSMA PET/MR metrics differentiate between normal and tumoural prostatic tissue. • A multi-parametric approach combining molecular and anatomical information might direct prostate biopsy. • PSMA PET/MR metrics are warranted for radiomics analysis.

TU-F-CAMPUS-J-04: Impact of Voxel Anisotropy On Statistic Texture Features of Oncologic PET: A Simulation Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, F; Byrd, D; Bowen, S

2015-06-15

Purpose: Texture metrics extracted from oncologic PET have been investigated with respect to their usefulness as definitive indicants for prognosis in a variety of cancer. Metric calculation is often based on cubic voxels. Most commonly used PET scanners, however, produce rectangular voxels, which may change texture metrics. The objective of this study was to examine the variability of PET texture feature metrics resulting from voxel anisotropy. Methods: Sinograms of NEMA NU-2 phantom for 18F-FDG were simulated using the ASIM simulation tool. The obtained projection data was reconstructed (3D-OSEM) on grids of cubic and rectangular voxels, producing PET images of resolutionmore » of 2.73x2.73x3.27mm3 and 3.27x3.27x3.27mm3, respectively. An interpolated dataset obtained from resampling the rectangular voxel data for isotropic voxel dimension (3.27mm) was also considered. For each image dataset, 28 texture parameters based on grey-level co-occurrence matrices (GLCOM), intensity histograms (GLIH), neighborhood difference matrices (GLNDM), and zone size matrices (GLZSM) were evaluated within lesions of diameter of 33, 28, 22, and 17mm. Results: In reference to the isotopic image data, texture features appearing on the rectangular voxel data varied with a range of -34-10% for GLCOM based, -31-39% for GLIH based, -80 -161% for GLNDM based, and −6–45% for GLZSM based while varied with a range of -35-23% for GLCOM based, -27-35% for GLIH based, -65-86% for GLNDM based, and -22 -18% for GLZSM based for the interpolated image data. For the anisotropic data, GLNDM-cplx exhibited the largest extent of variation (161%) while GLZSM-zp showed the least (<1%). As to the interpolated data, GLNDM-busy varied the most (86%) while GLIH-engy varied the least (<1%). Conclusion: Variability of texture appearance on oncologic PET with respect to voxel representation is substantial and feature-dependent. It necessitates consideration of standardized voxel representation for inter-institution studies attempting to validate prognostic values of PET texture features in cancer treatment.« less

“Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

PubMed Central

Croyle, Maria A.; Chirmule, Narendra; Zhang, Yi; Wilson, James M.

2001-01-01

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy. PMID:11312351

Gallium-68 DOTATOC PET/CT In Vivo Characterization of Somatostatin Receptor Expression in the Prostate

PubMed Central

Gajić, Milan M.; Obradović, Vladimir B.; Baum, Richard P.

2014-01-01

Abstract Aim: The aim was to investigate somatostatin receptor (sstr) expression in normal prostate by determining the maximum standardized uptake value (SUVmax) of 68Ga-DOTATOC PET/CT in neuroendocrine tumor (NET) patients, without NET involvement of the prostate gland, for establishing the reference standard. Methods: Sixty-four NET patients underwent 68Ga-DOTATOC PET/CT. SUVmax of the prostate gland, normal liver, testes, and gluteus muscles were evaluated. The prostate gland size was measured. Statistical analysis was performed using dedicated software (SPSS13). Results: Mean/median 68Ga-DOTATOC SUVmax values were as follows: normal prostate 2.6±0.0, slightly enlarged prostate 4.2±1.6, prostatic hypertrophy 4.9±1.6, prostatic hyperplasia 5.0±1.5, prostate cancer 9.5±2.1, normal liver 7.3±1.8, testes 1.8±0.5, and gluteus 1.0±0.2. The normal prostate gland had three times less sstr expression than normal liver tissue. Strong correlation was found between patient age and sstr expression in prostate/prostate size. No significant difference existed in sstr expression between prostatic hypertrophy and hyperplasia. Much higher sstr expression was found in prostatic cancer compared with normal prostate. Conclusion: 68Ga-DOTATOC PET/CT defines the baseline sstr uptake in prostate not affected by NET (significantly lower than in the liver). Higher values were established in prostatic hyperplasia and hypertrophy. Only concomitant prostate cancer was associated with higher SUVmax in comparison with non-neoplastic liver. PMID:24450327

An osteogenesis/angiogenesis-stimulation artificial ligament for anterior cruciate ligament reconstruction.

PubMed

Li, Hong; Li, Jinyan; Jiang, Jia; Lv, Fang; Chang, Jiang; Chen, Shiyi; Wu, Chengtie

2017-05-01

To solve the poor healing of polyethylene terephthalate (PET) artificial ligament in bone tunnel, copper-containing bioactive glass (Cu-BG) nanocoatings on PET artificial ligaments were successfully prepared by pulsed laser deposition (PLD). It was hypothesized that Cu-BG coated PET (Cu-BG/PET) grafts could enhance the in vitro osteogenic and angiogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and in vivo graft-bone healing after anterior cruciate ligament (ACL) reconstruction in a goat model. Scanning electron microscope and EDS mapping analysis revealed that the prepared nanocoatings had uniform element distribution (Cu, Ca, Si and P) and nanostructure. The surface hydrophilicity of PET grafts was significantly improved after depositing Cu-BG nanocoatings. The in vitro study displayed that the Cu-BG/PET grafts supported the attachment and proliferation of rBMSCs, and significantly promoted the expression of HIF-1α gene, which up-regulated the osteogenesis-related genes (S100A10, BMP2, OCN) and angiogenesis-related genes (VEGF) in comparison with PET or BG coated PET (BG/PET) grafts which do not contain Cu element. Meanwhile, Cu-BG/PET grafts promoted the bone regeneration at the graft-host bone interface and decreased graft-bone interface width, thus enhancing the bonding strength as well as angiogenesis (as indicated by CD31 expression) in the goat model as compared with BG/PET and pure PET grafts. The study demonstrates that the Cu-containing biomaterials significantly promote osteogenesis and angiogenesis in the repair of bone defects of large animals and thus offering a promising method for ACL reconstruction by using Cu-containing nanobioglass modified PET grafts. It remains a significant challenge to develop an artificial graft with distinct osteogenetic/angiogenetic activity to enhance graft-bone healing for ligament reconstruction. To solve these problems, copper-containing bioactive glass (Cu-BG) nanocoatings on PET artificial ligaments were successfully prepared by pulsed laser deposition (PLD). It was found that the prepared Cu-BG/PET grafts significantly stimulated the proliferation and osteogenic/angiogenic differentiation of bone marrow stromal cells (BMSCs) through activating HIF-1α/S100A10/Ca 2+ signal pathway. The most important is that the in vivo bone-forming ability of Cu-containing biomaterials was, for the first time, elucidated in a large animal model, revealing the enhanced capacity of osteogenesis and angiogenesis with incorporation of bioactive Cu element. It is suggested that the copper-containing biomaterials significantly promote osteogenesis and angiogenesis in large animal defects and thus offering a promising method for ACL reconstruction by using Cu-containing nanobioglass modification of PET grafts, paving the way to apply Cu-containing biomaterials for tissue engineering and regenerative medicine. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Optical Imaging of Mammaglobin Expression of Breast Cancer

DTIC Science & Technology

2003-05-01

monoclonal anti-MMG antibodies with a near infrared fluorescent probe for optical imaging and 64Cu -DOTA for positron emission tomography (mPET...Preliminary results indicate that the mPET imaging with 64Cu -DOTA-anti MMG monoclonal antibodies showed predominant liver uptake in mice. In contrast, the

Peninsula Humane Society Teacher's Packet. Secondary Level.

ERIC Educational Resources Information Center

Peninsula Humane Society, San Mateo, CA.

Activities in this teacher's packet are designed to familiarize secondary school students with the responsibilities involved in pet ownership. Teaching plans are provided for a total of 12 lessons grouped under social studies, language arts, math, and health sciences. Activities focus on pet overpopulation, expressions of social responses in…

Clinical Trials Using Anti-CD19/CD28/CD3zeta CAR Gammaretroviral Vector-transduced Autologous T Lymphocytes KTE-C19

Cancer.gov

NCI supports clinical trials that test new and more effective ways to treat cancer. Find clinical trials studying anti-cd19/cd28/cd3zeta car gammaretroviral vector-transduced autologous t lymphocytes kte-c19.

A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products.

PubMed

Haut, Larissa H; Gill, Amanda L; Kurupati, Raj K; Bian, Ang; Li, Yan; Giles-Davis, Wynetta; Xiang, Zhiquan; Zhou, Xiang Yang; Ertl, Hildegund C J

2016-10-01

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.

Synthesis and preclinical evaluation of [(11)C]PAQ as a PET imaging tracer for VEGFR-2.

PubMed

Samén, Erik; Thorell, Jan-Olov; Lu, Li; Tegnebratt, Tetyana; Holmgren, Lars; Stone-Elander, Sharon

2009-08-01

(R,S)-N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-((1-methyl-3-piperidinyl)methoxy)-4-quinazolinamine (PAQ) is a tyrosine kinase inhibitor with high affinity for the vascular endothelial growth factor receptor 2 (VEGFR-2), which plays an important role in tumour angiogenesis. The aim of this work was to develop and evaluate in mice the (11)C-labelled analogue as an in vivo tracer for VEGFR-2 expression in solid tumours. [(11)C]PAQ was synthesized by an N-methylation of desmethyl-PAQ using [(11)C]methyl iodide. The tracer's pharmacokinetic properties and its distribution in both subcutaneous and intraperitoneal tumour models were evaluated with positron emission tomography (PET). [(18)F]FDG was used as a reference tracer for tumour growth. PET results were corroborated by ex vivo and in vitro phosphor imaging and immunohistochemical analyses. In vitro assays and PET in healthy animals revealed low tracer metabolism, limited excretion over 60 min and a saturable and irreversible binding. Radiotracer uptake in subcutaneous tumour masses was low, while focal areas of high uptake (up to 8% ID/g) were observed in regions connecting the tumour to the host. Uptake was similarly high but more distributed in tumours growing within the peritoneum. The pattern of radiotracer uptake was generally different from that of the metabolic tracer [(18)F]FDG and correlated well with variations in VEGFR-2 expression determined ex vivo by immunohistochemical analysis. These results suggest that [(11)C]PAQ has potential as a noninvasive PET tracer for in vivo imaging of VEGFR-2 expression in angiogenic "hot spots".

Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli.

PubMed

Mirzaei, Maryam; Saffar, Behnaz; Shareghi, Behzad

2016-06-01

Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus. Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli . The Y. intermedia phytase gene was optimized according to the codon usage in E. coli . The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni 2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg -1 ) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.

Distant failure prediction for early stage NSCLC by analyzing PET with sparse representation

NASA Astrophysics Data System (ADS)

Hao, Hongxia; Zhou, Zhiguo; Wang, Jing

2017-03-01

Positron emission tomography (PET) imaging has been widely explored for treatment outcome prediction. Radiomicsdriven methods provide a new insight to quantitatively explore underlying information from PET images. However, it is still a challenging problem to automatically extract clinically meaningful features for prognosis. In this work, we develop a PET-guided distant failure predictive model for early stage non-small cell lung cancer (NSCLC) patients after stereotactic ablative radiotherapy (SABR) by using sparse representation. The proposed method does not need precalculated features and can learn intrinsically distinctive features contributing to classification of patients with distant failure. The proposed framework includes two main parts: 1) intra-tumor heterogeneity description; and 2) dictionary pair learning based sparse representation. Tumor heterogeneity is initially captured through anisotropic kernel and represented as a set of concatenated vectors, which forms the sample gallery. Then, given a test tumor image, its identity (i.e., distant failure or not) is classified by applying the dictionary pair learning based sparse representation. We evaluate the proposed approach on 48 NSCLC patients treated by SABR at our institute. Experimental results show that the proposed approach can achieve an area under the characteristic curve (AUC) of 0.70 with a sensitivity of 69.87% and a specificity of 69.51% using a five-fold cross validation.

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

PubMed

Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

1999-04-01

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

A meaty matter. Pet diet and the vegetarian's dilemma.

PubMed

Rothgerber, Hank

2013-09-01

The present research examined pet ownership, current pet diet, and guilt associated with pet diet among a fairly large sample of non-meat-eaters (n=515). It specifically focused on the conflict that pits feeding one's pet an animal-based diet that may be perceived as best promoting their well-being with concerns over animal welfare and environmental degradation threatened by such diets, here labeled the vegetarian's dilemma. Questionnaire responses indicated that ethically motivated meat abstainers were more likely to own pets and owned more of them than those motivated by health concerns or a combination of ethical and health concerns. Vegans and those resisting meat on ethical grounds were more likely to feed their pet a vegetarian diet and expressed the greatest concerns over feeding their pet an animal-based diet. For vegans and ethical meat abstainers, it is suggested that questions concerning what to feed their pet approaches a tragic tradeoff contrasting two sacred values: protecting the well-being of their pets and protecting the well-being of other animals and the environment. For meat abstainers motivated by health concerns, this constitutes a relatively easy moral problem because the primary concern for such individuals is the health of their pet with less or no regard for other ramifications of the decision, i.e., harming other animals or the environment. Copyright © 2013 Elsevier Ltd. All rights reserved.

68Ga-PSMA-11 Dynamic PET/CT Imaging in Primary Prostate Cancer.

PubMed

Sachpekidis, Christos; Kopka, Klaus; Eder, Matthias; Hadaschik, Boris A; Freitag, Martin T; Pan, Leyun; Haberkorn, Uwe; Dimitrakopoulou-Strauss, Antonia

2016-11-01

The aim of our study is to assess the pharmacokinetics and biodistribution of Ga-PSMA-11 in patients suffering from primary prostate cancer (PC) by means of dynamic and whole-body PET/CT. Twenty-four patients with primary, previously untreated PC were enrolled in the study. All patients underwent dynamic PET/CT (dPET/CT) scanning of the pelvis and whole-body PET/CT studies with Ga-PSMA-11. The evaluation of dPET/CT studies was based on qualitative evaluation, SUV calculation, and quantitative analysis based on two-tissue compartment modeling and a noncompartmental approach leading to the extraction of fractal dimension (FD). A total of 23/24 patients (95.8%) were Ga-PSMA-11 positive. In 9/24 patients (37.5%), metastatic lesions were detected. PC-associated lesions demonstrated the following mean values: SUVaverage = 14.3, SUVmax = 23.4, K1 = 0.24 (1/min), k3 = 0.34 (1/min), influx = 0.15 (1/min), and FD = 1.27. The parameters SUVaverage, SUVmax, k3, influx, and FD derived from PC-associated lesions were significantly higher than respective values derived from reference prostate tissue. Time-activity curves derived from PC-associated lesions revealed an increasing Ga-PSMA-11 accumulation during dynamic PET acquisition. Correlation analysis revealed a moderate but significant correlation between PSA levels and SUVaverage (r = 0.60) and SUVmax (r = 0.57), and a weak but significant correlation between Gleason score and SUVaverage (r = 0.33) and SUVmax (r = 0.28). Ga-PSMA-11 PET/CT confirmed its capacity in detecting primary PC with a detection rate of 95.8%. Dynamic PET/CT studies of the pelvis revealed an increase in tracer uptake in PC-associated lesions during the 60 minutes of dynamic PET acquisition, a finding with potential applications in anti-PSMA approaches.

Optimization of culturing conditions of recombined Escherichia coli to produce umami octopeptide-containing protein.

PubMed

Zhang, Yin; Wei, Xiong; Lu, Zhou; Pan, Zhongli; Gou, Xinhua; Venkitasamy, Chandrasekar; Guo, Siya; Zhao, Liming

2017-07-15

Using synthesized peptides to verify the taste of natural peptides was probably the leading cause for tasting disputes regarding umami peptides. A novel method was developed to prepare the natural peptide which could be used to verify the taste of umami peptide. A controversial octopeptide was selected and gene engineering was used to structure its Escherichia coli. expressing vector. A response surface method was adopted to optimize the expression conditions of the recombinant protein. The results of SDS-PAGE for the recombinant protein indicated that the recombinant expression system was successfully structured. The fitting results of the response surface experiment showed that the OD 600 value was the key factor which influenced the expression of the recombinant protein. The optimal culturing process conditions predicted with the fitting model were an OD 600 value of 0.5, an IPTG concentration of 0.6mM, a culturing temperature of 28.75°C and a culturing time of 5h. Copyright © 2017 Elsevier Ltd. All rights reserved.

Construction of siRNA/miRNA expression vectors based on a one-step PCR process

PubMed Central

Xu, Jun; Zeng, Jie Qiong; Wan, Gang; Hu, Gui Bin; Yan, Hong; Ma, Li Xin

2009-01-01

Background RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems. Results Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate. Conclusion This novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries. PMID:19490634

18F-FDG PET/CT as an Indicator of Survival in Ewing Sarcoma of Bone

PubMed Central

Salem, Usama; Amini, Behrang; Chuang, Hubert H.; Daw, Najat C.; Wei, Wei; Haygood, Tamara Miner; Madewell, John E.; Costelloe, Colleen M.

2017-01-01

Objective: The existing literature of 18 F-FDG PET/CT in Ewing sarcoma investigates mixed populations of patients with both soft tissue and bone primary tumors. The aim of our study was to evaluate whether the maximum standardized uptake value (SUVmax) obtained with 18F-FDG PET/CT before and after induction chemotherapy can be used as an indicator of survival in patients with Ewing sarcoma originating exclusively in the skeleton. Materials and Methods: A retrospective database search from 2004-2011 identified 28 patients who underwent 18 F-FDG PET/CT before (SUV1, n= 28) and after (SUV2, n=23) induction chemotherapy. Mean follow up was 3.3 years and median follow up for survivors was 6.3 years (range: 2.6-9.8 years). Multivariate and univariate Cox proportional hazard model was used to assess for correlation of SUV1, SUV2, and the change in SUVmax with overall survival (OS) and progression-free survival (PFS). Results: Mean SUVmax was 10.74 before (SUV1) and after 4.11 (SUV2) induction chemotherapy. High SUV1 (HR = 1.05, 95% CI: 1.0-1.1, P = 0.01) and SUV2 (HR =1.2, 95% CI: 1.0-1.4, P = 0.01) were associated with worse OS. A cut off point of 11.6 was identified for SUV1. SUV1 higher than 11.6 had significantly worse OS (HR = 5.71, 95% CI: 1.85 - 17.61, P = 0.003) and PFS (HR = 3.16, 95% CI: 1.13 - 8.79, P = 0.03, P < 0.05 is significant). Conclusion: 18F-FDG PET/CT can be used as a prognostic indicator for survival in primary Ewing sarcoma of bone. PMID:28928879

Mapping of Heavy Chain Genes for Mouse Immunoglobulins M and D

NASA Astrophysics Data System (ADS)

Liu, Chih-Ping; Tucker, Philip W.; Mushinski, J. Frederic; Blattner, Frederick R.

1980-09-01

A single DNA fragment containing both μ and δ immunoglobulin heavy chain genes has been cloned from normal BALB/c mouse liver DNA with a new λ phage vector Charon 28. The physical distance between the membrane terminal exon of μ and the first domain of δ is 2466 base pairs, with δ on the 3' side of μ . A single transcript could contain a variable region and both μ and δ constant regions. The dual expression of immunoglobulins M and D on spleen B cells may be due to alternate splicing of this transcript.

[18F]fluoroethylcholine-PET/CT imaging for radiation treatment planning of recurrent and primary prostate cancer with dose escalation to PET/CT-positive lymph nodes.

PubMed

Würschmidt, Florian; Petersen, Cordula; Wahl, Andreas; Dahle, Jörg; Kretschmer, Matthias

2011-05-01

At present there is no consensus on irradiation treatment volumes for intermediate to high-risk primary cancers or recurrent disease. Conventional imaging modalities, such as CT, MRI and transrectal ultrasound, are considered suboptimal for treatment decisions. Choline-PET/CT might be considered as the imaging modality in radiooncology to select and delineate clinical target volumes extending the prostate gland or prostate fossa. In conjunction with intensity modulated radiotherapy (IMRT) and imaged guided radiotherapy (IGRT), it might offer the opportunity of dose escalation to selected sites while avoiding unnecessary irradiation of healthy tissues. Twenty-six patients with primary (n = 7) or recurrent (n = 19) prostate cancer received Choline-PET/CT planned 3D conformal or intensity modulated radiotherapy. The median age of the patients was 65 yrs (range 45 to 78 yrs). PET/CT-scans with F18-fluoroethylcholine (FEC) were performed on a combined PET/CT-scanner equipped for radiation therapy planning. The majority of patients had intermediate to high risk prostate cancer. All patients received 3D conformal or intensity modulated and imaged guided radiotherapy with megavoltage cone beam CT. The median dose to primary tumours was 75.6 Gy and to FEC-positive recurrent lymph nodal sites 66,6 Gy. The median follow-up time was 28.8 months. The mean SUV(max) in primary cancer was 5,97 in the prostate gland and 3,2 in pelvic lymph nodes. Patients with recurrent cancer had a mean SUV(max) of 4,38. Two patients had negative PET/CT scans. At 28 months the overall survival rate is 94%. Biochemical relapse free survival is 83% for primary cancer and 49% for recurrent tumours. Distant disease free survival is 100% and 75% for primary and recurrent cancer, respectively. Acute normal tissue toxicity was mild in 85% and moderate (grade 2) in 15%. No or mild late side effects were observed in the majority of patients (84%). One patient had a severe bladder shrinkage (grade 4) after a previous treatment with TUR of the prostate and seed implantation. FEC-PET/CT planning could be helpful in dose escalation to lymph nodal sites of prostate cancer.

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

PubMed

Tagliavia, Marcello; Cuttitta, Angela

2016-01-01

High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

68Ga-PSMA-PET/CT in Patients With Biochemical Prostate Cancer Recurrence and Negative 18F-Choline-PET/CT.

PubMed

Bluemel, Christina; Krebs, Markus; Polat, Bülent; Linke, Fränze; Eiber, Matthias; Samnick, Samuel; Lapa, Constantin; Lassmann, Michael; Riedmiller, Hubertus; Czernin, Johannes; Rubello, Domenico; Bley, Thorsten; Kropf, Saskia; Wester, Hans-Juergen; Buck, Andreas K; Herrmann, Ken

2016-07-01

Investigating the value of Ga-PSMA-PET/CT in biochemically recurring prostate cancer patients with negative F-choline-PET/CT. One hundred thirty-nine consecutive patients with biochemical recurrence after curative (surgery and/or radiotherapy) therapy were offered participation in this sequential clinical imaging approach. Patients first underwent an F-choline-PET/CT. If negative, an additional Ga-PSMA-PET/CT was offered. One hundred twenty-five of 139 eligible patients were included in the study; 32 patients underwent additional Ga-PSMA-PET/CT. Patients with equivocal findings (n = 5) on F-choline-PET/CT and those who declined the additional Ga-PSMA-PET/CT (n = 9) were excluded. Images were analyzed visually for the presence of suspicious lesions. Findings on PET/CT were correlated with PSA level, PSA doubling time (dt), and PSA velocity (vel). The overall detection rates were 85.6% (107/125) for the sequential imaging approach and 74.4% (93/125) for F-choline-PET/CT alone. Ga-PSMA-PET/CT detected sites of recurrence in 43.8% (14/32) of the choline-negative patients. Detection rates of the sequential imaging approach and F-choline-PET/CT alone increased with higher serum PSA levels and PSA vel. Subgroup analysis of Ga-PSMA-PET/CT in F-choline negative patients revealed detection rates of 28.6%, 45.5%, and 71.4% for PSA levels of 0.2 or greater to less than 1 ng/mL, 1 to 2 ng/mL, and greater than 2 ng/mL, respectively. The sequential imaging approach designed to limit Ga-PSMA imaging to patients with negative choline scans resulted in high detection rates. Ga-PSMA-PET/CT identified sites of recurrent disease in 43.8% of the patients with negative F-choline PET/CT scans.

68Ga-PSMA-PET/CT in Patients With Biochemical Prostate Cancer Recurrence and Negative 18F-Choline-PET/CT

PubMed Central

Bluemel, Christina; Krebs, Markus; Polat, Bülent; Linke, Fränze; Eiber, Matthias; Samnick, Samuel; Lapa, Constantin; Lassmann, Michael; Riedmiller, Hubertus; Czernin, Johannes; Rubello, Domenico; Bley, Thorsten; Kropf, Saskia; Wester, Hans-Juergen; Buck, Andreas K.; Herrmann, Ken

2016-01-01

Purpose Investigating the value of 68Ga-PSMA-PET/CT in biochemically recurring prostate cancer patients with negative 18F-choline-PET/CT. Patients and Methods One hundred thirty-nine consecutive patients with biochemical recurrence after curative (surgery and/or radiotherapy) therapy were offered participation in this sequential clinical imaging approach. Patients first underwent an 18F-choline-PET/CT. If negative, an additional 68Ga-PSMA-PET/CTwas offered. One hundred twenty-five of 139 eligible patients were included in the study; 32 patients underwent additional 68Ga-PSMA-PET/CT. Patients with equivocal findings (n = 5) on 18F-choline-PET/CT and those who declined the additional 68Ga-PSMA-PET/CT (n = 9) were excluded. Images were analyzed visually for the presence of suspicious lesions. Findings on PET/CT were correlated with PSA level, PSA doubling time (dt), and PSA velocity (vel). Results The overall detection rates were 85.6% (107/125) for the sequential imaging approach and 74.4% (93/125) for 18F-choline-PET/CT alone. 68Ga-PSMA-PET/CT detected sites of recurrence in 43.8% (14/32) of the choline-negative patients. Detection rates of the sequential imaging approach and 18F-choline-PET/CT alone increased with higher serum PSA levels and PSA vel. Subgroup analysis of 68Ga-PSMA-PET/CT in 18F-choline negative patients revealed detection rates of 28.6%, 45.5%, and 71.4% for PSA levels of 0.2 or greater to less than 1 ng/mL, 1 to 2 ng/mL, and greater than 2 ng/mL, respectively. Conclusions The sequential imaging approach designed to limit 68Ga-PSMA imaging to patients with negative choline scans resulted in high detection rates. 68Ga-PSMA-PET/CT identified sites of recurrent disease in 43.8% of the patients with negative 18F-choline PET/CT scans. PMID:26975008

TU-H-CAMPUS-JeP2-03: Machine-Learning-Based Delineation Framework of GTV Regions of Solid and Ground Glass Opacity Lung Tumors at Datasets of Planning CT and PET/CT Images

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikushima, K; Arimura, H; Jin, Z

Purpose: In radiation treatment planning, delineation of gross tumor volume (GTV) is very important, because the GTVs affect the accuracies of radiation therapy procedure. To assist radiation oncologists in the delineation of GTV regions while treatment planning for lung cancer, we have proposed a machine-learning-based delineation framework of GTV regions of solid and ground glass opacity (GGO) lung tumors following by optimum contour selection (OCS) method. Methods: Our basic idea was to feed voxel-based image features around GTV contours determined by radiation oncologists into a machine learning classifier in the training step, after which the classifier produced the degree ofmore » GTV for each voxel in the testing step. Ten data sets of planning CT and PET/CT images were selected for this study. The support vector machine (SVM), which learned voxel-based features which include voxel value and magnitudes of image gradient vector that obtained from each voxel in the planning CT and PET/CT images, extracted initial GTV regions. The final GTV regions were determined using the OCS method that was able to select a global optimum object contour based on multiple active delineations with a level set method around the GTV. To evaluate the results of proposed framework for ten cases (solid:6, GGO:4), we used the three-dimensional Dice similarity coefficient (DSC), which denoted the degree of region similarity between the GTVs delineated by radiation oncologists and the proposed framework. Results: The proposed method achieved an average three-dimensional DSC of 0.81 for ten lung cancer patients, while a standardized uptake value-based method segmented GTV regions with the DSC of 0.43. The average DSCs for solid and GGO were 0.84 and 0.76, respectively, obtained by the proposed framework. Conclusion: The proposed framework with the support vector machine may be useful for assisting radiation oncologists in delineating solid and GGO lung tumors.« less